VIRAL VECTORS FOR THE TREATMENT OF DIABETES

Abstract

They are provided gene constructs comprising a nucleotide sequence encoding the Insulin-like growth factor 1 (IGF-1) of a mammal; and target sequences of a microRNA of a tissue where the expression of IGF-1 is wanted to be prevented, wherein the sequences (a) and (b) are operationally linked to a promoter of ubiquitous expression. Also provided are expression vectors comprising the gene construct and pharmaceutical compositions comprising them. They are useful in the treatment and/or prevention of diabetes mellitus in mammals, wherein a dysfunction and/or a loss of the beta-cells of the islets of Langerhans is present.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a divisional of U.S. application Ser. No. 15/532,958, filed Jun. 2, 2017, which is the 35 U.S.C. 371 National Stage of International Application Number PCT/EP2015/078878, filed Dec. 7, 2015, which claims priority from European patent application 14196536.8, filed Dec. 5, 2014, the contents of which are incorporated herein by reference.

SEQUENCE LISTING SUBMISSION VIA EFS-WEB

[0002] A computer readable text file, entitled "4525_0070002_Seqlisting_ST25.txt", created on or about Mar. 31, 2021, with a file size of about 169,312 bytes contains the sequence listing for this application and is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to the fields of medicine and gene therapy and, particularly, to gene constructs and expression vectors for the treatment and/or prevention of diabetes mellitus in mammals.

BACKGROUND ART

[0004] Diabetes

[0005] Diabetes comprises a group of metabolic diseases characterized by the presence of hyperglycemia as a result of defects in insulin secretion, in insulin action, or both. The chronic hyperglycemia of diabetes is associated with long-term damage, such as dysfunction and failure of various organs and especially the eyes, kidneys, nerves, heart and blood vessels. Currently it is estimated to exist in the world more than 380 million of people with diabetes and it is expected that this number will be increased to over 590 million by 2035 (International Diabetes Federation). Most cases of diabetes fall into two broad categories: type 1 and type 2 diabetes. Type 1 diabetes is caused by autoimmune destruction of pancreatic .beta. cells. In contrast, type 2 diabetes is caused by insulin resistance in peripheral tissues (mainly muscle, liver and adipose tissue) and by an inadequate secretion of the hormone insulin. Defects in insulin secretion observed in type 2 diabetes are probably caused by the combination of both cellular dysfunction and reduction in .beta.-cell mass. Thus, in both type 1 diabetes and type 2 diabetes, ultimately, there is an insufficient functional .beta.-cell mass (American Diabetes Association, 2014).

[0006] Type 1 Diabetes

[0007] Type 1 diabetes (T1D) represents 5-10% of diabetic patients and generally develops in childhood or adolescence individuals. T1D results from the autoimmune destruction of pancreatic .beta.-cells, which leads to insulin deficiency and hyperglycemia. The spontaneous onset of T1D in humans is preceded by a progressive leukocytic infiltration into the islets (insulitis), which persists for a relatively long period of time before the massive destruction of cells. This disease becomes clinically apparent after a preclinical period of variable duration during which the autoimmune destruction reduces the .beta.-cells mass in pancreatic islet so that glucose levels in blood cannot be maintained at physiological range. The lack of insulin leads to hyperglycemia and ketoacidosis, which can end up causing a coma and sometimes the death of the patient. T1D patients are diagnosed after loosing almost the entire mass of .beta.-cells, and require replacement therapy with insulin to survive. Therefore, interventions aimed to stop the immune attack on the islets and to promote .beta.-cell survival are of great interest for the treatment of diabetes.

[0008] Current Treatments for Type 1 Diabetes

[0009] Current treatments for T1D are based on insulin replacement therapies, which are required throughout the life of the patient to reduce blood glucose levels. However, these treatments do not prevent the development of secondary complications arising from long-term chronic hyperglycemia, which are the main cause of death of patients with T1D. In addition, patients are exposed to episodes of hypoglycemia, with risk of death, because this therapy does not allow reaching the sophisticated level of physiological regulation. Pancreas or islet transplant also offers a therapeutic option for T1D since they provide a source of physiologically regulated endogenous insulin. However, the eventual failure of most transplanted islets after recurrent autoimmune destruction, the shortage of donors, the immune rejection and the need of immunosuppression make these alternatives very limited.

[0010] On the other hand, therapies exist based on immunomodulators, which would delay the onset of T1D and/or would protect the residual .beta.-cells from the autoimmune attack thus maintaining endogenous insulin production. The first of these treatments showed limited efficacy but new approaches are being developed For example, there are anti-CD3 antibodies which reduce the number of circulating T lymphocytes in the patient. However, Phase III clinical trials with these monoclonal antibodies did not get the expected initial objectives (Sherry et al., 2011). Another antibody, anti-CD20, against B lymphocytes, is being tested on diabetics patients. After a year of treatment with anti-CD20 patients had improved function of .beta. cells and needed lower insulin doses compared to controls during the first six months, but afterwards, the treated group had finally lost the .beta. cell mass (Pescovitz et al., 2014).

[0011] Another area under development is the study of embryonic stem cells (ESC) and induced pluripotent cells (iPS), which through different strategies can be differentiated or reprogrammed to cells producing insulin and in the future could become a potential cellular therapy for T1D. It has been possible to reprogram adult fibroblasts from diabetic patients to insulin-producing cells by inserting only three transcription factors: octamer-binding transcription factor 4 (Oct-4), SRY (Sex Determining Region Y)-box 2 (Sox-2) and Kruppel-like factor 4 (KLF-4). There are many groups that are moving towards differentiating ESC and iPS cells to PDX-1+ capable of producing insulin or secreting peptide C. However, the biggest challenge in reprogramming is to get a fully differentiated state of .beta. like cells so that they can produce insulin in a finely regulated way and, even so, the reprogrammed cells would not be protected from autoimmune destruction. In addition, studies are still needed to assess the risks of autoimmune destruction of transplanted iPS- or ESC-derived cells and the tumorigenic potential of such cells.

[0012] Therefore there is a need to develeop new therapeutic strategies to counteract type 1 diabetes.

[0013] Gene therapy offers a new treatment tool with great potential. For example it can counteract the autoimmune attack against .beta. cells and/or to regenerate .beta. cell mass. Among possible candidate genes for the treatment of diabetes, insulin-like growth factor 1 (IGF-1) is known for its immunomodulatory properties (Smith, 2010) and its control over the proliferation and survival of .beta.-cells (Hill and Hogg, 1992).

[0014] Insulin-Like Growth Factor 1 (IGF-1)

[0015] IGF-1 is a major mediator of both pre-natal and post-natal growth and has shown an important role in the development of the pancreas as it stimulates the proliferation and differentiation of .beta.-cells. Additional, IGF-1 is considered to be one of the main interplayers of the endocrine and immune system crosstalk.

[0016] Approaches with Recombinant Protein Igf-1

[0017] Since IGF-1 and insulin share the same cell signaling pathway, factor IGF-1 has been used to try to treat diabetes showing benefits (Cheetham et al., 1995; Savage et al., 2004). The intravenous administration of recombinant protein IGF-1 in patients with marked insulin resistance produces similar effects to insulin, improving glycemic control and other metabolic parameters. However, the increase in circulating levels of IGF-1 involves the development of undesired side effects (Jabri et al., 1994).

[0018] Moreover, it was observed that daily subcutaneous administration of IGF-1 recombinant protein in pre-diabetic NOD mice reduced the severity of insulitis in the islets and the incidence of T1D (Bergerot et al., 1995; Kaino et al., 1996). Furthermore, the administration of IGF-1 alone or in complex with IGFBP-3 protected islets of insulitis and delayed the onset of T1D in NOD mouse. Similarly, the transfer of autoreactive T cells to NOD mice treated with IGF-1 reduced the incidence of diabetes due to a reduction in insulitis and to alterations in the activation of T cells of the immune system (Bergerot et al., 1996). Finally, it was observed that the plasmid-derived IGF-1 expression in the non-parenchymal liver cells was able to preserve .beta.-cell mass and prevent the development of autoimmune diabetes in transgenic mice for IFN-.beta. treated with Streptozotocin. This prevention was also due to an increase in regulatory T cells in the pancreas of these mice (Anguela et al., 2013).

[0019] Transgenic Animals Expressing IGF-1

[0020] It has been reported that local overexpression of IGF-1 in the .beta.-cell is able to prevent infiltration of the islet and .beta.-cell death in transgenic mouse RIP-1/IFN-.beta., a model of lymphocyte infiltration of the islets with increased susceptibility to the development of diabetes (Casellas et al., 2006). In addition, the transgenic expression of IGF-1 into .beta.-cells of diabetic mice induced by treatment with multiple doses of Streptozotocin (STZ, 5x50mg/kg) was able to regenerate the endocrine pancreas (Agudo et al., 2008; George et al., 2002). However, these models are far from becoming feasible therapies for the treatment of diabetes and do not demonstrate whether the local expression of IGF-1 in pancreatic .beta.-cells is also able to prevent or counteract T1D in a model of spontaneous onset of the disease.

[0021] Although existing treatments for both type 1 and type 2 diabetes have significantly improved the quality of life of patients, these therapies have certain drawbacks and are not able to cure the disease. Because T1D patients require insulin therapy throughout their lives and have a high risk of secondary complications, new preventive and curative therapies are needed.

[0022] "Adeno-associated viral (AAV) vectors have emerged as very safe and effective delivery vehicles to mediate long-term transgene expression of therapeutic genes in a wide range of tissues in vivo both in adult animal models and in humans (Mingozzi & High 2011). Promising results have been obtained using AAV vectors administered directly into the pancreatic duct to achieve widespread pancreas transduction in mice (Jimenez et al 2011). Additionally, recent progress has shown that endogen microRNA target sequences (miRTs) can be included in the AAV expression cassette to efficiently control transgene expression (Brown & Naldini, 2009) which opens the door to new approaches of sophisticated regulation of vector tropism."

SUMMARY OF THE INVENTION

[0023] One problem to be solved by the present invention may be seen as related to the provision of a therapeutical approach for the treatment and/or prevention of diabetes in mammals, preferably diabetes mellitus in mammals. The solution is based on the provision of a gene construct encoding Insulin-like growth factor 1 (IGF-1) and expression vectors for being used in gene therapy.

[0024] The inventors have developed a gene therapy strategy directed to pancreas to counteract diabetes in the NOD mouse, which has many similarities with human T1D as will be seen below. An AAV8 vector expressing IGF-1 under the control of the ubiquitous CAG promoter was generated. In order to restrict the expression of IGF-1 in pancreas, the target sequences of microRNA-122A (expressed mainly in the liver, see FIG. 4) and microRNA-1 (expressed mainly in the heart, see FIG. 5) were linked to the construct at the 3'-UTR region . The construct contained four copies of the target sequence of miRNA-122A and four copies of the target sequence of miRNA-1 in the 3'-UTR region (AAV8-CAG- IGF1-dmiRT), in both cases fully complementary to miRNA-122A or miRNA-1.

[0025] It has been observed that intraductal administration of this vector was able to prevent the onset of hyperglycemia in NOD mouse. Thus, most of the animals administered with the vector encoding IGF-1 remained normoglycemic throughout the 28 weeks of follow-up and the incidence of diabetes was significantly reduced. Therefore, gene transfer of IGF-1 in the pancreas using AAV vectors represents a new gene therapy approach for diabetes, and particularly type 1 diabetes. The intraductal administration of vectors AAV8-CAG-IGF1-dmiRT precluded liver and heart IGF-1 overexpression as shown by similar IGF-1 expression levels to those observed in the control group in contrast to animals administered with vector without the target sequences of microRNAs (AAV8-CAG-IGF1). In the context of the invention the "precluding effect" of the vector or gene construct could be replaced by reduction of IGF-1 expression as later defined herein. However, the expression in the pancreas was not altered by the presence of these sequences. The levels of expression of microRNAs were maintained regardless of the diabetic stage of animals indicating that the precluding effect of the AAV construct in the liver or heart NOD animals was not lost even during the diabetic process. In the case of the liver, the precluding effect was even more sustained due to increased levels of expression of microRNA-122A in hyperglycemic NOD individuals.

[0026] The main advantages achieved by the gene construct and vector of the invention are:

[0027] 1) The intraductal administration of the vector to NOD mice resulted in an efficient transduction of exocrine and endocrine pancreas. Pancreatic islets were transduced mainly in the periphery but also core cells were genetically engineered. The transduction of .beta. cells on the periphery of the islets is important to combat the autoimmune destruction in T1D, because the autoimmune attack by infiltrating lymphocytes actively takes place in the periphery of the islets. Moreover, the administration (preferably pancreatic administration) of AAV8 vectors with an ubiquitous expression promoter makes possible the expression of IGF-1 in beta-cells of the islets as well as maximizing the number of acinar cells providing IGF-1 to not transduced beta- cells. Thus, in front of an autoimmune attack and of the selective destruction of a percentage of the beta-cells in the NOD islets, the exocrine pancreas would continue providing the therapeutic IGF-1 to the remaining cells.

[0028] 2) NOD mice showed high levels of expression of microRNA-122A in liver and of microRNA-1 in heart, regardless of the diabetic stage. Furthermore, the expression of these microRNAs was not detectable in the pancreas of NOD mice. Rermarkably, it is believed that this is the first time that the target sequences of the miRNAs 122a and 1 are used with success in NOD mice. In an embodiment, these target sequences are used to decrease, reduce or even preclude transgene expression in liver and/or heart.

[0029] 3) Overexpression (preferably pancreatic overexpression) of IGF-1 mediated by the pancreas intraductal administration of the AAV8-CAG-IGF-1-dmiRT vectors, protected NOD mice of developing spontaneous diabetes and reduced the incidence of diabetes. NOD mice were also protected against lymphocytic infiltration of the islets and preserved beta-cell mass over time.

[0030] Moreover, the long-term and effective expression provided by a single administration of the vectors of the invention represents a significant advantage over other therapies. For example, due to the short half-life of IGF-1 in circulation, treatment with recombinant protein IGF-1 requires a constant and repeated administration and, moreover, its effects have a limited duration once treatment is interrupted. Furthermore, the intraductal administration can potentially be applied to larger animals and humans, through a non-surgical and less invasive clinical process called endoscopic retrograde cholangiopancreatography (ERCP) (Hendrick et al, 2011).

[0031] Thus, a first aspect of the invention is related to a gene or expression construct comprising:

[0032] (a) a nucleotide sequence encoding the Insulin-like growth factor 1 (IGF-1) of a mammal; and (b) at least one target sequence of a microRNA of a tissue where the expression of IGF-1 is wanted to be prevented; wherein the sequences (a) and (b) are operationally linked to a promoter of ubiquitous expression.

[0033] A second aspect of the invention is an expression vector comprising the gene or expression construct as defined above.

[0034] A third aspect of the invention is the vector and/or the gene construct as defined previously, for use as a medicament.

[0035] A fourth aspect of the invention is the vector and/or the gene construct as defined previously, for use in treatment and/or prevention of diabetes mellitus in mammals, wherein a dysfunction and/or a loss of the beta-cells of the islets of Langerhans is present.

[0036] Finally, a fifth aspect of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a gene construct and/or of a vector as defined previously, together with one or more pharmaceutically acceptable excipients or vehicles.

[0037] The detailed description and examples shown below are presented for the purposes of providing those skilled in the art with a sufficiently clear and complete explanation of this invention, but should not be considered limitations on the essential aspects contemplated therein, as presented in earlier sections of this description.

DETAILED DESCRIPTION OF THE INVENTION

[0038] First aspect of the invention: Gene construct

[0039] The first aspect of the invention is related to a gene construct comprising: (a) a nucleotide sequence encoding the Insulin-like growth factor 1 (IGF-1) of a mammal; and (b) at least one target sequence of a microRNA of a tissue where the expression of IGF-1 is wanted to be prevented; wherein the sequences (a) and (b) are operationally linked to a promoter of ubiquitous expression.

[0040] A gene construct according to the invention can also be called "expression cassette" or "expression construct" and refers to a gene or a group of genes, including the gene that encodes a protein of interest, which is operatively linked to a promoter that controls its expression. Being "operationally linked" should be understood, as the sequence of the gene is placed after the promoter sequence or relatively close in the event that restriction fragments or elements that provide stability to the construction are included. The construction can also comprise small gene fragments with useful sequences in order to adapt it to the desired expression systems, which the skilled in art will know. The general part of this application entitled "general definitions" comprises more detail as to said "gene construct".

[0041] The gene construct of the invention allows to express preferably, when it is placed in a suitable vector, IGF-1 of a mammal in a tissue of interest, preventing its expression in other tissues where the expression is wanted to be prevented. For this reason, the construct comprises target sequences of microRNAs that are specific to those tissues where the expression is not of interest. In a preferred embodiment, these microRNAs are expressed in those tissues where IGF-1 over expression is not of interest. IGF-1 expression may not be of interest for example when non-desired side-effects are expected. For example liver-overexpression may mediate increased circulating levels of IGF-1 which may lead to non-desired side effects (Jabri et al 1994).

[0042] As will be seen below, in the present invention, the expression (preferably the over-expression) of IGF-1 in mammals in heart and/or liver tissues, (preferably liver) should be prevented. The skilled person will understand that endogenous expression of IGF-1 may still occur. The present invention prevents expression (preferably over-expression) of IGF-1 in heart and/or liver tissues resulting from the gene construct or expression vector of the invention. In other words, the present invention prevents expression, preferably over-expression of IGF-1 in heart and/or liver tissues. They are tissues that are also transduced when an intraductal injection is performed to the biliar duct in order to achieve specific expression in the pancreas (Jimenez et al 2011). For this reason, in a particular embodiment, the target sequence of a microRNA of the gene construct is selected from those target sequences that bind to microRNAs expressed in heart and/or liver of the mammal.

[0043] The IGF-1 molecule is preferably expressed even more preferably over-expressed in the pancreatic tissue, more preferably solely in the pancreatic tissue and/or preferably the expression, (even more preferably the over-expression) of the IGF-1 molecule is to be prevented in non-pancreatic tissue.

[0044] In this context, the wording "non-pancreatic tissue" is preferably the liver or the heart. In this context, the wording "non-pancreatic tissue" is more preferably the liver or the heart when the vector is administered via intraductal injection.

[0045] In this context, "prevented" could be replaced by decreased or inhibited or precluded. The IGF-1 expression preferably over-expression is said to have been prevented in a non-pancreatic tissue/cell (i.e. preferably the liver and/or the heart when the vector is administered via intraductal injection) when the IGF-1 expression, preferably over-expression level in a given non-pancreatic tissue/cell is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% lower (or more preferably not detectable) than the expression preferably over-expression level of IGF-1 in the same non-pancreatic tissue/cell when a corresponding control vector without the target sequence of a microRNA is being administered. The skilled person may assess the expression level in a cell derived from said tissue. IGF-1 expression may be assessed at the transcript level via PCR or at the protein level via Western blotting. The skilled person knows which cells could be used. Preferred cells are derived from a subject and are hepatocytes for the liver tissue and cardiomyocytes for the heart tissue. Cell lines may also be used as Hub7 (human liver cell line) (Geisler 2011) and differentiated C2Cl2 (mouse myoblast) (Kang 2012).

[0046] Within the context of the invention, a target sequence that binds miRNAs that are specific to those tissues whose expression is not of interest could be replaced by a target sequence that binds miRNAs that are expressed in those tissues in which IGF-1 expression is to be prevented. The skilled person understands that such miRNA may be expressed in other tissues. For example miRNA-1 is expressed in brown adipose tissues. In an embodiment, when the vector is administered by intraductal injection, the target sequence preferably binds a miRNA that is expressed in the heart and/or the liver (preferably the liver). The skilled person is well aware of techniques allowing the identification of miRNAs specifically expressed in these tissues. Preferred miRNAs and associated target sequences are defined later herein. In the context of the invention, a target sequence of a miRNA preferably means at least one. It therefore means that one, two, three, four or at least one, at least two, at least three, at least four target sequence of a miRNA are present in said construct. The same holds for each target sequence of one miRNA as defined herein. Depending on the expression level of the miRNA used, the skilled person may have to fine tune the optimal number of target sequence to obtain a prevention of the expression, preferably over-expression of IGF-1 in the tissue concerned as earlier defined herein (i.e. IGF-1 expression preferably over-expression level in a given non-pancreatic tissue/cell is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% lower (or more preferably not detectable) than the expression preferably over-expression level of IGF-1 in the same non-pancreatic tissue/cell when a corresponding control vector without the target sequence of a microRNA is being administered).

[0047] In a particular embodiment, the nucleotide sequence encoding the IGF-1 is selected from a nucleotide sequence encoding the human IGF-1 protein, which comprises an amino acid sequence selected from the group consisting of sequences SEQ ID NOs: 23 to 27; and a nucleotide sequence encoding the murine IGF-1 protein, which comprises an amino acid sequence selected from the group consisting of sequences SEQ ID NOs: 28 to 29, and 44 to 47.

[0048] SEQ ID NO: 23 corresponds to the human protein of 137 amino acids with accession number at the Protein database from NCBI NP_001104754, version 1 of 25 May 2014. It is the form known as preprolGF-1, isoform 2 (isoform of IGF-1a).

[0049] SEQ ID NO: 24 corresponds to the human protein of 195 amino acids with accession number at the Protein database from NCBI NP_001104755, version 1 of 25 May 2014. It is the form known as preprolGF-1, isoform 3 (isoform of IGF-1b). This human isoform does not have any murine counterpart.

[0050] SEQ ID NO: 25 corresponds to the human protein of 158 amino acids with accession number at the Protein database from NCBI NP_001104753, version 1 of 25 May 2014. It is the form known as preprolGF-1, isoform 1 (isoform of IGF-1c/MGF). This human isoform is the equivalent of murine IGF-1b. It is also named MGF (Mechano Growth Factor).

[0051] SEQ ID NO: 26 corresponds to the human protein of 153 amino acids with accession number at the Protein database from NCBI NP_000609, version 1 of 25 May 2014. It is the form known as preprolGF-1, isoform 4 (isoform of IGF-1c/MGF).

[0052] SEQ ID NO: 27 corresponds to the human protein of 179 amino acids with accession number at the Protein database from NCBI XP_005268892, version 1 of 3 Feb. 2014. It is the form known as IGF-1, isoform X1. No isoform type has been attributed to this sequence.

[0053] SEQ ID NO: 28 corresponds to the murine protein of 137 amino acids with accession number at the Protein database from NCBI NP_001104746, version 1 of 18 May 2014. It is the form known as preprolGF-1, isoform 5 (isoform of IGF-1a).

[0054] SEQ ID NO: 29 corresponds to the murine protein of 133 amino acids with accession number at the Protein database from NCBI AAH12409, version 1 of 15 Jul. 2006 (isoform of IGF-1b/MGF).

[0055] SEQ ID NO: 44 corresponds to the murine protein of 159 amino acids with accession number at the Protein database from NCBI NP_034642, version 2 of 18 May 2014. It is the form known as transcription variant 1 (isoform of IGF-1b/MGF).

[0056] SEQ ID NO: 45 corresponds to the murine protein of 165 amino acids with accession number at the Protein database from NCBI NP_908941, version 1 of 18 May 2014. It is the form known as transcription variant 2. No isoform type has been attributed to this sequence.

[0057] SEQ ID NO: 46 corresponds to the murine protein of 143 amino acids with accession number at the Protein database from NCBI NP_001104744, version 1 of 18 May 2014. It is the form known as transcription variant 3 (isoform of IGF-1b/MGF).

[0058] SEQ ID NO: 47 corresponds to the murine protein of 153 amino acids with accession number at the Protein database from NCBI NP_001104745, version 1 of 18 May 2014. It is the form known as transcription variant 4 (isoform of IGF-1a).

[0059] In another particular embodiment, the gene construct according to the invention comprises a nucleotide sequence encoding the IGF-1 of a mammal, which is selected from the group consisting of sequences SEQ ID NOs: 1 to 7, and 48 to 51.

[0060] SEQ ID NO: 1 corresponds to the murine nucleotide sequence with accession number at the Nucleotide database from NCBI NM_001111276, version 1 of 18 May 2014. It encodes the amino acid sequence NP_001104746 (SEQ ID NO: 28) above mentioned.

[0061] SEQ ID NO: 2 corresponds to the murine nucleotide sequence with accession number at the Nucleotide database from NCBI BC012409, version 1 of 15 Jul. 2006. It encodes the amino acid sequence AAH12409 (SEQ ID NO: 29) above mentioned.

[0062] SEQ ID NO: 48 corresponds to the murine nucleotide sequence with accession number at the Nucleotide database from NCBI NM_010512, version 4 of 18 May 2014. It encodes the amino acid sequence NP_034642 (SEQ ID NO: 44) above mentioned.

[0063] SEQ ID NO: 49 corresponds to the murine nucleotide sequence with accession number at the Nucleotide database from NCBI NM_184052.3, version 3 of 18 May 2014. It encodes the amino acid sequence NP_908941 (SEQ ID NO: 45) above mentioned.

[0064] SEQ ID NO: 50 corresponds to the murine nucleotide sequence with accession number at the Nucleotide database from NCBI NM_001111274, version 1 of 18 May 2014. It encodes the amino acid sequence NP_001104744 (SEQ ID NO: 46) above mentioned.

[0065] SEQ ID NO: 51 corresponds to the murine nucleotide sequence with accession number at the Nucleotide database from NCBI NM_001111275, version 1 of 18 May 2014. It encodes the amino acid sequence NP_001104745 (SEQ ID NO: 47) above mentioned.

[0066] SEQ ID NO: 3 corresponds to the human nucleotide sequence with accession number at the Nucleotide database from NCBI NM_001111284, version 1 of 25 May 2014. It encodes the amino acid sequence NP_001104754 (SEQ ID NO: 23) above mentioned.

[0067] SEQ ID NO: 4 corresponds to the human nucleotide sequence with accession number at the Nucleotide database from NCBI NM_001111285, version 1 of 25 May 2014. It encodes the amino acid sequence NP_001104755 (SEQ ID NO: 24) above mentioned.

[0068] SEQ ID NO: 5 corresponds to the human nucleotide sequence with accession number at the Nucleotide database from NCBI NM_001111283, version 1 of 25 May 2014. It encodes the amino acid sequence NP_001104753 (SEQ ID NO: 25) above mentioned.

[0069] SEQ ID NO: 6 corresponds to the human nucleotide sequence with accession number at the Nucleotide database from NCBI NM_000618, version 3 of 25 May 2014. It encodes the amino acid sequence NP_000609 (SEQ ID NO: 26) above mentioned.

[0070] SEQ ID NO: 7 corresponds to the human nucleotide sequence with accession number at the Nucleotide database from NCBI XM_005268835, version 1 of 3 Feb. 2014. It encodes the amino acid sequence XP_005268892 (SEQ ID NO: 27) above mentioned.

[0071] The expert will understand that the invention comprises those variants of the nucleotide sequences encoding the proteins of interest and which have a percentage of identity with the sequences listed of at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% so that the proteins expressed have the same or similar activity to the IGF-1 of mammals. The same or similar activity means that at least one activity of IGF-1 is maintained to at least some extent in said variant. "At least one activity of IGF-1" means at least one anti-diabetic activity as later defined herein. "At least some extent" usually means at least 50%, 60%, 70%, 80%, 90% of the activity is maintained. The invention therefore contemplates the degeneracy of the genetic code and also includes nucleotide sequences with nucleotide variations that lead to silent or conservative mutations at protein level. Also included are amino acid mutations that do not affect the activity of the protein. The identity between two amino acid sequences is preferably determined by the algorithm Blastp, described in Altschul, S. F., et. al. 1997, and NCBI ncbi.nlm.nih.gov/BLAST. The invention encompasses variants of nucleic acids represented by nucleotide sequences encoding a protein and protein variants represented by an amino acid sequence. A more detailed explanation of the term variant is provided in the section entitled "general definition".

[0072] In another particular embodiment, optionally in combination with any of the embodiments above or below indicated, the gene construct comprises a target sequence of a microRNA, wherein the target is selected from a group consisting of sequences SEQ ID NOs: 8 to 22, 93, 94, 95 and/or combinations thereof:

[0073] This target sequence is preferably located in the 3'UTR region of the gene or expression construct. 3'UTR region could be replaced with 3'UTR end of said construct.

TABLE-US-00001 SEQ ID NO: 8 (5'caaacaccattgtcacactcca3'), target for the microRNA 122a (Accession Number to the miRBase database MI0000442); SEQ ID NO: 9 (5'AGTCACGTACTGTCTTGAACC3'), target for the microRNA 152 (MI0000462); SEQ ID NO: 10 (5'GGGTCACAAGTCTGATGGACAAG3'), target for the microRNA 199a-5p (MI0000242); SEQ ID NO: 11 (5'TGTCATCAGACGTGTAACCAAT3'), target for the microRNA 99a-3p (MI0000101); SEQ ID NO: 12 (5'TACTGGATACTTAACTGTCTG3'), target for the microRNA 215 (MI0000291); SEQ ID NO: 13 (5'ggctgtcaattcataggtcag3'), target for the microRNA 192 (MI0000234); SEQ ID NO: 14 (5'ACATTGTCGTTGAGGTACACCT3'), target for the microRNA 194 (MI0000488); SEQ ID NO: 15 (5'ttacatacttctttacattcca3'), target for the microRNA 1 (MI0000651); SEQ ID NO: 16 (5'AGTCACGTGATGTCTTGAAACA3'), target for the microRNA 148 (MI0000253); SEQ ID NO: 17 (5'AAACCAGGGGAAGTTGGTCGAC3'), target for the microRNA 133a (MI0000450); SEQ ID NO: 18 (5'ACCTTACATTCCTTCACACACC3'), target for the microRNA 206 (MI0000490); SEQ ID NO: 19 (5'ATTCCGTGCGCCACTTACGG3'), target for the microRNA 124 (MI0000443); SEQ ID NO: 20 (5'AGGGACTCTGGGAAATTGGACACT3'), target for the microRNA 125 (MI0000469); SEQ ID NO: 21 (5'ATTAGAGTCGACCGTTGACACT3'), target for the microRNA 216 (MI0000292); SEQ ID NO: 22 (5'GTCACGTTACAATTTTCCCGTA3') target for the microRNA 130 (MI0000448) (mirbase.org/, version 21 Jun. 2014). SEQ ID NO: 93 (5' TGTCATCAGACGTGTAACCAAT3'), target for the microRNA 199a-3p (MI0000242); SEQ ID NO: 94 (5'TATTCTGCTCGTTTTTCGAACA3'), target for the microRNA 208 (MI0000251); and SEQ ID NO: 95 (5'ATGTCATGACACTATTGACTT3'), target for the microRNA 101 (MI0000103) (mirbase.org/, version 4 Dec. 2015).

[0074] In an embodiment, the gene construct comprises at least one, at least two, at least three, or at least four copies or one or two or three or four copies of a target sequence of a microRNA which is selected from a group consisting of nucleotide sequences SEQ ID NO: 8 to 22, 93, 94, 95 and/or combinations thereof.

[0075] The skilled person will understand that a target of any new miRNA expressed in the tissues wherein the over-expression of IGF-1 needs to be prevented is also encompassed within the present invention. In an embodiment, the gene construct does not comprise as target sequence SEQ ID NO:21.

[0076] In a preferred embodiment, the gene construct comprises at least one, at least two, at least three, or at least four copies or one or two or three or four copies of a target sequence of a microRNA which is selected from a group consisting of nucleotide sequences selected from SEQ ID NO: 8 to 20, 22, 93, 94 and 95 and/or combinations thereof.

[0077] In a preferred embodiment, the gene construct comprises at least one, at least two, at least three, or at least four copies or one or two or three or four copies of a target sequence of a microRNA which is expressed in the liver (preferably selected from SEQ ID NO: 8-14, 16, 93, 95) and at least one, at least two, at least three, or at least four copies or one or two or three or four copies of a target sequence of a microRNA which is expressed in the heart (preferably selected from SEQ ID NO:15, 17, 18 and 94).

[0078] In a more particular embodiment, the gene construct comprises a target sequence of a microRNA, which is selected from a group consisting of nucleotide sequences SEQ ID NO: 8 and SEQ ID NO: 15, which are target of microRNA 122 and miRl, respectively.

[0079] In another particular embodiment, optionally in combination with any of the embodiments above or below indicated, the gene construct comprises at least one target sequence of the microRNA 122a and at least one target sequence of the microRNA 1. In a preferred embodiment, the gene construct comprises four copies of the target sequence of the microRNA 122a and four copies of the target sequence of the microRNA 1. In another preferred embodiment, the gene construct comprises at least one, at least two, at least three, at least four or one, two, three or four copies of the target sequence of the microRNA 122a and at least one, at least two, at least three, at least four or one, two, three or four copies of the target sequence of the microRNA 1.

[0080] As for the IGF-1 molecule and coding sequence, the invention is not limited to the specific target sequence of a miRNA as identified herein. The skilled person may identify variants of said target sequence that is still able to bind said miRNA to some extent. In this context, "some extent" means that the target sequence preferably retains at least 50%, 60%, 70%, 80%, 90%, 99% of the binding of the initial target sequence as assessed using an EMSA (Electrophoretic Mobility Shift Assay) and/or is still able to reduce, decrease, prevent or preclude the IGF-1 expression, preferably over-expression in the context of the gene construct or expression vector of the invention to at least 50%, 60%, 70%, 80%, 90%, 99% of the reduction of expression obtained using the initial target sequence in a similar gene construct or expression vector, preferably assessed in a cell as earlier explained herein. In this context a variant of a binding sequence may have at least 85%, at least 90%, at least 95%, at laest 96%, at least 97%, at least 98%, at least 99% identity with the specific binding sequence identified herein. Other particular embodiments of the invention are gene constructs comprising microRNA target sequences with homology or identity of at least 85% to 99% with the target of sequences SEQ ID NO: 8 to 22, 93, 94 and 95, preferably 8 to 20, 22, 93, 94 and 95 listed above. More particularly, the target sequences have a percentage of 85% homology with sequences SEQ ID NO:8 to 22, more preferably with SEQ ID NO:8 to 20, 22, 93, 94 and 95.

[0081] "MicroR" or "miRNA" or "microRNA" or "miR" as used herein, are small (.about.22 nucleotides) sequences evolutionarily conserved of regulatory RNAs involved in gene silencing of RNA at post-transcriptional level (See Bartel DP. 2004). Through the base pairing with complementary regions (most often in the untranslated region 3'(3'UTR) of the cellular messenger RNA (mRNA)), miRNAs may act to suppress mRNA translation or cause catalytic degradation of mRNA. Due to the differential expression among tissues, many cellular miRNAs can be exploited to mediate the differential expression of gene therapy vectors. Having multiple copies of target elements complementary to these tissue specific miRNAs (miRT) in vectors or viral vectors, the expression of the transgene (or gene from another species of interest) in an unwanted tissue can be efficiently inhibited. The complementarity of the miRNA target sequences may be complete or partial. In any case, while it is able to match with the miRNA is sufficient to prevent the expression of the transgene placed in a viral vector.

[0082] In another particular embodiment, optionally in combination with any of the embodiments above or below indicated, the gene construct comprises a constitutive and/or an ubiquitous promoter. In a preferred embodiment the promoter is an ubiquitous and constitutive promoter. The expression "a promoter of ubiquitous expression" could be replaced with "an ubiquitous promoter". Preferably, it comprises the CAG promoter, which is an ubiquitous promoter, which means that it can drive the expression in any or in many tissue(s) of a nucleotide sequence operatively linked to it. The CAG promoter refers to the combination based on the cytomegalovirus early enhancer element and the chicken beta actin promoter (see Alexopoulou A, et al, 2008). A preferred CAG promoter is represented by SEQ ID NO:52. A variant of said promoter (preferably represented by a sequence having at least 60% identity with SEQ ID NO:52) may also be used as long as said variant is said to be an active promoter as defined later herein.

[0083] A "constitutive" promoter is a promoter that is active under most physiological and developmental conditions. An "inducible" promoter is a promoter that is preferably regulated depending on physiological or developmental conditions. An inducible promoter may be active after drug delivery or light exposure. A "constitutive" promoter therefore is not regulated in the sense of an "inducible" promoter. A "tissue specific" promoter is preferably active in specific types of cells/tissues. As opposed to a "tissue-specific" promoter, the promoter used in the context of the invention is an "ubiquitous" promoter. An ubiquitous promoter may be defined as a promoter that is active in many or in any different tissue(s). Usually, "many" in this context may mean more than 5 or at least 6, 10, 15, 20 or in 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 different tissues. In a preferred embodiment, a promoter as used in the construct of the invention is ubiquitous and/or constitutive. In a more preferred embodiment, said promoter is ubiquitous and constitutive. In another preferred embodiment, the promoter as used herein is non-pancreas specific and not an ubiquitous promoter. It means it is not only or not solely or not exclusively active in pancreas. It may also be active in at least 1, 2, 3, 4, or 5 or more different tissues.

[0084] For "promoter" must be understood a nucleic acid fragment that functions to control the transcription of one or more polynucleotides, which is placed 5' upstream of the polynucleotide sequence(s), and which is structurally identified by the presence of a binding site for RNA dependent DNA polymerase, transcription initiation sites and, but not limited to, binding sites for transcription factors, repressors, and any other nucleotide sequences known in the art to act directly or indirectly to regulate the amount of transcription from the promoter.

[0085] A promoter is said to be active or is said to drive the expression of a nucleotide sequence operatively linked to it when it can initiate transcription of said nucleotide sequence in an expression system using a gene construct comprising said promoter operably linked to a nucleotide sequence of interest using a suitable assay such a RT-qPCR or Northern blotting (detection of the transcript). The activity of said promoter may also be assessed at the protein level using a suitable assay for the encoded protein such as Western blotting or an ELISA. A promoter is said to be capable to initiate transcription if a transcript can be detected or if an increase in a transcript or protein level is found of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 500%, 1000%, 1500% or 2000% as compared to transcription using a construct which only differs in that it is free of said promoter.

[0086] The term "polynucleotide" as used herein, refers to a nucleic acid molecule, either DNA or RNA, which contains deoxyribonucleotides or ribonucleotides. The nucleic acid may be double stranded, single stranded, or contain portions of both double stranded or single strand sequences. The term "polynucleotide" includes, but is not limited to, nucleic acid sequences with the ability to encode a polypeptide and nucleic acid sequences partially or completely complementary to a polynucleotide endogenous of the cell or of the subject administered with it so that, after the transcription of the same, generates a RNA molecule (e.g., microRNA, shRNA, siRNA) able to hybridize and inhibit the expression of the endogenous polynucleotide.

[0087] In another particular embodiment, optionally in combination with any of the embodiments listed below or above, the nucleotide sequence comprises a sequence that encodes any of the isoforms of IGF-1 of a mammal, particularly human, murine, dog or cat; the CAG promoter sequence and four copies of at least two targets of microRNA, particularly, four copies of the target sequence of miR122 and four copies of the target sequence of miR1.

[0088] The expert will know how to switch the nucleotide sequences encoding the murine protein IGF-1 in any of its isoforms, to the corresponding nucleotide sequences that encode the protein isoforms of human, canine (dog) or feline (cat) IGF-1. Within the context of the invention an IGF-1 or Igf1 refers to the nucleotide sequence encoding preprolGF-1a unless otherwise indicated. The invention is not limited to the use of preproIGF-1a. Some embodiments of the invention relate to the use of mechano growth factor (MGF), which corresponds to murine preprolGF-1b and human preprolGF1c. Other embodiments relate to the use of human preprolGF-1b.

[0089] Preferred IGF-1a proteins are represented by SEQ ID NO: 23, 28 and 47 or variants thereof. Preferred corresponding IGF-la nucleic acid are represented by SEQ ID NO: 1, 51 and 3 or variants thereof. Preferred MGF proteins are represented by SEQ ID NO: 25, 26, 29, 44 and 46 or variants thereof. Preferred corresponding MGF nucleic acid are represented by SEQ ID NO: 2, 48, 50, 5 and 6 or variants thereof.

[0090] Second Aspect of the Invention: Expression Vector

[0091] These gene constructs are placed in expression vectors. Thus, another aspect of the invention is an expression vector comprising the gene construct as defined in any of the preceding embodiments. The section entitled "general definitions" provides more detailed information as to the expression vector of the invention.

[0092] In a particular embodiment, the vector is characterized for being a viral vector, more preferably a viral vector selected from the group consisting of adenoviral vectors, adeno associated vectors or adeno associated viral vectors, retroviral vectors, and lentiviral vectors. These vectors are also known as adenovirus derived vector, adeno associated derived vector, retrovirus derived vector and lentivirus derived vector.

[0093] In a more particular embodiment, the vector is a viral adeno associated vector or adeno associated viral vector selected from the group consisting of adeno associated viral vector of serotype 6, adeno associated viral vector of serotype 7, adeno associated viral vector of serotype 8, adeno associated viral vector of serotype 9, adeno associated viral vector of serotype 10, adeno associated viral vector of serotype 11, adeno associated viral vector of serotype rh8, and adeno associated viral vector of serotype rh10. In a preferred embodiment, the vector is a adeno associated viral vector of serotype 8 (abbreviated AAV8). These serotypes of adeno associated viral vectors are known for their tropism in the pancreas.

[0094] The terms "adeno associated virus", "AAV virus", "AAV virion," "AAV viral particle" and "AAV particle", used as synonyms herein, refer to a viral particle composed of at least one capsid protein of AAV (preferably composed of all capsid protein of a particular AAV serotype) and an encapsulated polynucleotide of the AAV genome. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide different from a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell) flanked by AAV inverted terminal repeats, then they are typically known as a "AAV vector particle" or "AAV viral vector"or "AAV vector". AAV refers to a virus that belongs to the genus Dependovirus family Parvoviridae. The AAV genome is approximately 4.7 Kb in length and it consists of single strand deoxyribonucleic acid (ssDNA) that can be positive or negative detected. The invention also encompasses the use of double stranded AAV also called dsAAV or scAAV. The genome includes inverted terminal repeats (ITR) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. The frame rep is made of four overlapping genes that encode proteins Rep necessary for AAV lifecycle. The frame cap contains nucleotide sequences overlapping with capsid proteins: VP1, VP2 and VP3, which interact to form a capsid of icosahedral symmetry (see Carter and Samulski ., 2000, and Gao et al, 2004). More information is provided as to this type of virus, viral particle or viral vector in the section entitled "general definitions".

[0095] In a particular embodiment, the expression vector comprises a sequence selected among sequences SEQ ID NOs: 30 and 31.

[0096] SEQ ID NO: 30 comprises 7264 nucleotides and is named herein as pAAV-CAG-preprolGF1a-doble mirT122a-mirT1. It is used to express in pancreas the murine preprolGF-1 (isoform 5, SEQ ID NO: 28). The CAG promoter is located at bases 180 to 1824; the sequence encoding the murine preprolGF-1 is located at nucleotides 2112-2525 (signal peptide: nucleotides 2112-2197; mature IGF1: 2198-2417 nucleotides, peptide Ea: nucleotides 2418 to 2525); at nucleotides 2942-3460 there is the polyadenylation signal (PolyA, poliadenines) of the rabbit beta globin; the four copies of the target sequence of miR122a and four copies of the target sequence of miR1 (herein called as doublemiRT122a-miRT1, also called herein dmiRT) are at nucleotides 2592-2852 of the gene construct; and the inverted terminal repeat sequences (ITR) from the serotype 2 adeno associated virus (AAV2) are located at nucleotides 2-143 (AAV2 5' ITR) and at nucleotides 3519-3651 (AAV2 3' ITR).

[0097] The expression cassette comprised within SEQ ID NO: 30 consists of SEQ ID NO:96: CAG promoter: 180-1824bp, Mouse preproIGFla: 2112-2525 bp (signal peptide: 2112-2197 bp; mature IGF1: 2198-2417 bp; Ea peptide: 2418-2525 bp), doblemiRT122a-mirT1 (4 copies of the mirT122a and 4 copies of the mirT1): 2592-2852 bp, Rabbit .beta.-Globin polyA signal: 2942-3460 bp.

[0098] The viral vector comprised within SEQ ID NO:30 consists of SEQ ID NO: 97: AAV2 5' ITR: 2-143 bp, CAG promoter: 180-1824 bp, Mouse preprolGF1a: 2112-2525 bp (signal peptide: 2112-2197 bp; mature IGF1: 2198-2417 bp; Ea peptide: 2418-2525 bp), doblemiRT122a-mirT1 (4 copies of the mirT122a and 4 copies of the mirT1): 2592-2852 bp, Rabbit .beta.-Globin polyA signal: 2942-3460 bp, AAV2 3' ITR: 3519-3651 bp.

[0099] SEQ ID NO: 31 comprises 7200 nucleotides and is also called herein pAAV-CAG-preproIGF1b-doble miRT122a-mirT1. It is used to express in pancreas the murine preprolGF-1 (SEQ ID NO: 29). The CAG promoter is located at bases 180 to 1824; the sequence encoding the murine preprolGF-1 is located at nucleotides 2036 to 2434 (signal peptide: nucleotides 2036-2101; mature IGF1: nucleotides 2102-2311, peptide Eb: nucleotides 2312-2434); at nucleotides 2878-3396 there is the polyadenylation signal (PolyA, poliadenines) of the rabbit beta globin; the four copies of the target sequence of miR122a and the four copies of the target sequence of miR1 (herein called as doblemiRT122a-miRT1) are at nucleotides 2528-2788 of the gene construction; and the inverted terminal repeat sequences from the serotype 2 adeno associated virus (AAV2) are located at nucleotides 2-143 (AAV2 5'ITR) and at nucleotides 3455 to 3587 (AAV2 3' ITR).

[0100] The expression cassette comprised within SEQ ID NO: 31 consists of SEQ ID NO:98: CAG promoter: 180-1824 bp, Mouse preproIGF lb (MGF): 2036-2434 bp (signal peptide: 2036-2101 bp; mature IGF1: 2102-2311 bp; Eb peptide: 2312-2434 bp), doblemiRT122a-mirT1 (4 copies of the mirT122a and 4 copies of the mirT1): 2528-2788 bp, Rabbit .beta.-Globin polyA signal: 2878-3396 bp.

[0101] The viral vector comprised within SEQ ID NO:31 consists of SEQ ID NO: 99: AAV2 5' ITR: 2-143 bp, CAG promoter: 180-1824bp, Mouse preproIGF1b (MGF): 2036-2434 bp (signal peptide: 2036-2101 bp; mature IGF1: 2102-2311 bp; Eb peptide: 2312-2434 bp), doblemiRT122a-mirT1 (4 copies of the mirT122a and 4 copies of the mirT1): 2528-2788 bp, Rabbit .beta.-Globin polyA signal: 2878-3396 bp AAV2 3' ITR: 3455-3587 bp.

[0102] As can be seen throughout the description, the vectors of the invention are preferably for the expression of the gene construct in mammalian pancreas, preferably human or murine pancreas.

[0103] The expression vectors of the invention can be obtained by methods known to the expert. The invention also comprises methods of obtaining vectors comprising the steps of:

[0104] (a) providing a cell with: (i) a gene construct as defined above, flanked by adeno associated virus ITRs; (ii) cap and rep proteins of adeno associated virus; and (iii) adequate viral proteins for the replication of AAV;

[0105] (b) cultivating the cell in suitable conditions to produce the AAV assembly; and

[0106] (c) purifying AAV vector produced by the cell.

[0107] More detail is provided in the section entitled "general definitions".

[0108] In a particular embodiment of the method of obtaining the expression vectors, rep proteins of the adeno associated virus are of 2 serotype 2 (AAV2) and cap proteins derive from an AVV serotype selected from a group consisting of adeno associated viral vector of serotype 6, adeno associated viral vector of serotype 7, adeno associated viral vector of serotype 8, adeno associated viral vector of serotype 9, adeno associated viral vector of serotype 10, adeno associated viral vector of serotype 11, adeno associated viral vector of serotype rh8, and adeno associated viral vector of serotype rh10.

[0109] Third, Fourth and Fifth Aspects of the Invention: Therapeutic Applications and Pharmaceutical Compositions

[0110] Another aspect of the invention refers to the gene construct and/or the expression vector, both as defined above, for use as a medicament. This aspect may also be formulated as the use of the vector and/or of the gene construct in medicine.

[0111] Alternatively, it refers to a method of treatment and/or prevention of a subject in need thereof, wherein the treatment comprises administering a therapeutically effective amount of the vector or of the gene construct as described previously.

[0112] In a particular embodiment, the vector and/or the gene construct are used in treatment and/or prevention of diabetes in mammals, preferably diabetes mellitus in mammals, wherein a dysfunction and/or a loss of the beta-cells of the Langerhans islets is present. In a more particular embodiment, diabetes is type 1 diabetes mellitus in mammal (T1D). "Dysfunction" should be understood as the cells do not work properly even though the number of cells per volume of islet is maintained. "Loss" means that the number of cells is lower than that associated with a pancreas without diabetes. Lower may mean 10% lower, 20% lower, 30% lower, 40% lower, 50% lower, 60% lower, 70% lower, 80% lower or more.

[0113] During the autoimmune process characteristic of T1D, two stages can be clearly distinguished: a clinically occult phase, characterized by the generation of antibodies directed against a variety of antigens of .sub.R cells together with a gradual infiltration of autoreactive T lymphocytes and other inflammatory cells in the islets, called insulitis; and an open phase of diabetes where the destruction of .sub.R cells is extensive causing a deficiency in production of insulin and finally hyperglycemia. The period of time between the onset of T1D biomarkers and the onset of clinical symptoms is highly variable and can take several years before clinical disease occurs. Not until there is a loss of 70-80% of the .beta. cell mass that occurs the onset of elevated blood glucose values and T1D is diagnosed.

[0114] In this regard, the vector and/or the gene construct are used in treatment and/or prevention of diabetes, both the asymptomatic and the symptomatic phase. Within the context of the invention the "treatment" and the "prevention" of diabetes encompasses the prevention, the regression, the delay and/or curing diabetes. The vector and/or the gene construct as used herein preferably exhibit an anti-diabetes effect as later defined herein.

[0115] In even a more particular embodiment, the vector or the gene construct is for use in the treatment and/or prevention of T1D in a mammal selected from human, murine, feline (cats) and canine (dogs), preferably in humans.

[0116] These embodiments can also be formulated as the use of the vector and/or of the gene construct described above for the preparation of a medicament for the treatment and/or prevention of diabetes mellitus, in which there is dysfunction and/or loss of beta-cells of the islets of Langerhans. More preferably, they are used to prepare a medicament for the treatment of T1D in mammals, especially murine, humans, cats and dogs. Alternatively, these embodiments relate to methods for therapeutic and/or prophylactic treatment of diabetes mellitus, particularly T1D, comprising administering a therapeutically or prophylactically effective amount of the vector or of the gene construct as described previously in a subject (particularly a human) in need thereof.

[0117] In the context of the present invention is to be understood by "therapeutically effective amount" the amount of a compound (herein the vector or the gene construct) which, when administered, is sufficient to prevent the development of or alleviate to some extent one or more symptoms of the disease to which is directed (diabetes). The particular dose of the compound to be administered according to this invention will be determined by considering the particular circumstances surrounding the case, including the route of administration, the particular condition that it is treated and other similar considerations that the expert will know how to interpret.

[0118] An object of the present invention is a pharmaceutical composition comprising a gene construct as defined previously and/or of a vector as defined previously, together with one or more pharmaceutically acceptable excipients or vehicles.

[0119] Another object of the present invention is a pharmaceutical composition comprising a therapeutically effective amount of a gene construct as defined previously and/or of a vector as defined previously, together with one or more pharmaceutically acceptable excipients or vehicles.

[0120] In the context of the present invention, the term "pharmaceutically acceptable excipients or vehicles" refers to pharmaceutically acceptable materials, compositions or vehicles. Each component should be pharmaceutically acceptable in the sense that it must be compatible with the other ingredients of the pharmaceutical composition. It should also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications put into context with a reasonable relation benefit/risk. Such pharmaceutically acceptable excipients or vehicles may be pharmacetucially acceptable carrier, filler, preservative, solubilizer, diluent. They may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000.

[0121] A gene construct and/or an expression vector and/or a pharmaceutical composition of the invention comprising a gene construct and/or an expression vector is preferably said to be able to be used for preventing, delaying, reverting, curing and/or treating a diabetes, when said gene construct, expression vector and/or composition are able to exhibit an anti-diabetic effect in a treated individual. An anti-diabetic effect may be reached when glucose disposal is increased and/or when glucose tolerance is improved and/or circulating insulin is increased and/or hyperglycemia is delayed. Glucose and insulin circulating levels in blood/serum and glucose tolerance could be assessed using techniques known to the skilled person or as done in the experimental part. For humans, it is accepted that in fasting conditions (fasting is defined as no calorie intake for at least 8 h) in healthy human subjects, glucose is usually ranged from 70-125 mg/dl and insulin from 5-20 mcU/ml. Values of fasting plasma glucose (FPG) of 126 mg/dl or higher are criteria of diagnosis of Diabetes (ADA, 2010). In healthy mice, glucose is usually ranged from 60-180 mg/dl and insulin from 0.3 to 10 ng/ml. In this context, "increase" (respectively "improvement") means at least a detectable increase (respectively a detectable improvement) using an assay known to the skilled person or using assays as carried out in the experimental part. In this context, "decrease" means at least a detectable decrease using an assay known to the skilled person or using assays as carried out in the experimental part. A detectable decrease may be a decrease of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more by comparison to the corresponding level before treatment or to the corresponding level in a control subject.

[0122] The anti-diabetic effect may be assessed on a sample from a treated individual such as blood.

[0123] The expression "glucose disposal is increased" may mean increased glucose uptake by peripheral tissues (mainly liver, skeletal muscle and adipose tissue) resulting in decreased circulating levels of glucose. The expression "glucose tolerance is improved" may mean that circulating levels of glucose are decreased after glucose overload.

[0124] The expression "the hyperglycemia is delayed" preferably means that the delay may be of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or at least 1, 2, 3, 4, 5, 6, 7 weeks or at least 1, 2, 3, 4, 5, 6, 7, months or at least 1, 2, 3, 4, 5, 6, 7, years. Hyperglycemia is typically defined when circulating levels of glucose are higher than the ones defined above. In this context, "higher" means at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% higher.

[0125] An anti-diabetic effect may also be observed when the progression of a typical symptom (i.e. insulitis, beta cell loss, . . . ) has been slowed down as assessed by a physician. A decrease of a typical symptom may mean a slow down in progression of symptom development or a complete disappearance of symptoms. Symptoms, and thus also a decrease in symptoms, can be assessed using a variety of methods, to a large extent the same methods as used in diagnosis of diabetes, including clinical examination and routine laboratory tests. Such methods include both macroscopic and microscopic methods, as well as molecular methods, X-rays, biochemical, immunohistochemical and others.

[0126] A medicament as defined herein (gene construct, expression vector, composition) is preferably able to alleviate one symptom or one characteristic of a patient or of a cell, tissue or organ of said patient if after at least one week, one month, six month, one year or more of treatment using an expression vector or a composition of the invention, said symptom or characteristic is no longer detectable. A preferred organ is the pancreas and a preferred cell is a beta-cell of the pancreas.

[0127] A gene construct or an expression vector or a composition as defined herein for use according to the invention may be suitable for administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing a diabetes, and may be administered in vivo, ex vivo or in vitro. Said gene construct or expression vector or composition may be directly or indirectly administrated to a cell, tissue and/or an organ in vivo of an individual affected by or at risk of developing a diabetes, and may be administered directly or indirectly in vivo, ex vivo or in vitro. A preferred administration mode is intraductal as later defined herein.

[0128] A gene construct or an expression vector or a composition of the invention may be directly or indirectly administered using suitable means known in the art. Improvements in means for providing an individual or a cell, tissue, organ of said individual with a gene construct or an expression vector or a composition of the invention are anticipated, considering the progress that has already thus far been achieved. Such future improvements may of course be incorporated to achieve the mentioned effect of the invention. A gene construct or an expression vector or a composition can be delivered as is to an individual, a cell, tissue or organ of said individual. Depending on the disease or condition, a cell, tissue or organ of said individual may be as earlier defined herein. When administering a gene construct or an expression vector or a composition of the invention, it is preferred that such gene construct or vector or composition is dissolved in a solution that is compatible with the delivery method. For intravenous, subcutaneous, intramuscular, intrathecal, intraarticular and/or intraventricular administration it is preferred that the solution is a physiological salt solution.

[0129] As encompassed herein, a therapeutically effective dose of a gene construct, vector or composition as mentioned above is preferably administered in a single and unique dose hence avoiding repeated periodical administration.

[0130] A further compound may be present in a composition of the invention. Said compound may help in delivery of the composition. Below is provided a list of suitable compounds: compounds capable of forming complexes, nanoparticles, micelles and/or liposomes that deliver each constituent as defined herein, complexed or trapped in a vesicle or liposome through a cell membrane. Many of these compounds are known in the art. Suitable compounds comprise polyethylenimine (PEI), or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, synthetic amphiphiles (SAINT-18), lipofectin.TM., DOTAP.

[0131] Depending on their identity, the skilled person will know which type of formulation is the most appropriate for the composition as defined herein.

[0132] In this context a further compound may be insulin that could be regularly injected.

[0133] In a further aspect there is provided a method for preventing, delaying, reverting, curing and/or treating a diabetes wherein a gene construct or expression vector or composition as defined herein as defined herein is being used.

[0134] Such a method is preferably for alleviating one or more symptom(s) of diabetes in an individual, in a cell, tissue or organ of said individual or alleviate one or more characteristic(s) or symptom(s) of a cell, tissue or organ of said individual, the method comprising administering to said individual an expression construct or vector (preferably a viral expression construct or a viral vector) or a composition as defined herein.

[0135] In a further aspect there is provided a use of a gene construct or vector or a composition as defined herein for the manufacture of a medicament for preventing, delaying, reverting, curing and/or treating a diabetes.

[0136] Diabetes and the type of subject treated have been earlier defined herein.

[0137] In a preferred embodiment, a treatment in a use or in a method according to the invention does not have to be repeated. Alternatively in a use or a method according to the invention said administration of the gene construct or of said composition may be repeated each year or each 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 years.

[0138] The gene construct and/or the vector and/or the pharmaceutical compositions that comprise them, can be used in the treatment of diabetes, via systemic or local. In a preferred embodiment, they are used in the treatment by local intraductal administration via or through by the bile duct. The intraductal administration can potentially be applied to larger animals and humans, through a non-surgical and less invasive clinical process called endoscopic retrograde cholangiopancreatography (ERCP) (Hendrick et al., 2011).

[0139] Other Aspects of the Invention

[0140] The invention also has the object of methods for in vitro or ex vivo transduction of mammalian cells, where the methods comprise providing mammalian cells, particularly cells of murine, pig and human pancreas, with an expression vector or gene construct, as described above. Ex-vivo gene therapy delivering AAV vectors to mammalian cells may be carried out as described in Rehman K et al 2005.

[0141] The invention also has the object of in vitro methods of transduction of mammalian cells, wherein the methods comprise contacting the mammalian cell, particularly cells of murine, pig and human pancreas, with an expression vector as described above and/or a construction gene as described above.

[0142] In a particular embodiment, the gene construct and/or expression vector used in the method of transduction is an adeno associated viral vector, and the cell is a beta-cell from the islet of Langerhans or the complete islet of Langerhans of the pancreas of the mammal, preferably human.

[0143] Another object of the invention is, therefore, a mammalian cell transduced or a complete islet transduced. In a particular embodiment, the cell is a pancreatic cell, preferably a beta-cell of the islet of Langerhans or a complete islet of Langerhans.

[0144] Within the context of the invention a beta-cell could be replaced by a beta-cell of the islet of Langerhans or by a .beta.-cell or by a .beta.cell.

[0145] Throughout the description and claims the word "comprise" and its variations are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein. The following examples and drawings are provided herein for illustrative purposes, and without intending to be limiting to the present invention. In addition, this invention covers all possible combinations of particular and preferred embodiments herein indicated.

[0146] General Definitions

[0147] Expression Construct

[0148] An expression construct or gene construct carries a genome that is able to stabilize and remain episomal in a cell. However, an expression construct for gene construct may also integrate into the genome of the cell. Within the context of the invention, a cell may mean to encompass a cell used to make the construct or a cell wherein the construct will be administered. Alternatively a construct is capable of integrating into a cell's genome, e.g. through homologous recombination or otherwise. A particularly preferred expression construct is one wherein a nucleotide sequence encoding an IGF-1 as defined herein, is operably linked to a promoter and a target sequence of a miRNA as defined herein. An expression construct, preferably a viral expression construct is intended to be used in gene therapy. Expression construct may be used to introduce DNA/gene of interest to the pancreas via microbubble treatment with ultrasound (Chen et al., 2006). A viral expression construct is designed to comprise part of a viral genome as later defined herein.

[0149] Expression constructs disclosed herein could be prepared using recombinant techniques in which nucleotide sequences encoding said IGF-1 are expressed in a suitable cell, e.g. cultured cells or cells of a multicellular organism, such as described in Ausubel et al., "Current Protocols in Molecular Biology", Greene Publishing and Wiley-Interscience, New York (1987) and in Sambrook and Russell (2001, supra); both of which are incorporated herein by reference in their entirety. Also see, Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describing site directed mutagenesis) and Roberts et al. (1987) Nature 328:731-734 or Wells, J. A., et al. (1985) Gene 34: 315 (describing cassette mutagenesis).

[0150] Typically, a nucleic acid or nucleotide sequence encoding an IGF-1 is used in an expression construct or gene construct. The phrase "expression construct " generally refers to a nucleotide sequence that is capable of effecting expression of a gene in a host compatible with such sequences. These expression constructs typically include at least suitable promoter sequences and optionally, transcription termination signals. An additional factor necessary or helpful in effecting expression can also be used as described herein. A nucleic acid or DNA or nucleotide sequence encoding an IGF-1 is incorporated into a DNA construct capable of introduction into and expression in an in vitro cell culture. Specifically, a DNA construct is suitable for replication in a prokaryotic host, such as bacteria, e.g., E. coli, or can be introduced into a cultured mammalian, plant, insect, (e.g., Sf9), yeast, fungi or other eukaryotic cell lines.

[0151] A DNA construct or gene construct or expression construct prepared for introduction into a particular host may include a replication system recognized by the host, an intended DNA segment encoding a desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide-encoding segment. The term "operably linked" has already been defined herein. For example, a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence. A promoter has also already been defined herein. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of a polypeptide. Generally, a DNA sequence that is operably linked are contiguous, and, in the case of a signal sequence, both contiguous and in reading frame. However, enhancers need not be contiguous with a coding sequence whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof, or by gene synthesis.

[0152] The selection of an appropriate promoter sequence generally depends upon the host cell selected for the expression of a DNA segment. Examples of suitable promoter sequences include prokaryotic, and eukaryotic promoters well known in the art (see, e.g. Sambrook and Russell, 2001, supra). A transcriptional regulatory sequence typically includes a heterologous enhancer or promoter that is recognised by the host. The selection of an appropriate promoter depends upon the host, but promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available (see, e.g. Sambrook and Russell, 2001, supra). An expression construct or gene construct or expression vector includes the replication system and transcriptional and translational regulatory sequences together with the insertion site for the polypeptide encoding segment can be employed. In most cases, the replication system is only functional in the cell that is used to make the vector (bacterial cell as E. coli). Most plasmids and vectors do not replicate in the cells infected with the vector. Examples of workable combinations of cell lines and expression vectors are described in Sambrook and Russell (2001, supra) and in Metzger et al. (1988) Nature 334: 31-36. For example, suitable expression vectors can be expressed in, yeast, e.g. S.cerevisiae, e.g., insect cells, e.g., Sf9 cells, mammalian cells, e.g., CHO cells and bacterial cells, e.g., E. coli. A cell may thus be a prokaryotic or eukaryotic host cell. A cell may be a cell that is suitable for culture in liquid or on solid media.

[0153] Alternatively, a host cell is a cell that is part of a multicellular organism such as a transgenic plant or animal. An animal is preferably a mammal.

[0154] Viral Vector, Expression Vector

[0155] An expression vector is preferably a viral vector or a gene therapy vector. Such a viral vector is a vector that comprises a viral expression construct as defined above. A vector (preferably a viral vector) is preferably a gene therapy vector. A gene therapy vector is a vector that is suitable for gene therapy. Vectors that are suitable for gene therapy are described in Anderson 1998, Nature 392: 25-30; Walther and Stein, 2000, Drugs 60: 249-71; Kay et al., 2001, Nat. Med. 7: 33-40; Russell, 2000, J. Gen. Virol. 81: 2573-604; Amado and Chen, 1999, Science 285: 674-6; Federico, 1999, Curr. Opin. Biotechno1.10: 448-53; Vigna and Naldini, 2000, J. Gene Med. 2: 308-16; Marin et al., 1997, Mol. Med. Today 3: 396-403; Peng and Russell, 1999, Curr. Opin. Biotechnol. 10: 454-7; Sommerfelt, 1999, J. Gen. Virol. 80: 3049-64; Reiser, 2000, Gene Ther. 7: 910-3; and references cited therein.

[0156] A particularly suitable gene therapy vector includes an Adenoviral and Adeno-associated virus (AAV) vector. These vectors infect a wide number of dividing and non-dividing cell types including synovial cells and liver cells. The episomal nature of the adenoviral and AAV vectors after cell entry makes these vectors suited for therapeutic applications. (Russell, 2000, J. Gen. Virol. 81: 2573-2604; Goncalves, 2005, Virol J. 2(1):43) as indicated above. AAV vectors are even more preferred since they are known to result in very stable long term expression of transgene expression (up to 9 years in dog (Niemeyer et al, Blood. 2009 Jan 22;113(4):797-806) and .about.2 years in human (Nathwani et al, N Engl J Med. 2011 Dec. 22; 365(25):2357-65, Simonelli et al, Mol Ther. 2010 March; 18(3):643-50. Epub 2009 Dec. 1.)). Preferred adenoviral vectors are modified to reduce the host response as reviewed by Russell (2000, supra). Method for gene therapy using AAV vectors are described by Wang et al., 2005, J Gene Med. March 9 (Epub ahead of print), Mandel et al., 2004, Curr Opin Mol Ther. 6(5):482-90, and Martin et al., 2004, Eye 18(11):1049-55, Nathwani et al, N Engl J Med. 2011 Dec. 22; 365(25):2357-65, Apparailly et al, Hum Gene Ther. 2005 April; 16(4):426-34.

[0157] Another suitable gene therapy vector includes a retroviral vector. A preferred retroviral vector for application in the present invention is a lentiviral based expression construct. Lentiviral vectors have the ability to infect and to stably integrate into the genome of dividing and non-dividing cells (Amado and Chen, 1999 Science 285: 674-6). Methods for the construction and use of lentiviral based expression constructs are described in U.S. Pat. Nos. 6,165,782, 6,207,455, 6,218,181, 6,277,633 and 6,323,031 and in Federico (1999, Curr Opin Biotechnol 10: 448-53) and Vigna et al. (2000, J Gene Med 2000; 2: 308-16).

[0158] Other suitable gene therapy vectors include a herpes virus vector, a polyoma virus vector or a vaccinia virus vector.

[0159] A gene therapy vector comprises a nucleotide sequence encoding an IGF-1 to be expressed, whereby said nucleotide sequence is operably linked to the appropriate regulatory sequences. Such regulatory sequence will at least comprise a promoter sequence. Suitable promoters for expression of a nucleotide sequence encoding an IGF-1 from gene therapy vectors include e.g. cytomegalovirus (CMV) intermediate early promoter, viral long terminal repeat promoters (LTRs), such as those from murine moloney leukaemia virus (MMLV) rous sarcoma virus, or HTLV-1 , the simian virus 40 (SV 40) early promoter and the herpes simplex virus thymidine kinase promoter. Suitable promoters are described below. Several inducible promoter systems have been described that may be induced by the administration of small organic or inorganic compounds. Such inducible promoters include those controlled by heavy metals, such as the metallothionine promoter (Brinster et al. 1982 Nature 296: 39-42; Mayo et al. 1982 Cell 29: 99-108), RU-486 (a progesterone antagonist) (Wang et al. 1994 Proc. Natl. Acad. Sci. USA 91: 8180-8184), steroids (Mader and White, 1993 Proc. Natl. Acad. Sci. USA 90: 5603-5607), tetracycline (Gossen and Bujard 1992 Proc. Natl. Acad. Sci. USA 89: 5547-5551; U.S. Pat. No. 5,464,758; Furth et al. 1994 Proc. Natl. Acad. Sci. USA 91: 9302-9306; Howe et al. 1995 J. Biol. Chem. 270: 14168-14174; Resnitzky et al. 1994 Mol. Cell. Biol. 14: 1669-1679; Shockett et al. 1995 Proc. Natl. Acad. Sci. USA 92: 6522-6526) and the tTAER system that is based on the multi-chimeric transactivator composed of a tetR polypeptide, as activation domain of VP16, and a ligand binding domain of an estrogen receptor (Yee et al., 2002, U.S. Pat. No. 6,432,705).

[0160] A gene therapy vector may optionally comprise a further nucleotide sequence coding for a further polypeptide. A further polypeptide may be a (selectable) marker polypeptide that allows for the identification, selection and/or screening for cells containing the expression construct. Suitable marker proteins for this purpose are e.g. the fluorescent protein GFP, and the selectable marker genes HSV thymidine kinase (for selection on HAT medium), bacterial hygromycin B phosphotransferase (for selection on hygromycin B), Tn5 aminoglycoside phosphotransferase (for selection on G418), and dihydrofolate reductase (DHFR) (for selection on methotrexate), CD20, the low affinity nerve growth factor gene. Sources for obtaining these marker genes and methods for their use are provided in Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3.sup.rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York.

[0161] A gene therapy vector is preferably formulated in a pharmaceutical composition as defined herein. In this context, a pharmaceutical composition may comprise a suitable pharmaceutical carrier as earlier defined herein.

[0162] Adeno-associated virus vector (AAV vector) A preferred viral vector or a preferred gene therapy vector is an AAV vector. An AAV vector as used herein preferably comprises a recombinant AAV vector (rAAV). A "rAAV vector" as used herein refers to a recombinant vector comprising part of an AAV genome encapsidated in a protein shell of capsid protein derived from an AAV serotype as explained herein. Part of an AAV genome may contain the inverted terminal repeats (ITR) derived from an adeno-associated virus serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV rh8,

[0163] AAV rh10 and others. Preferred ITR are derived from an AAV2. Protein shell comprised of capsid protein may be derived from an AAV serotype such as AAV1, 2, 3, 4, 5, 6, 8, 9, 10, 11, rh8, rh10 and others. A preferred AAV capsid is a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV rh8, AAV rh10 capsid. Any capsid with a tropism for pancreas is preferably used in the context of the invention even if this capsid has not yet been discovered. The invention therefore also encompasses the use of viral vector encapsidated in a capsid protein with a tropism for pancreas. A preferred ITR is from the AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV rh8, AAV rh10. Preferred ITR are derived from an AAV2. A protein shell may also be named a capsid protein shell.

[0164] rAAV vector may have one or preferably all wild type AAV genes deleted, but may still comprise functional ITR nucleic acid sequences. Functional ITR sequences are necessary for the replication, rescue and packaging of AAV virions. The ITR sequences may be wild type sequences or may have at least 80%, 85%, 90%, 95, or 100% sequence identity with wild type sequences or may be altered by for example in insertion, mutation, deletion or substitution of nucleotides, as long as they remain functional. In this context, functionality refers to the ability to direct packaging of the genome into the capsid shell and then allow for expression in the host cell to be infected or target cell.In the context of the present invention a capsid protein shell may be of a different serotype than the rAAV vector genome ITR.

[0165] A nucleic acid molecule represented by a nucleic acid sequence of choice is preferably inserted between the rAAV genome or ITR sequences as identified above, for example an expression construct comprising an expression regulatory element operably linked to a coding sequence and a 3' termination sequence. Said nucleic acid molecule may also be called a transgene.

[0166] "AAV helper functions" generally refers to the corresponding AAV functions required for rAAV replication and packaging supplied to the rAAV vector in trans. AAV helper functions complement the AAV functions which are missing in the rAAV vector, but they lack AAV ITRs (which are provided by the rAAV vector genome). AAV helper functions include the two major ORFs of AAV, namely the rep coding region and the cap coding region or functional substantially identical sequences thereof. Rep and Cap regions are well known in the art, see e.g. Chiorini et al. (1999, J. of Virology, Vol 73(2): 1309-1319) or U.S. Pat. No. 5,139,941, incorporated herein by reference. The AAV helper functions can be supplied on a AAV helper construct. Introduction of the helper construct into the host cell can occur e.g. by transformation, transfection, or transduction prior to or concurrently with the introduction of the rAAV genome present in the rAAV vector as identified herein. The AAV helper constructs of the invention may thus be chosen such that they produce the desired combination of serotypes for the rAAV vector's capsid protein shell on the one hand and for the rAAV genome present in said rAAV vector replication and packaging on the other hand.

[0167] "AAV helper virus" provides additional functions required for AAV replication and packaging. Suitable AAV helper viruses include adenoviruses, herpes simplex viruses (such as HSV types 1 and 2) and vaccinia viruses. The additional functions provided by the helper virus can also be introduced into the host cell via vectors, as described in U.S. Pat. No. 6,531,456 incorporated herein by reference.

[0168] A "transgene" is herein defined as a gene or a nucleic acid molecule (i.e. a molecule encoding an IGF-1) that has been newly introduced into a cell, i.e. a gene that may be present but may normally not be expressed or expressed at an insufficient level in a cell. In this context, "insufficient" means that although said IGF-1 is expressed in a cell, a condition and/or disease as defined herein could still be developed. In this case, the invention allows the over-expression of an IGF-1. The transgene may comprise sequences that are native to the cell, sequences that naturally do not occur in the cell and it may comprise combinations of both. A transgene may contain sequences coding for an IGF-1 and/or additional proteins as earlier identified herein that may be operably linked to appropriate regulatory sequences for expression of the sequences coding for an IGF-1 in the cell. Preferably, the transgene is not integrated into the host cell's genome.

[0169] "Transduction" refers to the delivery of an IGF-1 into a recipient host cell by a vector, preferably a viral vector. For example, transduction of a target cell by a rAAV vector of the invention leads to transfer of the rAAV genome contained in that vector into the transduced cell. "Host cell" or "target cell" refers to the cell into which the DNA delivery takes place, such as the muscle cells of a subject. AAV vectors are able to transduce both dividing and non-dividing cells.

[0170] Production of an AAV Vector

[0171] The recombinant AAV vector, including all combinations of AAV serotype capsid and AAV genome ITRs, is produced using methods known in the art, as described in Pan et al. (J. of Virology 1999, Vol 73(4):3410-3417) and Clark et al. (Human Gene Therapy, 1999, 10:1031-1039), incorporated herein by reference. In short, the methods generally involve (a) the introduction of the rAAV genome into a host cell, (b) the introduction of an AAV helper construct into the host cell, wherein the helper construct comprises the viral functions missing from the rAAV genome and (c) introducing a helper virus or a helper plasmid into the host cell. All functions for rAAV vector replication and packaging need to be present, to achieve replication and packaging of the rAAV genome into rAAV vectors. The AAV vector may also be produced in baculovirus system. The introduction into the host cell can be carried out using standard virological techniques and can be simultaneously or sequentially. Finally, the host cells are cultured to produce rAAV vectors and are purified using standard techniques such as CsCl gradients (Xiao et al. 1996, J. Virol. 70: 8098-8108). Residual helper virus activity can be inactivated using known methods, such as for example heat inactivation. If helper activity has been provided using a plasmid, no inactivation is needed. The purified rAAV vector is preferably dialized to eliminate CsC1 traces. The purified rAAV vector is then ready for use in the methods. High titres of more than 10.sup.12 particles per ml and high purity (free of detectable helper and wild type viruses) can be achieved (Clark et al. supra and Flotte et al. 1995, Gene Ther. 2: 29-37).

[0172] The rAAV genome present in a rAAV vector comprises at least the nucleotide sequences of the inverted terminal repeat regions (ITR) of one of the AAV serotypes (preferably the ones of serotype AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV rh8, AAV rh10), or nucleotide sequences substantially identical thereto, and nucleotide sequence encoding an IGF-1 (under control of a suitable regulatory element) inserted between the two ITRs. Preferred ITR are derived from an AAV2. A vector genome requires the use of flanking 5' and a 3' ITR sequences to allow for efficient packaging of the vector genome into the rAAV capsid. The complete genome of several AAV serotypes and corresponding ITR has been sequenced (Chiorini et al. 1999, J. of Virology Vol. 73, No.2, p1309-1319). They can be either cloned or made by chemical synthesis as known in the art, using for example an oligonucleotide synthesizer as supplied e.g. by Applied Biosystems Inc. (Fosters, Calif., USA) or by standard molecular biology techniques. The ITRs can be cloned from the AAV viral genome or excised from a vector comprising the AAV ITRs. The ITR nucleotide sequences can be either ligated at either end to the nucleotide sequence encoding one or more therapeutic proteins using standard molecular biology techniques, or the wild type AAV sequence between the ITRs can be replaced with the desired nucleotide sequence.

[0173] Preferably, the rAAV genome as present in a rAAV vector does not comprise any nucleotide sequences encoding viral proteins, such as the rep (replication) or cap (capsid) genes of AAV. This rAAV genome may further comprise a marker or reporter gene, such as a gene for example encoding an antibiotic resistance gene, a fluorescent protein (e.g. gfp) or a gene encoding a chemically, enzymatically or otherwise detectable and/or selectable product (e.g. lacZ, aph, etc.) known in the art. The rAAV genome as present in said rAAV vector further comprises a promoter sequence operably linked to the nucleotide sequence encoding an IGF-1. Preferred promoter sequences have already been defined herein.

[0174] A suitable 3' untranslated sequence may also be operably linked to the nucleotide sequence encoding an IGF-1. Suitable 3' untranslated regions may be those naturally associated with the nucleotide sequence or may be derived from different genes such as those disclosed herein (i.e. target sequence of a miRNA).

[0175] Optionally, additional nucleotide sequences may be operably linked to the nucleotide sequence(s) encoding an IGF-1, such as nucleotide sequences encoding signal sequences, nuclear localization signals, expression enhancers, and the like.

[0176] Variants

[0177] In the context of the invention, a protein is represented by an amino acid sequence. In the context of this invention a preferred protein is an IGF-1.

[0178] In the context of the invention, a nucleic acid molecule as a nucleic acid molecule encoding an IGF-1 is represented by a nucleic acid or nucleotide sequence which encodes a protein or a polypeptide or a protein fragment or a peptide or a derived peptide. A nucleic acid molecule may comprise a regulatory region.

[0179] It is to be understood that each nucleic acid molecule or protein or protein fragment or peptide or derived peptide or polypeptide as identified herein by a given Sequence Identity Number (SEQ ID NO) is not limited to this specific sequence as disclosed. Each gene sequence or nucleotide sequence or nucleic acid sequence as identified herein encodes a given protein or polypeptide or protein fragment or peptide or derived peptide. Throughout this application, each time one refers to a specific nucleotide sequence SEQ ID NO (take SEQ ID NO: X as example) encoding a given polypeptide, one may replace it by:

[0180] i. a nucleotide sequence comprising a nucleotide sequence that has at least 60% sequence identity or similarity with SEQ ID NO: X;

[0181] ii. a nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence of (i);

[0182] iii. a nucleotide sequence the sequence of which differs from the sequence of a nucleic acid molecule of (i) or (ii) due to the degeneracy of the genetic code; or,

[0183] iv. a nucleotide sequence that encodes an amino acid sequence that has at least 60% amino acid identity or similarity with an amino acid sequence encoded by a nucleotide sequence SEQ ID NO: X.

[0184] Throughout this application, each time one refers to a specific amino acid sequence SEQ ID NO (take SEQ ID NO: Y as example), one may replace it by: a polypeptide comprising an amino acid sequence that has at least 60% sequence identity or similarity with amino acid sequence SEQ ID NO: Y.

[0185] Each nucleotide sequence or amino acid sequence described herein by virtue of its identity or similarity percentage (at least 60%) with a given nucleotide sequence or amino acid sequence respectively has in a further preferred embodiment an identity or a similarity of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identity or similarity with the given nucleotide or amino acid sequence respectively. In a preferred embodiment, sequence identity or similarity is determined by comparing the whole length of the sequences as identified herein. Unless otherwise indicated herein, identity or similarity with a given SEQ ID NO means identity or similarity based on the full length of said sequence (i.e. over its whole length or as a whole).

[0186] Each non-coding nucleotide sequence (i.e. of a promoter) could be replaced by a nucleotide sequence comprising a nucleotide sequence that has at least 60% sequence identity or similarity with SEQ ID NO: A. A preferred nucleotide sequence has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% identity with SEQ ID NO:A. Identity may be assessed over the whole SEQ ID NO or over part thereof as explained herein.

[0187] "Sequence identity" or "sequence homology" is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred embodiment, sequence identity is calculated based on the full length of two given SEQ ID NO or on part thereof. Part thereof preferably means at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.

[0188] "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

[0189] Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.

[0190] Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, WI. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).

[0191] Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons.

[0192] Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly to Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg; Gln or Glu; Met to Leu or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Val to Ile or Leu.

[0193] The NOD Mouse

[0194] NOD mice (Non-obese Diabetic) represent the main spontaneous mouse model to study the disease. This mouse model is characterized to develop diabetes without obesity, which is very similar to human T1D. The incidence of diabetes in NOD strain presents a marked difference depending on gender. At 30 weeks of age, the cumulative incidence of diabetic individuals is 60-80% in females and 20-30% in males NOD. The onset of diabetes occurs abruptly in both sexes, and usually begins after the age of 12-14 weeks, although it can occur at older ages. Diabetic symptoms are characterized biochemically by polyuria, polydipsia, hyperglycemia, hypercholesterolemia and glycosuria accompanied by a rapid loss of body weight. In no case a remission has been observed. However, the daily administration of insulin leads to increase body weight and prolongs the life of diabetic mice. The pathological examination of the pancreas shows a high frequency of infiltration of lymphocytes in the islets of Langerhans. Insulitis is present in both genders even in pre-diabetic stages (after 5 weeks) and progresses with age.

[0195] Multiple loci control genetic susceptibility to the development of diabetes in the NOD mouse. These mice show a unique haplotype for MHC, called H-2.sup.g7, which is the main genetic contribution to disease susceptibility. This MHC haplotype presents a series of defects that substantially alter the repertoire of peptides that can bind and be presented by MHC encoded by this allele. Surprisingly, a similar genetic alteration is also observed in genetic susceptibility loci to T1D of the MHC in humans. Besides MHC locus, many other loci contribute to the development of the disease, and they are called locus Idd.

[0196] In the NOD mouse, the antigen presenting cells against the islet appear first in the pancreatic lymph nodes at 2-4 weeks of age. Effector T cells present in nodules are activated and begin the process of autoimmunity against islets. At 4-6 weeks of age it can be observed a peri-vascular and peri-ductal accumulation of lymphocytes, and finally, at 6-8 weeks it appears infiltration to the islet perimeter (peri-insulitis). First, neurons and Schwann cells surrounding the islet are destroyed. T cells synthesize cytokines, particularly IFN-.gamma. and propagate autoimmune reaction that intensifies the destruction of these cells until it breaks up the basal membrane of the islet. The infiltrating lymphocytes specifically destroy insulin-producing beta-cells.

[0197] Mononuclear cells present in the infiltrated islet in the NOD mouse, as in humans, are CD4+ and CD8+ autoreactive (dendritic cells, macrophages, NK, B cells). In the early stages of activation of autoimmune response macrophages and dendritic cells are predominant. During the peri-insulitis, islets are infiltrated mostly by CD4+ cells, whereas CD8+ cells are recruited at a later stage, when the mononuclear infiltrate penetrates the interior of the islet. The inflammation produced by this process culminates with the progressive destruction of beta-cells. When the mass of destroyed beta-cell reaches values around 80%, it is produced the clinical manifestation of diabetes. At this stage, the islets are characterized by having completely lost its component of beta-cells and for the regression of insulitis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0198] Otherwise indicated IGF-1 refers to IGF-1a.

[0199] FIG. 1. Experimental design. Intraductal administration of the AAV8-CAG-GFP vector: AAV vector of serotype 8 encoding the green fluorescent protein GFP under the control of the CAG promoter (hybrid cytomegalovirus enhancer/chicken .beta.-actin constitutive promoter).

[0200] FIGS. 2A-2B. Pancreas transduction of female NOD mice intraductally administered with the AAV8-CAG-GFP vector. Pancreas immunohistochemistry against GFP (grey) and insulin (black) in samples from mice injected intraductally with 1.4.times.10.sup.12 vg at the age of 4 weeks and killed 1 month later. FIG. 2A Islet n-cell transduction. Original magnification .times.400. FIG. 2B Exocrine pancreas transduction. Two representative images of the exocrine pancreas immunostained against GFP. Original magnification .times.200.

[0201] FIGS. 3A-3D. Liver and heart transduction of female NOD mice intraductally administered with the AAV8-CAG-GFP vector. Samples from mice injected intraductally with 1.4.times.10.sup.12 vg at the age of 4 weeks and killed 1 month later. FIG. 3A and FIG. 3B. Liver immunohistochemistry against GFP. Original magnification .times.100. FIG. 3C and FIG. 3D. Heart immunohistochemistry against GFP. Original magnification .times.200.

[0202] FIG. 4. Expression of microRNA-122a in pancreas and liver. The levels of expression of microRNA-122a (miR-122a) were quantified by qPCR in pancreas and liver samples from female NOD mice. 8-week-old normoglycemic (NG) and 24-week-old hyperglycemic (HG) animals were analyzed. Results are expressed as mean.+-.SEM, n=4-6 animals per group. ND: Non-Detected. a.u.: arbitrary units.

[0203] FIG. 5. Expression of microRNA-1 in pancreas and heart. The levels of expression of microRNA-1 (miR-1) were quantified by qPCR in pancreas and heart samples from female NOD mice. 8-week-old normoglycemic (NG) and 24-week-old hyperglycemic (HG) animals were analyzed. Results are expressed as mean.+-.SEM, n=4-6 animals per group. ND: Non-Detected. a.u.: arbitrary units.

[0204] FIG. 6. Schematic diagram of the construct encoding murine IGF-1 protein pAAV-CAG-IGF1-dmiRT. ITR: Inverted Terminal Repeats; CAG: hybrid cytomegalovirus enhancer/chicken .beta.-actin constitutive promoter; mIGF-1: murine sequence of the Igf-1 gene; miRT-122a: target sequence of the microRNA-122a (4 copies); miRT-1: target sequence of the microRNA-1 (4 copies); pA: poly A, polyadenylation signal. The abbreviated form dmiRT refers to both target sequences for microRNA-122a and -1.

[0205] FIG. 7. In vitro testing of the plasmid pAAV-CAG-IGF1-dmiRT expression in INS-1 cells. Igf-1 expression levels 48 h post-transfection. 12-well culture plates were used and each well was transfected with 1 .mu.g of DNA. Control wells were not transfected with any DNA. Values are expressed in arbitrary units normalized for the Rplp0 gene and relative to control group. Results are expressed as mean.+-.SEM, n=3 wells per condition. ***p<0.001 vs. CONTROL.

[0206] FIG. 8. In vitro testing of the plasmid pAAV-CAG-IGF1-dmiRT expression in C2C12 cells. Igf-1 expression levels 6 days post-transfection and post-induction of the differentiation process of the cells. 6-well culture plates were used and each well was transfected with 4 .mu.g of DNA 6 h before the initiation of the differentiation process. Control wells were not transfected with any DNA. Values are expressed in arbitrary units normalized for the RplpO gene and relative to control group. Results are expressed as mean.+-.SEM, n=3 wells per condition. *p<0.05 vs. CONTROL; ***p<0.001 vs. CONTROL; ##p<0.01 vs. pAAV CAG IGF1.

[0207] FIG. 9. In vivo testing of the plasmid pAAV-CAG-IGF-1-dmiRT expression by hydrodynamic administration in ICR mice. Igf-1 expression levels in the liver 24 h post-hydrodynamic administration of 20 .mu.g of DNA diluted in saline. Control animals were administered with saline. Values are expressed in arbitrary units normalized for the Rplp0 gene and relative to control group. Results are expressed as mean.+-.SEM, n=3-5 male mice per group. **p<0.01 vs. CONTROL; ##p<0.01 vs. pAAV CAG IGF1.

[0208] FIGS. 10A-10C. Schematic diagram of the AAV8 vectors generated. FIG. 10A AAV8-CAG-IGF-1-dmiRT. Genome of the AAV8 vector expressing Igf-1 and the target sequences for the microRNA-122a and -1 (dmiRT). FIG. 10B AAV8-CAG-IGF-1. Genome of the AAV8 vector expressing Igf-1 without the dmiRT fragment. FIG. 10C AAV8-CAG-NULL. Genome of the AAV8 vector that does not encode any transgene. All the viral vectors contain the CAG promoter (hybrid cytomegalovirus enhancer/chicken .beta.-actin constitutive promoter) and the signal of polyadenylation (pA) flanked by the ITRs. The schematic representation is not to scale.

[0209] FIG. 11. Experimental design. Intraductal administration of AAV8-CAG-IGF-1-dmiRT, AAV8-CAG-IGF-1 and AAV8-CAG-NULL viral vectors in 4-week-old female NOD mice at a dose of 1.4.times.10.sup.12 vg. The animals were killed 1 month post-injection and the expression of Igf-1 and viral genomes were analyzed.

[0210] FIG. 12. In vivo testing of the AAV8-CAG-IGF-1-dmiRT viral vectors infectivity by intraductal administration in NOD mice. Igf-1 expression levels were quantified in pancreas, liver and heart RNA samples from NOD mice 1-month post-intraductal administration of 1.4.times.10.sup.12 vg per mouse. Values are expressed in arbitrary units normalized for the Rplp0 gene and relative to AAV8-CAG-NULL group. Results are expressed as mean.+-.SEM, n=4-5 animals per group. **p<0.01 vs. AAV8 CAG NULL; ##p<0.01 vs. AAV8 CAG IGF-1; ###p<0.001 vs. AAV8 CAG IGF-1.

[0211] FIG. 13. Viral genome quantification in liver and heart. Viral genomes corresponding to AAV8-CAG-NULL, AAV8-CAG-IGF-1 and AAV8-CAG-IGF-1-dmiRT vectors were quantified by qPCR in liver and heart samples of NOD mice 1-month post-intraductal administration of 1.4.times.10.sup.12 vg per mouse. Results are expressed as mean.+-.SEM, n=4-5 animals per group.

[0212] FIG. 14. Experimental design. Intraductal administration of AAV8-CAG-IGF-1-dmiRT and AAV8-CAG-NULL viral vectors in 4-week-old female NOD mice at a dose of 1.4.times.10.sup.12 vg. Glycaemia was followed-up in treated mice until 28 weeks of age and diabetes incidence was determined.

[0213] FIGS. 15A-15B. Individual glycaemic profiles in female NOD mice intraductally administered with AAV8 vectors. FIG. 15A. Glycaemia follow-up in AAV8-CAG-NULL treated NOD mice. n=10. Due to the death of one hyperglycemic mouse, one of the blood glucose monitoring lines is discontinued from 16 weeks of age onwards. FIG. 15B. Glycaemia follow-up in AAV8-CAG-IGF-1-dmiRT treated mice. n=16. The arrow indicates the age at which the corresponding viral vectors were administered (4 weeks). Results are expressed as mean.+-.SEM.

[0214] FIG. 16. Diabetes incidence in female NOD mice intraductally administered with AAV8 vectors. Mice were considered diabetic after two consecutives measurements of blood glucose .gtoreq.250 mg/dl. n=10 (AAV8-CAG-NULL); n=16 (AAV8-CAG-IGF-1-dmiRT). The arrow indicates the age at which the corresponding viral vectors were administered (4 weeks).

[0215] FIGS. 17A-17B. Pancreas transduction in female NOD mice intraductally administered with the AAV8-CAG-IGF1-dmiRT vector. FIG. 17A and FIG. 17B. Pancreas, liver and heart immunohistochemistry against IGF-1 in samples from NOD mice intraductally injected at 4 weeks of age with 1.4.times.10.sup.12 vg and killed 1 month later. FIG. 17A. Pancreatic islets transduction. NULL: AAV8-CAG-NULL; IGF-1: AAV8-CAG-IGF-1-dmiRT. Original magnification .times.400. FIG. 17B. Liver and heart representative image of AAV8-CAG-IGF1-dmiRT administered mice. Original magnification .times.100.

[0216] FIGS. 18A-18D. Intraductal delivery of AAV8-CAG-IGF-1a-dmiRT vectors protects NOD mice from autoimmune diabetes. FIG. 18A. Immunohistochemical detection of IGF-1 (black) (upper panel) and double immunostaining of insulin (grey) and IGF-1 (black) (lower panel) in islets at 8 and 28 weeks of age; Original magnification .times.400. FIG. 18B and FIG. 18C. Igf1a gene expression was analyzed in isolated islets (B) and total pancreas (C) at 28 weeks of age (n=4-6). FIG. 18D. Serum IGF-1 circulating levels at 28 weeks of age (n=8-10). Results are expressed as mean .+-.SEM. *p<0.05 vs. AAV8-CAG-NULL.

[0217] FIGS. 19A-19B. Liver and heart IGF-1 overproduction is prevented in AAV8-CAG-IGF1a-dmiRT treated mice. FIG. 19A. No IGF-1+ cells were detected neither in the liver nor in the heart of 28-week-old NOD mice intraductally injected with 1.times.10.sup.12vg of AAV8-CAG-IGF-la-dmiRT (AAV-IGF-1). Original magnification .times.400 (insets .times.1000). FIG. 19B. Igf1a gene expression analysis in liver and heart of AAV8-CAG-IGF-1a-dmiRT and AAV8-CAG-NULL-treated mice at 28 weeks of age (n=9-10).

[0218] FIGS. 20A-20F. AAV8-CAG-IGF1a-dmiRT treated mice show preservation of .beta.-cell mass by protection against autoimmune attack. FIG. 20A.

[0219] Immunohistochemical analysis of insulin (brown) in pancreas from AAV8-CAG-NULL (AAV-NULL) and AAV8-CAG-IGF-la-dmiRT (AAV-IGF-1) treated mice at 8 weeks and 28 weeks of age. NG: Normoglycemic HG: Hyperglycemic. FIG. 20B. .beta.-cell mass quantification. (n=3-5) FIG. 20C. Insulitis score was quantified in normoglycemic mice at 8 and 28 weeks of age. FIG. 20D. Fed serum concentration of insulin at 28 weeks of age. ND, Non-Detected. FIG. 20E and FIG. 20F. Relative gene expression of pro-inflammatory cytokines FIG. 20E. and antigen presenting molecules FIG. 20F. in isolated islets from normoglycemic mice at 28 weeks of age (n=4-6). Results are expressed as mean.+-.SEM. *p<0.05 vs. AAV8-CAG-NULL.

[0220] FIGS. 21A-21B. Absence of miR-122a repression in the liver upon intraductal administration of AAV8 vectors bearing miRT-122a sequences. FIG. 21A. Relative hepatic expression levels of target genes known to be regulated by endogenous miR-122a at 28 weeks of age. Gys1, glycogen synthase 1; AldoA, aldolase A/fructose-biphosphate; Slc7a1, solute carrier family 7; Ccng1, cyclin G1. FIG. 21B. Fed serum cholesterol levels at 28 weeks of age. NG: Normoglycemic. Results are expressed as mean.+-.SEM (n=4-7).

[0221] FIG. 22. Schematic representation of the AAV-CAG-IGF-1b-dmiRT expression cassette for the murine isoform IGF-lb. ITR: Inverted Terminal Repeats; CAG: hybrid promoter based on chicken beta-actin promoter and cytomegalovirus enhancer; mIGF-1b: sequence of murine IGF-1b (; splicing isoform of pro-Igf-1, also called mechano-growth factor-MGF); miRT-122A: microRNA-122A target sequence (4 copies); miRT-1: microRNA-1 target sequence (4 copies); pA: poly A polyadenylation sequence. The abbreviated form dmiRT refers to microRNA target sequences for both 122A and 1.

[0222] FIGS. 23A-23D. Intraductal delivery of AAV8-CAG-IGF-1b-dmiRT vectors protects NOD mice from autoimmune diabetes. FIG. 23A. Cumulative diabetes incidence of NOD mice intraductally injected with 1.4.times.10.sup.12 vg of AAV8-CAG-NULL or AAV8-CAG-IGF1b-dmiRT vectors at 4 weeks of age. FIG. 23B. Individual glycemic profiles (n=10, AAV8-CAG-NULL; n=17, AAV8-CAG-IGF1b-dmiRT). FIG. 23C. Immunohistochemical detection of IGF1 (black) (upper panel) in islets, liver and heart at 8 weeks of age; Original magnification .times.400 (islets) and .times.100 (liver, heart). AAV-NULL: AAV8-CAG-NULL; AAV-IGF- 1b: AAV8-CAG-IGF-1b-dmiRT. FIG. 23D. Serum IGF-1 circulating levels at 28 weeks of age (n=8-10). Results are expressed as mean.+-.SEM.

EXAMPLES

[0223] We developed an AAV8-mediated gene transfer strategy to overexpress IGF-1 specifically in the pancreas of NOD mice. In order to target .beta.-cells as well as to maximize the number of acinar cells that would supply IGF-1 to .beta.-cells, the ubiquitous CAG promoter was selected to drive IGF-1 expression (Otherwise indicated IGF-1 refers to IGF-1a).

[0224] Infectivity and Tropism of AAV8 Vectors Into NOD Mouse Pancreas

[0225] Most studies examining the tropism of AAV vectors have been conducted in adult male individuals from healthy mouse strains (ICR, BALB/c, C57BL/6, etc.). However, the development of a gene therapy strategy for the prevention or treatment of diabetes in the spontaneous model NOD model requires the transfer of therapeutic genes in female individuals. At 4 weeks of age, NOD mice still preserve beta-cell mass, which is free of insulitis. It is from this age onwards that begins the process of infiltration of the islets and massive loss of beta-cells, culminating in the appearance of the disease. Therefore, 4-weeks-old would be a convenient time for the administration of candidate genes in NOD females to preserve beta-cell mass. One such candidate gene would be IGF-1.

[0226] 1. Analysis of AAV8 Vector Transduction Locally Administered to 4-Week-Old Female NOD Mice

[0227] We used a single-stranded AAV8 vector that overexpressed the green fluorescent protein (GFP) under the control of the ubiquitous promoter CAG (promoter hybrid based on the (3-actin promoter of the cytomegalovirus enhancer and chicken) to asses the transduction of pancreatic islets from 4-week-old female NOD mice at a dose of 1.4.times.10.sup.12 viral genomes (vg) (AAV8-CAG-GFP, viral vector comprising the sequence SEQ ID NO: 32). At one month post-injection animals were killed and tissue transduction was examined (FIG. 1).

[0228] 1.1. Pancreas Transduction

[0229] It was observed that female NOD mice locally injected in the pancreas with AAV8-CAG-GFP showed efficient transduction of beta-cells of the islets and a wide distribution of the vector in the exocrine pancreatic tissue (FIGS. 2A-2B), similar to what had been described in other animal models (Jimenez et al., 2011). Transduction of pancreatic islets was examined by double immunohistochemical staining against GFP and against insulin (FIG. 2A). In addition, it was observed that the dose administered allowed the transduction of mostly the periphery of the islet cells but also cells from the core.

[0230] 1.2. Transduction of Peripheral Tissues

[0231] It has been described that when AAV8 vectors are administered locally by intraductal injection in the pancreas, part of the burden of the administered viral vector is able to reach the systemic circulation and transduce non-pancreatic tissues, mainly the liver and heart (Jimenez et al., 2011). Thus, the use of ubiquitous promoters such as CAG promoter, allows expression of the gene construct carried in the AAV8 vector in the heart and liver of intradctally injected mice.

[0232] The histological analysis of these tissues in female NOD mice injected with AAV8-CAG-GFP vectors confirmed the expression of green fluorescent protein GFP in liver and heart. Hepatocyte transduction was mostly dected around the central venules of the liver (FIGS. 3A-3D).

[0233] 1.3. Modulation of AAV Tropism by Means of microRNA Target Sequences

[0234] To preclude transgene expression in liver and heart and restrict AAV8-mediated transgene overexpression to the pancreas, we took advantage of the microRNAs (miRs) regulation network. miRs are small RNA sequences that negatively regulate mRNAs through effects on the stability and translation of mRNA transcripts (Bartel, 2004). Thus, by incorporating target sites for a specific miR (miRTs) into the 3'-UTR of a transgene, its expression is inhibited in cells in which that miR is highly expressed (Brown and Naldini 2009). In this work, the highly expressed liver-specific microRNA-122a (miR-122a) and heart-specific microRNA-1 were selected to modulate AAV tropism.

[0235] 1.3.1. Analysis of the Expression of microRNA-122a in the Liver of NOD Mice

[0236] To validate the incorporation of the target sequence for the microRNA-122a (miRT-122a) into the AAV construct as a strategy to block the expression of the transgene in the liver, the levels of expression of miR-122a were assessed in female NOD mice. This study was conducted in pre-diabetic individuals (normoglycemic) and individuals at the stage of overt diabetes (hyperglycemia) to study whether there were changes in the hepatic expression of miR-122a during the progressive advancement of the disease. In addition, we also analyzed the expression of miR-122a in the pancreas of NOD mice to rule out possible interference of transgene expression in the tissue of interest due to the presence of miRT-122a sequences in the AAV construct.

[0237] As expected, miR-122a expression was only detected in the liver of NOD animals. In addition, it was observed that the expression levels of miR-122a in the liver were higher in diabetic NOD mice compared with pre-diabetic individuals, although the differences did not reach statistical significance (FIG. 4). This could be due to the role of miR-122a in lipid metabolism in the liver (Esau et al., 2006), which is altered in diabetic mice. In any case, this suggested that even disease progression in NOD mice, the inhibition of the AAV construct in the liver would be sustained, or even increased. However, there was no significant detectable levels of expression of miR-122a in the pancreas, regardless of the stage of diabetic animal.

[0238] 1.3.2. Analysis of the Expression of microRNA-1 in the Heart of NOD Mice

[0239] Similarly, to validate the use of the target sequence for the microRNA-1 (miRT-1) as a strategy to block the expression of the transgene carried in the AAV vector specifically in the heart, it the levels of expression of miR-1 were assessed in pre-diabetic (normoglycemic) female NOD mice and NOD mice at the phase of overt diabetes (hyperglycemic). In addition, we also analyzed the expression of miR-1 in the pancreas to rule out possible interference of the expression of the transgene in this tissue. The results showed that miR-1 was expressed in the heart of NOD animals at similar levels regardless of the diabetic stage. Regarding the pancreas, expression of miR-1 was not detected for any of the conditions tested (FIG. 5).

[0240] Thus, the use of an AAV8 vector intraductally administered to 4-week-old NOD mice would be an appropriate approach to direct the expression of a transgene as IGF-1 in the pancreas of NOD mice. In addition, the incorporation of the target sequences miRT-122a and miRT-1 in the 3'-UTR region of the AAV construct may be an effective strategy to block the expression of the transgene in undesired tissues, such as liver and heart without altering the levels of expression in the pancreas. Thereby, overexpression of IGF-1 locally in the pancreas would be achieved. Moreover, the inhibition of transgene expression provided by the recognition of miRT-122a and miRT-1 in the liver and heart, respectively, would not be altered during diabetes progression in NOD mice.

[0241] 1.4. Construction of an Adeno-Associated Viral Vector Encoding Murine IGF-1

[0242] A construct for producing AAV8 vectors capable of directing the overexpression of IGF-1 specifically in the pancreas of NOD female individuals was prepared. With the aim of achieving high levels of overexpression of the transgene and transduce the largest possible number of cells in the pancreas, the ubiquitous CAG promoter was used. With the aim of blocking the expression of IGF-1 in undesired tissues such as liver or heart, four copies of the target sequence of the liver-specific miR-122a and four copies of the target sequence of the heart-specific miR-1 totally complementary (pAAV-CAG-IGF-1-4xmiRT122a-4xmiRT1, SEQ ID NO: 30 or SEQ ID NO: 31) were introduced in the region 3'-UTR of the construct. To simplify the nomenclature of the construct, the term dmiRT was used to refer to the presence of four copies of the target sequence for both microRNA-122A and -1 (pAAV-CAG-IGF-1-dmiRT) (FIG. 6).

[0243] 1.4.1. Checking the Expression of the Construct pAAV-CAG-IGF-1-dmiRT In Vitro and In Vivo.

[0244] Before producing the serotype 8 viral vectors AAV8-CAG-IGF-1-dmiRT the in vitro expression of the generated construct pAAV-CAG-IGF-1-dmiRT was verified. To this end, INS-1 cells (from rat insulinoma) and C2C12 cells (mouse myoblasts) were transfected and the levels of IGF-1 were quantified. Furthermore, the expression construct pAAV-CAG-IGF-1-dmiRT was also evaluated in vivo by hydrodynamic administration to ICR mice.

[0245] 1.4.1.1. Transfection of the Construct pAAV-CAG-IGF-1-dmiRT Into INS-1 Cells

[0246] INS-1 cells were transfected with plasmid pAAV-CAG-IGF-1-dmiRT (SEQ ID NO: 30 or SEQ ID NO: 31) to evaluate whether the generated construct correctly expressed IFG-1. In addition, in order to assess the possible influence of target sequences of microRNAs 122A and 1 in the expression of the construct, INS-1 cells were also transfected with a vector containing the same expression cassette without the target sequences of microRNAs 122A and 1 (pAAV-CAG-IGF-1, SEQ ID NO: 33). At 48 hours post-transfection, the mRNA was isolated from the cells and the levels of IFG-1 quantified by PCR. The result showed a high expression of IGF-1 transcript in transfected cells compared to control cells not transfected, thus verifying the functionality of the generated expression vector. In addition, the levels of overexpression of IGF-1 from construct pAAV-CAG-IGF-1-dmiRT were indistinguishable from the levels obtained with the construct pAAV-CAG-IGF-1 (SEQ ID NO: 33) (FIG. 7). These data indicated that the plasmid was functional in vitro and that the target sequences of microRNAs 122A and 1 did not interfere with the expression of IGF-1 in INS-1 cells.

[0247] 1.4.1.2. Transfection of the Construct pAAV-CAG-IGF-1-dmiRT Into C2C12 Cells: Functionality of the Target Sequence of the microRNA-1

[0248] C2C12 cells, an immortalized line of mouse myoblasts, proliferate rapidly under conditions of high concentration of serum, and differentiate and fuse into myotubes in low serum concentration. In order to check the functionality of the target sequence of microRNA-1 in the construct pAAV-CAG-IGF-1-dmiRT, C2C12 cells were transfected and the differentiation process of these cells was induced. To assess the effect of the microRNA target sequence-1 in the construct, cells were also transfected with plasmid pAAV-CAG-IGF-1. After six days of transfection and having induced differentiation to myotubes, mRNA was isolated from the cells and the levels of Igf-1 quantified by PCR. The results showed a marked overexpression of Igf-1 in C2C12 cells transfected with plasmid pAAV-CAG-IGF-1. However, the presence of the target sequence of microRNA-1 in the plasmid construct pAAV-CAG-IGF-1-dmiRT caused a decrease of approximately 50% in the expression of IGF-1 compared to the levels obtained from construct pAAV-CAG-IGF-1 (FIG. 8). These results validate the use of microRNA target sequence-1 to reduce the levels of Igf-1 in muscle cells.

[0249] 1.4.1.3. Hydrodynamic Administration of the Construct pAAV-CAG-IGF-1-dmiRT in ICR Mice: Gunctionality of the Target Sequence of the microRNA-122a in Liver.

[0250] The cell line mainly used for the in vitro study of liver tissue, cells derived from a hepatocellular carcinoma HepG2, do not express the microRNA-122a. Therefore, to assess the functionality of the target sequence of microRNA-122A, expressed in the liver, and validate the expression construct pAAV-CAG-IGF-1-dmiRT in vivo, an hydrodynamic injection of plasmid in mice ICR was carried out. The hydrodynamic administration through the tail vein (HTV injection) of plasmid DNA is an invaluable tool for gene transfer in mouse. It consists of a fast injection, in about 5 seconds, of a saline solution containing the plasmid DNA in a volume equivalent to 8-10% of body weight of the mouse. ICR male mice of 8 weeks of age were administered by HTV injection with the construct pAAV-CAG-IGF-1-dmiRT. To compare the effect of the microRNA target sequence-122A, animals were also injected with pAAV-CAG-IGF-1 plasmid. The control group was administered with saline solution without any plasmid.

[0251] At 24 hours post-administration, the liver was recovered, hepatocytes mRNA was isolated and the levels of Igf-1 were quantified by quantitative PCR. The results showed that the presence of the microRNA-122a target sequence in the construct pAAV-122A-CAG-IGF-1-dmiRT was able to completely block Igf-1 expression in the liver of mice. In contrast, in animals injected with plasmid pAAV-CAG-IGF-1 a marked overexpression of Igf-1 was obtained in this tissue (FIG. 9). These results indicated that the expression vector pAAV-CAG-IGF-1-dmiRT was also functional in vivo and validated the use of the microRNA target sequence-122A to reduce the levels of Igf-1 in liver cells.

[0252] 1.4.2. Production of Vectors AAV8-CAG-IGF-1-dmiRT for the Gene Transfer in Pancreas of NOD Mice

[0253] Once validated the expression of the construct pAAV-CAG-IGF-1-dmiRT validated both in vitro and in vivo, the production of vectors AAV of serotype 8 AAV8-CAG-IGF-1-dmiRT was carried out. In addition, to confirm that the target sequences of microRNAs 122A and 1- contained in AAV8-CAG-IGF-1-dmiRT were also functional in the NOD mouse and did not alter the expression in the pancreas, vectors encoding the same expression construct without the dmiRT fragment (AAV8-CAG-IGF-1) were generated. Finally, in order to rule out any effect produced by the administration of a vector AAV8, vectors not encoding any transgene (AAV8-CAG-NUL, vector comprising SEQ ID NO: 34) were also generated (FIGS. 10A-10C). All-viral vectors AAV8-CAG-IGF-1-dmiRT, AAV8-CAG-IGF and AAV8-CAG-NUL were produced using the system triple transfection in HEK-293 cells as specified in Material and Methods section. Once generated, they were administered to NOD mice to evaluate their infectivity.

[0254] 1.4.2.1. In Vivo Analysis of Infectivity and Expression of Vectors AAV8-CAG-IGF-1-dmiRT in NOD Mice

[0255] Female NOD mice of 4 weeks old were intraductal administered with vectors AAV8-CAG-IFGF-1-dmiRT, AAV8-CAG-IGF-1 and AAV8-CAG-NUL at a dose of 1.4.times.10.sup.12 vg (vg: viral genomes) per animal to assess their infectivity. One month post-administration, the animals were killed and samples were extracted from the pancreas, liver and heart to quantify the expression of Igf-1 and the presence of viral genomes in these tissues (FIG. 11).

[0256] Quantitative PCR analysis of the expression of Igf-1 in pancreas showed a significant increase in Igf-1 mRNA both in animals administered with vectors AAV8-CAG-1IGF1 and in animals with AAV8-CAG-IGF1-dmiRT, compared with the control group injected with the vector AAV8-CAG-NUL. In addition, levels of overexpression in pancreas were similar between the two groups, which corroborated the non-interference of sequences miRT-122a and miRT-1 in the expression of the transgene. However, when measuring the expression of Igf-1 in liver and heart, only increased levels of Igf-1 mRNA were detected in animals injected with the vectors AAV8-CAG-IGF-1. Animals that were administered with the vector containing the target sequences of microRNAs 122A and 1 (AAV8-CAG-IFG-1-dmiRT) showed levels of Igf-1 expression similar to those obtained in animals injected with the vector AAV8-CAG-NUL (FIG. 12).

[0257] To confirm that the reduced expression of Igf-1 in liver and in heart of animals injected with the vector AAV8-CAG-IGF-1-dmiRT was not due to a different infectivity of viral preparations AAV8-CAG-IGF-1 and AAV8-CAG-IGF-1-dmiRT, a quantification of viral genomes in these tissues was done. The presence of viral genomes was determined by quantitative PCR, as specified in the Materials and Methods section. The result allowed to observe similar levels of viral genomes present in liver and in heart of the animals injected, regardless of the viral preparation. This indicated that the viral preparations AAV8-CAG-IGF-1-dmiRT, AAV8-CAG-IGF-1 and AAV8-CAG-NUL, administered at the same dose, had a similar infectious power in liver and heart in animals injected intraductally. Therefore, this confirmed that the lower Igf-1 expression observed in these tissues was due to the presence of the target sequences of the microRNA-122A, in liver, and of the microRNA-1 in heart, in the vector AAV8-CAG-IGF-1-dmiRT. In addition, it was observed that the tropism of vectors AAV8 was much higher in the liver, compared with the heart, where a much lower amount of viral genomes per cell was found (FIG. 13).

[0258] These results confirmed that the presence of the sequence of recognition of miR-122A, in liver, and miR-1 in heart, in the construct AAV8-CAG-IGF-1-dmiRT, blocked the expression of Igf-1 induced by vector AAV8 in the tissues mentioned. Thus, the vector AAV8-CAG-IGF-1-dmiRT administered intraductal represented an appropriate strategy to direct the overexpression of Igf-1 locally in pancreas of NOD mice.

[0259] 2. Study of Prevention of Diabetes in NOD Mice Administered with Vector AAV8-CAG-IGF-1-dmiRT

[0260] To examine whether AAV-mediated specific pancreatic overexpression of Igf1 may prevent the development of autoimmune diabetes, 1.times.10.sup.12 vg of AAV8-CAG-IGF-1-dmiRT vectors were intraductally delivered to 4-week-old NOD mice. Control NOD mice received the same dose of non-coding AAV8-CAG-NULL vectors (FIG. 14). Long-term follow-up of blood glucose levels in AAV8-CAG-IGF-1-dmiRT treated mice revealed that whereas 60% of the animals in the control group became diabetic by 28 weeks of age, 75% of the IGF-1-treated mice remained normoglycemic (FIGS. 15A-15B and FIG. 16).

[0261] 2.1. IGF-1 Expression in Animals Administered with AAV8-CAG-IGF-1-dmiRT

[0262] One month after intraductal administration of AAV8-CAG-IGF-1-dmiRT vectors IGF-1 overexpression was found in the pancreas of NOD mice. Islets of animals treated with AAV8-CAG-IGF-1-dmiRT vectors showed a clear overexpression of IGF-1 compared to the islets of animals administered with AAV8-CAG-NULL vectors. Immunohistochemical staining against IGF-1 in the pancreas did not allowed to observe transduction of acinar cells possibly due to the rapid secretion of IGF-1. On the other hand, IGF-1 overproduction was not detected in the liver or the heart of animals administered with AAV8-CAG-IGF-1-dmiRT vectors (FIGS. 17A-17B).

[0263] Long-term overproduction of IGF-1 was predominantly detected in beta-cells of the mice receiving AAV8-CAG-IGF-1a-dmiRT vectors but not in the AAV8-CAG-NULL-treated animals (FIG. 18A). Accordingly, at 28 weeks of age, a 30-fold increase in Igf1a mRNA levels was found in islets from AAV8-CAG-IGF-1a-dmiRT-treated mice (FIG. 18B). No IGF-1.sup.+ exocrine cells were detected probably due to the rapid secretion of the factor (FIG. 18A). However, a 10-fold increase in Igf1a mRNA levels confirmed IGF1 overexpression in total pancreas of AAV8-CAG-IGF-1a-dmiRT treated mice at 28 weeks of age (FIG. 18C), very similarly to the results reported at 1 month after vector administration (FIG. 12). Consistent with the presence of the dmiRT sequence in the AAV construct, neither IGF-1.sup.+ cells nor increased Igf1a mRNA were observed in the liver or the heart of AAV8-CAG-IGF-1a-dmiRT treated mice (FIGS. 19A-19B). In agreement, no differences in circulating IGF-1 levels were found between groups (FIG. 18D), suggesting that prevention of diabetes was mediated by IGF-1 locally produced in the pancreas.

[0264] 2.2. AAV8-CAG-IGF-1-dmiRT-Treated Mice Show Preservation of Beta-cell Mass by Protection Against Autoimmune Attack.

[0265] NOD mice treated with AAV8-CAG-IGF-1a-dmiRT vectors were significantly protected against lymphocytic infiltration of the islets and preserved beta-cell mass over time (FIG. 20A-C). Insulitis quantification was not possible in hyperglycemic mice, as beta-cells could barely be detected and the inflammatory infiltrate had disappeared. In agreement with preserved beta-cell mass, normal insulin circulating levels were detected in AAV8-CAG-IGF-1a-dmiRT treated mice at 28 weeks of age (FIG. 20D). Furthermore, islets from AAV8-CAG-IGF-1a-dmiRT mice showed a reduction in the expression of the inflammatory mediators Mip-lalpha, mip-1beta, Mig, IP-10 and Rantes as well as in the expression of Ifn-gamma, Tnf-alpha, and Il-1beta that exert direct cytotoxic effects against beta-cells (FIG. 20E). Additionally, the expression levels of the beta.sub.2-microglobulin (.beta..sub.2-m), H2-Aa, B7.1 and B7.2 genes, involved in antigen presentation, were also decreased in islets of IGF-la-treated animals (FIG. 20F).

[0266] 2.3 Absence of miR-122a Repression in the Liver Upon Intraductal Administration of AAV8 Vectors Bearing miRT-122a Sequences.

[0267] Given that vectors bearing miRTs have been used as competitive inhibitors to repress endogenous miRs, we investigated specific inhibition of miR-122a in the liver of AAV8-CAG-IGF-1-dmiRT treated mice. To exclude diabetes-related alterations, potential miR-122a repression was analyzed in normoglycemic animals. No interference in the expression levels of various miR-122a-regulated genes (Tsai et al 2012) was confirmed in 28-week-old NOD mice treated with AAV8-CAG-IGF-1-dmiRT compared to mice injected with AAV8-CAG-NULL vectors, which do not encode for any target site for miR122a (FIG. 21A). Additionally, serum cholesterol levels, reported to be regulated by miR-122a (Tsai et al 2012), were unchanged in the AAV8-CAG-IGF-1-dmiRT group (FIG. 21B). These results suggest that intraductal administration of AAV8-CAG-IGF-1-dmiRT vectors did not alter microRNA regulation network in the liver.

[0268] 2.4. Intraductal Delivery of AAV8-CAG-IGF-1b-dmiRT Vector Protects Against Diabetes Development in NOD Mice.

[0269] To examine whether AAV-mediated specific pancreatic overexpression of Igf-lb may prevent the development of autoimmune diabetes, 1.times.10.sup.12 vg of AAV8-CAG-IGF-1b-dmiRT vectors (FIG. 22) were intraductally delivered to 4-week-old NOD mice. Control NOD mice received the same dose of non-coding AAV8-CAG-NULL vectors. Long-term follow-up of blood glucose levels in AAV8-CAG-IGF-1b-dmiRT treated mice revealed that whereas 60% of the animals in the control group became diabetic by 28 weeks of age, 47% of the IGF-1b-treated mice remained normoglycemic (FIG. 23A, FIG. 23B). Overproduction of IGF-1 was predominantly detected in beta-cells of the mice receiving AAV8-CAG-IGF-1b-dmiRT vectors but not in the AAV8-CAG-NULL-treated animals (FIG. 23C). Consistent with the presence of the dmiRT sequence in the AAV construct, no IGF-1.sup.+ cells were detected in the liver or the heart of AAV8-CAG-IGF-1b-dmiRT treated mice (FIG. 23C). In agreement, no differences in circulating IGF-1 levels were found between groups (FIG. 23D), suggesting that prevention of diabetes was mediated by IGF-1 locally produced in the pancreas.

[0270] 3. Materials

[0271] 3.1. Animals

[0272] Female NOD mice were purchased to Charles River Laboratories, Barcelona, Spain. To study the function of the target sequence of microRNA-122a, male ICR mice of 8 weeks old were used (Harlan Teklad, Barcelona, Spain).

[0273] Mice were housed in pathogen-free facilities (SER-CBATEG, Centre de Biotecnologia Animal i Terapia Genica, Barcelona) under controlled conditions of temperature (22.+-.2.degree. C.) and light (cycles of 12 hours of light and 12 hours of darkness) and fed ad libitum i.e., without restricting access to food, with a standard diet (2019S Teklad Global; Harlan Teklad, Madison, Wis., USA). For sampling, animals were anesthetized using anesthetic inhalators (Isoflurane, IsoFlo.RTM., Abbott Animal Health, Illinois, USA) and were eutanized by beheading. Blood and tissue samples were taken between 9:00 and 11:00 a.m. and immediately frozen with liquid N.sub.2 and stored at -80.degree. C. (blood and tissues) and preserved in formaldehyde (tissues). The Ethics Committee in Human and Animal Research and the Autonomous University of Barcelona (UAB) approved all experimental procedures.

[0274] 3.2. Bacterial Strains

[0275] XL2Bue E. coli strains (Stratagene-Agilent Technologies, Santa Clara, Calif., USA) were used to obtain the different plasmid constructs. All plasmids contain the gene for resistance to ampicillin to be selected. The bacterial culture was grown in solid LB media (Miller's LB Broth, Conda, Madrid, Spain) with 2% agar and 50 .mu.g/ml ampicillin.

[0276] 3.3. Antibodies

[0277] The tissue samples were fixed with a buffered solution of 4% formaldehyde, included in paraffin blocks and subsequently, sections of 2-3 microns were obtained to perform incubation with the corresponding antibodies. The antibodies used for the detection of proteins using immunohistochemical techniques are summarized in the following table 1:

TABLE-US-00002 TABLE 1 Antibodies Antibody Host Provider Ref. Anti-insulin Guinea Pig Sigma-Aldrich I-8510 Anti-IGF-1 Goat R&D Systems AF791 Anti-GFP Goat Abcam ab6673 Anti-IgG of Guinea Goat Molecular Probes A-11075 pig conjugated with Alexa Fluor 568 Anti-IgG goat, Donkey Santa Cruz sc-2042 byotinylated

[0278] 3,3-diaminobenzidine (DAB) (Sigma-Aldrich D5637-1G) and counterstaining with Mayer hematoxylin (Merck 109 249) were used for the preparations for light field imaging. For fluorescence images, streptavidin conjugated with Alexa Fluor 488 (Molecular Probes S-11223) was used to amplify the signal of antibody conjugated with biotin.

[0279] 3.4. Reagents

[0280] Molecular biology reagents were obtained from Roche (Roche Diagnostics Corp., IN, USA), Invitrogen Corporation/Life Technologies (San Diego, CA, USA), Bio-Rad Laboratories (Hercules, Calif., USA), Amersham Biosciences (Piscataway, N.J., USA), Sigma (St.Louis, Mo., USA), Promega Corporation (Madison, Wis., USA), BASF (Barcelona, Spain), Qiagen (Hilden, Germany), QBIOgen/MP Biomedicals (Irvine, Calif., USA) and Fermentas (St. Leon-Rot, Germany). Culture media and antibodies were obtained from PAA (Pasching, Austria) and serum (FBS) from Gibco (Invitrogen, Life Technologies).

[0281] 3.5. Plasmids

[0282] Plasmids used are identified in table 2.

TABLE-US-00003 TABLE 2 Plasmids used Gene of Name Promoter interest PolyA pAAV-CAG-GFP CAG GFP rabbit .beta.-globin (SEQ ID NO: 32) pAAV-CAG-NULL CAG -- rabbit .beta.-globin (SEQ ID NO: 34) pAAV-CAG-IGF1-dmiRT CAG mIGF1 rabbit .beta.-globin (SEQ ID NO: 30 or miRT122a SEQ ID NO: 31) miRT1 pAAV-CAG-IGF1 CAG mIGF1 rabbit .beta.-globin (SEQ ID NO: 33)

[0283] The CAG promoter (SEQ ID NO: 52) is a hybrid promoter consisting of the chicken .beta.-actin promoter and the enhancer of Cytomegalovirus and has a ubiquitous expression.

[0284] The cloning strategies used for the generation of different plasmids are summarized in the following table 3.

TABLE-US-00004 TABLE 3 cloning strategies used Name Cloning strategy pAAV-CAG-IGF1-dmiRT vector pAAV-CAG-GFP-dmiRT - digested with HindIII and NotI. insert pAAV-CAG-IGF-1 digested with HindIII and NotI. pAAV-CAG-IGF1 vector pAAV-CAG-IGF-1-dmiRT digested with BamHI/KpnI and blunt extremes

[0285] 3.6. Methods

[0286] 3.6.1. Basic DNA Techniques

[0287] 3.6.1.1. Preparation of Plasmid DNA

[0288] To obtain small amounts of plasmid DNA (3-4 .mu.g) minipreparacions (minipreps) were performed according to the alkaline lysis protocol originally described by Bionboim and collaborators (Birnboim and Doly, 1979).

[0289] To obtain large amounts of DNA (1-2.5 mg) maxipreparacions (maxipreps) or megapreparacions (megapreps) were performed from 250 or 500 ml of culture medium, respectively. The method is also based on alkaline lysis but in this case, DNA purification was performed by adsorption columns (PureYield.TM. plasmid MaxiPrep System, Promega Corporation, for maxipreps or EndoFree Plasmid Mega Kit, Qiagen, for the megapreps). The purity and concentration of the obtained DNA was determined by using a Nanodrop device (ND-1000, ThermoCientific).

[0290] 3.6.1.2. DNA Digestion with Restriction Enzymes

[0291] Each restriction enzyme requires specific reaction conditions of pH, ionic strength and temperature. In each case, the instructions of the manufacturer were followed (New England Biolabs, Roche, Promega and Fermentas). In general, DNA was digested with 0.5 units enzyme per 1 .mu.g of DNA in the buffer supplied by the manufacturer for 1-2 hours at the optimum temperature of each enzyme. The reaction product was analyzed on 1-2% agarose gels. When DNA should be digested with two or more restriction enzymes, digestions were carried out jointly if buffers and temperature conditions were compatible. If the enzymes had different requirements, after the first digestion DNA was purified by using the Geneclean.RTM. kit (QBIOgene) according to the manufacturer's instructions. DNA was eluted with 30 .mu.L of elution buffer provided by the manufacturer to subsequently perform the second digestion.

[0292] 3.6.1.3. Dephosphorylation of DNA Fragments

[0293] The plasmid DNA, once digested, can be religated. This process can be avoided by removing the phosphate residues at the 5' end of the vector. For dephosphorylation, one unit of alkaline phosphatase (Shrimp Alkaline phosphatase, Promega) per 1 .mu.g of DNA was used in the commercial buffer 1.times.. The dephosphorylation reaction was performed for 30 min at 37.degree. C. Subsequently, the enzyme was inactivated at 65.degree. C. for 15 min to avoid any interaction of the phosphatase in the ligation reaction.

[0294] 3.6.1.4. Generation of DNA Fragments with Blunt Ends

[0295] When blunt ends were needed for cloning, the digested DNA fragment was treated with the enzyme Klenow DNA polymerase I (New England Biolabs). The reaction was carried out following the manufacturer's instructions.

[0296] 3.6.1.5. Generation of Hybrid DNA Molecules: Ligation

[0297] The combination of hybrid DNA molecules from different fragments can be made by the action of the enzyme ligase (ligation). The DNA fragments of interest were mixed in various ratios of vector and insert (1:1, 1:5, 1:10) with the enzyme DNA ligase of bacteriophage T4 (New England Biolabs) and the corresponding buffer according to the protocol established by the trading house. The products resulting from the ligation were transformed into competent E. coli cells of the XL2-blue strain (Stratagene-Agilent Technologies, Santa Clara, Calif., USA).

[0298] 3.6.1.6. Transformation of Competent XL2-blue E.coli Cells

[0299] The plasmid DNA can be introduced into competent bacterial cells via transformation. In this study, the electroporation method was chosen to carry out the transformation of XL2-blue E.coli cells. 40 .mu.l of competent cells (2.times.10.sup.10 cells/ml) were thawed on ice until use. 1 .mu.l (approximately 10 ng) of the ligation reaction of DNA or control DNA was added directly to the electrocompetent cells. After incubation on ice for 5 min, cells were electroporated at 2500 V with an electroporador (Bio-Rad). Later, diluted .mu.l LB 200, were sown on LB plates with appropriate antibiotics and were incubated at 37.degree. C. O/N (overnight). The next morning DNA was extracted from recombinant colonies. By using restriction enzymes the presence of the hybrid molecules of DNA was analyzed.

[0300] 3.6.1.7. DNA Purification from Agarose Gels

[0301] Electrophoresis in agarose gel is the standard method used to separate, identify and purify DNA fragments. To separate DNA fragments between 0.2 and 7 kb 1% agarose gels were used. To separate fragments <0.2 kb 2% agarose gels were used. The visualization of the DNA in the gel was achieved by adding low concentrations of the fluorescent dye ethidium bromide (0.5 .mu.g/ml), which is sandwiched between two strands of DNA. DNA was visualized using low wavelength (310 nm) ultraviolet light (UV) using a transiluminator and a camera system (Syngene). 1 kb DNA ladder (Invitrogen) was used as molecular weight marker.

[0302] The agarose gels were prepared dissolving agarose in 1.times. TAE electrophoresis buffer (40 mM Tris-acetate pH 8.3 and 1 mM EDTA) with 0.5 .mu.g/ml of ethidium bromide. DNA samples were loaded in the agarose gel with lx loading buffer (Fermentas) and ran within 1.times. TAE electrophoresis buffer at 80 V. In order to obtain and purify DNA fragments of interest from the agarose gel, the GeneJET.TM._9 Gel Extraction Kit (Fermentas) was used. The DNA was quantified using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific Inc., USA).

[0303] 3.6.2. Eukaryotic Cells in Culture

[0304] 3.6.2.1. INS-1 Cells

[0305] INS-1 cells (ATCC) are from a rat insulinoma. INS-1 cells were incubated at 37.degree. C., 5% CO.sub.2 and medium RPMI-1640 (PAA) (10 mM glucose) with 2 mM glutamine, supplemented with FBS (Fetal Bovine Serum, PAA) at 10% (heat inactivated), 10 mM HEPES, 1 mM sodium pyruvate and 50 .mu.M 2-mercaptoethanol. To perform the maintenance steps, INS-1 cells were trypsinated to cause plate desadhesion, and then cells were plated at different dilutions.

[0306] 3.6.2.2. C2C12 Cells

[0307] C2C12 cell line (ATCC) is from an immortalized line of mouse myoblasts. The maintenance of the cells was conducted in medium DMEM (PAA) with 2 .mu.M glutamine and supplemented with 10% FBS (heat inactivated). To induce their differentiation to myotubes, the same medium was used but supplemented with HS (Horse Serum, PAA) 2% (inactivated by heat) instead of 10% FBS. In both cases they were grown in the incubator at 37.degree. C. and 8.5% CO.sub.2.

[0308] 3.6.2.3. HEK-293 Cells

[0309] HEK-293 or 293 cells (ATCC) are human embryonic kidney cells that present the adenoviral gene of Ad5 El stably integrated into the genome of cells. These cells were used for the amplification of viral vectors. They were kept in culture medium DMEM (PAA) with 2 mM glutamine, supplemented with 10% FBS (heat inactivated) in an incubator set at 8.5% CO.sub.2 and 37.degree. C. When they had a 70% confluence, cells were trypsinated and plated in different dilutions.

[0310] 3.6.3. DNA Transfection in Cultured Cells

[0311] To carry out the analysis of the in vitro expression of the constructs obtained, plasmid transfections were performed in different cell lines (INS-1, C2C12). For this purpose, the technique of transfection with Lipofectamina (Lipofectamine.TM.2000, Invitrogen) was used. The proportion of Lipofectamina/DNA used was 2.5 .mu.l of Lipofectamina (1 mg / ml) to 1 .mu.g DNA in INS-1 cells and 10 .mu.l of Lipofectamina (1 mg / ml) for four .mu.g DNA C2C12 cells. Upon transfection, INS-1 cells and C2C12 were 70-80% confluence. After 4-6 h post-transfection, the medium was changed from INS-1 cells to fresh culture medium. For C2C12 cells, the medium used was differentiation medium DMEM (PAA) with 2 mM glutamine and supplemented with HS (Horse Serum, PAA) 2% (inactivated by heat). The culture of the cells was stopped at 48 h post-transfection, in the case of INS-1 and 6 days post-transfection and induction of differentiation process in the case of C2C12 cells for analyzing the samples.

[0312] 3.6.4. Production, Purification and Titration of Adeno Associated Viral Vectors

[0313] 3.6.4.1. Production and Purification

[0314] Infective viral particles of AAV8 vectors were generated in HEK-293 cell cultures grown in Roller Bottles (RB) using a triple transfection protocol (Ayuso et al., 2010) which involves the use of three plasmids. This protocol is based on the precipitation of the virus using polyethylene glycol (PEG) and gradient ultracentrifugation with CsC1 that eliminates capsids content and lower protein impurities. The co-transfection of each RB (roller bottle) was carried out in 30 ml of calcium phosphate, 150 .mu.g of plasmid DNA of interest (with the ITR sequences and the corresponding expression cassette), together with 150 .mu.g of accessory plasmid rep2/cap8 (expression plasmid coding for the capsid proteins of the virus particles and for proteins necessary for viral replication, Plasmid Factory) and 150 .mu.g of plasmid helper pWEAD (expression plasmid coding for necessary adenoviral proteins; Plasmid Factory).

[0315] A total of 10 RB for each vector AAV8-CAG-IGF-1, AAV8-CAG-NULL and AAV8-CAG-IGF-1-dmiRT, and 20 RB for AAV8-CAG-GFP, were used for viral production. 48 h post-transfection, cells were collected and centrifuged at 2500.times.g for 10 min. The culture medium was stored at 4.degree. C. The cell pellet was reconstituted in TMS (50 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl.sub.2, pH 8.0) and was sonicated to lyse the cells and release the virus from the inside. The lysate was centrifuged for 30 min at 2500.times.g and the supernatant of this centrifugation was added to the culture medium previously stored at 4.degree. C. Then the viral particles were precipitated by an incubation of 15 hours in PEG 8000 8% (Sigma) at 4.degree. C. After that, vectors were precipitated by centrifugation at 4000.times.g for 30 min. This new precipitate, which contained viral vectors from the culture medium and from the cells, were reconstituted in TMS treated with benzonase (Merck) for 1 h at 37.degree. C. and then centrifuged for 10 min at 10000.times.g. The resulting supernatant was loaded into 37.5 ml tubes Ultra clear (Beckman) containing a CsCl discontinuous gradient of density 1.5 (5 ml) and 1.3 g/ml (10 ml). Then, they were centrifuged for 17h at 27000 rpm in a SW28 rotor (Beckman). The virus bands were collected using 18G needles and were transferred to Ultra clear tubes of 12.5 ml. The remaining of the 12.5 ml was filled with CsCl to 1.379 g/ml to generate a continuous gradient. These tubes were centrifuged at 38000 rpm in a SW40Ti rotor (Beckman) for 48 h. Finally, the bands corresponding to the full virus were collected and dialyzed through a 10 kDa membrane (Slide-A-Lyzer Dialysis Products, Pierce) and then filtered through 0.22 .mu.m filters (Millipore).

[0316] 3.6.4.2. Titration of Viral Genomes

[0317] The AAV8 viral genomes were determined by quantitative PCR (qPCR) adapting the protocol described by AAV2 and AAV8 Reference Standard Material (Lock et al., 2010) to vectors used in this study. Quantification of each vector was made in parallel with a reference vector of known concentration to ensure the validity of results. As a standard curve a linearized plasmid was used and quantified by measuring the absorbance at 260 nm. To ensure that the title of the viral vector will not be overestimated due to the presence in the final viral preparation of remaining DNA plasmids from the transfection, a DNAse treatment was performed prior to quantification. Only encapsidated genomes are resistant to digestion with DNase. 5 .mu.l of each preparation of the viral vector in 5 .mu.l of DNAse buffer 10.times. (130 mM Tris-HCl, 50 mM MgCl.sub.2, 1.2 mM CaCl.sub.2, pH 7.5), 1 .mu.l of DNAse (10 U/mL) and 36 .mu.l Milli-Q water. The digestion was incubated 60 min at 37.degree. C. After digestion of the samples, they were diluted to obtain an amplification value within the range of standard curve. Each TaqMan qPCR reaction contained in a final volume of 10 TaqMan LightCycler.RTM. 480 Probe Master 5 Primer forward (10 .mu.M) 0.2 .mu.l, Primer reverse (10 .mu.M) 0.2 .mu.l, probe (10 .mu.M) 0.1 .mu.l, H.sub.2O Milli-Q 2 .mu.l, diluted vector 2.5 .mu.l. The reaction involved an initial incubation for 10 min at 95.degree. C. (activation of the polymerase and denaturation of viral capsids, allowing the release of genomes) followed by 40 cycles of 30 s at 95.degree. C. (denaturation) and 30 s at 60.degree. C. (alignment and elongation). The primers used in the quantification of viral genomes of AAV hybridized to the common zone of the poly A (.beta.-globin intron):

TABLE-US-00005 Forward: (SEQ ID NO: 38) 5'CTT GAG CAT CTG ACT TCT GGC TAA T 3' Reverse: (SEQ ID NO: 39) 5'GGA GAG GAG GAA AAA TCT GGC TAG 3' Probe: (SEQ ID NO: 40) 5'CCG AGT GAG AGA CAC AAA AAA TTC CAA CAC 3'

[0318] The viral title was the result of the median of three quantification performed on different days as identified in table 4.

TABLE-US-00006 TABLE 4 viral titres Viral vector Titre (vg/ml) AAV8-CAG-GFP 8.7 .times. 10.sup.13 AAV8-CAG-NULL 1.4 .times. 10.sup.13 AAV8-CAG-IGF-1 9.8 .times. 10.sup.13 AAV8-CAG-IGF-1-dmiRT 1.3 .times. 10.sup.14

[0319] 3.6.4.3. Quantification of Viral Particles by Silver Staining

[0320] The analysis of the viral preparations by protein electrophoresis SDS-PAGE and subsequent staining with silver nitrate allows quantification of the viral capsids, which compared with the value of viral genomes obtained by qPCR allows to calculate the percentage of empty capsids in each preparation (ratio: viral particles/viral genomes). Moreover, this method allows display on the gel the degree of contamination of non-viral protein that could affect the transduction efficiency in vivo. The appropriate volumes of the vector of interest, of the vector reference (control also used in the quantification by qPCR) and of different dilutions of K208 vector (of known concentration and used as a standard curve) were mixed with the buffer 4.times. Novex.RTM. Tris-Glycine LDS Sample Buffer (Invitrogen) and 10.times. NuPAGE Sample Reducing Agent (Invitrogen) to a final volume of 20 .mu.l. After 5 minutes of boiling, samples were loaded on a gel 10% Bis-Tris Gel 1.5 mm 15 well (Invitrogen) and ran to 120 V for 2 h. Proteins of the gel were fixed with a mixture of Milli-Q H.sub.2O/ethanol/acetic acid. Then the gel was sensitized with a mixture of Na.sub.2S.sub.2O.sub.3/sodium acetate/ethanol/H.sub.2O Milli-Q. Finally, the gel was stained with silver nitrate and the bands were revealed using a mixture of Na.sub.2CO.sub.3/Formaldehyde/Milli-Q H.sub.2O. The title of viral particles was obtained by densitometry. From the intensity of the VP3 of each dilution of vector K208 a standard curve was generated to quantify viral particles of different preparations.

[0321] 3.6.4.4. Determination of Viral Genomes in Tissues by qPCR

[0322] The final value of the number of viral genomes that have transduced a given cell is also representative of the transgene copy number per cell. The determination of viral genomes was obtained from the comparison of 20 ng of genomic DNA extracted from various tissues of mice with a calibration curve generated from linearized plasmid serial dilutions.

[0323] 3.6.4.4.1.Generation of the Calibration Curve

[0324] The calibration curve was generated with linearized plasmid DNA pAAV-CAG-GFP-WPRE. Therefore, knowing the concentration of the plasmid and the number of base pairs of the construct (7772 bp) the number of copies of transgene per .mu.l was obtained. This plasmid DNA was serially diluted so as to achieve a calibration curve of logarithmic dilutions 10.sup.7:10.sup.6:10.sup.5:10.sup.4:10.sup.3:10.sup.2 copies of the transgene/.mu.l.

[0325] 3.6.4.4.2. Quantification of the Number of Copies of the Transgene by qPCR

[0326] Once the calibration curve generated and samples diluted, a qPCR was performed for calculating the number of copies of the transgene. Each qPCR reaction (TaqMan LightCycler.RTM. 480 Probe Master, Roche) was performed in 20 .mu.l final volume: TaqMan LightCycler.RTM. 480 Probe Master 10 Primer forward (10 .mu.M) 1 .mu.l, Primer reverse (10 .mu..M) 1 probe (10 .mu.M) 0.2 H.sub.2O Milli-Q 6.8 diluted vector 1 .mu.l. The reaction involved an initial denaturation for 10 min at 95.degree. C. and 45 cycles of 10 s at 95.degree. C., alignment (30 s at 60.degree. C.) and elongation (ls at 72.degree. C.). The oligonucleotides used are the following:

TABLE-US-00007 Forward: (SEQ ID NO: 41) 5' CTT GAG CAT CTG ACT TCT GGC TAA T 3' Reverse: (SEQ ID NO: 42) 5' GGA GAG GAG GAA AAA TCT GGC TAG 3' Probe: (SEQ ID NO: 43) 5' CCG AGT GAG AGA CAC AAA AAA TTC CAA CAC 3'.

[0327] The values obtained in the qPCR relative to the calibration curve, provided the number of copies of the transgene that was initially in 20 ng of total genomic DNA. Knowing that in mice, 20 ng of genomic DNA correspond to 3115.26 diploid cells, the number of copies of the transgene per diploid cell was obtained.

TABLE-US-00008 TABLE 5 TaqMan reaction TaqMan Reaction Volume TaqMan LightCycler .RTM. 480 Probe Master 10 .mu.l Primer forward (10 .mu.M) 1 .mu.l Primer reverse (10 .mu.M) 1 .mu.l Sonda (10 .mu.M) 0.2 .mu.l H.sub.2O Milli-Q 6.8 .mu.l Vector dilution 1 .mu.l

[0328] 3.6.4.5. In Vivo Injection of the Viral Vectors

[0329] 3.6.4.5.1. Retrograde Administration Through Pancreatic Biliary Duct

[0330] The retrograde injection via pancreatic biliary duct was conducted following the protocol described by Loiler and collaborators (Loiler et al., 2005) with minor modifications. The animals were anesthetized by an intraperitonial injection of ketamine (100 mg/kg) and xylacine (10 mg/kg). Once the zone shaved and an incision of 2-3 cm done, the abdomen was opening through an incision through the alba line, putting an abdominal separator. The bile duct was identified. Liver lobes were separated and the bile duct was clamped in the bifurcation of the hepatic tryad to prevent the spread of viral vector to the liver. Later, a 30G needle was introduced through the Vater papilla and retrogrally followed through biliary duct. The needle was fixed clamping the duct at the point of the intestine to secure its position and prevent the escape of viral vectors in the intestine . Slowly, a total volume of 100 .mu.l was injected with the corresponding dose of viral vectors. 1 min after injection, the clip which fixed the needle was pulled from and a drop of surgical veterinary adhesive Histoacryl (Braun, TS1050044FP) was applied at the entry point of the needle. Approximately 2 min later the clip of the biliar duct was pulled from and the abdominal wall and skin were sutured. The mice were left to recover from anesthesia on a heating mantle to prevent heat loss.

[0331] 3.6.4.5.2. Hydrodynamic Administration

[0332] The hydrodynamic administration via tail vein was performed as previously described (Liu et al., 1999). The DNA plasmid was diluted in saline volume (ml) equal to .about.10% of the average body weight (g) of the animals and it was manually injected through the tail lateral vein in less than 5 seconds. Before injection, the animals were exposed to infrared light under a 250 W (Philips) for a few minutes to dilate blood vessels and facilitate the visualization and access to the vein of the tail. The animals were placed in a plastic restrainer (Harvard Apparatus) to immobilize them and to facilitate the injection. 30G needles (BD) were used. In general, the mice tolerated well the hydrodynamic injections. Immediately after injection the mice normally remained motionless and showed forced breath that persisted for a few minutes, reaction which is caused by the nature of the injection. Apart from these immediate effects due to the hydrodynamic injection, the animals did not suffer known consequences during treatment.

[0333] 3.6.4.6. Isolation of Pancreatic Islets

[0334] The pancreatic islets were extracted from pancreas digestion and subsequent isolation of pancreatic islets. In order to digest the pancreas, mice were sacrificed, the abdominal cavity was exposed and 3 ml of a solution of Liberase (Roche, 0104 mg/ml medium without serum M199 (Gibco-Life Technologies 10012-037)) was perfused to the pancreas via the common biliar duct. During perfusion, circulation through the Vatter ampoule was blocked by placing a clamp. Once perfused, the pancreas were isolated from the animal remained in a tube in gel before being digested at 37.degree. C. for 19 min. To stop digestion and dilute the Liberase solution, 35 ml of cold medium M199 with 10% serum (Biowest S0250-500) were added and the tube stirred for 30 s to completely disintegrate the tissue. Then, two washes with 30 ml and 10 ml respectively of M199 medium supplemented with serum were done. Then, the solution of disintegrated tissue was filtered (450 mm PGI 34-1800-09) and collected into a new tube. The filtrate with 20 ml of medium with serum was centrifuged (Eppendorf 5810R rotor A-4 -62) at 200-230.times.g for 5 min at 4.degree. C. The supernatant was discarded and after carefully removing all traces of the medium, the pellet was resuspended in 13 ml of Histopaque-1077 (Sigma 10771) and M199 medium without serum was added to a volume of 25 ml avoiding mixing the two phases. Then it was centrifuged (Eppendorf 5810R) at 1000.times.g for 24 min at 4.degree. C. to obtain the pancreatic islets at the interface between the medium and the Histopaque and thus, they were collected with the pipette. Once isolated, the islets were washed twice with 40 ml of medium with serum and centrifuged at 1400 rpm, 2.5 min at room temperature. In the final wash the pellet with islets was resuspended in 15 ml of M199 medium. In this step, and to help their identification under the microscope, the islets were stained by adding a solution of 200 .mu.l Dithizone to the medium (for 10 ml volume: 30 mg Dithizone (Fluka 43820), 9 ml absolute EtOH, 150 .mu.l NH.sub.4OH and 850 .mu.l H.sub.2O). After 5 min of incubation, islets were transferred to a petri dish to be caught under the binocular microscope.

[0335] Once islets recovered, they were centrifuged at 300.times.g, 5 min and 4.degree. C. to remove the remains of medium, and were processed to RNA extraction.

[0336] 3.6.5. Analysis of the mRNA Expression by qPCR

[0337] 3.6.5.1. Total RNA Extraction

[0338] The tissue samples to obtain total RNA were obtained from freshly sacrificed animals and were quickly frozen in liquid nitrogen. Frozen tissues were homogenized (Polytron.RTM. MICCRA D-KIT-9 Prozess ART & Labortechnik GmbH & Co. KG, Mullheim, Germany) in 1 ml solution TriPure Isolation Reagent (Roche, 11667), and following the protocol of RNA purification in column RNeasy Mini Kit de QIAGEN (Cat.No.74104, QIAGEN, Invitrogen), total RNA was obtained. All samples were treated with DNasel purification columns (RNase-Free DNase Set supplied with the columns, Qiagen) and after removing the enzyme with the buffer supplied by the manufactured, the RNA was eluted in 30 .mu.l of distilled water free of RNases (DEPC). Finally, the concentration of RNA obtained was determined using a device Nanodrop (ND-1000, ThermoCientific).

[0339] 3.6.5.2. RNA Extraction from Pancreatic Islets

[0340] The obtention of RNA from pancreatic islets was performed by adding 1 ml of the solution TriPure Isolation Reagent (Roche, 11667) to resuspend the islets and following the commercial protocol of RNA purification in column RNeasy Micro Kit by QIAGEN (Cat.No.74004, QIAGEN, Invitrogen). The RNA was finally eluted in a volume of 14 .mu.l of water free of RNases. The concentration and purity of the obtained RNA was determined using a device Nanodrop (ND-1000, ThermoCientific).

[0341] 3.6.5.3. cDNA Synthesis

[0342] One .mu.g of total RNA was retrotranscripted to cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) following the manufacturer's instructions. Oligo-dT and random hexamer oligonucleotides were used as primers in the reaction with the presence of RNA inhibitor.

[0343] 3.6.5.4. Quantitative PCR

[0344] The qPCR was done in LightCycler.RTM. 480 (Roche) using LightCycler.RTM. 480 SYBR Green I Master (Roche). The following table 6 shows the different primers of mouse (m) or rat (r) used in qPCR:

TABLE-US-00009 TABLE 6 primers used in qPCR Forward sequence Reverse sequence Gene (5'-3') (5'-3') mIGF-1a TGGATGCTCTTCAGTTCGTGT CAACACTCATCCACAATGCCT mCc13 (Mip-1.alpha.) GCAACCAAGTCTTCTCAGCG AGCAAAGGCTGCTGGTTTCA mCc14 (Mip-1.beta.) CCATGAAGCTCTGCGTGTCT GAGAAACAGCAGGAAGTGGGA mCxcl9 (MIG) CGAGGCACGATCCACTACAA AGTCCGGATCTAGGCAGGTT mCxcl10 (IP-10) CCAAGTGCTGCCGTCATTTT AGCTTCCCTATGGCCCTCAT mCc15 (RANTES) GTGCCCACGTCAAGGAGTATT CCCACTTCTTCTCTGGGTTGG mCCL2 (MCP-1) ATGCAGTTAACGCCCCACTC GCTTCTTTGGGACACCTGCT mIFN-.gamma. AGACAATCAGGCCATCAGCA TGGACCTGTGGGTTGTTGAC mTNF-.alpha. TCTTCTCATTCCTGCTTGTGG GGTCTGGGCCATAGAACTGA mIL-1.beta. TGCCACCTTTTGACAGTGATG TGATGTGCTGCTGCGAGATT mH2-Aa CTCTGATTCTGGGGGTCCT ACCATAGGTGCCTACGTGGT m.beta.2-microglobulin CCGGAGAATGGGAAGC GTAGACGGTCTTGGGC mCD80 (B7.1) ATACGACTCGCAACCACACC GAATCCTGCCCCAAAGAGCA mCD86 (B7.2) GCTTCAGTTACTGTGGCCCT TGTCAGCGTTACTATCCCGC mSlc7a1 AAA CAC CCG TAA TCG CCA CT GGC TGG TAC CGT AAG ACC AA mCcng1 TGA CTG CAA GAT TAC GGG ACT CCC AAG ATG CTT CGC CTG TA mGys1 CCG CTA ACT CTA CCG GTC AC CCC CAT TCA TCC CCT GTC AC mAldoA GCG TTC GCT CCT TAG TCC TT AAT GCA GGG ATT CAC ACG GT mRplp0 TCCCACCTTGTCTCCAGTCT ACTGGTCTAGGACCCGAGAAG rRplp0 GATGCCCAGGGAAGACAG CACAATGAAGCATTTTGGGTAG (identified as SEQ ID NO: 53-92)

[0345] Each qPCR reaction contained 20 .mu.l of total volume: SYBR Green LightCycler.RTM. 480 Probe Master 10 .mu.l, Primer forward (10 .mu.M) 0.4 .sub.11.1, Primer reverse (10 .mu.M) 0.4 .mu.l, H.sub.2O Milli-Q 7.2 .mu.l, cDNA (dil.1/10) 2 .mu.l. The reaction consisted of 5 min at 95.degree. C. for initial denaturation and 45 cycles of four phases: denaturation (10 s at 95.degree. C.), alignment (10 s at 60.degree. C.), elongation (10 s at 72.degree. C.) and 30 s at 60.degree. C. Before cooling the reaction to 4.degree. C., it was kept 5 s at 95 C and 1 min at 65.degree. C. to determine the melting temperature. The method delta-delta-Ct (2-.DELTA..DELTA.Ct) described by Livak (Livak and Schmittgen 2001) was used to quantify the relative expression of genes of interest.

[0346] 3.6.6. Analysis of miRNA expression by qPCR

[0347] 3.6.6.1. miRNA Extraction from Tissues

[0348] Tissues were mecanically disintegrated with a polytron (Polytron.RTM. MICCRA-KIT D-9, ART Prozess & Labortechnik GmbH & Co. KG, Mullheim, Germany) with a lysis solution following the manufacturer's instructions of the comercial kit miRVana.TM. miRNA Isolation Kit (Ambion by Life Technologies, Madrid, Spain).

[0349] 3.6.6.2. cDNA Synthesis

[0350] The obtained miRNA were diluted to a concentration of 5 ng/.mu.l, from which 2 .mu.l were retrotranscripted into cDNA using miRCURY LNA.TM. Universal RT microRNA PCR--Universal cDNA synthesis kit II (Exiqon, Vedbaek, Denmark) following manufacturer's instructions.

[0351] 3.6.6.3. Quantitative PCR

[0352] Differently to the aforementioned quantitative PCR mRNA method, for the miRNA, ExilLENT SYBR.RTM. Green Master mix (Exiqon, Vedbaek, Denmark) was used following the manufacturer's instructions. Each qPCR reaction contained 10 mL total volume: PCR Master mix 5 .mu.l, Primer mix 1 .mu.l, cDNA (dil. 1/80) 4 .mu.l. The results of Ct were processed as explained in section 7.4, but in this case a different housekeeping gene, the U6, was used.

[0353] 3.6.7. Determination of Serum Parameters

[0354] Serum was obtained from blood samples obtained from the tail vein, or after the decapitation of mice in studies to final time. In both cases, blood was collected in heparine tubes and was kept for 1 h at 4.degree. C. Later, it was centrifuged for 10 min at 4.degree. C. 12000xg to obtain the serum, which was kept frozen at -80.degree. C. until the time of the determination of different parameters.

[0355] 3.6.7.1. Glucose

[0356] Serum glucose levels were determined from a drop of blood (5 .mu.l) from the tail of mice, through the system Glucometer Elite.TM. (Bayer Leverkusen, Germany). An animal was considered as diabetic, with two consecutive measurements of a glycemia .gtoreq.250 mg/dl.

[0357] 3.6.7.2. Insulin

[0358] The circulating insulin levels were determined from 100 .mu.l of serum by radioimmunoassay (RIA) using the Rat Insulin kit (Millipore) following the manufacturer's instructions. Mouse Insulin has a cross-reactivity of 100% compared to the one of rat.

[0359] 3.6.7.3. IGF-1

[0360] Circulating IGF-1 levels were determined from 10 .mu.l of serum using a commercial ELISA kit for mouse/rat IGF-1 (AC-18F1, Novozymes, Denmark) and following the manufacturer instructions. The detection limit of the kit is 63 ng/ml.

[0361] 3.6.7.4. Cholesterol

[0362] Total cholesterol in serum was quantified spectrophotometrically using an enzymatic assay kit (Horiba-ABX, Montpellier, France) and determined by Pentra 400 Analyzer (Horiba-ABX).

[0363] 3.6.8. Immunohistochemical Analysis of the Tissues

[0364] Tissues were fixed with a buffered solution of formalin 10% for 24 h at 4.degree. C., they were included in paraffin blocks (inclusor type Histokinette) and sections were obtained (2-3 p.m) with the help of a microtome (RM2135, Leica Biosystems, Barcelona). Subsequently, tissue sections were deparaffined (2 washes with xylol 10 min, 2 washes with 100% ethanol 5 min and 2 washes with 96% ethanol 5 min) and proceeded to the stain. The sections were incubated O/N at 4.degree. C. with primary antibodies (see section 3.3.), three washes of 5 min with PBS were done, and then incubated with the corresponding secondary antibody (see section 3.3) for lh at room temperature. For fluorescence immunohistochemistry, sections were subsequently incubated with streptavidin conjugated with fluorophores. For immunohistochemistry in light field, they were revealed with diaminobenzidine (DAB) (see section 3.3.). The images were obtained with an Eclipse 90i microscope (Nikon Instruments Inc., Tokyo, Japan). The program Nis Elements Advanced Research 2:20 was used to quantify the marked areas.

[0365] 3.6.9. Determination of Beta-Cell Mass

[0366] Beta cell mass was calculated by multiplying the total weight of the pancreas by the percentage of the beta cell area. The beta cell area of the pancreas was calculated from three sections separated 200 .mu.m and immunostaining against insulin by dividing the area of all insulin positive cells in each section between the total area of the corresponding section.

[0367] 3.6.10. Determination of the Degree of Insulitis

[0368] The incidence and severity of insulitis was analyzed in 3 pancreatic paraffin sections, each separately 100-150 microns and immunostained against insulin. The degree of insulitis (lymphocyte infiltration) in the pancreatic islets was determined according to the following classification criteria: no infiltration (0%), periinsulitis (mononuclear cells surrounding ducts and islets, but without major infiltration of the architecture of the islet, <25%); moderate insulitis (mononuclear cell infiltrate <50% of the surface of the islet); severe insulitis (>50% of the area of the islet infiltrated by lymphocytes and/or loss of islet architecture).

[0369] 3.6.11. Statistical Analysis

[0370] Results were expressed as mean.+-.standard error of the mean (SEM). Comparison of the results was performed using the Student t test of impaired data or through the table ANOVA of two factors. The differences were considered statistically significant with */190 p<0.05, **/##p<0.01 and ***/###p<0.001.

TABLE-US-00010 TABLE 7 list of SEQ ID Nos identified in the sequence listing SEQ ID NO Type of sequence 1 Murine nucleic acid coding for IGF-1a (SEQ ID NO: 28) 2 Murine nucleic acid coding for IGF-1b (SEQ ID NO: 29) 3 Human nucleic acid coding for IGF-1a (SEQ ID NO: 23) 4 Human nucleic acid coding for IGF-1b (SEQ ID NO: 24) 5 Human nucleic acid coding for IGF-1c/MGF (SEQ ID NO: 25) 6 Human nucleic acid coding for IGF-1c/MGF (SEQ ID NO: 26) 7 Human nucleic acid (SEQ ID NO: 27) 8-22 Nucleic acid sequences target for a given miRNA 23 Human protein IGF-1a (isoform 2) 24 Human protein IGF-1b (isoform 3) 25 Human protein IGF-1c/MGF (isoform 1) 26 Human protein IGF-1c/MGF (isoform 4) 27 Human protein IGF-1 (isoform XI) 28 Murine protein IGF-1a (isoform 5) 29 Murine protein IGF-1b 30 pAAV-CAG-preproIGF-1a-doble-miRT122a-miRT1 (SEQ ID NO: 28) 31 pAAV-CAG-preproIGF-1b-doble-miRT122a-miRT1 (SEQ ID NO: 29) 32 pAAV-CAG-GFP 33 pAAV-CAG-IGF-1 34 pAAV-CAG-NULL 35-43 primers 44 Murine protein IGF-1b/MGF (variant 1) 45 Murine protein IGF-1 (variant 2) 46 Murine protein IGF-1b/MGF (variant 3) 47 Murine protein IGF-1a (variant 4) 48 Murine nucleic acid coding for IGF-1b/MGF (SEQ ID NO: 44) 49 Murine nucleic acid coding for IGF-1 (SEQ ID NO: 45) 50 Murine nucleic acid coding for IGF-1b (SEQ ID NO: 46) 51 Murine nucleic acid coding for IGF-1a (SEQ ID NO: 47) 52 CAG promoter 53-92 primers 93-95 Nucleic acid sequences target for a given miRNA 96 Expression cassette of SEQ ID NO: 30 97 Viral vector of SEQ ID NO: 30 98 Expression cassette of SEQ ID NO: 31 99 Viral vector of SEQ ID NO: 31

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Sequence CWU 1

1

9916987DNAMus musculus 1acaatggaaa tgagtggctt cccttggggg aaaaagacgg actccaactc ccagctgtgc 60aatttactca ttgtttaaat ggacaaaagg cagtttaccc aggctcagag catacctgcc 120tgggtgtcca aatgtaacta gatgctttca caaaccccac ccacaaaaca acacctgttc 180ttaagtcctc agttttgtgt tcacctcggc ctcatagtac ccactctgac ctgctgtgta 240aacgacccgg acctaccaaa atgaccgcac ctgcaataaa gatacacatc atgtcgtctt 300cacacctctt ctacctggcg ctctgcttgc tcaccttcac cagctccacc acagctggac 360cagagaccct ttgcggggct gagctggtgg atgctcttca gttcgtgtgt ggaccgaggg 420gcttttactt caacaagccc acaggctatg gctccagcat tcggagggca cctcagacag 480gcattgtgga tgagtgttgc ttccggagct gtgatctgag gagactggag atgtactgtg 540ccccactgaa gcctacaaaa gcagcccgct ctatccgtgc ccagcgccac actgacatgc 600ccaagactca gaaggaagta catttgaaga acacaagtag aggaagtgca ggaaacaaga 660cctacagaat gtaggaggag cctcccacgg agcagaaaat gccacatcac cgcaggatcc 720tttgctgctt gagcaacctg caaaacatcg aaacacctac caaataacaa taataagtcc 780aataacatta caaagatggg catttccccc aatgaaatat acaagtaaac attccaacat 840cgtctttagg agtgtttgtt taaaaagctt tgcaccttgc aaaagtggtc ctggcgtggg 900tagattgctg ttggtccttt atcaataaca ttctatagag aaaaaaaata tatatataac 960tatatctcct agtccctgcc tctaaagagc cgaaaatgca tggatgttgt agagatccag 1020ttgctctaag tttctctctg aattttggct gctgaagcca ttcatttagc aactgtgtag 1080aggtggttta tgaatggttc ccttatcttc acctcttccc acgtagctca agctgcttgt 1140tttacagagt ctaatcatct tgtctagctg cattagacac accctttcct aacacttgta 1200tttgttgaat ttggcctcct taagagcaat agcaaataag tagtcaagtg gcctaccaag 1260ttttaacgta cctgactcca tctgtggcat ttgtaccaaa tataagttga atgcatttat 1320tttagacaca aagctttatt ttttttgaca ttgtgtttca agaaaaaaaa tagaataaca 1380ataactacaa ctttgaggcc aatcattttt aggtgtgtgt ttgaagcata gaacgtctct 1440taaactctca atggtttctt caaatgataa gttagtatgt aacctaagta tagcagtttc 1500tctctttttt atttttttcc atatagagca ctatgtaaag ttagtatatc aataatacag 1560gaaatatcaa acagtatgta aaactctgtt gttgttgttt tttagtacaa tggtgctatt 1620ttgtagtttg ttatatgaaa gaatctagtc aacacagtaa aaggagaaag caaagcaaaa 1680acaacaaacg aaagcctgga gcctaagatg acaaaacgag gaagggaact gaaaaaaaaa 1740atccttcctc ttgggagatg caaaggcctc cccaattatg ccttccaaga agaacttaag 1800atatagagtc cattaagacg cacttacttg tcaagtccag agaggaagct atggagtggg 1860aaaagcaaga ggctagggat ttgggagtcc tggtttcttt ttaatcactg aagaagtaag 1920tatttgcaac ctgggtcaca caaactcacc accctgtgac ctcagtcaaa tcactccacc 1980tctcggtgcc tcagttttcc tcatctgcaa aatgggggca atatgtcatc tacctacctc 2040aaaggggtgg tatgaagatt aaaaagtaga ccttcagatt tttgttctgg gtttccagga 2100gggtgcaaca tcagaaccct tgaattgcta ggatgcaagg aattctgtaa ataacccact 2160aacaatgtag ctccaaggat cattcatctg tcactgggat gccaccacaa tatccaagtt 2220cttattggtg aagctgtgca actaattagt gacaagctaa ggactcagtc tccccagcat 2280gtcacacggc aggagacatt tgatttgcag ttttatttaa cttctgcatt tgagcttatg 2340actataaaga ctagtgaaaa gaagggagag aggagaaaga agatccttgc caagtaaagg 2400gtaattaatt attattccat ttatccactc tcattaaagg gtaattaatt attccatgta 2460tccactctca ttaatccttc cagtcactta gtatctagaa ataactctaa cattgtcaat 2520gagactctac tcagtttgcc aaacacaatt ctccttcccc atagcatatg aaaaaaaggc 2580gctgacattc ttaaattttg aaatagtatc tattacaatc acaggttgct gtagcagatg 2640tagtcttgcc cttgtttgta catgcatgta tttttttttt aattttatga aaatgtgcta 2700gcaagaattg ctacttgagg ggcaaaattc ttccttctca agcctgaggt tctccctagt 2760gtctgcttag aaggaaggat ccagcttcct ggaaatgtgt tggatgcatt caactgggca 2820ttgctaacca aaaacattta gaaaaatgtt ctctatgtat atagcaagat tgtctccctc 2880ttttaaaaac aaaatccaat attcacatct tattacctac aaccttgatt ctctattgca 2940agcttcctta atattcttat aaaatgtatt aagaaaaaca aaaaggacac ctttagctct 3000ccttccgcca ggttgcctct agaatctctg gggaaatgca gaaggtgctg ttgagtaaag 3060ccctcagaag gattggattt aggaacatca ggcacgctgt acatcccctg attactgtag 3120aaatgtaaat ggaataagag gtcagctgac catccacctg cttccccaga aggatacagg 3180gaaaagttag gccctcacac accctgggtg acacttctga cttctagttc ttgttcacag 3240tgtgtacttt ttcaaattgg taattcccag aaaaacacat aggtggcctt ctccagatct 3300gtgggcttcc tgccatggtt ggatttggtg attccaagtg tctatcacat attttgttca 3360cttaattcta tccacagtca gaaattcttt caatgaggaa agtttaaata tgcaatcctt 3420tatccaatac ctaattctct ccaactgcat cataaatcaa gtaataaaaa ttaattgtac 3480taattaatca taataatgta ccattgtact tttaaatgaa tgaacactgc aagacaaatc 3540tatgtaaact ctgaaaagta actgatcatt atatggtgaa tcaaaatgac tcaagattga 3600tagaaaggga catttaaaat tttacaactc aaaattttgt agactttgct atggaggtaa 3660attgttttag tgcctagaga tggagcggtt ttaataaatt tacaaaagaa ctataaagat 3720aggtaggaag gaattttcat ttgataggat tgttgctgat ttacttactc aatacctagg 3780tcaaatgttg atcctattct ccaaagacta tcaagtgctt gaacattgta agatgagtct 3840gctccactga aaatgtaata catctctcca ttataatcta ttttcctggg gtaaaaaaat 3900ccttttttta aatatccacc tacatatacc taccctacat gtgcatttgc acatgcgtgc 3960atacgctcat gcgccccacc ccacacacac ctattcaccc taagactaag aagaaatcat 4020ttctttgaaa gtcttatctt tcaaaaaagg cagcggtgcc ccttgagact ccttctcctt 4080ctttgaatgt caatgtgaaa tgtggcatgt ctgtgtacat gaaaccatct cataccctat 4140ggctccaggg tttctttatg gtttgtgcac ttgggaggat gcgcagaaga caggatgcag 4200cctgttttgc tttccccttt actgtttggc cagctacgcc aatgtggtgc tattgtttct 4260ttaagaaagt acttgactaa aaaaaaagaa aaaaagaaaa aaagaaaaga aaaagaaaaa 4320agaaaaaaaa agaaagcata gacctatttt tttaaagtct gaaaacaaca gttctatagt 4380agatggctta ctgagatagc attagatcta gccaccaccc tagccaccac ctttcaacta 4440tgtgtcactc acaagtagaa tattgttcac caagttgtga gtttgggggt tcagagacaa 4500aggatggaaa agttttaaag ttagatggct caatcatttc attggctctc aaatttaaca 4560aaattggcaa tacttcaccc aatctgaagt gttggtcaat aacttgaact gggggcaaaa 4620ataacttcag gcaaatggca gaagaaaata attaacttac ttcttgcttt ttttgttgat 4680tgtttggttt cctgttgatt tttggttttg gttttgctgt gggtgggtga gtacatgtgt 4740gtaagtacgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgttccact caaaaacaaa 4800tactcagaaa gtggagaaaa tacaacgatt ttaagagcat agacttacct actactagaa 4860ccagcttctg tcacatcctc tggagaaggc actgatttct tgttttgtag aggttgctct 4920tccatcagtg acctgaaaga gtgaccagtc tcctagagta gacatggatc tcattaggag 4980aagacagaag tatttcctta tgaattgggc ttatctactg acaaagaaag ggaagagttt 5040atgagaagtt attgaagaag atggctaaca gtctgtgaag attttgttct ggtttttttt 5100gttgttgttg ttgttgggtt tgggttttga tttttttttt ttttttttta ctttatacaa 5160tctttatgaa tggaaatctt aatgctcaaa aagacttggt ctttttttct ctttcgtaac 5220agaatggaag atgacaaact cacatagact ctttctaggc tggctagcaa aggtgtggtt 5280tgacttattt gaatcagacc attttaaatg ttcctctcta tttttaatca taaaaggctg 5340tcataattta ttagcgtagg ccctttttgg cacttctcaa atgaatgagc attcccattc 5400aaagcatggc tttccccatg gttccaaaac atgaatgatt aatattaagg aattatttac 5460ttcaaaatac agtagaagtg tgagtctctg ttcccattcc ccacaaagat cattaagtcc 5520tgaatcgggg gcgggggtgg ggcgcctgga tactaaggga atttttttgt tgcttgtttt 5580ttgttttcaa tgctagtgct taatcctata gtatacagat ttgcttcttg ctattgtgat 5640attctgtaag actttcctgt taggtattag aaattgatac ataaatacct tttttgtgtg 5700gtttctattt aaaaggaaag agataagact gtctgaacct taaattcgta aggcacatga 5760taaagagatc acattaaata acaagccata tctggttcaa tcctttcttt cttatcattt 5820taaggaaaac ttgcccagat aagacagagg cccaggggac ttttgaaact ctctttgttc 5880cgccaattca ttttggctgg tgatggtttt tccccagtgt ctgcctcaga atcttttaga 5940ggctggccag actaaagact gtcttttaaa acacatttca catggttcct cttaatgaat 6000gattacactt atgtagaaca tgattttttt ttctctccac ttattttttt tttccccatc 6060attgataagg gttcttaagg agaagaattc attaacaaaa ctcaagaaag cgtacaaaaa 6120aaaaattcta aatgtcactg cccaattgaa atacgagcta aaatggaaat actttctcct 6180acttaaaacc cagactgaat caccttcaaa atgacctttc acaatctttc caatttgcct 6240ttgtttaaac tgtctgggcc taaaagcaag cattattcat tttctcttgc ccaaagtgaa 6300cttgtgtaaa gtaggaaaat taaaagaaac tgctagaaat cccttccaac cagtggctga 6360cccctctcac tagctcacag caaagtctcc tctgttgatc tatcacctag tctcatttcg 6420tttgaatatt tacattgtac ctactgctaa acacttggca ggaggctcca tccatatctc 6480ctatcggtgt ctctgtatcc ttaaaccttg caaacatcat acagtgtata ttaagtttac 6540aggaaagctc caaatagcat atcagacctg gtctctcttt gttaaagatt taaggagcta 6600tgggaatctg gattacaacg cacattttgc ttcatttatt tttatcacac tttaaaggcc 6660aagggtgatg attaacttac agacactgaa ttgatttccc tactgaaacc tgaaagtaat 6720atttggtcat tcattgtatg tgttttacac aaaaaaaaca tcttctatca aattactcct 6780gattgtattt gaagtggtta ttcaattcat ttatggcaga gcaatatctg tcctaatgac 6840tcttataaaa tgtaactaac tgaatcatta tcttacattt actgtttagt aagcatattt 6900tgaaattgta tggctagagt gtcataataa aatggtatat ctttctttag taattacatt 6960aaaattaatc atgtttgatt aactggt 698721536DNAMus musculus 2gacttcttga agataaagat acacatcatg tcgtcttcac acctcttcta cctggcgctc 60tgcttgctca ccttcaccag ctccaccaca gctggaccag agaccctttg cggggctgag 120ctggtggatg ctcttcagtt cgtgtgtgga ccgaggggct tttacttcaa caagcccaca 180ggctatggct ccagcattcg gagggcacct cagacaggca ttgtggatga gtgttgcttc 240cggagctgtg atctgaggag actggagatg tactgtgccc cactgaagcc tacaaaagca 300gcccgctcta tccgtgccca gcgccacact gacatgccca agactcagaa gtccccgtcc 360ctatcgacaa acaagaaaac gaagctgcaa aggagaagga aaggaagtac atttgaagaa 420cacaagtaga ggaagtgcag gaaacaagac ctacagaatg taggaggagc ctcccacgga 480gcagaaaatg ccacatcacc gcaggatcct ttgctgcttg agcaacctgc aaaacatcga 540aacacctacc aaataacaat aataagtcca ataacattac aaagatgggc atttccccca 600atgaaatata caagtaaaca ttccaacatc gtctttagga gtgtttgttt aaaaagcttt 660gcaccttgca aaagtggtcc tggcgtgggt agattgctgt tgatccttta tcaataacat 720tctatagaga aaaaaatata tatatataac tatatctcct agtccctgcc tctaaagagc 780cgaaaatgca tggatgttgt agagatccag ttgctctaag tttctctctg aattttggct 840gctgaagcca ttcatttagc aactgtgtag aggtggttta tgaatggttc ccttatcttc 900acctcttccc acgtagctca agctgcttgt tttacagagt ctaatcatct tgtctagctc 960cattagacac accctttcct aacacttgta tttgttgaat ttggcctcct taagagcaat 1020agcaaataag tagtcaagtg gcctaccaag ttttaacgta cctgactcca tctgtggcat 1080ttgtaccaaa tataagttga atgcatttat tttagacaca aagctttatt ttttttgaca 1140ttgtgtttca agaaaaaaaa tagaataaca ataactacaa ctttgaggcc aatcattttt 1200aggtgtgtgt ttgaagcata gaacgtctct taaactctca atggttcctt caaatgataa 1260gttagtatgt aacctaagta tagcagtttc tctctttttt atttttttcc atatagagca 1320ctatgtaaag ttagtatatc aataatacag gaaatatcaa acagtatgta aaactctgtt 1380gttgttgttt tttagtacaa tggtgctatt ttgtagtttg ttatatgaaa gaatctagtc 1440aacacagtaa aaggagaaag caaagcaaaa acaacaaacg aaagcctgga gcctaagatg 1500acaaaacgag gaagggaact gaaaaaaaaa aaaaaa 153637204DNAHomo sapiens 3gcatacctgc ctgggtgtcc aaatgtaact agatgctttc acaaacccca cccacaaagc 60agcacatgtt tttaagactt cagttttcta ttcacatcgg cctcataata cccaccctga 120cctgctgtaa aagacctgga acaaacaaaa atgattacac ctacagtgaa gatgcacacc 180atgtcctcct cgcatctctt ctacctggcg ctgtgcctgc tcaccttcac cagctctgcc 240acggctggac cggagacgct ctgcggggct gagctggtgg atgctcttca gttcgtgtgt 300ggagacaggg gcttttattt caacaagccc acagggtatg gctccagcag tcggagggcg 360cctcagacag gcatcgtgga tgagtgctgc ttccggagct gtgatctaag gaggctggag 420atgtattgcg cacccctcaa gcctgccaag tcagctcgct ctgtccgtgc ccagcgccac 480accgacatgc ccaagaccca gaaggaagta catttgaaga acgcaagtag agggagtgca 540ggaaacaaga actacaggat gtaggaagac cctcctgagg agtgaagagt gacatgccac 600cgcaggatcc tttgctctgc acgagttacc tgttaaactt tggaacacct accaaaaaat 660aagtttgata acatttaaaa gatgggcgtt tcccccaatg aaatacacaa gtaaacattc 720caacattgtc tttaggagtg atttgcacct tgcaaaaatg gtcctggagt tggtagattg 780ctgttgatct tttatcaata atgttctata gaaaagaaaa aaaaaatata tatatatata 840tatcttagtc cctgcctctc aagagccaca aatgcatggg tgttgtatag atccagttgc 900actaaattcc tctctgaatc ttggctgctg gagccattca ttcagcaacc ttgtctaagt 960ggtttatgaa ttgtttcctt atttgcactt ctttctacac aactcgggct gtttgtttta 1020cagtgtctga taatcttgtt agtctatacc caccacctcc cttcataacc tttatatttg 1080ccgaatttgg cctcctcaaa agcagcagca agtcgtcaag aagcacacca attctaaccc 1140acaagattcc atctgtggca tttgtaccaa atataagttg gatgcatttt attttagaca 1200caaagcttta tttttccaca tcatgcttac aaaaaagaat aatgcaaata gttgcaactt 1260tgaggccaat catttttagg catatgtttt aaacatagaa agtttcttca actcaaaaga 1320gttccttcaa atgatgagtt aatgtgcaac ctaattagta actttcctct ttttattttt 1380tccatataga gcactatgta aatttagcat atcaattata caggatatat caaacagtat 1440gtaaaactct gttttttagt ataatggtgc tattttgtag tttgttatat gaaagagtct 1500ggccaaaacg gtaatacgtg aaagcaaaac aataggggaa gcctggagcc aaagatgaca 1560caaggggaag ggtactgaaa acaccatcca tttgggaaag aaggcaaagt ccccccagtt 1620atgccttcca agaggaactt cagacacaaa agtccactga tgcaaattgg actggcgagt 1680ccagagagga aactgtggaa tggaaaaagc agaaggctag gaattttagc agtcctggtt 1740tctttttctc atggaagaaa tgaacatctg ccagctgtgt catggactca ccactgtgtg 1800accttgggca agtcacttca cctctctgtg cctcagtttc ctcatctgca aaatgggggc 1860aatatgtcat ctacctacct caaaggggtg gtataaggtt taaaaagata aagattcaga 1920ttttttttac cctgggttgc tgtaagggtg caacatcagg gcgcttgagt tgctgagatg 1980caaggaattc tataaataac ccattcatag catagctaga gattggtgaa ttgaatgctc 2040ctgacatctc agttcttgtc agtgaagcta tccaaataac tggccaacta gttgttaaaa 2100gctaacagct caatctctta aaacactttt caaaatatgt gggaagcatt tgattttcaa 2160tttgattttg aattctgcat ttggttttat gaatacaaag ataagtgaaa agagagaaag 2220gaaaagaaaa aggagaaaaa caaagagatt tctaccagtg aaaggggaat taattactct 2280ttgttagcac tcactgactc ttctatgcag ttactacata tctagtaaaa cctcgtttaa 2340tactataaat aatattctat tcattttgaa aaacacaatg attccttctt ttctaggcaa 2400tataaggaaa gtgatccaaa atttgaaata ttaaaataat atctaataaa aagtcacaaa 2460gttatcttct ttaacaaact ttactcttat tcttagctgt atatacattt ttttaaaagt 2520ttgttaaaat atgcttgact agagtttcca gttgaaaggc aaaaacttcc atcacaacaa 2580gaaatttccc atgcctgctc agaagggtag cccctagctc tctgtgaatg tgttttatcc 2640attcaactga aaattggtat caagaaagtc cactggttag tgtactagtc catcatagcc 2700tagaaaatga tccctatctg cagatcaaga ttttctcatt agaacaatga attatccagc 2760attcagatct ttctagtcac cttagaactt tttggttaaa agtacccagg cttgattatt 2820tcatgcaaat tctatatttt acattcttgg aaagtctata tgaaaaacaa aaataacatc 2880ttcagttttt ctcccactgg gtcacctcaa ggatcagagg ccaggaaaaa aaaaaaaaag 2940actccctgga tctctgaata tatgcaaaaa gaaggcccca tttagtggag ccagcaatcc 3000tgttcagtca acaagtattt taactctcag tccaacatta tttgaattga gcacctcaag 3060catgcttagc aatgttctaa tcactatgga cagatgtaaa agaaactata catcattttt 3120gccctctgcc tgttttccag acatacaggt tctgtggaat aagatactgg actcctcttc 3180ccaagatggc acttcttttt atttcttgtc cccagtgtgt accttttaaa attattccct 3240ctcaacaaaa ctttataggc agtcttctgc agacttaacg tgttttctgt catagttaga 3300tgtgataatt ctaagagtgt ctatgactta tttccttcac ttaattctat ccacagtcaa 3360aaatccccca aggaggaaag ctgaaagatg cactgccata ttatctttct taactttttc 3420caacacataa tcctctccaa ctggattata aataaattga aaataactca ttataccaat 3480tcactatttt attttttaat gaattaaaac tagaaaacaa attgatgcaa accctggaag 3540tcagttgatt actatatact acagcagaat gactcagatt tcatagaaag gagcaaccaa 3600aatgtcacaa cccaaaactt tacaagcttt gcttcagaat tagattgctt tataattctt 3660gaatgaggca atttcaagat atttgtaaaa gaacagtaaa cattggtaag aatgagcttt 3720caactcatag gcttatttcc aatttaattg accatactgg atacttaggt caaatttctg 3780ttctctcttc cccaaataat attaaagtat tatttgaact ttttaagatg aggcagttcc 3840cctgaaaaag ttaatgcagc tctccatcag aatccactct tctagggata tgaaaatctc 3900ttaacaccca ccctacatac acagacacac acacacacac acacacacac acacacacac 3960acattcaccc taaggatcca atggaatact gaaaagaaat cacttccttg aaaattttat 4020taaaaaacaa acaaacaaac aaaaagcctg tccacccttg agaatccttc ctctccttgg 4080aacgtcaatg tttgtgtaga tgaaaccatc tcatgctctg tggctccagg gtttctgtta 4140ctattttatg cacttgggag aaggcttaga ataaaagatg tagcacattt tgctttccca 4200tttattgttt ggccagctat gccaatgtgg tgctattgtt tctttaagaa agtacttgac 4260taaaaaaaaa agaaaaaaag aaaaaaaaga aagcatagac atattttttt aaagtataaa 4320aacaacaatt ctatagatag atggcttaat aaaatagcat taggtctatc tagccaccac 4380cacctttcaa ctttttatca ctcacaagta gtgtactgtt caccaaattg tgaatttggg 4440ggtgcagggg caggagttgg aaatttttta aagttagaag gctccattgt tttgttggct 4500ctcaaactta gcaaaattag caatatatta tccaatcttc tgaacttgat caagagcatg 4560gagaataaac gcgggaaaaa agatcttata ggcaaataga agaatttaaa agataagtaa 4620gttccttatt gatttttgtg cactctgctc taaaacagat attcagcaag tggagaaaat 4680aagaacaaag agaaaaaata catagattta cctgcaaaaa atagcttctg ccaaatcccc 4740cttgggtatt ctttggcatt tactggttta tagaagacat tctcccttca cccagacatc 4800tcaaagagca gtagctctca tgaaaagcaa tcactgatct catttgggaa atgttggaaa 4860gtatttcctt atgagatggg ggttatctac tgataaagaa agaatttatg agaaattgtt 4920gaaagagatg gctaacaatc tgtgaagatt ttttgtttct tgtttttgtt tttttttttt 4980ttttacttta tacagtcttt atgaatttct taatgttcaa aatgacttgg ttcttttctt 5040ctttttttat atcagaatga ggaataataa gttaaaccca catagactct ttaaaactat 5100aggctagata gaaatgtatg tttgacttgt tgaagctata atcagactat ttaaaatgtt 5160ttgctatttt taatcttaaa agattgtgct aatttattag agcagaacct gtttggctct 5220cctcagaaga aagaatcttt ccattcaaat cacatggctt tccaccaata ttttcaaaag 5280ataaatctga tttatgcaat ggcatcattt attttaaaac agaagaattg tgaaagttta 5340tgcccctccc ttgcaaagac cataaagtcc agatctggta ggggggcaac aacaaaagga 5400aaatgttgtt gattcttggt tttggatttt gttttgtttt caatgctagt gtttaatcct 5460gtagtacata tttgcttatt gctattttaa tattttataa gaccttcctg ttaggtatta 5520gaaagtgata catagatatc ttttttgtgt aatttctatt taaaaaagag agaagactgt 5580cagaagcttt aagtgcatat ggtacaggat aaagatatca atttaaataa ccaattccta 5640tctggaacaa tgcttttgtt ttttaaagaa acctctcaca gataagacag aggcccaggg 5700gatttttgaa gctgtcttta ttctgccccc atcccaaccc agcccttatt attttagtat 5760ctgcctcaga attttataga gggctgacca agctgaaact ctagaattaa aggaacctca 5820ctgaaaacat atatttcacg tgttccctct tttttttttt cctttttgtg agatggggtc 5880tcgcactgtc ccccaggctg gagtgcagtg gcatgatctc ggctcactgc aacctccacc 5940tcctgggttt aagcgattct cctgcctcag cctcctgagt agctgggatt acaggcaccc 6000accactatgc ccggctaatt ttttggattt ttaatagaga cggggtttta ccatgttggc 6060caggttggtc tcaaactcct gaccttgtga tttgcccgcc tcagcctccc aaattgctgg 6120gattacaggc atgagccacc acaccctgcc catgtgttcc ctcttaatgt atgattacat 6180ggatcttaaa catgatcctt ctctcctcat tcttcaacta tctttgatgg ggtctttcaa 6240ggggaaaaaa atccaagctt ttttaaagta aaaaaaaaaa aagagaggac acaaaaccaa 6300atgttactgc tcaactgaaa tatgagttaa gatggagaca gagtttctcc taataaccgg 6360agctgaatta cctttcactt tcaaaaacat gaccttccac aatccttaga atctgccttt

6420ttttatatta ctgaggccta aaagtaaaca ttactcattt tattttgccc aaaatgcact 6480gatgtaaagt aggaaaaata aaaacagagc tctaaaatcc ctttcaagcc acccattgac 6540cccactcacc aactcatagc aaagtcactt ctgttaatcc cttaatctga ttttgtttgg 6600atatttatct tgtacccgct gctaaacaca ctgcaggagg gactctgaaa cctcaagctg 6660tctacttaca tcttttatct gtgtctgtgt atcatgaaaa tgtctattca aaatatcaaa 6720acctttcaaa tatcacgcag cttatattca gtttacataa aggccccaaa taccatgtca 6780gatctttttg gtaaaagagt taatgaacta tgagaattgg gattacatca tgtattttgc 6840ctcatgtatt tttatcacac ttataggcca agtgtgataa ataaacttac agacactgaa 6900ttaatttccc ctgctacttt gaaaccagaa aataatgact ggccattcgt tacatctgtc 6960ttagttgaaa agcatatttt ttattaaatt aattctgatt gtatttgaaa ttattattca 7020attcacttat ggcagaggaa tatcaatcct aatgacttct aaaaatgtaa ctaattgaat 7080cattatctta catttactgt ttaataagca tattttgaaa atgtatggct agagtgtcat 7140aataaaatgg tatatctttc tttagtaatt acattaaaat tagtcatgtt tgattaatta 7200gttc 72044949DNAHomo sapiens 4ttttgtagat aaatgtgagg attttctcta aatccctctt ctgtttgcta aatctcactg 60tcactgctaa attcagagca gatagagcct gcgcaatgga ataaagtcct caaaattgaa 120atgtgacatt gctctcaaca tctcccatct ctctggattt ctttttgctt cattattcct 180gctaaccaat tcattttcag actttgtact tcagaagcaa tgggaaaaat cagcagtctt 240ccaacccaat tatttaagtg ctgcttttgt gatttcttga aggtgaagat gcacaccatg 300tcctcctcgc atctcttcta cctggcgctg tgcctgctca ccttcaccag ctctgccacg 360gctggaccgg agacgctctg cggggctgag ctggtggatg ctcttcagtt cgtgtgtgga 420gacaggggct tttatttcaa caagcccaca gggtatggct ccagcagtcg gagggcgcct 480cagacaggca tcgtggatga gtgctgcttc cggagctgtg atctaaggag gctggagatg 540tattgcgcac ccctcaagcc tgccaagtca gctcgctctg tccgtgccca gcgccacacc 600gacatgccca agacccagaa gtatcagccc ccatctacca acaagaacac gaagtctcag 660agaaggaaag gttggccaaa gacacatcca ggaggggaac agaaggaggg gacagaagca 720agtctgcaga tcagaggaaa gaagaaagag cagaggaggg agattggaag tagaaatgct 780gaatgcagag gcaaaaaagg aaaatgaagg acaggaggat taaacagaca gaggcaagga 840tgatgagaga ggagcagaca gcaagaatga aaagcagaaa atacaataga ggaaatgaag 900aaaagtaggc ctgctggagc tagatgatga tgtgatggaa atagaagta 94957370DNAHomo sapiens 5ttttgtagat aaatgtgagg attttctcta aatccctctt ctgtttgcta aatctcactg 60tcactgctaa attcagagca gatagagcct gcgcaatgga ataaagtcct caaaattgaa 120atgtgacatt gctctcaaca tctcccatct ctctggattt ctttttgctt cattattcct 180gctaaccaat tcattttcag actttgtact tcagaagcaa tgggaaaaat cagcagtctt 240ccaacccaat tatttaagtg ctgcttttgt gatttcttga aggtgaagat gcacaccatg 300tcctcctcgc atctcttcta cctggcgctg tgcctgctca ccttcaccag ctctgccacg 360gctggaccgg agacgctctg cggggctgag ctggtggatg ctcttcagtt cgtgtgtgga 420gacaggggct tttatttcaa caagcccaca gggtatggct ccagcagtcg gagggcgcct 480cagacaggca tcgtggatga gtgctgcttc cggagctgtg atctaaggag gctggagatg 540tattgcgcac ccctcaagcc tgccaagtca gctcgctctg tccgtgccca gcgccacacc 600gacatgccca agacccagaa gtatcagccc ccatctacca acaagaacac gaagtctcag 660agaaggaaag gaagtacatt tgaagaacgc aagtagaggg agtgcaggaa acaagaacta 720caggatgtag gaagaccctc ctgaggagtg aagagtgaca tgccaccgca ggatcctttg 780ctctgcacga gttacctgtt aaactttgga acacctacca aaaaataagt ttgataacat 840ttaaaagatg ggcgtttccc ccaatgaaat acacaagtaa acattccaac attgtcttta 900ggagtgattt gcaccttgca aaaatggtcc tggagttggt agattgctgt tgatctttta 960tcaataatgt tctatagaaa agaaaaaaaa aatatatata tatatatatc ttagtccctg 1020cctctcaaga gccacaaatg catgggtgtt gtatagatcc agttgcacta aattcctctc 1080tgaatcttgg ctgctggagc cattcattca gcaaccttgt ctaagtggtt tatgaattgt 1140ttccttattt gcacttcttt ctacacaact cgggctgttt gttttacagt gtctgataat 1200cttgttagtc tatacccacc acctcccttc ataaccttta tatttgccga atttggcctc 1260ctcaaaagca gcagcaagtc gtcaagaagc acaccaattc taacccacaa gattccatct 1320gtggcatttg taccaaatat aagttggatg cattttattt tagacacaaa gctttatttt 1380tccacatcat gcttacaaaa aagaataatg caaatagttg caactttgag gccaatcatt 1440tttaggcata tgttttaaac atagaaagtt tcttcaactc aaaagagttc cttcaaatga 1500tgagttaatg tgcaacctaa ttagtaactt tcctcttttt attttttcca tatagagcac 1560tatgtaaatt tagcatatca attatacagg atatatcaaa cagtatgtaa aactctgttt 1620tttagtataa tggtgctatt ttgtagtttg ttatatgaaa gagtctggcc aaaacggtaa 1680tacgtgaaag caaaacaata ggggaagcct ggagccaaag atgacacaag gggaagggta 1740ctgaaaacac catccatttg ggaaagaagg caaagtcccc ccagttatgc cttccaagag 1800gaacttcaga cacaaaagtc cactgatgca aattggactg gcgagtccag agaggaaact 1860gtggaatgga aaaagcagaa ggctaggaat tttagcagtc ctggtttctt tttctcatgg 1920aagaaatgaa catctgccag ctgtgtcatg gactcaccac tgtgtgacct tgggcaagtc 1980acttcacctc tctgtgcctc agtttcctca tctgcaaaat gggggcaata tgtcatctac 2040ctacctcaaa ggggtggtat aaggtttaaa aagataaaga ttcagatttt ttttaccctg 2100ggttgctgta agggtgcaac atcagggcgc ttgagttgct gagatgcaag gaattctata 2160aataacccat tcatagcata gctagagatt ggtgaattga atgctcctga catctcagtt 2220cttgtcagtg aagctatcca aataactggc caactagttg ttaaaagcta acagctcaat 2280ctcttaaaac acttttcaaa atatgtggga agcatttgat tttcaatttg attttgaatt 2340ctgcatttgg ttttatgaat acaaagataa gtgaaaagag agaaaggaaa agaaaaagga 2400gaaaaacaaa gagatttcta ccagtgaaag gggaattaat tactctttgt tagcactcac 2460tgactcttct atgcagttac tacatatcta gtaaaacctc gtttaatact ataaataata 2520ttctattcat tttgaaaaac acaatgattc cttcttttct aggcaatata aggaaagtga 2580tccaaaattt gaaatattaa aataatatct aataaaaagt cacaaagtta tcttctttaa 2640caaactttac tcttattctt agctgtatat acattttttt aaaagtttgt taaaatatgc 2700ttgactagag tttccagttg aaaggcaaaa acttccatca caacaagaaa tttcccatgc 2760ctgctcagaa gggtagcccc tagctctctg tgaatgtgtt ttatccattc aactgaaaat 2820tggtatcaag aaagtccact ggttagtgta ctagtccatc atagcctaga aaatgatccc 2880tatctgcaga tcaagatttt ctcattagaa caatgaatta tccagcattc agatctttct 2940agtcacctta gaactttttg gttaaaagta cccaggcttg attatttcat gcaaattcta 3000tattttacat tcttggaaag tctatatgaa aaacaaaaat aacatcttca gtttttctcc 3060cactgggtca cctcaaggat cagaggccag gaaaaaaaaa aaaaagactc cctggatctc 3120tgaatatatg caaaaagaag gccccattta gtggagccag caatcctgtt cagtcaacaa 3180gtattttaac tctcagtcca acattatttg aattgagcac ctcaagcatg cttagcaatg 3240ttctaatcac tatggacaga tgtaaaagaa actatacatc atttttgccc tctgcctgtt 3300ttccagacat acaggttctg tggaataaga tactggactc ctcttcccaa gatggcactt 3360ctttttattt cttgtcccca gtgtgtacct tttaaaatta ttccctctca acaaaacttt 3420ataggcagtc ttctgcagac ttaacgtgtt ttctgtcata gttagatgtg ataattctaa 3480gagtgtctat gacttatttc cttcacttaa ttctatccac agtcaaaaat cccccaagga 3540ggaaagctga aagatgcact gccatattat ctttcttaac tttttccaac acataatcct 3600ctccaactgg attataaata aattgaaaat aactcattat accaattcac tattttattt 3660tttaatgaat taaaactaga aaacaaattg atgcaaaccc tggaagtcag ttgattacta 3720tatactacag cagaatgact cagatttcat agaaaggagc aaccaaaatg tcacaaccca 3780aaactttaca agctttgctt cagaattaga ttgctttata attcttgaat gaggcaattt 3840caagatattt gtaaaagaac agtaaacatt ggtaagaatg agctttcaac tcataggctt 3900atttccaatt taattgacca tactggatac ttaggtcaaa tttctgttct ctcttcccca 3960aataatatta aagtattatt tgaacttttt aagatgaggc agttcccctg aaaaagttaa 4020tgcagctctc catcagaatc cactcttcta gggatatgaa aatctcttaa cacccaccct 4080acatacacag acacacacac acacacacac acacacacac acacacacat tcaccctaag 4140gatccaatgg aatactgaaa agaaatcact tccttgaaaa ttttattaaa aaacaaacaa 4200acaaacaaaa agcctgtcca cccttgagaa tccttcctct ccttggaacg tcaatgtttg 4260tgtagatgaa accatctcat gctctgtggc tccagggttt ctgttactat tttatgcact 4320tgggagaagg cttagaataa aagatgtagc acattttgct ttcccattta ttgtttggcc 4380agctatgcca atgtggtgct attgtttctt taagaaagta cttgactaaa aaaaaaagaa 4440aaaaagaaaa aaaagaaagc atagacatat ttttttaaag tataaaaaca acaattctat 4500agatagatgg cttaataaaa tagcattagg tctatctagc caccaccacc tttcaacttt 4560ttatcactca caagtagtgt actgttcacc aaattgtgaa tttgggggtg caggggcagg 4620agttggaaat tttttaaagt tagaaggctc cattgttttg ttggctctca aacttagcaa 4680aattagcaat atattatcca atcttctgaa cttgatcaag agcatggaga ataaacgcgg 4740gaaaaaagat cttataggca aatagaagaa tttaaaagat aagtaagttc cttattgatt 4800tttgtgcact ctgctctaaa acagatattc agcaagtgga gaaaataaga acaaagagaa 4860aaaatacata gatttacctg caaaaaatag cttctgccaa atcccccttg ggtattcttt 4920ggcatttact ggtttataga agacattctc ccttcaccca gacatctcaa agagcagtag 4980ctctcatgaa aagcaatcac tgatctcatt tgggaaatgt tggaaagtat ttccttatga 5040gatgggggtt atctactgat aaagaaagaa tttatgagaa attgttgaaa gagatggcta 5100acaatctgtg aagatttttt gtttcttgtt tttgtttttt tttttttttt actttataca 5160gtctttatga atttcttaat gttcaaaatg acttggttct tttcttcttt ttttatatca 5220gaatgaggaa taataagtta aacccacata gactctttaa aactataggc tagatagaaa 5280tgtatgtttg acttgttgaa gctataatca gactatttaa aatgttttgc tatttttaat 5340cttaaaagat tgtgctaatt tattagagca gaacctgttt ggctctcctc agaagaaaga 5400atctttccat tcaaatcaca tggctttcca ccaatatttt caaaagataa atctgattta 5460tgcaatggca tcatttattt taaaacagaa gaattgtgaa agtttatgcc cctcccttgc 5520aaagaccata aagtccagat ctggtagggg ggcaacaaca aaaggaaaat gttgttgatt 5580cttggttttg gattttgttt tgttttcaat gctagtgttt aatcctgtag tacatatttg 5640cttattgcta ttttaatatt ttataagacc ttcctgttag gtattagaaa gtgatacata 5700gatatctttt ttgtgtaatt tctatttaaa aaagagagaa gactgtcaga agctttaagt 5760gcatatggta caggataaag atatcaattt aaataaccaa ttcctatctg gaacaatgct 5820tttgtttttt aaagaaacct ctcacagata agacagaggc ccaggggatt tttgaagctg 5880tctttattct gcccccatcc caacccagcc cttattattt tagtatctgc ctcagaattt 5940tatagagggc tgaccaagct gaaactctag aattaaagga acctcactga aaacatatat 6000ttcacgtgtt ccctcttttt ttttttcctt tttgtgagat ggggtctcgc actgtccccc 6060aggctggagt gcagtggcat gatctcggct cactgcaacc tccacctcct gggtttaagc 6120gattctcctg cctcagcctc ctgagtagct gggattacag gcacccacca ctatgcccgg 6180ctaatttttt ggatttttaa tagagacggg gttttaccat gttggccagg ttggtctcaa 6240actcctgacc ttgtgatttg cccgcctcag cctcccaaat tgctgggatt acaggcatga 6300gccaccacac cctgcccatg tgttccctct taatgtatga ttacatggat cttaaacatg 6360atccttctct cctcattctt caactatctt tgatggggtc tttcaagggg aaaaaaatcc 6420aagctttttt aaagtaaaaa aaaaaaaaga gaggacacaa aaccaaatgt tactgctcaa 6480ctgaaatatg agttaagatg gagacagagt ttctcctaat aaccggagct gaattacctt 6540tcactttcaa aaacatgacc ttccacaatc cttagaatct gccttttttt atattactga 6600ggcctaaaag taaacattac tcattttatt ttgcccaaaa tgcactgatg taaagtagga 6660aaaataaaaa cagagctcta aaatcccttt caagccaccc attgacccca ctcaccaact 6720catagcaaag tcacttctgt taatccctta atctgatttt gtttggatat ttatcttgta 6780cccgctgcta aacacactgc aggagggact ctgaaacctc aagctgtcta cttacatctt 6840ttatctgtgt ctgtgtatca tgaaaatgtc tattcaaaat atcaaaacct ttcaaatatc 6900acgcagctta tattcagttt acataaaggc cccaaatacc atgtcagatc tttttggtaa 6960aagagttaat gaactatgag aattgggatt acatcatgta ttttgcctca tgtattttta 7020tcacacttat aggccaagtg tgataaataa acttacagac actgaattaa tttcccctgc 7080tactttgaaa ccagaaaata atgactggcc attcgttaca tctgtcttag ttgaaaagca 7140tattttttat taaattaatt ctgattgtat ttgaaattat tattcaattc acttatggca 7200gaggaatatc aatcctaatg acttctaaaa atgtaactaa ttgaatcatt atcttacatt 7260tactgtttaa taagcatatt ttgaaaatgt atggctagag tgtcataata aaatggtata 7320tctttcttta gtaattacat taaaattagt catgtttgat taattagttc 737067321DNAHomo sapiens 6ttttgtagat aaatgtgagg attttctcta aatccctctt ctgtttgcta aatctcactg 60tcactgctaa attcagagca gatagagcct gcgcaatgga ataaagtcct caaaattgaa 120atgtgacatt gctctcaaca tctcccatct ctctggattt ctttttgctt cattattcct 180gctaaccaat tcattttcag actttgtact tcagaagcaa tgggaaaaat cagcagtctt 240ccaacccaat tatttaagtg ctgcttttgt gatttcttga aggtgaagat gcacaccatg 300tcctcctcgc atctcttcta cctggcgctg tgcctgctca ccttcaccag ctctgccacg 360gctggaccgg agacgctctg cggggctgag ctggtggatg ctcttcagtt cgtgtgtgga 420gacaggggct tttatttcaa caagcccaca gggtatggct ccagcagtcg gagggcgcct 480cagacaggca tcgtggatga gtgctgcttc cggagctgtg atctaaggag gctggagatg 540tattgcgcac ccctcaagcc tgccaagtca gctcgctctg tccgtgccca gcgccacacc 600gacatgccca agacccagaa ggaagtacat ttgaagaacg caagtagagg gagtgcagga 660aacaagaact acaggatgta ggaagaccct cctgaggagt gaagagtgac atgccaccgc 720aggatccttt gctctgcacg agttacctgt taaactttgg aacacctacc aaaaaataag 780tttgataaca tttaaaagat gggcgtttcc cccaatgaaa tacacaagta aacattccaa 840cattgtcttt aggagtgatt tgcaccttgc aaaaatggtc ctggagttgg tagattgctg 900ttgatctttt atcaataatg ttctatagaa aagaaaaaaa aaatatatat atatatatat 960cttagtccct gcctctcaag agccacaaat gcatgggtgt tgtatagatc cagttgcact 1020aaattcctct ctgaatcttg gctgctggag ccattcattc agcaaccttg tctaagtggt 1080ttatgaattg tttccttatt tgcacttctt tctacacaac tcgggctgtt tgttttacag 1140tgtctgataa tcttgttagt ctatacccac cacctccctt cataaccttt atatttgccg 1200aatttggcct cctcaaaagc agcagcaagt cgtcaagaag cacaccaatt ctaacccaca 1260agattccatc tgtggcattt gtaccaaata taagttggat gcattttatt ttagacacaa 1320agctttattt ttccacatca tgcttacaaa aaagaataat gcaaatagtt gcaactttga 1380ggccaatcat ttttaggcat atgttttaaa catagaaagt ttcttcaact caaaagagtt 1440ccttcaaatg atgagttaat gtgcaaccta attagtaact ttcctctttt tattttttcc 1500atatagagca ctatgtaaat ttagcatatc aattatacag gatatatcaa acagtatgta 1560aaactctgtt ttttagtata atggtgctat tttgtagttt gttatatgaa agagtctggc 1620caaaacggta atacgtgaaa gcaaaacaat aggggaagcc tggagccaaa gatgacacaa 1680ggggaagggt actgaaaaca ccatccattt gggaaagaag gcaaagtccc cccagttatg 1740ccttccaaga ggaacttcag acacaaaagt ccactgatgc aaattggact ggcgagtcca 1800gagaggaaac tgtggaatgg aaaaagcaga aggctaggaa ttttagcagt cctggtttct 1860ttttctcatg gaagaaatga acatctgcca gctgtgtcat ggactcacca ctgtgtgacc 1920ttgggcaagt cacttcacct ctctgtgcct cagtttcctc atctgcaaaa tgggggcaat 1980atgtcatcta cctacctcaa aggggtggta taaggtttaa aaagataaag attcagattt 2040tttttaccct gggttgctgt aagggtgcaa catcagggcg cttgagttgc tgagatgcaa 2100ggaattctat aaataaccca ttcatagcat agctagagat tggtgaattg aatgctcctg 2160acatctcagt tcttgtcagt gaagctatcc aaataactgg ccaactagtt gttaaaagct 2220aacagctcaa tctcttaaaa cacttttcaa aatatgtggg aagcatttga ttttcaattt 2280gattttgaat tctgcatttg gttttatgaa tacaaagata agtgaaaaga gagaaaggaa 2340aagaaaaagg agaaaaacaa agagatttct accagtgaaa ggggaattaa ttactctttg 2400ttagcactca ctgactcttc tatgcagtta ctacatatct agtaaaacct cgtttaatac 2460tataaataat attctattca ttttgaaaaa cacaatgatt ccttcttttc taggcaatat 2520aaggaaagtg atccaaaatt tgaaatatta aaataatatc taataaaaag tcacaaagtt 2580atcttcttta acaaacttta ctcttattct tagctgtata tacatttttt taaaagtttg 2640ttaaaatatg cttgactaga gtttccagtt gaaaggcaaa aacttccatc acaacaagaa 2700atttcccatg cctgctcaga agggtagccc ctagctctct gtgaatgtgt tttatccatt 2760caactgaaaa ttggtatcaa gaaagtccac tggttagtgt actagtccat catagcctag 2820aaaatgatcc ctatctgcag atcaagattt tctcattaga acaatgaatt atccagcatt 2880cagatctttc tagtcacctt agaacttttt ggttaaaagt acccaggctt gattatttca 2940tgcaaattct atattttaca ttcttggaaa gtctatatga aaaacaaaaa taacatcttc 3000agtttttctc ccactgggtc acctcaagga tcagaggcca ggaaaaaaaa aaaaaagact 3060ccctggatct ctgaatatat gcaaaaagaa ggccccattt agtggagcca gcaatcctgt 3120tcagtcaaca agtattttaa ctctcagtcc aacattattt gaattgagca cctcaagcat 3180gcttagcaat gttctaatca ctatggacag atgtaaaaga aactatacat catttttgcc 3240ctctgcctgt tttccagaca tacaggttct gtggaataag atactggact cctcttccca 3300agatggcact tctttttatt tcttgtcccc agtgtgtacc ttttaaaatt attccctctc 3360aacaaaactt tataggcagt cttctgcaga cttaacgtgt tttctgtcat agttagatgt 3420gataattcta agagtgtcta tgacttattt ccttcactta attctatcca cagtcaaaaa 3480tcccccaagg aggaaagctg aaagatgcac tgccatatta tctttcttaa ctttttccaa 3540cacataatcc tctccaactg gattataaat aaattgaaaa taactcatta taccaattca 3600ctattttatt ttttaatgaa ttaaaactag aaaacaaatt gatgcaaacc ctggaagtca 3660gttgattact atatactaca gcagaatgac tcagatttca tagaaaggag caaccaaaat 3720gtcacaaccc aaaactttac aagctttgct tcagaattag attgctttat aattcttgaa 3780tgaggcaatt tcaagatatt tgtaaaagaa cagtaaacat tggtaagaat gagctttcaa 3840ctcataggct tatttccaat ttaattgacc atactggata cttaggtcaa atttctgttc 3900tctcttcccc aaataatatt aaagtattat ttgaactttt taagatgagg cagttcccct 3960gaaaaagtta atgcagctct ccatcagaat ccactcttct agggatatga aaatctctta 4020acacccaccc tacatacaca gacacacaca cacacacaca cacacacaca cacacacaca 4080ttcaccctaa ggatccaatg gaatactgaa aagaaatcac ttccttgaaa attttattaa 4140aaaacaaaca aacaaacaaa aagcctgtcc acccttgaga atccttcctc tccttggaac 4200gtcaatgttt gtgtagatga aaccatctca tgctctgtgg ctccagggtt tctgttacta 4260ttttatgcac ttgggagaag gcttagaata aaagatgtag cacattttgc tttcccattt 4320attgtttggc cagctatgcc aatgtggtgc tattgtttct ttaagaaagt acttgactaa 4380aaaaaaaaga aaaaaagaaa aaaaagaaag catagacata tttttttaaa gtataaaaac 4440aacaattcta tagatagatg gcttaataaa atagcattag gtctatctag ccaccaccac 4500ctttcaactt tttatcactc acaagtagtg tactgttcac caaattgtga atttgggggt 4560gcaggggcag gagttggaaa ttttttaaag ttagaaggct ccattgtttt gttggctctc 4620aaacttagca aaattagcaa tatattatcc aatcttctga acttgatcaa gagcatggag 4680aataaacgcg ggaaaaaaga tcttataggc aaatagaaga atttaaaaga taagtaagtt 4740ccttattgat ttttgtgcac tctgctctaa aacagatatt cagcaagtgg agaaaataag 4800aacaaagaga aaaaatacat agatttacct gcaaaaaata gcttctgcca aatccccctt 4860gggtattctt tggcatttac tggtttatag aagacattct cccttcaccc agacatctca 4920aagagcagta gctctcatga aaagcaatca ctgatctcat ttgggaaatg ttggaaagta 4980tttccttatg agatgggggt tatctactga taaagaaaga atttatgaga aattgttgaa 5040agagatggct aacaatctgt gaagattttt tgtttcttgt ttttgttttt tttttttttt 5100tactttatac agtctttatg aatttcttaa tgttcaaaat gacttggttc ttttcttctt 5160tttttatatc agaatgagga ataataagtt aaacccacat agactcttta aaactatagg 5220ctagatagaa atgtatgttt gacttgttga agctataatc agactattta aaatgttttg 5280ctatttttaa tcttaaaaga ttgtgctaat ttattagagc agaacctgtt tggctctcct 5340cagaagaaag aatctttcca ttcaaatcac atggctttcc accaatattt tcaaaagata 5400aatctgattt atgcaatggc atcatttatt ttaaaacaga agaattgtga aagtttatgc 5460ccctcccttg caaagaccat aaagtccaga tctggtaggg gggcaacaac aaaaggaaaa 5520tgttgttgat tcttggtttt ggattttgtt ttgttttcaa tgctagtgtt taatcctgta 5580gtacatattt gcttattgct attttaatat tttataagac cttcctgtta ggtattagaa 5640agtgatacat agatatcttt tttgtgtaat ttctatttaa aaaagagaga agactgtcag 5700aagctttaag tgcatatggt acaggataaa gatatcaatt taaataacca attcctatct 5760ggaacaatgc ttttgttttt taaagaaacc tctcacagat aagacagagg cccaggggat

5820ttttgaagct gtctttattc tgcccccatc ccaacccagc ccttattatt ttagtatctg 5880cctcagaatt ttatagaggg ctgaccaagc tgaaactcta gaattaaagg aacctcactg 5940aaaacatata tttcacgtgt tccctctttt tttttttcct ttttgtgaga tggggtctcg 6000cactgtcccc caggctggag tgcagtggca tgatctcggc tcactgcaac ctccacctcc 6060tgggtttaag cgattctcct gcctcagcct cctgagtagc tgggattaca ggcacccacc 6120actatgcccg gctaattttt tggattttta atagagacgg ggttttacca tgttggccag 6180gttggtctca aactcctgac cttgtgattt gcccgcctca gcctcccaaa ttgctgggat 6240tacaggcatg agccaccaca ccctgcccat gtgttccctc ttaatgtatg attacatgga 6300tcttaaacat gatccttctc tcctcattct tcaactatct ttgatggggt ctttcaaggg 6360gaaaaaaatc caagcttttt taaagtaaaa aaaaaaaaag agaggacaca aaaccaaatg 6420ttactgctca actgaaatat gagttaagat ggagacagag tttctcctaa taaccggagc 6480tgaattacct ttcactttca aaaacatgac cttccacaat ccttagaatc tgcctttttt 6540tatattactg aggcctaaaa gtaaacatta ctcattttat tttgcccaaa atgcactgat 6600gtaaagtagg aaaaataaaa acagagctct aaaatccctt tcaagccacc cattgacccc 6660actcaccaac tcatagcaaa gtcacttctg ttaatccctt aatctgattt tgtttggata 6720tttatcttgt acccgctgct aaacacactg caggagggac tctgaaacct caagctgtct 6780acttacatct tttatctgtg tctgtgtatc atgaaaatgt ctattcaaaa tatcaaaacc 6840tttcaaatat cacgcagctt atattcagtt tacataaagg ccccaaatac catgtcagat 6900ctttttggta aaagagttaa tgaactatga gaattgggat tacatcatgt attttgcctc 6960atgtattttt atcacactta taggccaagt gtgataaata aacttacaga cactgaatta 7020atttcccctg ctactttgaa accagaaaat aatgactggc cattcgttac atctgtctta 7080gttgaaaagc atatttttta ttaaattaat tctgattgta tttgaaatta ttattcaatt 7140cacttatggc agaggaatat caatcctaat gacttctaaa aatgtaacta attgaatcat 7200tatcttacat ttactgttta ataagcatat tttgaaaatg tatggctaga gtgtcataat 7260aaaatggtat atctttcttt agtaattaca ttaaaattag tcatgtttga ttaattagtt 7320c 73217877DNAHomo sapiens 7ttctattcac atcggcctca taatacccac cctgacctgc tgtaaaagac ctggaacaaa 60caaaaatgat tacacctaca gtgaagatgc acaccatgtc ctcctcgcat ctcttctacc 120tggcgctgtg cctgctcacc ttcaccagct ctgccacggc tggaccggag acgctctgcg 180gggctgagct ggtggatgct cttcagttcg tgtgtggaga caggggcttt tatttcaaca 240agcccacagg gtatggctcc agcagtcgga gggcgcctca gacaggcatc gtggatgagt 300gctgcttccg gagctgtgat ctaaggaggc tggagatgta ttgcgcaccc ctcaagcctg 360ccaagtcagc tcgctctgtc cgtgcccagc gccacaccga catgcccaag acccagaagt 420atcagccccc atctaccaac aagaacacga agtctcagag aaggaaaggt tggccaaaga 480cacatccagg aggggaacag aaggagggga cagaagcaag tctgcagatc agaggaaaga 540agaaagagca gaggagggag attggaagta gaaatgctga atgcagaggc aaaaaaggaa 600aatgaaggac aggaggatta aacagacaga ggcaaggatg atgagagagg agcagacagc 660aagaatgaaa agcagaaaat acaatagagg aaatgaagaa aagtaggcct gctggagcta 720gatgatgatg tgatggaaat agaagtaacc ttttagagaa tctcgctaag aaacatggag 780aaaacggaaa agaaaaatgt aatgccctag aaagcgcaaa gaaagacagt ggcaaaaatg 840aaaaaaaaaa ataaaaatta taaaagaggc aaaaaaa 877822DNAArtificial Sequencetarget sequence for microRNA 122a (miRBase database accession number MI0000442) 8caaacaccat tgtcacactc ca 22921DNAArtificial Sequencetarget sequence for microRNA 152 (MI0000462) 9agtcacgtac tgtcttgaac c 211023DNAArtificial Sequencesequence target microRNA 199a-5p (MI0000242) 10gggtcacaag tctgatggac aag 231122DNAArtificial Sequencesequence target for microRNA 99a-3p (MI0000101) 11tgtcatcaga cgtgtaacca at 221221DNAArtificial Sequencetarget sequence for microRNA 215 (MI0000291) 12tactggatac ttaactgtct g 211321DNAArtificial Sequencetarget sequence for microRNA 192 (MI0000234) 13ggctgtcaat tcataggtca g 211422DNAArtificial Sequencetarget sequence for microRNA 194 (MI0000488) 14acattgtcgt tgaggtacac ct 221522DNAArtificial Sequencetarget sequence for microRNA 1 (MI0000651) 15ttacatactt ctttacattc ca 221622DNAArtificial Sequencetarget sequence for microRNA 148 (MI0000253) 16agtcacgtga tgtcttgaaa ca 221722DNAArtificial Sequencetarget sequence for microRNA 133a (MI0000450) 17aaaccagggg aagttggtcg ac 221822DNAArtificial Sequencetarget sequence microRNA 206 (MI0000490) 18accttacatt ccttcacaca cc 221920DNAArtificial Sequencetarget sequence for microRNA 124 (MI0000443) 19attccgtgcg ccacttacgg 202024DNAArtificial Sequencetarget sequence for microRNA 125 (MI0000469) 20agggactctg ggaaattgga cact 242122DNAArtificial Sequencetarget sequence for microRNA 216 (MI0000292) 21attagagtcg accgttgaca ct 222222DNAArtificial Sequencetarget sequence for microRNA 130 (MI0000448) 22gtcacgttac aattttcccg ta 2223137PRTHomo sapiens 23Met Ile Thr Pro Thr Val Lys Met His Thr Met Ser Ser Ser His Leu1 5 10 15Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala 20 25 30Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 35 40 45Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly 50 55 60Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys65 70 75 80Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 85 90 95Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp 100 105 110Met Pro Lys Thr Gln Lys Glu Val His Leu Lys Asn Ala Ser Arg Gly 115 120 125Ser Ala Gly Asn Lys Asn Tyr Arg Met 130 13524195PRTHomo sapiens 24Met Gly Lys Ile Ser Ser Leu Pro Thr Gln Leu Phe Lys Cys Cys Phe1 5 10 15Cys Asp Phe Leu Lys Val Lys Met His Thr Met Ser Ser Ser His Leu 20 25 30Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala 35 40 45Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 50 55 60Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly65 70 75 80Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys 85 90 95Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 100 105 110Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp 115 120 125Met Pro Lys Thr Gln Lys Tyr Gln Pro Pro Ser Thr Asn Lys Asn Thr 130 135 140Lys Ser Gln Arg Arg Lys Gly Trp Pro Lys Thr His Pro Gly Gly Glu145 150 155 160Gln Lys Glu Gly Thr Glu Ala Ser Leu Gln Ile Arg Gly Lys Lys Lys 165 170 175Glu Gln Arg Arg Glu Ile Gly Ser Arg Asn Ala Glu Cys Arg Gly Lys 180 185 190Lys Gly Lys 19525158PRTHomo sapiens 25Met Gly Lys Ile Ser Ser Leu Pro Thr Gln Leu Phe Lys Cys Cys Phe1 5 10 15Cys Asp Phe Leu Lys Val Lys Met His Thr Met Ser Ser Ser His Leu 20 25 30Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala 35 40 45Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 50 55 60Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly65 70 75 80Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys 85 90 95Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 100 105 110Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp 115 120 125Met Pro Lys Thr Gln Lys Tyr Gln Pro Pro Ser Thr Asn Lys Asn Thr 130 135 140Lys Ser Gln Arg Arg Lys Gly Ser Thr Phe Glu Glu Arg Lys145 150 15526153PRTHomo sapiens 26Met Gly Lys Ile Ser Ser Leu Pro Thr Gln Leu Phe Lys Cys Cys Phe1 5 10 15Cys Asp Phe Leu Lys Val Lys Met His Thr Met Ser Ser Ser His Leu 20 25 30Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala 35 40 45Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 50 55 60Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly65 70 75 80Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys 85 90 95Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 100 105 110Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp 115 120 125Met Pro Lys Thr Gln Lys Glu Val His Leu Lys Asn Ala Ser Arg Gly 130 135 140Ser Ala Gly Asn Lys Asn Tyr Arg Met145 15027179PRTHomo sapiens 27Met Ile Thr Pro Thr Val Lys Met His Thr Met Ser Ser Ser His Leu1 5 10 15Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala 20 25 30Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 35 40 45Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly 50 55 60Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys65 70 75 80Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 85 90 95Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp 100 105 110Met Pro Lys Thr Gln Lys Tyr Gln Pro Pro Ser Thr Asn Lys Asn Thr 115 120 125Lys Ser Gln Arg Arg Lys Gly Trp Pro Lys Thr His Pro Gly Gly Glu 130 135 140Gln Lys Glu Gly Thr Glu Ala Ser Leu Gln Ile Arg Gly Lys Lys Lys145 150 155 160Glu Gln Arg Arg Glu Ile Gly Ser Arg Asn Ala Glu Cys Arg Gly Lys 165 170 175Lys Gly Lys28137PRTMus musculus 28Met Thr Ala Pro Ala Ile Lys Ile His Ile Met Ser Ser Ser His Leu1 5 10 15Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Thr Thr Ala 20 25 30Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 35 40 45Val Cys Gly Pro Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly 50 55 60Ser Ser Ile Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys65 70 75 80Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 85 90 95Lys Pro Thr Lys Ala Ala Arg Ser Ile Arg Ala Gln Arg His Thr Asp 100 105 110Met Pro Lys Thr Gln Lys Glu Val His Leu Lys Asn Thr Ser Arg Gly 115 120 125Ser Ala Gly Asn Lys Thr Tyr Arg Met 130 13529133PRTMus musculus 29Met Ser Ser Ser His Leu Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe1 5 10 15Thr Ser Ser Thr Thr Ala Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu 20 25 30Val Asp Ala Leu Gln Phe Val Cys Gly Pro Arg Gly Phe Tyr Phe Asn 35 40 45Lys Pro Thr Gly Tyr Gly Ser Ser Ile Arg Arg Ala Pro Gln Thr Gly 50 55 60Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu65 70 75 80Met Tyr Cys Ala Pro Leu Lys Pro Thr Lys Ala Ala Arg Ser Ile Arg 85 90 95Ala Gln Arg His Thr Asp Met Pro Lys Thr Gln Lys Ser Pro Ser Leu 100 105 110Ser Thr Asn Lys Lys Thr Lys Leu Gln Arg Arg Arg Lys Gly Ser Thr 115 120 125Phe Glu Glu His Lys 130307264DNAArtificial Sequencepolynucleotide herewith called pAAV-CAG- preproIGF1a-doble mirT122a-mirT1, expressing in pancreas murine preproIGF-1 isoform 5 (with NCBI accesion number NP_001104746). 30cactgaggcc cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg 60tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc 120caactccatc actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac 180tcgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 240cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 300caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 360gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 420tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 480ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 540attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat 600ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc 660gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 720gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 780tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 840gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 900ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 960gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag 1020ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt 1080gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg 1140cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg 1200cggtgccccg cggtgcgggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1260tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc 1320ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg 1380cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc 1440cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc gccggcggct 1500gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1560gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct 1620ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc 1680ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg 1740gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg 1800gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg 1860gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattgatt aattcgagcg 1920aacgcgtcga gtcgctcggt acgatttaaa ttgaattggc ctcgagcgca agcttgatat 1980cgaattccgt agatgctttc acaaacccca cccacaaaac aacacatgtt cttaagtcct 2040cagttttgtg ttcacctcgg cctcatagta cccactctga cctgctgtgt aaacgacccg 2100gacctaccaa aatgaccgca cctgcaataa agatacacat catgtcgtct tcacacctct 2160tctacctggc gctctgcttg ctcaccttca ccagctccac cacagctgga ccagagaccc 2220tttgcggggc tgagctggtg gatgctcttc agttcgtgtg tggaccgagg ggcttttact 2280tcaacaagcc cacaggctat ggctccagca ttcggagggc acctcagaca ggcattgtgg 2340atgagtgttg cttccggagc tgtgatctga ggagactgga gatgtactgt gccccactga 2400agcctacaaa agcagcccgc tctatccgtg cccagcgcca cactgacatg cccaagactc 2460agaaggaagt acatttgaag aacacaagta gaggaagtgc aggaaacaag acctacagaa 2520tgtaggagga gcctcccacg gagcagaaaa tgccacatca ccgcaggatc cactagttct 2580agagcggccg ctaattctag atcgcgaaca aacaccattg tcacactcca gtatacacaa 2640acaccattgt cacactccag atatcacaaa caccattgtc acactccaag gcgaacaaac 2700accattgtca cactccaagg ctattctaga tcgcgaatta catacttctt tacattccag 2760tatacattac atacttcttt acattccaga tatcattaca tacttcttta cattccaagg 2820cgaattacat acttctttac attccaaggc tacctgaggc ccgggggtac ctcttaatta 2880actggcctca tgggccttcc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 2940gtcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca 3000aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg 3060agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt 3120ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg 3180agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt 3240ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata 3300gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt 3360taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac 3420tactcccagt catagctgtc cctcttctct tatggagatc cctcgacctg cagcccaagc 3480tgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt 3540ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg 3600acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct ggcgtaatag 3660cagtgagcga gcgagcgcgc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 3720tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 3780gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg

3840ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 3900ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 3960acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4020tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4080ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4140ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4200ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4260actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4320gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 4380tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4440caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4500atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4560acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 4620ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 4680ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 4740tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 4800tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 4860gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 4920tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 4980tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 5040ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 5100tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 5160ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 5220gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 5280ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 5340cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 5400ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 5460ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 5520gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 5580ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 5640gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt 5700aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg 5760tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc 5820cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct 5880taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc 5940gcacagatgc gtaaggagaa aataccgcat caggcgattc caacatccaa taaatcatac 6000aggcaaggca aagaattagc aaaattaagc aataaagcct cagagcataa agctaaatcg 6060gttgtaccaa aaacattatg accctgtaat acttttgcgg gagaagcctt tatttcaacg 6120caaggataaa aatttttaga accctcatat attttaaatg caatgcctga gtaatgtgta 6180ggtaaagatt caaacgggtg agaaaggccg gagacagtca aatcaccatc aatatgatat 6240tcaaccgttc tagctgataa attcatgccg gagagggtag ctatttttga gaggtctcta 6300caaaggctat caggtcattg cctgagagtc tggagcaaac aagagaatcg atgaacggta 6360atcgtaaaac tagcatgtca atcatatgta ccccggttga taatcagaaa agccccaaaa 6420acaggaagat tgtataagca aatatttaaa ttgtaagcgt taatattttg ttaaaattcg 6480cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc 6540cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga 6600gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg 6660atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag 6720cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga 6780acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg 6840tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg 6900cgtactatgg ttgctttgac gagcacgtat aacgtgcttt cctcgttaga atcagagcgg 6960gagctaaaca ggaggccgat taaagggatt ttagacagga acggtacgcc agaatcctga 7020gaagtgtttt tataatcagt gaggccaccg agtaaaagag tctgtccatc acgcaaatta 7080accgttgtcg caatacttct ttgattagta ataacatcac ttgcctgagt agaagaactc 7140aaactatcgg ccttgctggt aatatccaga acaatattac cgccagccat tgcaacggaa 7200tcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc 7260gcta 7264317200DNAArtificial Sequencepolynucleotide herewith called pAAV-CAG- preproIGF1b-doble mirT122a-mirT1, expressing in pancreas murine preproIGF1 with NCBI accesion number l'NCBI AAH12409. 31cactgaggcc cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg 60tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc 120caactccatc actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac 180tcgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 240cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 300caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 360gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 420tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 480ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 540attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat 600ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc 660gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 720gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 780tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 840gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 900ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 960gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag 1020ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt 1080gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg 1140cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg 1200cggtgccccg cggtgcgggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1260tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc 1320ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg 1380cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc 1440cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc gccggcggct 1500gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1560gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct 1620ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc 1680ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg 1740gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg 1800gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg 1860gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattgatt aattcgagcg 1920aacgcgtcga gtcgctcggt acgatttaaa ttgaattggc ctcgagcgca agcttgagct 1980agctcgatat cgtcgaccca cgcgtccgga cttcttgaag ataaagatac acatcatgtc 2040gtcttcacac ctcttctacc tggcgctctg cttgctcacc ttcaccagct ccaccacagc 2100tggaccagag accctttgcg gggctgagct ggtggatgct cttcagttcg tgtgtggacc 2160gaggggcttt tacttcaaca agcccacagg ctatggctcc agcattcgga gggcacctca 2220gacaggcatt gtggatgagt gttgcttccg gagctgtgat ctgaggagac tggagatgta 2280ctgtgcccca ctgaagccta caaaagcagc ccgctctatc cgtgcccagc gccacactga 2340catgcccaag actcagaagt ccccgtccct atcgacaaac aagaaaacga agctgcaaag 2400gagaaggaaa ggaagtacat ttgaagaaca caagtagagg aagtgcagga aacaagacct 2460acagaatgta ggaggagcct cccacggagc agaaaatgcc acatcaccgc aggatccgcg 2520cggccgctaa ttctagatcg cgaacaaaca ccattgtcac actccagtat acacaaacac 2580cattgtcaca ctccagatat cacaaacacc attgtcacac tccaaggcga acaaacacca 2640ttgtcacact ccaaggctat tctagatcgc gaattacata cttctttaca ttccagtata 2700cattacatac ttctttacat tccagatatc attacatact tctttacatt ccaaggcgaa 2760ttacatactt ctttacattc caaggctacc tgaggcccgg gggtacctct taattaactg 2820gcctcatggg ccttccgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagtca 2880ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc aatgccctgg ctcacaaata 2940ccactgagat ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca 3000tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt 3060gtgtctctca ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta 3120tttggtttag agtttggcaa catatgccca tatgctggct gccatgaaca aaggttggct 3180ataaagaggt catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa 3240agccttgact tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac 3300atccctaaaa ttttccttac atgttttact agccagattt ttcctcctct cctgactact 3360cccagtcata gctgtccctc ttctcttatg gagatccctc gacctgcagc ccaagctgta 3420gataagtagc atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc 3480actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc 3540ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctggcg taatagcagt 3600gagcgagcga gcgcgcagct gcattaatga atcggccaac gcgcggggag aggcggtttg 3660cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 3720cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 3780aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 3840gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 3900tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 3960agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 4020ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 4080taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 4140gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 4200gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 4260ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 4320ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 4380gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 4440caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 4500taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 4560aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 4620tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 4680tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 4740gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 4800gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 4860aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 4920gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 4980ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 5040tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 5100atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 5160ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 5220ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 5280ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 5340atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 5400gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 5460tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 5520ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 5580acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc 5640tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa 5700aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg 5760agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac 5820tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac 5880agatgcgtaa ggagaaaata ccgcatcagg cgattccaac atccaataaa tcatacaggc 5940aaggcaaaga attagcaaaa ttaagcaata aagcctcaga gcataaagct aaatcggttg 6000taccaaaaac attatgaccc tgtaatactt ttgcgggaga agcctttatt tcaacgcaag 6060gataaaaatt tttagaaccc tcatatattt taaatgcaat gcctgagtaa tgtgtaggta 6120aagattcaaa cgggtgagaa aggccggaga cagtcaaatc accatcaata tgatattcaa 6180ccgttctagc tgataaattc atgccggaga gggtagctat ttttgagagg tctctacaaa 6240ggctatcagg tcattgcctg agagtctgga gcaaacaaga gaatcgatga acggtaatcg 6300taaaactagc atgtcaatca tatgtacccc ggttgataat cagaaaagcc ccaaaaacag 6360gaagattgta taagcaaata tttaaattgt aagcgttaat attttgttaa aattcgcgtt 6420aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta 6480taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc 6540actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg 6600cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact 6660aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt 6720ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc 6780ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgta 6840ctatggttgc tttgacgagc acgtataacg tgctttcctc gttagaatca gagcgggagc 6900taaacaggag gccgattaaa gggattttag acaggaacgg tacgccagaa tcctgagaag 6960tgtttttata atcagtgagg ccaccgagta aaagagtctg tccatcacgc aaattaaccg 7020ttgtcgcaat acttctttga ttagtaataa catcacttgc ctgagtagaa gaactcaaac 7080tatcggcctt gctggtaata tccagaacaa tattaccgcc agccattgca acggaatcgc 7140cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta 7200327127DNAArtificial Sequencepolynucleotide for expressing in pancreas green fluorescent protein (GFP) 32tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa 60ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga 120gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt 180tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga 240gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg 300gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag 360aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt 420aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact 480actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacctgc agcccaagct 540gtagataagt agcatggcgg gttaatcatt aactacaagg aacccctagt gatggagttg 600gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga 660cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg gcgtaatagc 720gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcga 780ttccgttgca atggctggcg gtaatattgt tctggatatt accagcaagg ccgatagttt 840gagttcttct actcaggcaa gtgatgttat tactaatcaa agaagtattg cgacaacggt 900taatttgcgt gatggacaga ctcttttact cggtggcctc actgattata aaaacacttc 960tcaggattct ggcgtaccgt tcctgtctaa aatcccttta atcggcctcc tgtttagctc 1020ccgctctgat tctaacgagg aaagcacgtt atacgtgctc gtcaaagcaa ccatagtacg 1080cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 1140cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 1200tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 1260ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat 1320cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 1380tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag 1440ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg 1500cgaattttaa caaaatatta acgcttacaa tttaaatatt tgcttataca atcttcctgt 1560ttttggggct tttctgatta tcaaccgggg tacatatgat tgacatgcta gttttacgat 1620taccgttcat cgattctctt gtttgctcca gactctcagg caatgacctg atagcctttg 1680tagagacctc tcaaaaatag ctaccctctc cggcatgaat ttatcagcta gaacggttga 1740atatcatatt gatggtgatt tgactgtctc cggcctttct cacccgtttg aatctttacc 1800tacacattac tcaggcattg catttaaaat atatgagggt tctaaaaatt tttatccttg 1860cgttgaaata aaggcttctc ccgcaaaagt attacagggt cataatgttt ttggtacaac 1920cgatttagct ttatgctctg aggctttatt gcttaatttt gctaattctt tgccttgcct 1980gtatgattta ttggatgttg gaatcgcctg atgcggtatt ttctccttac gcatctgtgc 2040ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta 2100agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg 2160gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca 2220ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt 2280aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc 2340ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 2400taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 2460cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 2520acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 2580ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 2640atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 2700gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 2760acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 2820atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 2880accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 2940ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 3000acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 3060gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 3120tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 3180ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 3240actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 3300taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 3360tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 3420gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 3480cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 3540gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 3600gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac 3660tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 3720ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 3780cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 3840gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 3900gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 3960gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 4020cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 4080tttttacggt tcctggcctt ttgctggcct

tttgctcaca tgttctttcc tgcgttatcc 4140cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 4200cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 4260ccgcctctcc ccgcgcgttg gccgattcat taatgcagct gcgcgctcgc tcgctcactg 4320aggcccagct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg 4380cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact 4440ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta tctactcgac 4500attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 4560atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 4620acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 4680tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 4740tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 4800attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 4860tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc 4920ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg 4980gggcgggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg 5040gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt 5100ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag 5160tcgctgcgtt gccttcgccc cgtgccccgc tccgcgccgc ctcgcgccgc ccgccccggc 5220tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct 5280gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt gaaagccttg 5340aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg 5400tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc 5460gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg 5520ccccgcggtg cggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg 5580gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc 5640cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg 5700ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct 5760cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga 5820ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt 5880cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc 5940gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt 6000gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac 6060ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc 6120tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac 6180gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tgattaattc gagcgaacgc 6240gtcgagtcgc tcggtacgat ttaaattgaa ttggcctcga gcgcaagctt gagctagcgc 6300taccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc 6360tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg 6420gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg 6480tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc 6540ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg 6600agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg 6660agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca 6720acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg 6780acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca 6840gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc 6900tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc 6960gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg 7020agctgtacaa gtccggactc agatctcgag ctcaagcttc gaattctgca gtcgacggta 7080ccgcgggccc gggatccacc ggatctagat aactgatccg cgcggcc 7127336964DNAArtificial Sequencepolynucleotide for expression of murine preproIGF1 33ctcttaatta actggcctca tgggccttcc gctcactgcc cgctttccag tcgggaaacc 60tgtcgtgcca gtcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc 120ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg 180aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg 240tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa 300catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg 360aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc 420ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta 480tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc 540ctctcctgac tactcccagt catagctgtc cctcttctct tatggagatc cctcgacctg 600cagcccaagc tgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag 660tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 720aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct 780ggcgtaatag cagtgagcga gcgagcgcgc agctgcatta atgaatcggc caacgcgcgg 840ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 900cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 960cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 1020accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 1080acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 1140cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1200acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 1260atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 1320agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 1380acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 1440gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 1500gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 1560gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 1620gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 1680acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 1740tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 1800ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 1860catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 1920ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 1980caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 2040ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 2100tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 2160cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 2220aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 2280tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 2340gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 2400cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 2460aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 2520tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 2580tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 2640gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 2700atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 2760taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta 2820tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg 2880gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt 2940aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc 3000ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt 3060gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgattc caacatccaa 3120taaatcatac aggcaaggca aagaattagc aaaattaagc aataaagcct cagagcataa 3180agctaaatcg gttgtaccaa aaacattatg accctgtaat acttttgcgg gagaagcctt 3240tatttcaacg caaggataaa aatttttaga accctcatat attttaaatg caatgcctga 3300gtaatgtgta ggtaaagatt caaacgggtg agaaaggccg gagacagtca aatcaccatc 3360aatatgatat tcaaccgttc tagctgataa attcatgccg gagagggtag ctatttttga 3420gaggtctcta caaaggctat caggtcattg cctgagagtc tggagcaaac aagagaatcg 3480atgaacggta atcgtaaaac tagcatgtca atcatatgta ccccggttga taatcagaaa 3540agccccaaaa acaggaagat tgtataagca aatatttaaa ttgtaagcgt taatattttg 3600ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc 3660ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt 3720tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc 3780tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg 3840tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga 3900aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg 3960ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg 4020ctacagggcg cgtactatgg ttgctttgac gagcacgtat aacgtgcttt cctcgttaga 4080atcagagcgg gagctaaaca ggaggccgat taaagggatt ttagacagga acggtacgcc 4140agaatcctga gaagtgtttt tataatcagt gaggccaccg agtaaaagag tctgtccatc 4200acgcaaatta accgttgtcg caatacttct ttgattagta ataacatcac ttgcctgagt 4260agaagaactc aaactatcgg ccttgctggt aatatccaga acaatattac cgccagccat 4320tgcaacggaa tcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc 4380gggcctcttc gctacactga ggcccagctg cgcgctcgct cgctcactga ggccgcccgg 4440gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc 4500agagagggag tggccaactc catcactagg ggttccttgt agttaatgat taacccgcca 4560tgctacttat ctactcgaca ttgattattg actagttatt aatagtaatc aattacgggg 4620tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg 4680cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 4740gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 4800cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 4860ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 4920cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc 4980ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa 5040ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg cggggcgggg 5100cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc 5160gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga 5220agcgcgcggc gggcgggagt cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc 5280tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac 5340ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg 5400tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg 5460gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc 5520ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg 5580agcgcggccg ggggcggtgc cccgcggtgc ggggggctgc gaggggaaca aaggctgcgt 5640gcggggtgtg tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc 5700ccccctgcac ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg 5760tacggggcgt ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc 5820gggcggggcg gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg 5880gagcgccggc ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg 5940cgagagggcg cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg 6000ccgccgcacc ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat 6060gggcggggag ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg 6120gggctgtccg cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt 6180ctggcgtgtg accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt 6240cctacagctc ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt 6300gattaattcg agcgaacgcg tcgagtcgct cggtacgatt taaattgaat tggcctcgag 6360cgcaagcttg atatcgaatt ccgtagatgc tttcacaaac cccacccaca aaacaacaca 6420tgttcttaag tcctcagttt tgtgttcacc tcggcctcat agtacccact ctgacctgct 6480gtgtaaacga cccggaccta ccaaaatgac cgcacctgca ataaagatac acatcatgtc 6540gtcttcacac ctcttctacc tggcgctctg cttgctcacc ttcaccagct ccaccacagc 6600tggaccagag accctttgcg gggctgagct ggtggatgct cttcagttcg tgtgtggacc 6660gaggggcttt tacttcaaca agcccacagg ctatggctcc agcattcgga gggcacctca 6720gacaggcatt gtggatgagt gttgcttccg gagctgtgat ctgaggagac tggagatgta 6780ctgtgcccca ctgaagccta caaaagcagc ccgctctatc cgtgcccagc gccacactga 6840catgcccaag actcagaagg aagtacattt gaagaacaca agtagaggaa gtgcaggaaa 6900caagacctac agaatgtagg aggagcctcc cacggagcag aaaatgccac atcaccgcag 6960gatc 6964346321DNAArtificial Sequencepolynucleotide containing adenoassociated virus genome elements without any transgene 34aattcgagct cggtacccgg gaatcaattc actcctcagg tgcaggctgc ctatcagaag 60gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct ttttccctct 120gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg 180aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca 240tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca 300tatgcccata tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg 360aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt 420ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat 480gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt 540ctcttatgga gatccctcga cctgcagccc aagctgtaga taagtagcat ggcgggttaa 600tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct 660cgctcactga ggccgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag 720ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 780gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 840cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 900tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 960cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 1020aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 1080cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 1140gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 1200ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 1260cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 1320aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 1380tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 1440ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 1500tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 1560ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 1620agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 1680atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 1740cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 1800ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 1860ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 1920agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 1980agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 2040gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 2100cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 2160gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 2220tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 2280tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 2340aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 2400cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 2460cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 2520aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 2580ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 2640tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 2700ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc 2760acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag 2820ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag 2880ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag 2940attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa 3000taccgcatca ggcgattcca acatccaata aatcatacag gcaaggcaaa gaattagcaa 3060aattaagcaa taaagcctca gagcataaag ctaaatcggt tgtaccaaaa acattatgac 3120cctgtaatac ttttgcggga gaagccttta tttcaacgca aggataaaaa tttttagaac 3180cctcatatat tttaaatgca atgcctgagt aatgtgtagg taaagattca aacgggtgag 3240aaaggccgga gacagtcaaa tcaccatcaa tatgatattc aaccgttcta gctgataaat 3300tcatgccgga gagggtagct atttttgaga ggtctctaca aaggctatca ggtcattgcc 3360tgagagtctg gagcaaacaa gagaatcgat gaacggtaat cgtaaaacta gcatgtcaat 3420catatgtacc ccggttgata atcagaaaag ccccaaaaac aggaagattg tataagcaaa 3480tatttaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 3540ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 3600cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 3660ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 3720accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 3780gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 3840gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 3900caccacaccc gccgcgctta atgcgccgct acagggcgcg tactatggtt gctttgacga 3960gcacgtataa cgtgctttcc tcgttagaat cagagcggga gctaaacagg aggccgatta 4020aagggatttt agacaggaac ggtacgccag aatcctgaga agtgttttta taatcagtga 4080ggccaccgag taaaagagtc tgtccatcac gcaaattaac cgttgtcgca atacttcttt 4140gattagtaat aacatcactt gcctgagtag aagaactcaa actatcggcc ttgctggtaa 4200tatccagaac aatattaccg ccagccattg caacggaatc gccattcgcc attcaggctg 4260cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctgcgcgct 4320cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg 4380gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc 4440ttgtagttaa tgattaaccc gccatgctac ttatctactc gacattgatt attgactagt 4500tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt 4560acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 4620tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 4680gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 4740acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 4800accttatggg actttcctac

ttggcagtac atctacgtat tagtcatcgc tattaccatg 4860gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca 4920attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg 4980gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag gcggagaggt 5040gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg 5100cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gttgccttcg 5160ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt 5220actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt 5280ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg 5340ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc 5400cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg 5460tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg 5520gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg 5580tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc cctccccgag ttgctgagca 5640cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg 5700cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg ccggggaggg 5760ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg cggcgagccg 5820cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat 5880ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa 5940gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg 6000ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct gccttcgggg 6060gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc 6120taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt 6180gctgtctcat cattttggca aagaattgat taattcgagc gaacgcgtcg agtcgctcgg 6240tacgatttaa attgaattgg cctcgagcgc aagcttgagc tagctcgata tcggcctagg 6300ctggatccgc gcggccgcaa g 63213520DNAArtificial SequenceForward primer 35tggatgctct tcagttcgtg 203620DNAArtificial SequenceReverse primer 36gagggtggct gttagccata 203720DNAArtificial SequenceReverse primer 37gcaacactca tccacaatgc 203825DNAArtificial SequenceForward primer 38cttgagcatc tgacttctgg ctaat 253924DNAArtificial SequenceReverse primer 39ggagaggagg aaaaatctgg ctag 244030DNAArtificial SequenceProbe 40ccgagtgaga gacacaaaaa attccaacac 304130DNAArtificial SequenceForward primer 41ccgagtgaga gacacaaaaa attccaacac 304230DNAArtificial SequenceReverse primer 42ccgagtgaga gacacaaaaa attccaacac 304330DNAArtificial SequenceProbe 43ccgagtgaga gacacaaaaa attccaacac 3044159PRTMus musculus 44Met Gly Lys Ile Ser Ser Leu Pro Thr Gln Leu Phe Lys Ile Cys Leu1 5 10 15Cys Asp Phe Leu Lys Ile Lys Ile His Ile Met Ser Ser Ser His Leu 20 25 30Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Thr Thr Ala 35 40 45Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 50 55 60Val Cys Gly Pro Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly65 70 75 80Ser Ser Ile Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys 85 90 95Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 100 105 110Lys Pro Thr Lys Ala Ala Arg Ser Ile Arg Ala Gln Arg His Thr Asp 115 120 125Met Pro Lys Thr Gln Lys Ser Pro Ser Leu Ser Thr Asn Lys Lys Thr 130 135 140Lys Leu Gln Arg Arg Arg Lys Gly Ser Thr Phe Glu Glu His Lys145 150 15545165PRTMus musculus 45Met Thr Ala Pro Ala Ile Lys Ile His Ile Met Ser Ser Ser His Leu1 5 10 15Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Thr Thr Ala 20 25 30Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 35 40 45Val Cys Gly Pro Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly 50 55 60Ser Ser Ile Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys65 70 75 80Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 85 90 95Lys Pro Thr Lys Ala Ala Arg Ser Ile Arg Ala Gln Arg His Thr Asp 100 105 110Met Pro Lys Thr Gln Lys Ser Pro Ser Leu Ser Thr Asn Lys Lys Thr 115 120 125Lys Leu Gln Arg Arg Arg Lys Gly Glu Pro Lys Thr His Pro Glu Gly 130 135 140Glu Gln Glu Glu Val Thr Glu Ala Thr Arg Lys Ile Arg Gly Pro Arg145 150 155 160Glu Lys Arg Leu Gly 16546143PRTMus musculus 46Met Thr Ala Pro Ala Ile Lys Ile His Ile Met Ser Ser Ser His Leu1 5 10 15Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Thr Thr Ala 20 25 30Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 35 40 45Val Cys Gly Pro Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly 50 55 60Ser Ser Ile Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys65 70 75 80Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 85 90 95Lys Pro Thr Lys Ala Ala Arg Ser Ile Arg Ala Gln Arg His Thr Asp 100 105 110Met Pro Lys Thr Gln Lys Ser Pro Ser Leu Ser Thr Asn Lys Lys Thr 115 120 125Lys Leu Gln Arg Arg Arg Lys Gly Ser Thr Phe Glu Glu His Lys 130 135 14047153PRTMus musculus 47Met Gly Lys Ile Ser Ser Leu Pro Thr Gln Leu Phe Lys Ile Cys Leu1 5 10 15Cys Asp Phe Leu Lys Ile Lys Ile His Ile Met Ser Ser Ser His Leu 20 25 30Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Thr Thr Ala 35 40 45Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 50 55 60Val Cys Gly Pro Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly65 70 75 80Ser Ser Ile Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys 85 90 95Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 100 105 110Lys Pro Thr Lys Ala Ala Arg Ser Ile Arg Ala Gln Arg His Thr Asp 115 120 125Met Pro Lys Thr Gln Lys Glu Val His Leu Lys Asn Thr Ser Arg Gly 130 135 140Ser Ala Gly Asn Lys Thr Tyr Arg Met145 150487121DNAMus musculus 48gataactttg ccagaagagg gagagagaga gaaggcgaat gttcccccag ctgtttcctg 60tctacagtgt ctgtgttttg tagataaatg tgaggatttt ctctaaatcc ctcttctgct 120tgctaaatct cactgtcact gctaaattca gagcagatag agcctgcgca atggaataaa 180gtcctcaaaa ttgaaatgtg acattgctct aacatctccc atctctctgg atttcttttt 240cgcctcatta tccctgccca ccaattcatt tccagacttt gtacttcaga agcgatgggg 300aaaatcagca gccttccaac tcaattattt aagatctgcc tctgtgactt cttgaagata 360aagatacaca tcatgtcgtc ttcacacctc ttctacctgg cgctctgctt gctcaccttc 420accagctcca ccacagctgg accagagacc ctttgcgggg ctgagctggt ggatgctctt 480cagttcgtgt gtggaccgag gggcttttac ttcaacaagc ccacaggcta tggctccagc 540attcggaggg cacctcagac aggcattgtg gatgagtgtt gcttccggag ctgtgatctg 600aggagactgg agatgtactg tgccccactg aagcctacaa aagcagcccg ctctatccgt 660gcccagcgcc acactgacat gcccaagact cagaagtccc cgtccctatc gacaaacaag 720aaaacgaagc tgcaaaggag aaggaaagga agtacatttg aagaacacaa gtagaggaag 780tgcaggaaac aagacctaca gaatgtagga ggagcctccc acggagcaga aaatgccaca 840tcaccgcagg atcctttgct gcttgagcaa cctgcaaaac atcgaaacac ctaccaaata 900acaataataa gtccaataac attacaaaga tgggcatttc ccccaatgaa atatacaagt 960aaacattcca acatcgtctt taggagtgtt tgtttaaaaa gctttgcacc ttgcaaaagt 1020ggtcctggcg tgggtagatt gctgttggtc ctttatcaat aacattctat agagaaaaaa 1080aatatatata taactatatc tcctagtccc tgcctctaaa gagccgaaaa tgcatggatg 1140ttgtagagat ccagttgctc taagtttctc tctgaatttt ggctgctgaa gccattcatt 1200tagcaactgt gtagaggtgg tttatgaatg gttcccttat cttcacctct tcccacgtag 1260ctcaagctgc ttgttttaca gagtctaatc atcttgtcta gctgcattag acacaccctt 1320tcctaacact tgtatttgtt gaatttggcc tccttaagag caatagcaaa taagtagtca 1380agtggcctac caagttttaa cgtacctgac tccatctgtg gcatttgtac caaatataag 1440ttgaatgcat ttattttaga cacaaagctt tatttttttt gacattgtgt ttcaagaaaa 1500aaaatagaat aacaataact acaactttga ggccaatcat ttttaggtgt gtgtttgaag 1560catagaacgt ctcttaaact ctcaatggtt tcttcaaatg ataagttagt atgtaaccta 1620agtatagcag tttctctctt ttttattttt ttccatatag agcactatgt aaagttagta 1680tatcaataat acaggaaata tcaaacagta tgtaaaactc tgttgttgtt gttttttagt 1740acaatggtgc tattttgtag tttgttatat gaaagaatct agtcaacaca gtaaaaggag 1800aaagcaaagc aaaaacaaca aacgaaagcc tggagcctaa gatgacaaaa cgaggaaggg 1860aactgaaaaa aaaaatcctt cctcttggga gatgcaaagg cctccccaat tatgccttcc 1920aagaagaact taagatatag agtccattaa gacgcactta cttgtcaagt ccagagagga 1980agctatggag tgggaaaagc aagaggctag ggatttggga gtcctggttt ctttttaatc 2040actgaagaag taagtatttg caacctgggt cacacaaact caccaccctg tgacctcagt 2100caaatcactc cacctctcgg tgcctcagtt ttcctcatct gcaaaatggg ggcaatatgt 2160catctaccta cctcaaaggg gtggtatgaa gattaaaaag tagaccttca gatttttgtt 2220ctgggtttcc aggagggtgc aacatcagaa cccttgaatt gctaggatgc aaggaattct 2280gtaaataacc cactaacaat gtagctccaa ggatcattca tctgtcactg ggatgccacc 2340acaatatcca agttcttatt ggtgaagctg tgcaactaat tagtgacaag ctaaggactc 2400agtctcccca gcatgtcaca cggcaggaga catttgattt gcagttttat ttaacttctg 2460catttgagct tatgactata aagactagtg aaaagaaggg agagaggaga aagaagatcc 2520ttgccaagta aagggtaatt aattattatt ccatttatcc actctcatta aagggtaatt 2580aattattcca tgtatccact ctcattaatc cttccagtca cttagtatct agaaataact 2640ctaacattgt caatgagact ctactcagtt tgccaaacac aattctcctt ccccatagca 2700tatgaaaaaa aggcgctgac attcttaaat tttgaaatag tatctattac aatcacaggt 2760tgctgtagca gatgtagtct tgcccttgtt tgtacatgca tgtatttttt ttttaatttt 2820atgaaaatgt gctagcaaga attgctactt gaggggcaaa attcttcctt ctcaagcctg 2880aggttctccc tagtgtctgc ttagaaggaa ggatccagct tcctggaaat gtgttggatg 2940cattcaactg ggcattgcta accaaaaaca tttagaaaaa tgttctctat gtatatagca 3000agattgtctc cctcttttaa aaacaaaatc caatattcac atcttattac ctacaacctt 3060gattctctat tgcaagcttc cttaatattc ttataaaatg tattaagaaa aacaaaaagg 3120acacctttag ctctccttcc gccaggttgc ctctagaatc tctggggaaa tgcagaaggt 3180gctgttgagt aaagccctca gaaggattgg atttaggaac atcaggcacg ctgtacatcc 3240cctgattact gtagaaatgt aaatggaata agaggtcagc tgaccatcca cctgcttccc 3300cagaaggata cagggaaaag ttaggccctc acacaccctg ggtgacactt ctgacttcta 3360gttcttgttc acagtgtgta ctttttcaaa ttggtaattc ccagaaaaac acataggtgg 3420ccttctccag atctgtgggc ttcctgccat ggttggattt ggtgattcca agtgtctatc 3480acatattttg ttcacttaat tctatccaca gtcagaaatt ctttcaatga ggaaagttta 3540aatatgcaat cctttatcca atacctaatt ctctccaact gcatcataaa tcaagtaata 3600aaaattaatt gtactaatta atcataataa tgtaccattg tacttttaaa tgaatgaaca 3660ctgcaagaca aatctatgta aactctgaaa agtaactgat cattatatgg tgaatcaaaa 3720tgactcaaga ttgatagaaa gggacattta aaattttaca actcaaaatt ttgtagactt 3780tgctatggag gtaaattgtt ttagtgccta gagatggagc ggttttaata aatttacaaa 3840agaactataa agataggtag gaaggaattt tcatttgata ggattgttgc tgatttactt 3900actcaatacc taggtcaaat gttgatccta ttctccaaag actatcaagt gcttgaacat 3960tgtaagatga gtctgctcca ctgaaaatgt aatacatctc tccattataa tctattttcc 4020tggggtaaaa aaatcctttt tttaaatatc cacctacata tacctaccct acatgtgcat 4080ttgcacatgc gtgcatacgc tcatgcgccc caccccacac acacctattc accctaagac 4140taagaagaaa tcatttcttt gaaagtctta tctttcaaaa aaggcagcgg tgccccttga 4200gactccttct ccttctttga atgtcaatgt gaaatgtggc atgtctgtgt acatgaaacc 4260atctcatacc ctatggctcc agggtttctt tatggtttgt gcacttggga ggatgcgcag 4320aagacaggat gcagcctgtt ttgctttccc ctttactgtt tggccagcta cgccaatgtg 4380gtgctattgt ttctttaaga aagtacttga ctaaaaaaaa agaaaaaaag aaaaaaagaa 4440aagaaaaaga aaaaagaaaa aaaaagaaag catagaccta tttttttaaa gtctgaaaac 4500aacagttcta tagtagatgg cttactgaga tagcattaga tctagccacc accctagcca 4560ccacctttca actatgtgtc actcacaagt agaatattgt tcaccaagtt gtgagtttgg 4620gggttcagag acaaaggatg gaaaagtttt aaagttagat ggctcaatca tttcattggc 4680tctcaaattt aacaaaattg gcaatacttc acccaatctg aagtgttggt caataacttg 4740aactgggggc aaaaataact tcaggcaaat ggcagaagaa aataattaac ttacttcttg 4800ctttttttgt tgattgtttg gtttcctgtt gatttttggt tttggttttg ctgtgggtgg 4860gtgagtacat gtgtgtaagt acgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgttc 4920cactcaaaaa caaatactca gaaagtggag aaaatacaac gattttaaga gcatagactt 4980acctactact agaaccagct tctgtcacat cctctggaga aggcactgat ttcttgtttt 5040gtagaggttg ctcttccatc agtgacctga aagagtgacc agtctcctag agtagacatg 5100gatctcatta ggagaagaca gaagtatttc cttatgaatt gggcttatct actgacaaag 5160aaagggaaga gtttatgaga agttattgaa gaagatggct aacagtctgt gaagattttg 5220ttctggtttt ttttgttgtt gttgttgttg ggtttgggtt ttgatttttt tttttttttt 5280tttactttat acaatcttta tgaatggaaa tcttaatgct caaaaagact tggtcttttt 5340ttctctttcg taacagaatg gaagatgaca aactcacata gactctttct aggctggcta 5400gcaaaggtgt ggtttgactt atttgaatca gaccatttta aatgttcctc tctattttta 5460atcataaaag gctgtcataa tttattagcg taggcccttt ttggcacttc tcaaatgaat 5520gagcattccc attcaaagca tggctttccc catggttcca aaacatgaat gattaatatt 5580aaggaattat ttacttcaaa atacagtaga agtgtgagtc tctgttccca ttccccacaa 5640agatcattaa gtcctgaatc gggggcgggg gtggggcgcc tggatactaa gggaattttt 5700ttgttgcttg ttttttgttt tcaatgctag tgcttaatcc tatagtatac agatttgctt 5760cttgctattg tgatattctg taagactttc ctgttaggta ttagaaattg atacataaat 5820accttttttg tgtggtttct atttaaaagg aaagagataa gactgtctga accttaaatt 5880cgtaaggcac atgataaaga gatcacatta aataacaagc catatctggt tcaatccttt 5940ctttcttatc attttaagga aaacttgccc agataagaca gaggcccagg ggacttttga 6000aactctcttt gttccgccaa ttcattttgg ctggtgatgg tttttcccca gtgtctgcct 6060cagaatcttt tagaggctgg ccagactaaa gactgtcttt taaaacacat ttcacatggt 6120tcctcttaat gaatgattac acttatgtag aacatgattt ttttttctct ccacttattt 6180tttttttccc catcattgat aagggttctt aaggagaaga attcattaac aaaactcaag 6240aaagcgtaca aaaaaaaaat tctaaatgtc actgcccaat tgaaatacga gctaaaatgg 6300aaatactttc tcctacttaa aacccagact gaatcacctt caaaatgacc tttcacaatc 6360tttccaattt gcctttgttt aaactgtctg ggcctaaaag caagcattat tcattttctc 6420ttgcccaaag tgaacttgtg taaagtagga aaattaaaag aaactgctag aaatcccttc 6480caaccagtgg ctgacccctc tcactagctc acagcaaagt ctcctctgtt gatctatcac 6540ctagtctcat ttcgtttgaa tatttacatt gtacctactg ctaaacactt ggcaggaggc 6600tccatccata tctcctatcg gtgtctctgt atccttaaac cttgcaaaca tcatacagtg 6660tatattaagt ttacaggaaa gctccaaata gcatatcaga cctggtctct ctttgttaaa 6720gatttaagga gctatgggaa tctggattac aacgcacatt ttgcttcatt tatttttatc 6780acactttaaa ggccaagggt gatgattaac ttacagacac tgaattgatt tccctactga 6840aacctgaaag taatatttgg tcattcattg tatgtgtttt acacaaaaaa aacatcttct 6900atcaaattac tcctgattgt atttgaagtg gttattcaat tcatttatgg cagagcaata 6960tctgtcctaa tgactcttat aaaatgtaac taactgaatc attatcttac atttactgtt 7020tagtaagcat attttgaaat tgtatggcta gagtgtcata ataaaatggt atatctttct 7080ttagtaatta cattaaaatt aatcatgttt gattaactgg t 7121491087DNAMus musculus 49acaatggaaa tgagtggctt cccttggggg aaaaagacgg actccaactc ccagctgtgc 60aatttactca ttgtttaaat ggacaaaagg cagtttaccc aggctcagag catacctgcc 120tgggtgtcca aatgtaacta gatgctttca caaaccccac ccacaaaaca acacctgttc 180ttaagtcctc agttttgtgt tcacctcggc ctcatagtac ccactctgac ctgctgtgta 240aacgacccgg acctaccaaa atgaccgcac ctgcaataaa gatacacatc atgtcgtctt 300cacacctctt ctacctggcg ctctgcttgc tcaccttcac cagctccacc acagctggac 360cagagaccct ttgcggggct gagctggtgg atgctcttca gttcgtgtgt ggaccgaggg 420gcttttactt caacaagccc acaggctatg gctccagcat tcggagggca cctcagacag 480gcattgtgga tgagtgttgc ttccggagct gtgatctgag gagactggag atgtactgtg 540ccccactgaa gcctacaaaa gcagcccgct ctatccgtgc ccagcgccac actgacatgc 600ccaagactca gaagtccccg tccctatcga caaacaagaa aacgaagctg caaaggagaa 660ggaaaggtga gccaaagaca cacccagaag gggaacagga ggaggtaacg gaggcaactc 720ggaaaatcag aggtcccaga gaaaaaagac tgggctagga actgtgagca agcaggcaaa 780gagggacatg cgggaacagg gatgaaggac gtgcgggaac agggatgaag aaggagcaga 840caggagccca ggaaagccgc agaggagctg aagcaaggca gtcctcacta agctagataa 900tgtctgtgac ggaagtaaga aagtcctcct ctgggatacg gcacttacac atgggaagaa 960tggtacgggg aagtgtaaca cctcagaaag tgacaagtga ccaggatgga acatcaacaa 1020caataacaac cattaaaaac atgccaccaa gaccttccct ccccttctta aaaatataaa 1080tcagagt 1087507039DNAMus musculus 50acaatggaaa tgagtggctt cccttggggg aaaaagacgg actccaactc ccagctgtgc 60aatttactca ttgtttaaat ggacaaaagg cagtttaccc aggctcagag catacctgcc 120tgggtgtcca aatgtaacta gatgctttca caaaccccac ccacaaaaca acacctgttc 180ttaagtcctc agttttgtgt tcacctcggc ctcatagtac ccactctgac ctgctgtgta 240aacgacccgg acctaccaaa atgaccgcac ctgcaataaa gatacacatc atgtcgtctt 300cacacctctt ctacctggcg ctctgcttgc tcaccttcac cagctccacc acagctggac 360cagagaccct ttgcggggct gagctggtgg atgctcttca gttcgtgtgt ggaccgaggg 420gcttttactt caacaagccc acaggctatg gctccagcat tcggagggca cctcagacag 480gcattgtgga tgagtgttgc ttccggagct

gtgatctgag gagactggag atgtactgtg 540ccccactgaa gcctacaaaa gcagcccgct ctatccgtgc ccagcgccac actgacatgc 600ccaagactca gaagtccccg tccctatcga caaacaagaa aacgaagctg caaaggagaa 660ggaaaggaag tacatttgaa gaacacaagt agaggaagtg caggaaacaa gacctacaga 720atgtaggagg agcctcccac ggagcagaaa atgccacatc accgcaggat cctttgctgc 780ttgagcaacc tgcaaaacat cgaaacacct accaaataac aataataagt ccaataacat 840tacaaagatg ggcatttccc ccaatgaaat atacaagtaa acattccaac atcgtcttta 900ggagtgtttg tttaaaaagc tttgcacctt gcaaaagtgg tcctggcgtg ggtagattgc 960tgttggtcct ttatcaataa cattctatag agaaaaaaaa tatatatata actatatctc 1020ctagtccctg cctctaaaga gccgaaaatg catggatgtt gtagagatcc agttgctcta 1080agtttctctc tgaattttgg ctgctgaagc cattcattta gcaactgtgt agaggtggtt 1140tatgaatggt tcccttatct tcacctcttc ccacgtagct caagctgctt gttttacaga 1200gtctaatcat cttgtctagc tgcattagac acaccctttc ctaacacttg tatttgttga 1260atttggcctc cttaagagca atagcaaata agtagtcaag tggcctacca agttttaacg 1320tacctgactc catctgtggc atttgtacca aatataagtt gaatgcattt attttagaca 1380caaagcttta ttttttttga cattgtgttt caagaaaaaa aatagaataa caataactac 1440aactttgagg ccaatcattt ttaggtgtgt gtttgaagca tagaacgtct cttaaactct 1500caatggtttc ttcaaatgat aagttagtat gtaacctaag tatagcagtt tctctctttt 1560ttattttttt ccatatagag cactatgtaa agttagtata tcaataatac aggaaatatc 1620aaacagtatg taaaactctg ttgttgttgt tttttagtac aatggtgcta ttttgtagtt 1680tgttatatga aagaatctag tcaacacagt aaaaggagaa agcaaagcaa aaacaacaaa 1740cgaaagcctg gagcctaaga tgacaaaacg aggaagggaa ctgaaaaaaa aaatccttcc 1800tcttgggaga tgcaaaggcc tccccaatta tgccttccaa gaagaactta agatatagag 1860tccattaaga cgcacttact tgtcaagtcc agagaggaag ctatggagtg ggaaaagcaa 1920gaggctaggg atttgggagt cctggtttct ttttaatcac tgaagaagta agtatttgca 1980acctgggtca cacaaactca ccaccctgtg acctcagtca aatcactcca cctctcggtg 2040cctcagtttt cctcatctgc aaaatggggg caatatgtca tctacctacc tcaaaggggt 2100ggtatgaaga ttaaaaagta gaccttcaga tttttgttct gggtttccag gagggtgcaa 2160catcagaacc cttgaattgc taggatgcaa ggaattctgt aaataaccca ctaacaatgt 2220agctccaagg atcattcatc tgtcactggg atgccaccac aatatccaag ttcttattgg 2280tgaagctgtg caactaatta gtgacaagct aaggactcag tctccccagc atgtcacacg 2340gcaggagaca tttgatttgc agttttattt aacttctgca tttgagctta tgactataaa 2400gactagtgaa aagaagggag agaggagaaa gaagatcctt gccaagtaaa gggtaattaa 2460ttattattcc atttatccac tctcattaaa gggtaattaa ttattccatg tatccactct 2520cattaatcct tccagtcact tagtatctag aaataactct aacattgtca atgagactct 2580actcagtttg ccaaacacaa ttctccttcc ccatagcata tgaaaaaaag gcgctgacat 2640tcttaaattt tgaaatagta tctattacaa tcacaggttg ctgtagcaga tgtagtcttg 2700cccttgtttg tacatgcatg tatttttttt ttaattttat gaaaatgtgc tagcaagaat 2760tgctacttga ggggcaaaat tcttccttct caagcctgag gttctcccta gtgtctgctt 2820agaaggaagg atccagcttc ctggaaatgt gttggatgca ttcaactggg cattgctaac 2880caaaaacatt tagaaaaatg ttctctatgt atatagcaag attgtctccc tcttttaaaa 2940acaaaatcca atattcacat cttattacct acaaccttga ttctctattg caagcttcct 3000taatattctt ataaaatgta ttaagaaaaa caaaaaggac acctttagct ctccttccgc 3060caggttgcct ctagaatctc tggggaaatg cagaaggtgc tgttgagtaa agccctcaga 3120aggattggat ttaggaacat caggcacgct gtacatcccc tgattactgt agaaatgtaa 3180atggaataag aggtcagctg accatccacc tgcttcccca gaaggataca gggaaaagtt 3240aggccctcac acaccctggg tgacacttct gacttctagt tcttgttcac agtgtgtact 3300ttttcaaatt ggtaattccc agaaaaacac ataggtggcc ttctccagat ctgtgggctt 3360cctgccatgg ttggatttgg tgattccaag tgtctatcac atattttgtt cacttaattc 3420tatccacagt cagaaattct ttcaatgagg aaagtttaaa tatgcaatcc tttatccaat 3480acctaattct ctccaactgc atcataaatc aagtaataaa aattaattgt actaattaat 3540cataataatg taccattgta cttttaaatg aatgaacact gcaagacaaa tctatgtaaa 3600ctctgaaaag taactgatca ttatatggtg aatcaaaatg actcaagatt gatagaaagg 3660gacatttaaa attttacaac tcaaaatttt gtagactttg ctatggaggt aaattgtttt 3720agtgcctaga gatggagcgg ttttaataaa tttacaaaag aactataaag ataggtagga 3780aggaattttc atttgatagg attgttgctg atttacttac tcaataccta ggtcaaatgt 3840tgatcctatt ctccaaagac tatcaagtgc ttgaacattg taagatgagt ctgctccact 3900gaaaatgtaa tacatctctc cattataatc tattttcctg gggtaaaaaa atcctttttt 3960taaatatcca cctacatata cctaccctac atgtgcattt gcacatgcgt gcatacgctc 4020atgcgcccca ccccacacac acctattcac cctaagacta agaagaaatc atttctttga 4080aagtcttatc tttcaaaaaa ggcagcggtg ccccttgaga ctccttctcc ttctttgaat 4140gtcaatgtga aatgtggcat gtctgtgtac atgaaaccat ctcataccct atggctccag 4200ggtttcttta tggtttgtgc acttgggagg atgcgcagaa gacaggatgc agcctgtttt 4260gctttcccct ttactgtttg gccagctacg ccaatgtggt gctattgttt ctttaagaaa 4320gtacttgact aaaaaaaaag aaaaaaagaa aaaaagaaaa gaaaaagaaa aaagaaaaaa 4380aaagaaagca tagacctatt tttttaaagt ctgaaaacaa cagttctata gtagatggct 4440tactgagata gcattagatc tagccaccac cctagccacc acctttcaac tatgtgtcac 4500tcacaagtag aatattgttc accaagttgt gagtttgggg gttcagagac aaaggatgga 4560aaagttttaa agttagatgg ctcaatcatt tcattggctc tcaaatttaa caaaattggc 4620aatacttcac ccaatctgaa gtgttggtca ataacttgaa ctgggggcaa aaataacttc 4680aggcaaatgg cagaagaaaa taattaactt acttcttgct ttttttgttg attgtttggt 4740ttcctgttga tttttggttt tggttttgct gtgggtgggt gagtacatgt gtgtaagtac 4800gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgttcca ctcaaaaaca aatactcaga 4860aagtggagaa aatacaacga ttttaagagc atagacttac ctactactag aaccagcttc 4920tgtcacatcc tctggagaag gcactgattt cttgttttgt agaggttgct cttccatcag 4980tgacctgaaa gagtgaccag tctcctagag tagacatgga tctcattagg agaagacaga 5040agtatttcct tatgaattgg gcttatctac tgacaaagaa agggaagagt ttatgagaag 5100ttattgaaga agatggctaa cagtctgtga agattttgtt ctggtttttt ttgttgttgt 5160tgttgttggg tttgggtttt gatttttttt tttttttttt tactttatac aatctttatg 5220aatggaaatc ttaatgctca aaaagacttg gtcttttttt ctctttcgta acagaatgga 5280agatgacaaa ctcacataga ctctttctag gctggctagc aaaggtgtgg tttgacttat 5340ttgaatcaga ccattttaaa tgttcctctc tatttttaat cataaaaggc tgtcataatt 5400tattagcgta ggcccttttt ggcacttctc aaatgaatga gcattcccat tcaaagcatg 5460gctttcccca tggttccaaa acatgaatga ttaatattaa ggaattattt acttcaaaat 5520acagtagaag tgtgagtctc tgttcccatt ccccacaaag atcattaagt cctgaatcgg 5580gggcgggggt ggggcgcctg gatactaagg gaattttttt gttgcttgtt ttttgttttc 5640aatgctagtg cttaatccta tagtatacag atttgcttct tgctattgtg atattctgta 5700agactttcct gttaggtatt agaaattgat acataaatac cttttttgtg tggtttctat 5760ttaaaaggaa agagataaga ctgtctgaac cttaaattcg taaggcacat gataaagaga 5820tcacattaaa taacaagcca tatctggttc aatcctttct ttcttatcat tttaaggaaa 5880acttgcccag ataagacaga ggcccagggg acttttgaaa ctctctttgt tccgccaatt 5940cattttggct ggtgatggtt tttccccagt gtctgcctca gaatctttta gaggctggcc 6000agactaaaga ctgtctttta aaacacattt cacatggttc ctcttaatga atgattacac 6060ttatgtagaa catgattttt ttttctctcc acttattttt tttttcccca tcattgataa 6120gggttcttaa ggagaagaat tcattaacaa aactcaagaa agcgtacaaa aaaaaaattc 6180taaatgtcac tgcccaattg aaatacgagc taaaatggaa atactttctc ctacttaaaa 6240cccagactga atcaccttca aaatgacctt tcacaatctt tccaatttgc ctttgtttaa 6300actgtctggg cctaaaagca agcattattc attttctctt gcccaaagtg aacttgtgta 6360aagtaggaaa attaaaagaa actgctagaa atcccttcca accagtggct gacccctctc 6420actagctcac agcaaagtct cctctgttga tctatcacct agtctcattt cgtttgaata 6480tttacattgt acctactgct aaacacttgg caggaggctc catccatatc tcctatcggt 6540gtctctgtat ccttaaacct tgcaaacatc atacagtgta tattaagttt acaggaaagc 6600tccaaatagc atatcagacc tggtctctct ttgttaaaga tttaaggagc tatgggaatc 6660tggattacaa cgcacatttt gcttcattta tttttatcac actttaaagg ccaagggtga 6720tgattaactt acagacactg aattgatttc cctactgaaa cctgaaagta atatttggtc 6780attcattgta tgtgttttac acaaaaaaaa catcttctat caaattactc ctgattgtat 6840ttgaagtggt tattcaattc atttatggca gagcaatatc tgtcctaatg actcttataa 6900aatgtaacta actgaatcat tatcttacat ttactgttta gtaagcatat tttgaaattg 6960tatggctaga gtgtcataat aaaatggtat atctttcttt agtaattaca ttaaaattaa 7020tcatgtttga ttaactggt 7039517069DNAMus musculus 51gataactttg ccagaagagg gagagagaga gaaggcgaat gttcccccag ctgtttcctg 60tctacagtgt ctgtgttttg tagataaatg tgaggatttt ctctaaatcc ctcttctgct 120tgctaaatct cactgtcact gctaaattca gagcagatag agcctgcgca atggaataaa 180gtcctcaaaa ttgaaatgtg acattgctct aacatctccc atctctctgg atttcttttt 240cgcctcatta tccctgccca ccaattcatt tccagacttt gtacttcaga agcgatgggg 300aaaatcagca gccttccaac tcaattattt aagatctgcc tctgtgactt cttgaagata 360aagatacaca tcatgtcgtc ttcacacctc ttctacctgg cgctctgctt gctcaccttc 420accagctcca ccacagctgg accagagacc ctttgcgggg ctgagctggt ggatgctctt 480cagttcgtgt gtggaccgag gggcttttac ttcaacaagc ccacaggcta tggctccagc 540attcggaggg cacctcagac aggcattgtg gatgagtgtt gcttccggag ctgtgatctg 600aggagactgg agatgtactg tgccccactg aagcctacaa aagcagcccg ctctatccgt 660gcccagcgcc acactgacat gcccaagact cagaaggaag tacatttgaa gaacacaagt 720agaggaagtg caggaaacaa gacctacaga atgtaggagg agcctcccac ggagcagaaa 780atgccacatc accgcaggat cctttgctgc ttgagcaacc tgcaaaacat cgaaacacct 840accaaataac aataataagt ccaataacat tacaaagatg ggcatttccc ccaatgaaat 900atacaagtaa acattccaac atcgtcttta ggagtgtttg tttaaaaagc tttgcacctt 960gcaaaagtgg tcctggcgtg ggtagattgc tgttggtcct ttatcaataa cattctatag 1020agaaaaaaaa tatatatata actatatctc ctagtccctg cctctaaaga gccgaaaatg 1080catggatgtt gtagagatcc agttgctcta agtttctctc tgaattttgg ctgctgaagc 1140cattcattta gcaactgtgt agaggtggtt tatgaatggt tcccttatct tcacctcttc 1200ccacgtagct caagctgctt gttttacaga gtctaatcat cttgtctagc tgcattagac 1260acaccctttc ctaacacttg tatttgttga atttggcctc cttaagagca atagcaaata 1320agtagtcaag tggcctacca agttttaacg tacctgactc catctgtggc atttgtacca 1380aatataagtt gaatgcattt attttagaca caaagcttta ttttttttga cattgtgttt 1440caagaaaaaa aatagaataa caataactac aactttgagg ccaatcattt ttaggtgtgt 1500gtttgaagca tagaacgtct cttaaactct caatggtttc ttcaaatgat aagttagtat 1560gtaacctaag tatagcagtt tctctctttt ttattttttt ccatatagag cactatgtaa 1620agttagtata tcaataatac aggaaatatc aaacagtatg taaaactctg ttgttgttgt 1680tttttagtac aatggtgcta ttttgtagtt tgttatatga aagaatctag tcaacacagt 1740aaaaggagaa agcaaagcaa aaacaacaaa cgaaagcctg gagcctaaga tgacaaaacg 1800aggaagggaa ctgaaaaaaa aaatccttcc tcttgggaga tgcaaaggcc tccccaatta 1860tgccttccaa gaagaactta agatatagag tccattaaga cgcacttact tgtcaagtcc 1920agagaggaag ctatggagtg ggaaaagcaa gaggctaggg atttgggagt cctggtttct 1980ttttaatcac tgaagaagta agtatttgca acctgggtca cacaaactca ccaccctgtg 2040acctcagtca aatcactcca cctctcggtg cctcagtttt cctcatctgc aaaatggggg 2100caatatgtca tctacctacc tcaaaggggt ggtatgaaga ttaaaaagta gaccttcaga 2160tttttgttct gggtttccag gagggtgcaa catcagaacc cttgaattgc taggatgcaa 2220ggaattctgt aaataaccca ctaacaatgt agctccaagg atcattcatc tgtcactggg 2280atgccaccac aatatccaag ttcttattgg tgaagctgtg caactaatta gtgacaagct 2340aaggactcag tctccccagc atgtcacacg gcaggagaca tttgatttgc agttttattt 2400aacttctgca tttgagctta tgactataaa gactagtgaa aagaagggag agaggagaaa 2460gaagatcctt gccaagtaaa gggtaattaa ttattattcc atttatccac tctcattaaa 2520gggtaattaa ttattccatg tatccactct cattaatcct tccagtcact tagtatctag 2580aaataactct aacattgtca atgagactct actcagtttg ccaaacacaa ttctccttcc 2640ccatagcata tgaaaaaaag gcgctgacat tcttaaattt tgaaatagta tctattacaa 2700tcacaggttg ctgtagcaga tgtagtcttg cccttgtttg tacatgcatg tatttttttt 2760ttaattttat gaaaatgtgc tagcaagaat tgctacttga ggggcaaaat tcttccttct 2820caagcctgag gttctcccta gtgtctgctt agaaggaagg atccagcttc ctggaaatgt 2880gttggatgca ttcaactggg cattgctaac caaaaacatt tagaaaaatg ttctctatgt 2940atatagcaag attgtctccc tcttttaaaa acaaaatcca atattcacat cttattacct 3000acaaccttga ttctctattg caagcttcct taatattctt ataaaatgta ttaagaaaaa 3060caaaaaggac acctttagct ctccttccgc caggttgcct ctagaatctc tggggaaatg 3120cagaaggtgc tgttgagtaa agccctcaga aggattggat ttaggaacat caggcacgct 3180gtacatcccc tgattactgt agaaatgtaa atggaataag aggtcagctg accatccacc 3240tgcttcccca gaaggataca gggaaaagtt aggccctcac acaccctggg tgacacttct 3300gacttctagt tcttgttcac agtgtgtact ttttcaaatt ggtaattccc agaaaaacac 3360ataggtggcc ttctccagat ctgtgggctt cctgccatgg ttggatttgg tgattccaag 3420tgtctatcac atattttgtt cacttaattc tatccacagt cagaaattct ttcaatgagg 3480aaagtttaaa tatgcaatcc tttatccaat acctaattct ctccaactgc atcataaatc 3540aagtaataaa aattaattgt actaattaat cataataatg taccattgta cttttaaatg 3600aatgaacact gcaagacaaa tctatgtaaa ctctgaaaag taactgatca ttatatggtg 3660aatcaaaatg actcaagatt gatagaaagg gacatttaaa attttacaac tcaaaatttt 3720gtagactttg ctatggaggt aaattgtttt agtgcctaga gatggagcgg ttttaataaa 3780tttacaaaag aactataaag ataggtagga aggaattttc atttgatagg attgttgctg 3840atttacttac tcaataccta ggtcaaatgt tgatcctatt ctccaaagac tatcaagtgc 3900ttgaacattg taagatgagt ctgctccact gaaaatgtaa tacatctctc cattataatc 3960tattttcctg gggtaaaaaa atcctttttt taaatatcca cctacatata cctaccctac 4020atgtgcattt gcacatgcgt gcatacgctc atgcgcccca ccccacacac acctattcac 4080cctaagacta agaagaaatc atttctttga aagtcttatc tttcaaaaaa ggcagcggtg 4140ccccttgaga ctccttctcc ttctttgaat gtcaatgtga aatgtggcat gtctgtgtac 4200atgaaaccat ctcataccct atggctccag ggtttcttta tggtttgtgc acttgggagg 4260atgcgcagaa gacaggatgc agcctgtttt gctttcccct ttactgtttg gccagctacg 4320ccaatgtggt gctattgttt ctttaagaaa gtacttgact aaaaaaaaag aaaaaaagaa 4380aaaaagaaaa gaaaaagaaa aaagaaaaaa aaagaaagca tagacctatt tttttaaagt 4440ctgaaaacaa cagttctata gtagatggct tactgagata gcattagatc tagccaccac 4500cctagccacc acctttcaac tatgtgtcac tcacaagtag aatattgttc accaagttgt 4560gagtttgggg gttcagagac aaaggatgga aaagttttaa agttagatgg ctcaatcatt 4620tcattggctc tcaaatttaa caaaattggc aatacttcac ccaatctgaa gtgttggtca 4680ataacttgaa ctgggggcaa aaataacttc aggcaaatgg cagaagaaaa taattaactt 4740acttcttgct ttttttgttg attgtttggt ttcctgttga tttttggttt tggttttgct 4800gtgggtgggt gagtacatgt gtgtaagtac gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 4860gtgtgttcca ctcaaaaaca aatactcaga aagtggagaa aatacaacga ttttaagagc 4920atagacttac ctactactag aaccagcttc tgtcacatcc tctggagaag gcactgattt 4980cttgttttgt agaggttgct cttccatcag tgacctgaaa gagtgaccag tctcctagag 5040tagacatgga tctcattagg agaagacaga agtatttcct tatgaattgg gcttatctac 5100tgacaaagaa agggaagagt ttatgagaag ttattgaaga agatggctaa cagtctgtga 5160agattttgtt ctggtttttt ttgttgttgt tgttgttggg tttgggtttt gatttttttt 5220tttttttttt tactttatac aatctttatg aatggaaatc ttaatgctca aaaagacttg 5280gtcttttttt ctctttcgta acagaatgga agatgacaaa ctcacataga ctctttctag 5340gctggctagc aaaggtgtgg tttgacttat ttgaatcaga ccattttaaa tgttcctctc 5400tatttttaat cataaaaggc tgtcataatt tattagcgta ggcccttttt ggcacttctc 5460aaatgaatga gcattcccat tcaaagcatg gctttcccca tggttccaaa acatgaatga 5520ttaatattaa ggaattattt acttcaaaat acagtagaag tgtgagtctc tgttcccatt 5580ccccacaaag atcattaagt cctgaatcgg gggcgggggt ggggcgcctg gatactaagg 5640gaattttttt gttgcttgtt ttttgttttc aatgctagtg cttaatccta tagtatacag 5700atttgcttct tgctattgtg atattctgta agactttcct gttaggtatt agaaattgat 5760acataaatac cttttttgtg tggtttctat ttaaaaggaa agagataaga ctgtctgaac 5820cttaaattcg taaggcacat gataaagaga tcacattaaa taacaagcca tatctggttc 5880aatcctttct ttcttatcat tttaaggaaa acttgcccag ataagacaga ggcccagggg 5940acttttgaaa ctctctttgt tccgccaatt cattttggct ggtgatggtt tttccccagt 6000gtctgcctca gaatctttta gaggctggcc agactaaaga ctgtctttta aaacacattt 6060cacatggttc ctcttaatga atgattacac ttatgtagaa catgattttt ttttctctcc 6120acttattttt tttttcccca tcattgataa gggttcttaa ggagaagaat tcattaacaa 6180aactcaagaa agcgtacaaa aaaaaaattc taaatgtcac tgcccaattg aaatacgagc 6240taaaatggaa atactttctc ctacttaaaa cccagactga atcaccttca aaatgacctt 6300tcacaatctt tccaatttgc ctttgtttaa actgtctggg cctaaaagca agcattattc 6360attttctctt gcccaaagtg aacttgtgta aagtaggaaa attaaaagaa actgctagaa 6420atcccttcca accagtggct gacccctctc actagctcac agcaaagtct cctctgttga 6480tctatcacct agtctcattt cgtttgaata tttacattgt acctactgct aaacacttgg 6540caggaggctc catccatatc tcctatcggt gtctctgtat ccttaaacct tgcaaacatc 6600atacagtgta tattaagttt acaggaaagc tccaaatagc atatcagacc tggtctctct 6660ttgttaaaga tttaaggagc tatgggaatc tggattacaa cgcacatttt gcttcattta 6720tttttatcac actttaaagg ccaagggtga tgattaactt acagacactg aattgatttc 6780cctactgaaa cctgaaagta atatttggtc attcattgta tgtgttttac acaaaaaaaa 6840catcttctat caaattactc ctgattgtat ttgaagtggt tattcaattc atttatggca 6900gagcaatatc tgtcctaatg actcttataa aatgtaacta actgaatcat tatcttacat 6960ttactgttta gtaagcatat tttgaaattg tatggctaga gtgtcataat aaaatggtat 7020atctttcttt agtaattaca ttaaaattaa tcatgtttga ttaactggt 7069521649DNAArtificial SequenceCAG promoter 52tctactcgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt 60catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga 120ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 180atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca 240gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 300cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 360tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc 420cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt 480gcagcgatgg gggcgggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag 540gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga 600aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg 660cgggcgggag tcgctgcgtt gccttcgccc cgtgccccgc tccgcgccgc ctcgcgccgc 720ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct 780cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt 840gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg 900tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag 960cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc 1020gggggcggtg ccccgcggtg cggggggctg cgaggggaac aaaggctgcg tgcggggtgt 1080gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca 1140cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg 1200tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc 1260ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg 1320cggctgtcga ggcgcggcga

gccgcagcca ttgcctttta tggtaatcgt gcgagagggc 1380gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac 1440cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga 1500gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc 1560gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt 1620gaccggcggc tctagagcct ctgctaacc 16495321DNAArtificial SequencePrimer mIGF-1a forward sequence 53tggatgctct tcagttcgtg t 215421DNAArtificial SequencePrimer mIGF-1a, reverse sequence 54caacactcat ccacaatgcc t 215520DNAArtificial SequencePrimer mCcl3 (Mip-1alpha), forward sequence 55gcaaccaagt cttctcagcg 205620DNAArtificial SequencePrimer mCcl3 (Mip-1alpha), reverse sequence 56agcaaaggct gctggtttca 205720DNAArtificial SequencePrimer mCcl4 (Mip-1beta), forward sequence 57ccatgaagct ctgcgtgtct 205821DNAArtificial SequencePrimer mCcl4 (Mip-1beta), reverse sequence 58gagaaacagc aggaagtggg a 215920DNAArtificial SequencePrimer mCxcl9 (MIG), forward sequence 59cgaggcacga tccactacaa 206020DNAArtificial SequencePrimer mCxcl9 (MIG), reverse sequence 60agtccggatc taggcaggtt 206120DNAArtificial SequencePrimer mCxcl10 (IP-10), forward sequence 61ccaagtgctg ccgtcatttt 206220DNAArtificial SequencePrimer mCxcl10 (IP-10), reverse sequence 62agcttcccta tggccctcat 206321DNAArtificial SequencePrimer mCcl5 (RANTES), forward sequence 63gtgcccacgt caaggagtat t 216421DNAArtificial SequencePrimer mCcl5 (RANTES), reverse sequence 64cccacttctt ctctgggttg g 216520DNAArtificial SequencePrimer mCCL2 (MCP-1), forward sequence 65atgcagttaa cgccccactc 206620DNAArtificial SequencePrimer mCCL2 (MCP-1), reverse sequence 66gcttctttgg gacacctgct 206720DNAArtificial SequencePrimer mIFN-gamma, forward sequence 67agacaatcag gccatcagca 206820DNAArtificial SequencePrimer mIFNgamma, reverse sequence 68tggacctgtg ggttgttgac 206921DNAArtificial SequencePrimer mTNFalpha, forward sequence 69tcttctcatt cctgcttgtg g 217020DNAArtificial SequencePrimer mTNFalpha, reverse sequence 70ggtctgggcc atagaactga 207121DNAArtificial SequencePrimer mIL-1beta, forward sequence 71tgccaccttt tgacagtgat g 217220DNAArtificial SequencePrimer mIL-1beta, reverse sequence 72tgatgtgctg ctgcgagatt 207319DNAArtificial SequencePrimer mH2-Aa, forward sequence 73ctctgattct gggggtcct 197420DNAArtificial SequencePrimer mH2Aa, reverse sequence 74accataggtg cctacgtggt 207516DNAArtificial SequencePrimer mbeta2-microglobulin, forward sequence 75ccggagaatg ggaagc 167616DNAArtificial SequencePrimer mbeta2-microglobulin, reverse sequence 76gtagacggtc ttgggc 167720DNAArtificial SequencePrimer mCD80 (B 7.1), forward sequence 77atacgactcg caaccacacc 207820DNAArtificial SequencePrimer mCD80 (B 7.1), reverse sequence 78gaatcctgcc ccaaagagca 207920DNAArtificial SequencePrimer mCD86 (B 7.2), forward sequence 79gcttcagtta ctgtggccct 208020DNAArtificial SequencePrimer mCD86 (B 7.2), reverse sequence 80tgtcagcgtt actatcccgc 208120DNAArtificial SequencePrimer, mSlc7a1, forward sequence 81aaacacccgt aatcgccact 208220DNAArtificial SequencePrimer mSlc7a1, reverse sequence 82ggctggtacc gtaagaccaa 208321DNAArtificial SequencePrimer mCcng1, forward sequence 83tgactgcaag attacgggac t 218420DNAArtificial SequencePrimer, mCcng1, reverse sequence 84cccaagatgc ttcgcctgta 208520DNAArtificial SequencePrimer mGys1, forward sequence 85ccgctaactc taccggtcac 208620DNAArtificial SequencePrimer mGys1, reverse sequence 86ccccattcat cccctgtcac 208720DNAArtificial SequencePrimer mAldoA, forward sequence 87gcgttcgctc cttagtcctt 208820DNAArtificial SequencePrimer mAldoA, reverse sequence 88aatgcaggga ttcacacggt 208920DNAArtificial SequencePrimer mRplp0, forward sequence 89tcccaccttg tctccagtct 209042DNAArtificial SequencePrimer mRplp0, reverse sequence 90actggtctag gacccgagaa actggtctag gacccgagaa gg 429118DNAArtificial SequencePrimer rRplp0, forward sequence 91gatgcccagg gaagacag 189222DNAArtificial SequencePrimer rRplp0, reverse sequence 92cacaatgaag cattttgggt ag 229322DNAArtificial Sequencetarget sequence of miRNA199A-3p 93tgtcatcaga cgtgtaacca at 229422DNAArtificial SequenceTarget sequence of miRNA208 94tattctgctc gtttttcgaa ca 229521DNAArtificial Sequencetarget sequence of miRNA101 95atgtcatgac actattgact t 21963281DNAArtificial Sequenceexpression cassette present in SEQ ID NO30 96ctcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca 420tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag 480cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc 540ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt 600ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg 660cgggagtcgc tgcgttgcct tcgccccgtg ccccgctccg cgccgcctcg cgccgcccgc 720cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc 780cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa 840gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg 900tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct 960gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg 1020gcggtgcccc gcggtgcggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc 1080gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc 1140cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc 1200gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc 1320tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag 1380ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc 1440tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc 1500cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg 1560ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc 1620ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg 1680ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgat taattcgagc 1740gaacgcgtcg agtcgctcgg tacgatttaa attgaattgg cctcgagcgc aagcttgata 1800tcgaattccg tagatgcttt cacaaacccc acccacaaaa caacacatgt tcttaagtcc 1860tcagttttgt gttcacctcg gcctcatagt acccactctg acctgctgtg taaacgaccc 1920ggacctacca aaatgaccgc acctgcaata aagatacaca tcatgtcgtc ttcacacctc 1980ttctacctgg cgctctgctt gctcaccttc accagctcca ccacagctgg accagagacc 2040ctttgcgggg ctgagctggt ggatgctctt cagttcgtgt gtggaccgag gggcttttac 2100ttcaacaagc ccacaggcta tggctccagc attcggaggg cacctcagac aggcattgtg 2160gatgagtgtt gcttccggag ctgtgatctg aggagactgg agatgtactg tgccccactg 2220aagcctacaa aagcagcccg ctctatccgt gcccagcgcc acactgacat gcccaagact 2280cagaaggaag tacatttgaa gaacacaagt agaggaagtg caggaaacaa gacctacaga 2340atgtaggagg agcctcccac ggagcagaaa atgccacatc accgcaggat ccactagttc 2400tagagcggcc gctaattcta gatcgcgaac aaacaccatt gtcacactcc agtatacaca 2460aacaccattg tcacactcca gatatcacaa acaccattgt cacactccaa ggcgaacaaa 2520caccattgtc acactccaag gctattctag atcgcgaatt acatacttct ttacattcca 2580gtatacatta catacttctt tacattccag atatcattac atacttcttt acattccaag 2640gcgaattaca tacttcttta cattccaagg ctacctgagg cccgggggta cctcttaatt 2700aactggcctc atgggccttc cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 2760agtcaggtgc aggctgccta tcagaaggtg gtggctggtg tggccaatgc cctggctcac 2820aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt 2880gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat 2940tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat 3000gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat gaacaaaggt 3060tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat 3120agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt atttttttct 3180ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga 3240ctactcccag tcatagctgt ccctcttctc ttatggagat c 3281973650DNAArtificial SequenceViral vector present in SEQ ID NO30 97actgaggccc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt 60cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc 120aactccatca ctaggggttc cttgtagtta atgattaacc cgccatgcta cttatctact 180cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 240ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 300aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 360actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 420caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 480tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 540ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc 600tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg 660atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg 720ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt 780ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg 840ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg ccgcccgccc 900cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg 960ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc 1020cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg 1080tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc 1140gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc 1200ggtgccccgc ggtgcggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt 1260gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc 1320tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc 1380ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc 1440gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg 1500tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg 1560acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc 1620tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct 1680tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg 1740ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg 1800cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg 1860caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattgatta attcgagcga 1920acgcgtcgag tcgctcggta cgatttaaat tgaattggcc tcgagcgcaa gcttgatatc 1980gaattccgta gatgctttca caaaccccac ccacaaaaca acacatgttc ttaagtcctc 2040agttttgtgt tcacctcggc ctcatagtac ccactctgac ctgctgtgta aacgacccgg 2100acctaccaaa atgaccgcac ctgcaataaa gatacacatc atgtcgtctt cacacctctt 2160ctacctggcg ctctgcttgc tcaccttcac cagctccacc acagctggac cagagaccct 2220ttgcggggct gagctggtgg atgctcttca gttcgtgtgt ggaccgaggg gcttttactt 2280caacaagccc acaggctatg gctccagcat tcggagggca cctcagacag gcattgtgga 2340tgagtgttgc ttccggagct gtgatctgag gagactggag atgtactgtg ccccactgaa 2400gcctacaaaa gcagcccgct ctatccgtgc ccagcgccac actgacatgc ccaagactca 2460gaaggaagta catttgaaga acacaagtag aggaagtgca ggaaacaaga cctacagaat 2520gtaggaggag cctcccacgg agcagaaaat gccacatcac cgcaggatcc actagttcta 2580gagcggccgc taattctaga tcgcgaacaa acaccattgt cacactccag tatacacaaa 2640caccattgtc acactccaga tatcacaaac accattgtca cactccaagg cgaacaaaca 2700ccattgtcac actccaaggc tattctagat cgcgaattac atacttcttt acattccagt 2760atacattaca tacttcttta cattccagat atcattacat acttctttac attccaaggc 2820gaattacata cttctttaca ttccaaggct acctgaggcc cgggggtacc tcttaattaa 2880ctggcctcat gggccttccg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 2940tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa 3000ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga 3060gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt 3120tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga 3180gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg 3240gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag 3300aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt 3360aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact 3420actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacctgc agcccaagct 3480gtagataagt agcatggcgg gttaatcatt aactacaagg aacccctagt gatggagttg 3540gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga 3600cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg 3650983217DNAArtificial SequenceExperssion cassette present in SEQ ID NO31 98ctcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca 420tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag 480cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc 540ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt 600ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg 660cgggagtcgc tgcgttgcct tcgccccgtg ccccgctccg cgccgcctcg cgccgcccgc 720cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc 780cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa 840gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg 900tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct 960gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg 1020gcggtgcccc gcggtgcggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc 1080gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc 1140cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc 1200gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc 1320tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag 1380ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc 1440tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc 1500cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg 1560ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc 1620ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg 1680ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgat taattcgagc 1740gaacgcgtcg agtcgctcgg tacgatttaa attgaattgg cctcgagcgc aagcttgagc 1800tagctcgata tcgtcgaccc acgcgtccgg acttcttgaa gataaagata cacatcatgt 1860cgtcttcaca cctcttctac ctggcgctct gcttgctcac cttcaccagc tccaccacag 1920ctggaccaga gaccctttgc ggggctgagc tggtggatgc tcttcagttc gtgtgtggac 1980cgaggggctt ttacttcaac aagcccacag gctatggctc cagcattcgg agggcacctc 2040agacaggcat tgtggatgag tgttgcttcc ggagctgtga tctgaggaga ctggagatgt 2100actgtgcccc actgaagcct acaaaagcag cccgctctat ccgtgcccag cgccacactg 2160acatgcccaa gactcagaag tccccgtccc tatcgacaaa caagaaaacg aagctgcaaa 2220ggagaaggaa aggaagtaca tttgaagaac acaagtagag gaagtgcagg aaacaagacc 2280tacagaatgt aggaggagcc tcccacggag cagaaaatgc cacatcaccg caggatccgc 2340gcggccgcta attctagatc gcgaacaaac accattgtca cactccagta tacacaaaca 2400ccattgtcac actccagata tcacaaacac cattgtcaca ctccaaggcg aacaaacacc

2460attgtcacac tccaaggcta ttctagatcg cgaattacat acttctttac attccagtat 2520acattacata cttctttaca ttccagatat cattacatac ttctttacat tccaaggcga 2580attacatact tctttacatt ccaaggctac ctgaggcccg ggggtacctc ttaattaact 2640ggcctcatgg gccttccgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagtc 2700aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg gctcacaaat 2760accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc 2820atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt 2880tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt 2940atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac aaaggttggc 3000tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa 3060aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa 3120catccctaaa attttcctta catgttttac tagccagatt tttcctcctc tcctgactac 3180tcccagtcat agctgtccct cttctcttat ggagatc 3217993586DNAArtificial SequenceViral vector present in SEQ ID NO 31 99actgaggccc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt 60cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc 120aactccatca ctaggggttc cttgtagtta atgattaacc cgccatgcta cttatctact 180cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 240ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 300aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 360actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 420caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 480tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 540ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc 600tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg 660atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg 720ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt 780ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg 840ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg ccgcccgccc 900cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg 960ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc 1020cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg 1080tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc 1140gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc 1200ggtgccccgc ggtgcggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt 1260gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc 1320tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc 1380ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc 1440gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg 1500tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg 1560acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc 1620tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct 1680tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg 1740ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg 1800cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg 1860caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattgatta attcgagcga 1920acgcgtcgag tcgctcggta cgatttaaat tgaattggcc tcgagcgcaa gcttgagcta 1980gctcgatatc gtcgacccac gcgtccggac ttcttgaaga taaagataca catcatgtcg 2040tcttcacacc tcttctacct ggcgctctgc ttgctcacct tcaccagctc caccacagct 2100ggaccagaga ccctttgcgg ggctgagctg gtggatgctc ttcagttcgt gtgtggaccg 2160aggggctttt acttcaacaa gcccacaggc tatggctcca gcattcggag ggcacctcag 2220acaggcattg tggatgagtg ttgcttccgg agctgtgatc tgaggagact ggagatgtac 2280tgtgccccac tgaagcctac aaaagcagcc cgctctatcc gtgcccagcg ccacactgac 2340atgcccaaga ctcagaagtc cccgtcccta tcgacaaaca agaaaacgaa gctgcaaagg 2400agaaggaaag gaagtacatt tgaagaacac aagtagagga agtgcaggaa acaagaccta 2460cagaatgtag gaggagcctc ccacggagca gaaaatgcca catcaccgca ggatccgcgc 2520ggccgctaat tctagatcgc gaacaaacac cattgtcaca ctccagtata cacaaacacc 2580attgtcacac tccagatatc acaaacacca ttgtcacact ccaaggcgaa caaacaccat 2640tgtcacactc caaggctatt ctagatcgcg aattacatac ttctttacat tccagtatac 2700attacatact tctttacatt ccagatatca ttacatactt ctttacattc caaggcgaat 2760tacatacttc tttacattcc aaggctacct gaggcccggg ggtacctctt aattaactgg 2820cctcatgggc cttccgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagtcag 2880gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac 2940cactgagatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat 3000ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg 3060tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat 3120ttggtttaga gtttggcaac atatgcccat atgctggctg ccatgaacaa aggttggcta 3180taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt ccatagaaaa 3240gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt ttctttaaca 3300tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc ctgactactc 3360ccagtcatag ctgtccctct tctcttatgg agatccctcg acctgcagcc caagctgtag 3420ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca 3480ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc 3540cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctg 3586

Claims

1. A gene construct comprising: (a) a nucleotide sequence encoding the Insulin-like growth factor 1 (IGF-1) of a mammal; and (b) at least one target sequence of a microRNA of a tissue where the expression of IGF-1 is to be prevented, wherein the sequences (a) and (b) are operationally linked to a ubiquitous promoter.

2. The gene construct according to claim 1, wherein the nucleotide sequence encoding the IGF-1 protein is selected from a nucleotide sequence encoding the human IGF-1 protein, which comprises an amino acid sequence selected from the group consisting of sequences having at least 70% identity with SEQ ID NOs: 23 to 27.

3. The gene construct according to claim 1, wherein the nucleotide sequence encoding the IGF-1 of a mammal, is selected from the group consisting of sequences having at least 70% identity with SEQ ID NOs: 3 to 7.

4. The gene construct according to claim 1, wherein the target sequence of a microRNA is selected from a group consisting of sequences SEQ ID NOs: 8 to 20, 22, 93, 94 and 95 and/or combinations thereof.

5. The gene construct according to claim 1, wherein the gene construct comprises at least one target sequence of the microRNA-122a and at least one target sequence of the microRNA-1.

6. The gene construct according to claim 1, wherein the gene construct comprises four copies of the target sequence or of the target sequences of a microRNA.

7. The gene construct according to claim 1, wherein the promoter is a constitutive promoter.

8. The gene construct according claim 7, wherein the promoter is the CAG promoter.

9. An expression vector that comprises the gene construct comprising: (a) a nucleotide sequence encoding the Insulin-like growth factor 1 (IGF-1) of a mammal; and (b) at least one target sequence of a microRNA of a tissue where the expression of IGF-1 is to be prevented, wherein the sequences (a) and (b) are operationally linked to a ubiquitous promoter.

10. The vector according to claim 9, characterized for being a viral vector selected from the group consisting of adenoviral vectors, adeno associated vectors, retroviral vectors, and lentiviral vectors.

11. The vector according to claim 9, characterized for being an adeno associated viral vector selected from the group consisting of adeno associated viral vector of serotype 6, adeno associated viral vector of serotype 7, adeno associated viral vector of serotype 8, adeno associated viral vector of serotype 9, adeno associated viral vector of serotype 10, adeno associated viral vector of serotype 11, adeno associated viral vector of serotype rh8, and adeno associated viral vector of serotype rh10.

12. (canceled)

13. The vector according to claim 9, which is for the expression of the gene construct in the pancreas.

14. (canceled)

15. A method of treatment and/or prevention of diabetes comprising administering a therapeutically effective amount of the vector of claim 9, wherein a dysfunction and/or a loss of the beta-cells of the islets of Langerhans is present.

16. The method of claim 15 wherein the diabetes is type 1 diabetes mellitus.

17. A pharmaceutical composition comprising a therapeutically effective amount of the vector of claim 9 and one or more pharmaceutically acceptable excipients or vehicles.

18. An adeno-associated viral (AAV) vector comprising a gene construct comprising: (a) a nucleotide sequence encoding the Insulin-like growth factor 1 (IGF-1) of a mammal; and (b) at least one target sequence of a microRNA of a tissue where the expression of IGF-1 is to be prevented, wherein the sequences (a) and (b) are operationally linked to a ubiquitous promoter.

19. The AAV vector of claim 18, wherein the at least one target sequence of a microRNA is selected from sequences that bind to microRNAs expressed in heart and/or liver.

20. The AAV vector of claim 18, characterized as AAV vector selected from the group consisting of adeno associated viral vector of serotype 6, adeno associated viral vector of serotype 7, adeno associated viral vector of serotype 8, adeno associated viral vector of serotype 9, adeno associated viral vector of serotype 10, adeno associated viral vector of serotype 11, adeno associated viral vector of serotype rh8, and adeno associated viral vector of serotype rh10.

21. A method of treatment and/or prevention of diabetes comprising administering a therapeutically effective amount of the AAV vector of claim 18, preferably diabetes mellitus in mammals, wherein a dysfunction and/or a loss of the beta-cells of the islets of Langerhans is present.

22. A method of obtaining an AAV vector as defined in claim 18 comprising: (a) providing a cell with: (i) a gene construct comprising (a) a nucleotide sequence encoding the Insulin-like growth factor 1 (IGF-1) of a mammal and (b) at least one target sequence of a microRNA of a tissue where the expression of IGF-1 is to be prevented, wherein the sequences (a) and (b) are operationally linked to a ubiquitous promoter, flanked by AAV ITRs; (ii) cap and rep proteins of adeno associated virus; and (iii) adequate viral proteins for the replication of AAV; (b) cultivating the cell in suitable conditions to produce the AAV assembly; and (c) purifying the AAV vector produced by the cell.