Patent application title: GENE THERAPY COMPOSITION FOR USE IN DIABETES TREATMENT
Inventors:
IPC8 Class: AC12N1586FI
USPC Class:
1 1
Class name:
Publication date: 2020-07-09
Patent application number: 20200216861
Abstract:
New gene therapy constructions and compositions are the subject of
present invention. The gene therapy compositions consist in
adeno-associated vectors which jointly express insulin (Ins) and
glucokinase (Gck) genes. The new gene therapy constructions are useful
for treatment of diabetes either in dosgs or human beings.Claims:
1. Gene therapy composition which comprises a single vector carrying and
allowing the expression of both an insulin gene (Ins) and a glucokinase
gene (Gck) operatively linked, wherein the vector contains a coding
sequence of the insulin gene and a coding sequence of the glucokinase
gene that are mutated with the purpose of increasing protein production
in a subject and in such a way that the codon adaptation index of these
mutated coding sequences is higher than 0.9.
2. The gene therapy composition of claim 1, wherein the vector is an adeno associated virus based vector.
3. The gene therapy composition of claim 1, wherein the vector is an adeno associated virus vector of serotype 1 (AAV1).
4. The gene therapy composition of claim 1, wherein the insulin gene encodes for human insulin and the glucokinase gene encodes for human glucokinase.
Description:
FIELD OF THE INVENTION
[0001] The invention pertains to the medical field, comprising gene therapy compositions for use in the treatment of Diabetes Type 1 or 2 (TD1 or T2D), either in higher mammals, particularly pets and more particularly dogs; or in human beings.
STATE OF THE ART
[0002] The two main forms of diabetes mellitus are type 1 (T1D) and type 2 (T2D) (1). T1D is characterized by a severe lack of insulin production due to specific destruction of the pancreatic .beta.-cells. .beta.-cell loss in T1D is the result of an autoimmune mediated process, where a chronic inflammation called insulitis causes .beta.-cell destruction (2, 3).
[0003] T1D is one of the most common endocrine and metabolic conditions in childhood; incidence is rapidly increasing, especially among young children. T1D is diagnosed when the autoimmune-mediated .beta.-cell destruction is almost complete and patients need insulin-replacement therapy to survive. T1D in an adult may present itself as T2D, with a slow deterioration in metabolic control, and subsequent progression to insulin dependency. This form is called latent autoimmune diabetes mellitus in adults (LADA) (6).
[0004] Lifelong insulin treatment is the therapy of choice for T1D. While lifelong treatment with exogenous insulin successfully manages diabetes, correct maintenance of a normoglycemic state can be challenging, Chronic hyperglycemia leads to severe microvascular (retinopathy and nephropathy), macrovascular (stroke, myocardial infarction), and neurological complications. These devastating complications can be prevented by normalization of blood glucose levels. Brittle diabetes is one example of a difficult-to-manage disease. Additionally, in many underdeveloped countries, especially in less privileged families, access to self-care tools and also to insulin is limited and this may lead to severe handicap and early death in diabetic children (6-8). The most common cause of death in a child with diabetes, from a global perspective, is lack of access to insulin; thus the availability of a one-time gene therapy approach could make a difference in terms of prognosis when access to insulin is limited (9).
[0005] The reduction of hyperglycemia and maintenance of normoglycemia is a goal of any therapeutic approach to T1D. The current therapy for most diabetic patients is based on regular subcutaneous injections of mixtures of soluble (short-acting) insulin and lente (long-acting) insulin preparations. Suspensions of soluble insulin particles of different size that give intermediate acting and long-acting components with more sustained action profiles are administered to achieve a constant basal level of the hormone (10). However, one of the major deficiencies of delayed-action insulin is the variable absorption from subcutaneous tissue (11), mainly because the formulation is a suspension. Moreover, the delayed-action preparations do not generally produce smooth background levels of insulin, resulting in either hyperglycemia or hypoglycemia. Intensive insulin therapy can delay the onset and slow the progression of retinopathy, nephropathy, and neuropathy in T1D patients (12). However, this kind of treatment is not suitable for all diabetic patients, especially the very young or the old ones. In addition, patients under intensive insulin treatment present a high risk for hypoglycemia. Hypoglycemia is caused by inappropriately raised insulin concentrations or enhanced insulin effect, because of excessive insulin dosage, increased bioavailability, increased sensitivity, and/or inadequate carbohydrate intake (13, 14).
[0006] To maintain normoglycemia, especially in cases of brittle diabetes, a form of diabetes not easily managed with exogenous insulin administration, one alternative approach is cell-based therapy that involves transplantation of pancreatic islets or .beta.-cells mainly from cadaveric donors. While some clinical success has been achieved with this approach, particularly with the Edmonton protocol (15, 16), there are still considerable obstacles to be overcome before these strategies will achieve widespread clinical acceptance and improved long-lasting efficacy. In particular, transplanted patients must receive life-long immunosuppression to avoid graft rejection, while the existing autoimmunity (the underlying cause of diabetes) may contribute to diminished graft survival or limit effectiveness of this treatment approach to only a few years at most (17). Another limitation of the approach comes from the fact that several donors are needed to treat a single patient. As a possible solution to the limited availability of human islets, pig islets may offer an abundant source of tissue and encapsulated islets have been xenotransplanted to non-human primates and, recently, to humans (18, 19). However, in addition to lack of long-term efficacy in terms of insulin production and the obvious safety concerns related to the use of non-human material that may carry unknown infectious diseases, the use of pig islets face difficulties for health authorities' regulatory approval and general public aversion. Also stem cell-based technologies have emerged in recent years as a possible approach to treat diabetes; besides issues related to the underlying autoimmune disease, which may require lifelong immunosuppression, these technologies are still too young to see them applied in the clinical arena in the next few years. Thus, while clinical and research efforts are needed to improve existing therapeutic strategies, it is clear that there is considerable need for new alternative approaches for the treatment of T1D.
[0007] To maintain normoglycemia, studies have also focused on the use of surrogate non-.beta.-cells to deliver insulin (20, 21). These approaches aim to lower blood glucose by delivering insulin under the control of glucose-responsive promoters, such as pyruvate kinase in the liver (22). However, the slow transcriptional control by glucose delays the insulin secretory response, which may lead to hyperglycemia immediately after meals and to hypoglycemia several hours later. To some extent, this can be circumvented by the use of cells that process and store insulin, such as gut K cells (23), or by inducing .beta.-cell neogenesis in the liver by expression of key transcription factors (24, 25). These strategies present other restrictions, such as feasibility, safety and long-term efficacy.
[0008] Unlike conventional insulin replacement therapy, gene therapy would offer the potential advantage of a single viral vector administration, which could ideally provide the necessary insulin through the lifetime of the diabetic subject.
[0009] To develop an alternative approach to diabetes therapy, the inventors had previously examined the ability of genetic manipulation of skeletal muscle to counteract diabetic hyperglycemia. Skeletal muscle is the most important site of glucose removal from blood, accounting for about 70% of glucose disposal after a meal. In addition, skeletal muscle is an excellent target tissue for gene transfer because of its accessibility and its capacity to secrete proteins. Glucose utilization by skeletal muscle is controlled by insulin-stimulated glucose transport through GLUT4 (26) and its phosphorylation by hexokinase II (HK-II) (27). HK-II has a low Km for glucose and is inhibited by glucose-6-phosphate, which limits glucose uptake. During diabetes, because of the lack of insulin, GLUT4 translocation to the plasma membrane and HKII mRNA levels and activity are decrease (28, 29). Expression of basal levels of insulin in skeletal muscle of transgenic mice increases glucose uptake (30), since insulin receptors are widely distributed in muscle fibers (31). When diabetic, insulin-expressing transgenic mice are normoglycemic during fasting but remain hyperglycemic in fed conditions (30). To increase glucose phosphorylation, the hepatic glucose phosphorylating enzyme Gck has also been expressed in skeletal muscle (32). In contrast to HK-II, Gck has a high Km for glucose (about 8 mM), it is not inhibited by glucose 6-phosphate, and it shows kinetic cooperativity with glucose (27). These features allow glucose to be taken up only when it is at high concentrations, as already reported in pancreatic .beta.-cells (33). Expression of Gck in skeletal muscle increases glucose disposal and reduces diabetic hyperglycemia (32, 34, 35). However, expression of Gck alone cannot normalize glycemia in type 1 diabetes because of the lack of insulin-mediated glucose transport. In this regard, we have found that the expression of Gck in skeletal muscle of fed diabetic transgenic mice in conjunction with the administration of low doses of soluble, short acting, insulin leads to the normalization of glycemia (32).
[0010] The invention departs from the hypothesis that basal production of insulin, by genetically engineered skeletal muscle, may provide the levels of insulin required to maintain normoglycemia between meals. After feeding, blood glucose levels rise and the insulin produced by skeletal muscle, acting in an autocrine/paracrine manner, may lead to GLUT4 translocation to plasma membrane and glucose transport into muscle fibers while expression of Gck may increase glucose utilization and normalization of glycemia. Thus, an approach combining insulin and Gck may prevent chronic hyperglycemia and avoid hypoglycemic events. In this regard, the inventors have recently shown that co-expression of Gck and insulin in mouse skeletal muscle reverts diabetic alterations (36). Double transgenic mice expressing both Gck and insulin in skeletal muscle counteract hyperglycemia and restores fluid and food intake after treatment with streptozotocin.
[0011] The inventors have also demonstrated in the past the feasibility of this approach in T1D mice by using Adeno-associated virus (AAV)-based vectors of serotype 1 (AAV1) to transfer the insulin and Gck genes into skeletal muscle of diabetic mice (36). AAV vectors are one of the preferred tools for gene transfer. The high transduction efficiency in vivo in a variety of post-mitotic tissues and the relatively low immunogenicity contributed to the AAV vectors use in a variety of preclinical studies (37). Translation of preclinical results into the clinical arena resulted in promising results (38-44), confirming the ability of AAV vectors to safely transduce liver, muscle, and neurological tissue in humans. Importantly, several groups showed that a single administration of AAV vectors to the liver, muscle, retina, and other tissues leads to long-term expression of the transgene product (45-47).
[0012] The inventors previously disclosed that expression of Gck and insulin genes into skeletal muscle of diabetic mice by using AAV1 vectors leads to complete normalization of glycemia (36). In addition, these mice present normal blood glucose levels when fasted and hypoglycemia is not observed. Insulin+Gck-treated diabetic mice also show increased skeletal muscle glucose uptake, normalization of liver glucose metabolism (increased glucose uptake and glycogen synthesis and reduced hepatic glucose production) and glucose tolerance test. Moreover, these mice present with normal food and fluid intake and normalization of abdominal fat pad and skeletal muscle weights. These results suggest that secretion of basal levels of insulin, in conjunction with increased glucose uptake by the skeletal muscle, may permit tight regulation of glycemia (36). Furthermore, in contrast to diabetic non-treated mice, preliminary results suggest that normalization of glycemia in Insulin+Gck-treated diabetic mice prevented development of secondary complications. However, there is still need of gene therapy compositions that may be proven useful in the treatment of diabetes in mammals of higher taxonomy, like pets (dogs) or even human beings.
[0013] T1 D is one of the most common endocrine and metabolic conditions in childhood; T1 D is diagnosed when the autoimmune-mediated .beta.-cell destruction is almost complete and patients need insulin-replacement therapy to survive. T2D results from the reduced ability of the pancreatic .beta.-cells to secrete enough insulin to stimulate glucose utilization by peripheral tissues; defects in both insulin secretion and action contribute to the pathogenesis of T2D, but it is now recognized that insulin deficiency is crucial to T2D pathogenesis. While lifelong treatment with exogenous insulin successfully manages diabetes, correct maintenance of a normoglycemic state can be challenging, exposing diabetic patients to life threatening hypoglycemia and long-term complications of hyperglycemia. Sub-optimal regulation of glycemia leads to severe microvascular (retinopathy and nephropathy), macrovascular (stroke, myocardial infarction), and neurological complications, which are hallmarks of both T1D and T2D. Alternative strategies involving transplantation of pancreatic islets or .beta.-cells, present still considerable obstacles to overcome before they achieve widespread clinical acceptance and improved long-lasting efficacy, probably including life-long immunosuppression to avoid graft rejection.
SUMMARY OF THE INVENTION
[0014] The invention herein presents an innovative alternative to treat T1D and T2D, based on gene therapy delivered to the skeletal muscle to counteract diabetic hyperglycemia. Muscle was selected as target tissue due to his easy accessibility, capacity to secrete proteins and because of its relevance in the pathophisiology of diabetes, being accountable for about a 70% of glucose disposal after a meal.
[0015] Adeno-associated viral vectors (AAV1) were selected as vehicles for delivering insulin and glucokinase genes into the muscle tissue (local delivery). These vectors have proven to be safe and are already used in clinical testing (38, 44). Basal production of insulin, by genetically engineered skeletal muscle, may provide the levels of insulin required to maintain normoglycemia between meals. After feeding, blood glucose levels rise and the insulin produced by skeletal muscle, acting in an autocrine/paracrine manner, may lead to GLUT4 translocation to plasma membrane and glucose transport into muscle fibers, while expression of Gck may increase glucose utilization and normalization of glycemia. Thus, an approach combining insulin and Gck may prevent chronic hyperglycemia and avoid hypoglycemic events. This approach was shown to be effective to normalize glycemia in diabetic mice (36).
[0016] The invention shows, by experiments carried out in Beagle dogs, that a single administration of AAV1-human insulin (vector that comprises the human insulin sequence gene cloned in the pGG2 plasmid, resulting in the plasmido pGG2 hIns, FIG. 20) and AAV1-rat glucokinase (vector that comprises the human glucokinase sequence gene cloned in the pGG2 plasmid, resulting in the plasmido pGG2 hGcK, FIG. 19) (AAV1-hIns, AAV1-rGck, respectively) was able to normalize fasting plasma glucose and improve glucose disposal after oral glucose tolerance test, for periods of time longer than 2 years. Normalization of body weight and elevated liver enzymes was also achieved in one dog with severe diabetes. Serious adverse events have not been observed in all (five) animals treated, suggesting a good safety profile of this approach. In conclusion, the invention shows that expression of human insulin and rat Gck in skeletal muscle is a valuable and safe approach that allows long-term survival in animal suffering from diabetes for long time (>2 years); along with body weight maintenance, normal physical performance and normalization of serum parameters.
[0017] Additionally, the present invention also discloses that a single administration of AAV-mhIns (vector that comprises the mutated human insulin sequence gene cloned in the pAAV-MCS plasmid, FIG. 5, resulting in the plasmid pAAVmhINS) and AAV-mhGcK (vector that comprises the human glucokinase sequence gene cloned in the pAAV-MCS plasmid, FIG. 5, resulting in the plasmid pAAVmhGeK) in diabetic mice showed a significant reduction in blood glucose levels in fasted and fed conditions compared with AAV-null-treated mices or single treatment with AAV-mhIns or AAV-mhGcK.
[0018] The plasmids disclosed in the present invention, pAAV-MCS and pGG2, are for illustrative purposes only and are not intended, nor should they be interpreted, to limit the scope of the invention. Persons skill in the art can be used any plasmid known in the art capable of producing AAV by conventional methods known by persons skilled in the art (Sambrook et al., "Molecular cloning, a Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory Press, N.Y., 1989 Vol 1-3).
[0019] The impact of the gene transfer approach of present invention, consisting of co-expression of low levels of insulin together with the enzyme glucokinase in skeletal muscle, implies normalization of glycemia with a one-time intervention what results in a great improvement of patients' quality of life and prevention of severe and costly secondary complications of diabetes. It should be noted that, compared to other experimental therapeutic approach to diabetes, the gene therapy compositions and the method discloses in the present invention are based on engineering skeletal muscle, a readily accessible tissue that does not require any invasive procedure to be manipulated. This is a considerable advantage over other approaches disclosed in the state of the art, such as engineering the liver or transplanting insulin-producing .beta.-cells. It should also be pointed out that the method disclosed in the present invention has the advantage of not requiring immunosuppression, as diabetic subjects are naturally immunologically tolerant to insulin and glucokinase; additionally, even basal (low) levels of expression of insulin and glucokinase may result in a dramatic improvement of the disease profile in terms of quality of life (better glycemic control) and reduction of insulin requirements. Thus, the use of two genes acting synergistically on glycemic control potentially represents a major advance in the management of T1D and T2D diabetes.
[0020] Furthermore, the present invention also disclosed that gene therapy with AAV-mhGcK could be combined with regular exogenous insulin injections to improve the conventional treatment of T1D. Additionally, AAV-GcK gene therapy per se could be considered as a treatment for diabetic patients in which insulin production is still present, such as in early phases of T2D development.
[0021] Therefore, the main embodiment of the invention corresponds to a gene therapy composition which comprises at least a first vector carrying and allowing the expression of insulin gene (Ins) and at least a second vector carrying and allowing the expression of glucokinase gene (Gck).
[0022] Further embodiments of the invention concern to gene therapy compositions wherein the first vector contains the CDS of SEQ ID NO. 1 or the CDS of SEQ ID NO. 3.
[0023] Other embodiments of the invention relate to gene therapy compositions, wherein the second vector contains the CDS of SEQ ID NO. 2 or the CDS of SEQ ID NO. 4.
[0024] More precisely, alternative embodiments of the invention consist in gene therapy compositions comprising either, a first vector containing the CDS of SEQ ID NO. 1 and a second vector containing the CDS of SEQ ID NO. 2; or a first vector containing the CDS of SEQ ID NO. 3 and a second vector containing the CDS of SEQ ID NO. 4.
[0025] In the gene therapy composition according to the invention, the first and the second vectors carrying genes can be the same, specifically that same vector can be a plasmid and more precisely a plasmid selected from: pGG2 (FIGS. 19 and 20) or pAAV (FIG. 5). Adeno-associated virus based vectors (AAV) are particularly preferred for working out present invention. Most preferred are the type 1 (AAV1).
[0026] The gene therapy compositions of the invention can be used in the treatment of diabetes in mammals, as a way of example, in dogs or pets in general and in human beings.
[0027] Also a last embodiment of the invention is to provide a method of treatment of diabetes in mammals, which comprise the administration to a subject in need of it, of a therapeutically effective dose of a gene therapy composition as mentioned above. Moreover, the gene therapy composition is administered, according to the method of invention, in a single and unique dose for all the treatment hence avoiding repeated periodical administration. More precisely, the single dose is administered to muscle tissue, accordingly to the method of invention, by means of an unique multi-needle injection.
[0028] Other embodiment of the invention relate to a mutated human insulin (mhIns) gene characterized by the CDS of SEQ ID NO: 3 and a mutated human glucokinase (mhGcK) gene characterized by the CDS of SEQ ID NO: 4. Also the invention relate the mutated human insulin (mhIns) and the mutated human glucokinase (mhGcK) genes, as disclosed previously, for the treatment of diabetes.
[0029] Present invention also disclosed a method of treatment of diabetes which comprises the administration to a subject in need of it, of a therapeutically effective dose of a gene therapy composition according to the present invention. Moreover, the gene therapy composition disclosed herein, is administered in a single dose for all the treatment, to the muscle tissue by means of an unique multi-needle injection.
[0030] Other embodiment of the invention relate to a method of treatment of diabetes which comprises the administration to a subject in need of it, of a therapeutically effective dose of a gene therapy composition which comprises at least a vector carrying and allowing the expression of glucokinase gene (Gck). Moreover, the vector is an adeno-associated virus based vector that contains the CDS of SEQ ID NO: 2 or the CDS of SEQ ID NO: 4. More preferably, the method disclosed herein is the plasmid pAAV. Furthermore, the gene therapy composition used in the present method is administered in a single dose for all the treatment, to muscle tissue by means of an unique multi-needle injection. The present method further comprises exogenous insulin injections.
[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, for ease of reference, some of these terms will now be defined.
[0032] The term "mutated genes" refers to the introduction of mutations in the coding sequence of the genes with the purpose of increasing protein production. The criteria used for these mutations are exposed in Example 2.
[0033] The term "AAV-null-treated mice" as used throughout the present specification is to be understood to mean an adeno-associated virus (AAV)-based vector capsid and genome but without expression of any coding sequence.
FIGURE LEGENDS
[0034] FIG. 1. Efficient transduction and secretion of insulin from dog skeletal muscle. (A) Unique 5 needle injection system used to inject dog muscle. (B) AAV1-GFP can efficiently transduce large numbers of dog muscle fibres. (C) Human insulin was detected by Northern blot from autopsy samples in Dog 1, but not in uninjected control (Con) muscle.
[0035] FIG. 2. Fasting glycemia profiles. (A) Dog 2 treated with 1.0.times.10.sup.12 vg (vector genomes)/kg AAV1-humanIns. (B) Dog 3 and 4 treated with 1.0.times.10.sup.12 vg/kg AAV1-humanIns and 1.0.times.10.sup.12 vg/kg AAV1-ratGck. Arrows indicate AAV injection. Time after diabetes induction is shown and grey bars indicate range of normoglycemia.
[0036] FIG. 3. Improved glucose disposal after oral glucose tolerance test (GTT) in dogs treated with AAV1-hIns+rGck. GTT was performed in the same dog before, after diabetes induction and at several time points after and AAV1-humanIns+ratGck treatment. Upper panel: GTT representative curves when healthy, diabetic non-treated and diabetic AAV-treated are shown; Lower panel: area under the curve. In the AAV-treated dogs, results are means+SEM of eight GTT. (A) Dog 2 showed no significant improvement to glucose disposal after treatment. (B) Dog 3 and (C) Dog 4 demonstrated improved ability to dispose of glucose during GTT. (D) Dog 5 after AAV1-humanIns+ratGck treatment showed a GTT profile similar to Dogs 3 and 4.
[0037] FIG. 4. Fasting glycemia profile, body weight and serum transaminase profile of Dog 5. (A) Dog 5 diabetic was treated with 1.0.times.10.sup.12 vg/kg AAV1-humanIns and 1.0.times.10.sup.12 vg/kg AAV1-ratGck. (B) Body weight profile. (C) Serum profile of alanine transaminase (ALT) activity. Arrows indicate AAV injection. Time after diabetes induction is shown and grey bars indicate range of normoglycemia (A) and normal ALT activity (C).
[0038] FIG. 5. Plasmid pAAV-MCS (Stratagene, Cedar Creek, Tex., USA). This plasmid contain the CMV promoter (pCMV) and polyA signal from growth hormone (hGHpA) flanked by the two Inverted Terminal Repeats (ITR) of AAV2.
[0039] FIG. 6. Quantification of mRNA by Northern Blot in HEK293 cells transfected with pAAVmhINS and pAAVmhGK plasmids. pAAVmhINS is a plasmid that express mutated human insulin (mhINS) under the control of CMV promoter and contain ITR sequences from AAV2. pAAVmhGcK is a plasmid that express mutated human Glucokinase (mhGK) under the control of CMV promoter and contain ITR sequences from AAV2. HEK293 cells were transfected with the appropiate plasmid and total RNA was isolated 48 h after transfection. Northern Blot was performed with 10 ug of RNA and hibridized with the mhINS and the mhGck cDNA, respectively. Remarkable mhINS (a) and mhGck (b) expression was detected.
[0040] FIG. 7. Quantification of mRNA by Northern Blot in HEK293 cells transduced with AAV1-mhINS and AAV1-mhGK plasmids. AAV1 vectors were generated by triple transfection following standard methods using pAAVmhINS and pAAVmhGK plasmids as viral backbone. HEK293 cells were tranduced with AAV1-mhINS or AAV1-mhGK and total RNA was isolated 48 h after transection. Northern Blot was performed with 10 ug of RNA and hibridized with the mhINS and the mhGck cDNA, respectively. Remarkable mhINS (a) and mhGck (b) expression was detected.
[0041] FIG. 8. Glucokinase protein levels measured by western blot analysis. (A) Densitometric analysis of Gck protein in westerns blots (n=3 per group) of GcK protein of HEK293 cells transduced with AAV1-rGck, AAV1-hGck and AAV1-mhGck. Values are represented as % of protein vs rGck vector. (B) Densitometric analysis of Gck protein in westerns blots (n=3 per group) of GcK protein of HEK293 cells transduced with AAV1-rGck, AAV1-hGck and AAV1-mhGck. Values are represented as % of protein vs hGck. # # p<0.01 vs hGck, ** p<0.01 vs rGck.
[0042] FIG. 9. Glucokinase activity in vitro. HEK293 cells were transduced with AAV1 null, AAV1-rGck, AAV1-hGck or AAV1-mhGck at a MOI=10E5 vg/cell. Glucokinase activity was measured in cell extracts and values are represented as .mu.U/mg total protein (n=3 per group). **p<0.01 vs hGck; # p<0.05 vs rGck; # # # p<0.001 vs rGck.
[0043] FIG. 10. Glucokinase activity in vivo. CD-1 healthy mice were injected in both hindlimbs: (A) quadriceps, (B) gastrocnemius and (C) tibialis, with AAV1 null, AAV1-rGck, AAV1-hGck or AAV1-mhGck (10E12 vg/kg). Glucokinase activity was measured in skeletal muscle extracts and values arc represented as .mu.U/mg total protein (n=5 per group). # p<0.05 vs rGck; p<0.01 vs hGck.
[0044] FIG. 11. Human C-peptide levels in culture media after HEK293 cells transduction with AAV1 vectors coding for mutated human insulin and non-mutated human insulin. HEK293 cells were transduced at different MOI with adenoasociated vectors (AAV1) coding for the transgenes. Human C-peptide measured by RIA in culture medium 72 h after viral transduction. Significative increase in human C-peptide levels is observed in mutated insulin versus non-mutated insulin at MOI of 10E5 vg/cell. *** p<0.001 vs hINS
[0045] FIG. 12. Insulin levels in culture media after HEK293 cells transduction with AAV1 vectors coding for mutated human insulin and non-mutated human insulin. HEK293 cells were transduced at different MOI with adenoasociated vectors (AAV1) coding for the transgenes. Insulin was measured by RIA in culture medium 72 h after viral transduction. Significative increase in human insulin levels was observed in mutated insulin versus non-mutated insulin vectors at MOI of 10E5 vector genomes/cell. *p<0.01 hINS
[0046] FIG. 13. Blood glycemia in mice treated with AAV1 vectors coding for mutated human insulin and non-mutated human insulin. C57bl6 healthy mice were injected with AAV1-hINS or AAV1-mhINS in both hindlimbs (quadriceps, gastrocnemius and tibialis) at a dose of 1.4E11 vg/mouse. Fed glycemia was measured two weeks after viral injection (n=3 per group). * p<0.05 vs hINS.
[0047] FIG. 14. Circulating levels of Human insulin and human C-peptide in mice treated with AAV1 vectors coding for mutated human insulin and non-mutated human insulin. C57bl6 healthy mice were injected with AAV1-hINS or AAV1-mhINS in both hindlimbs (quadriceps, gastrocnemius and tibialis) at a dose of 1.4E11 vg/mouse and human insulin (A) and human C-peptide (B) were measured by RIA in serum two weeks after viral transduction. * p<0.05 vs hINS.
[0048] FIG. 15. Fasted blood glycemia in mice treated with AAV1 vectors coding for mutated human insulin and Gck. C57bl6 healthy mice were injected with AAV1-mhINS, AAV1-mhGck or both vectors in hindlimbs (quadriceps, gastrocnemius and tibialis) at a dose of 10E12 vg/kg. Fasted glycemia was measured one month after viral injection (n=20 per group). *** p<0.001 vs AAV1 null; # p<0.05 vs mhINS; # # p<0.01 vs mhINS; $ $ $ p<0.001 vs mhGcK.
[0049] FIG. 16. Fed blood glycemia in mice treated with AAV1 vectors coding for mutated human insulin and Gck. C57bl6 healthy mice were injected with AAV1-mhINS, AAV1-mhGck or both vectors in hindlimbs (quadriceps, gastrocnemius and tibialis) at a dose of 10E12 vg/kg. Fed glycemia was measured one month after viral injection (n=20 per group). *** p<0.001 vs AAV1 null; # p<0.05 vs mhINS; $$$ p<0.001 vs mhGK.
[0050] FIG. 17. Insulin tolerance test in T1D animals treated with AAV1-mhGcK. Experimental diabetes was induced in 2 month-old c57bl6 mice by 5 daily consecutive dosis of STZ (50 mg/kg). Two weeks after STZ, AAV1-mhGck or AAV1-null vectors were injected into the hindlimbs at a dose of 10E12 vg/kg. One month after viral administration an intraperitoneal insulin tolerance test was performed (0.375 U/kg) (n=7 per group). * p<0.05 vs null.
[0051] FIG. 18. Insulin tolerance test in T2D animals treated with AAV1-rGcK. AAV1-rGck or AAV1-null (control) vectors were injected into the hindlimbs of 2 month-old c57bl6 mice at a dose of 10E12 vg/kg. Three months after viral administration an intraperitoneal insulin tolerance test was performed (0.75 U/kg) (n=10 per group). $ p<0.05 high fat diet (HFD) Gck vs AAV1 null-HFD; * p<0.05 Control-Chow vs AAV1 null-HFD.
[0052] FIG. 19. Plasmid pGG2-rGK. This plasmid contain the CMV promoter, the CDS of the rat Gck and polyA signal from SV40 flanked by the two Inverted Terminal Repeats (ITR) of AAV2.
[0053] FIG. 20. Plasmid pGG2-Ins. This plasmid contain the CMV promoter, the CDS of the human INS gene and polyA signal from SV40 flanked by the two Inverted Terminal Repeats (ITR) of AAV2.
DETAILED DESCRIPTION OF THE INVENTION
[0054] The significance and potential impact of the gene therapy invention approach, consisting of co-expression of low levels of insulin together with the enzyme glucokinase in skeletal muscle, are potentially enormous. Normalization of glycemia with a one-time intervention would result in a great improvement of patients' quality of life and prevention of severe and costly secondary complications of diabetes. The data disclosed in the present invention show that this is feasible and safe. It should be noted that, compared to other experimental therapeutic approach to diabetes, the strategy displayed in the invention is based on engineering skeletal muscle, a readily accessible tissue that do not require any invasive procedures to be manipulated. This is a considerable advantage over other approaches, such as engineering the liver or transplanting insulin-producing .beta.-cells. It should also be pointed out that the gene therapy composition and the method disclosed herein have the advantage of not requiring immunosuppression, as diabetic subjects are naturally immunologically tolerant to insulin and glucokinase; additionally, even basal (low) levels of expression of insulin and glucokinase may result in a dramatic improvement of the disease profile in terms of quality of life (better glycemic control) and reduction of insulin requirements. Thus, the use of two genes acting synergistically on glycemic control potentially represents a major advance in the management of T1D and T2D diabetes worldwide.
[0055] Therefore, the present invention relates gene therapy compositions which comprise at least a first vector carrying and allowing the expression of insulin gene (Ins) and at least a second vector carrying and allowing the expression of glucokinase gene (Gck). As alternative, the gene therapy compositions of present invention comprise a single vectors carrying and allowing the expression of both genes (Ins and GcK) operatively linked. Moreover, Ins and/or GcK genes can be, any of them independently, autologous or heterologous genes with regard to the species wherein are being expressed.
[0056] In a particular embodiment of the gene therapy compositions of the invention are characterized by the vectors are adeno associated virus based vector.
[0057] In another particular embodiment of the gene therapy composition disclosed in the present invention, the first vector contains the CDS of SEQ ID NO. 1 or the CDS of SEQ ID NO. 3.
[0058] In another particular embodiment of the gene therapy composition disclosed in the present invention, the second vector contains the CDS of SEQ ID NO. 2 or the CDS of SEQ ID NO. 4.
[0059] In another particular embodiment of the gene therapy composition, the first and the second carrying gene vectors are the same.
[0060] In another particular embodiment of the gene therapy composition, comprises a first vector containing the CDS of SEQ ID NO. 1 and a second vector containing the CDS of SEQ ID NO. 2.
[0061] In another particular embodiment of the gene therapy composition disclosed herein, the first vector is AAV-Ins and the second vector is AAV-GcK.
[0062] In another particular embodiment, the gene therapy composition of the invention comprises a first vector containing the CDS of SEQ ID NO. 3 and a second vector containing the CDS of SEQ ID NO. 4.
[0063] In another particular embodiment of the gene therapy composition disclosed in the present invention, the first vector is AAV-mhIns and the second vector is AAV-mhGcK.
[0064] In another particular embodiment, the gene therapy composition of the invention comprises a first vector containing the CDS of SEQ ID NO. 1 or the CDS of SEQ ID NO: 3 and a second vector containing the CDS of SEQ ID NO. 2 or the CDS of SEQ ID NO: 4.
[0065] In another particular embodiment, the gene therapy composition of the invention is characterized by the first vector is selected from AAV-Ins or AAV-mhIns and the second vector is selected from AAV-GcK or AAV-mhGcK.
[0066] Present invention also relates gene therapy compositions for use in the treatment of diabetes in mammals.
[0067] In a particular embodiment of the gene therapy compositions disclosed herein, the mammal is a rodent, preferably mice, rats, gerbils and guinea pigs and more preferably mice and rats.
[0068] In another preferred embodiment of the gene therapy compositions disclosed herein, the mammal is a dog.
[0069] In another preferred embodiment of the gene therapy compositions disclosed herein, the mammal is a human being.
[0070] Present invention also disclosed a mutated human insulin (mhIns) gene characterized by comprising the CDS having SEQ ID NO: 3 and a mutated human glucokinase (mhGcK) gene characterized by comprising the CDS having SEQ ID NO: 4.
[0071] Another object disclosed in the present invention is the mutated human insulin (mhIns) gene, as disclosed previously, for use in the treatment of diabetes.
[0072] Present invention also disclosed the use of the mutated human insulin (mhIns) gene disclosed herein for the manufacture of a medicament and/or a gene therapy composition for use in the treatment of diabetes.
[0073] Another object disclosed in the present invention is the mutated human glucokinase (mhGcK) gene, as disclosed previously, for use in the treatment of diabetes.
[0074] Present invention also disclosed the use of the mutated human glucokinase (mhGcK) gene disclosed herein for the manufacture of a medicament and/or a gene therapy composition for use in the treatment of diabetes.
[0075] Present invention also disclosed a method of treatment of diabetes which comprises the administration to a subject in need of it, of a therapeutically effective dose of a gene therapy composition according to the present invention.
[0076] In a preferred embodiment of the invention, the method comprises the administration of the gene therapy composition disclosed herein, in a single dose for all the treatment.
[0077] In another preferred embodiment of the invention, the method disclosed that the single dose is administered to muscle tissue by means of an unique multi-needle injection.
[0078] Present invention also disclosed a method of treatment of diabetes which comprises the administration to a subject in need of it, of a therapeutically effective dose of a gene therapy composition which comprises at least a vector carrying and allowing the expression of glucokinase gene (Gck).
[0079] In a preferred embodiment of the method of the present invention, the vector is an adeno-associated virus based vector.
[0080] In another preferred embodiment of the method disclosed herein, the vector comprises the CDS having either SEQ ID NO: 2 or SEQ ID NO: 4.
[0081] In another preferred embodiment of the method disclosed herein, the vector is selected from AAV-mhGcK or AAV-GcK.
[0082] In another preferred embodiment of the method disclosed herein, the gene therapy composition is administered in a single dose for all the treatment.
[0083] In another preferred embodiment of the method disclosed herein, the single dose is administered to muscle tissue by means of an unique multi-needle injection.
[0084] In another preferred embodiment of the invention the method further comprises exogenous insulin injections.
[0085] The invention will now be described in more detail by way of examples. The following examples are for illustrative purposes only and are not intended, nor should they be interpreted, to limit the scope of the invention.
EXAMPLE 1: INS+GCK GENE TRANSFER TO SKELETAL MUSCLE IN DIABETIC DOGS
[0086] Studies in diabetic Beagle dogs used a unique 5-point needle (FIG. 1A) to obtain widespread expression of a GFP reporter in skeletal muscle (FIG. 1B). Subsequently, 2.5.times.10.sup.12 vg/kg of AAV1-human Ins was injected into Dog 1 three days after diabetes induction with streptozotocin+alloxan (50). Low levels of circulating human C-peptide were observed 4 days later, peaking after 2 weeks in association with hypoglycemia. Dog 1 was sacrificed 21 days after treatment and strong insulin expression was detected in biopsies of the treated area (FIG. 1C). These results indicated that AAV vectors injected in multiple sites can efficiently deliver the insulin gene to widespread areas and that AAV-mediated gene transfer of insulin to a large animal model of diabetes was feasible, resulting in large amounts of insulin produced and secreted from the dog skeletal muscle.
[0087] Next goal of present invention was to determine the optimum dose able to achieve therapeutic efficacy without causing hypoglycemia. To this end, Dog 2 was injected with 1.0.times.10.sup.2 vg/kg of AAV1-human Ins after diabetes induction. After gene transfer, fasting glycemia decreased to reach normoglycemia without becoming hypoglycemic (FIG. 2A). After .about.300 days, the fasting glycemia values became slightly hyperglycemic and have since remained stable. However, even when normoglycemic, we did not see a significant improvement in the ability of this dog to dispose glucose (FIG. 2A). This was despite detecting human C-peptide .about.70 days after treatment, with stable levels achieved after 130 days those have lasted for more than 800 days, suggesting the long-term potential of this treatment. Muscle biopsies taken 14 and 270 days after treatment showed detectable insulin RNA at both time points, whereas a pancreas biopsy at day 270 showed less than 10% residual .beta.-cell mass and no sign of regeneration. Dog 2 demonstrated no adverse events, no signs of toxicity and had a normal weight gain profile suggesting that even modest levels of circulating insulin can have beneficial effects.
[0088] Dog 3 and Dog 4 were made diabetic and treated with the same dose of AAV1-human Ins as Dog 2 and an equal dose (1.0.times.10.sup.2 vg/kg) of AAV1-rat Gck. Both Dog 3 and 4 showed a more accelerated return to fasting normoglycemia (FIG. 2B). These dogs remained normoglycemic for a long period (>2 years). Circulating human insulin and C-peptide levels in these dogs were detectable after treatment and, importantly, both Dogs 3 and 4 showed an improved GTT profile compared with Dog 2 (FIG. 3B, C). Muscle biopsies 15 and 113 days after viral injection revealed strong expression of both insulin and Gck, whereas a pancreas biopsy at 113 days confirmed <5% residual .beta.-cell mass. No muscle damage was seen and, like Dog 2, we observed normal weight gain and no toxicity. Together, these data suggests that the combined treatment with human Ins and rat Gck leads to more beneficial effects in terms of improvement of glycemic control; these effects were not observed in Dog 2 despite the expression of insulin.
[0089] Then experimental diabetes in Dog 5 was induced and followed long-term progression of diabetes. Despite the complete absence of exogenous insulin treatment, this dog showed a gradual return to fasted normoglycemia, also coinciding with summer times. About six months after diabetes induction, we observed a severe rise in glycemia (FIG. 4A) parallel with a strong decrease in body weight (>30%) and marked increase in liver transaminases (FIG. 4B, C). At that moment, Dog 5 was treated with the same doses of AAV1-Ins and AAV1-Gck as Dog 3 and 4, which resulted in dramatic improvements of its metabolic profile. Fasting glycemia dropped sharply within 30 days of treatment (FIG. 4A), coinciding with a rise in circulating human C-peptide and a persistent weight gain (FIG. 4B). Biochemical signs of liver damage also normalized (FIG. 4C) and, most importantly, we observed an improved glucose disposal by GTT reminiscent of Dog 3 and 4 (FIG. 3D).
[0090] These results clearly demonstrate the beneficial effects of combined Ins+Gck therapy in long-term diabetic dogs. Therefore, joint expression of insulin and Gck in skeletal muscle is a safe approach that allows long-term survival in large diabetic animals (>2 years), body weight maintenance, normal physical performance and normalization of serum parameters.
EXAMPLE 2: CONSTRUCTION OF MUTATED VECTORS FOR EFFICIENT EXPRESSION OF HUMAN INSULIN AND HUMAN GLUCOKINASE
[0091] The coding sequence of either human insulin gene (hIns), containing specific sites for furin processing (36), or human glucokinase gene (hGcK) was modified to obtain codon mutated sequences (mhIns or mhGcK, respectively) following GeneArt procedures (48). GeneArt process involves avoiding cis-acting sequence motifs as:
[0092] Internal TATA-boxes, chi-sites and ribosomal entry sites
[0093] AR-rich or GC-rich sequence stretches
[0094] RNA instability motifs
[0095] Repeat sequences and RNA secondary structures
[0096] (Crytic) splice donor and acceptor sites in higher eukaryotes
[0097] The codon usage was adapted in GeneArt process to codon bias of Mus musculus genes. In addition, regions of very high (>80%) or very low (<30%) GC content were avoided when possible. The mutated gene constructs obtained showed CAT (codon adaptation index) of 0.96 what means high and stable expression rates in Mus musculus. GC-content adjustment made by the process of GenArt, prolongs mRNA half-life of the mutated construct achieved. The mutated human insulin and GcK genes described herein are then called mutated human genes. The mutated insulin and GcK cDNA was cloned in the multicloning site of the pAAV-MCS plasmid (Stratagene; FIG. 5) resulting in the plasmids pAAV-mhlns and pAAV-mhGcK respectively. This plasmid contains the CMV promoter and polyA signal from growth hormone flanked by the two Inverted terminal repeats (ITR) of AAV2. ITR sequences are required for packaging of the AAV genome into the AAV capsid, and are required for replication of the AAV genome during AAV production. Adeno-associated vectors were generated by triple transfection of Human Embryonic Kidney 293 cells (HEK293) cells according to standard methods.
[0098] HEK293 are cells from human origin that are stable transfected with the adenovirus E1 gene. The adenovirus E1 gene is required for adenovirus replication and also acts as a helper gene for AAV replication. The invention uses HEK293 cells for several purposes:
[0099] 1.--AAV production using triple transfection method. For AAV production, it is required to have the cassette of expression flanked by ITR (plasmid 1), a plasmid coding for Rep and Cap genes from the AAV (plasmid 2; provides replication functions for AAV genome and the capsid proteins depending on the desired serotype), a third plasmid coding for the essential genes of adenovirus required to provide helper function and support replication of AAV (plasmid 3, also named as adenovirus helper plasmid wich code for E2, E4 and VA genes). In addition to E2, E4 and VA, E1 gene is necessary for replication of AAV, in this case E1 gene is provided by the HEK293 cells instead of being in the adenovirus helper plasmid.
[0100] 2.--For DNA transfection. The inventors have used HEK293 to study expression, processing and secretion of insulin and expression of GK because they are very efficiently transfected with plasmid using calcium phosphate method.
[0101] 3.--HEK293 cells were also used to study expression, processing and secretion of insulin and expression of GK from AAV1 vectors, because this cell line (and not others) are permissive for AAV1-transduction.
[0102] Cells were cultured in roller bottles (RB) (Corning, Lowell, Mass.) in DMEM 10% FBS to 80% confluence and co-transfected with a plasmid carrying the expression cassette flanked by the viral ITRs (described above), a helper plasmid carrying the AAV rep2 and cap1 genes, and a plasmid carrying the adenovirus helper functions (both plasmids kindly provided by K.A. High, Children's Hospital of Philadelphia). Vectors were purified with an optimized method based on two consecutives cesium chloride gradients (49), dialyzed against PBS, filtered, titred by qPCR and stored at -80.degree. C. until use.
EXAMPLE 3: `IN VITRO` EXPRESSION OF MRNA FROM MUTATED TRANGENES
[0103] HEK293 cells were transfected with pAAVmhINS and pAAVmhGck using calcium phospate standard method. For experiments using AAV vectors, HEK293 cells were infected with AAV1 mhINS and AAV1 mhGck at different MOI (i.e. 10E4, 10E5, 10E6 vg/cell). Two days after transfection, cells were lysated with 1 ml of Tripure (Roche) and total RNA was extracted with RNAEasy Mini Kit (Qiagen). A Northern Blot was performed with 10 ug of RNA and hibridized with the mhINS (CDS of SEQ ID NO: 3) or the mhGck (CDS of SEQ ID NO: 4) cDNA, respectively (FIG. 6). Since these plasmids showed a high expression level of the gene of interest, adenoassociated type 1 viral vectors carrying these constructs were produced. Subsequently, AAV vectors were tested for their mRNA expression in HEK293 cells 96 h after transduction. High levels of transgene expression were detected by Northern Blot both with AAV1-mhINS and AAV1-mhGcK (FIG. 7).
EXAMPLE 4: `IN VITRO` EXPRESSION OF MHGCK PROTEIN FROM MUTATED TRANGENES
[0104] In addition to increased RNA expression, the present invention has also detected a substantial increase in mhGcK protein production by the mutated construct (FIG. 8). Codon mutated human Gck construct produce 600% more protein than the rat Gck construct and 300% more protein than the human Gck transgene (=non codon mutated). This data, together with data disclose in Example 3 (FIGS. 6 and 7) of the present invention demonstrate that mhGck contruct result in higher RNA and protein production compare with construct carrying rGcK or the wild type human Gck gene.
[0105] To demonstrate functionality of these novel constructs, AAV1 vectors coding for rat Gck (rGck, NM_012565), wild type human Gck (hGck, NM_033507) or codon mutated human Gck (mhGck, CDS of SEQ ID NO: 4) were produced as disclosed in the previous Example 3 and 4. HEK293 cells were transduced with the 3 different vectors and Gck activity was measured. As shown in FIG. 9, the Gck activity of codon mutated (mhGcK) construct was higher than wild type human (hGcK) construct and rat Gck (rGcK) contruct.
EXAMPLE 5: `IN VIVO` EXPRESSION OF GCK PROTEIN FROM MUTATED TRANGENES
[0106] To provide in vivo evidences of Gck function, the inventors injected AAV1 vectors coding for rGck, hGck and mhGck into 3 different muscles in the hindlimbs of healthy mice. One month after the injection these muscles were harvested and analyzed for Gck activity. As shown in FIG. 10, muscles treated with mhGck vectors disclosed higher Gck activity compare with hGck and rGck.
[0107] These results clearly demostrated superior effect of AAV1-mhGck vectors vs AAV1-rGck or AAV1-hGck and suggested that lower doses of codon mutated insulin vectors will be required to achieve same therapeutic effect than non-mutated vectors.
EXAMPLE 6: MUTATED CONSTRUCT SHOWED AN IN VITRO AND IN VIVO INCREASED INSULIN AND C-PEPTIDE PRODUCTION COMPARE TO STANDARD VECTORS
[0108] We aimed to compare the ability of the mutated insulin gene versus the non mutated insulin gene to produce human c-peptide and human insulin production. To this end, we transduced HEK293 cells with two different adenoassociated vectors (AAV1 mhINS) at 3 different MOIs (10E4, 10E5 and 10E6 vg/cell). Four wells per MOI and vector were used. Two days after the infection, standard culture media (DMEM+10%FBS) was changed to a serum-free media to avoid the RIA detection of the media containing insulin. Next day (three days after the infection) medium was collected and was analyzed by RIA for the human C-peptide and insulin quantification.
[0109] Then it was observed a significant increase in human C-peptide levels (FIG. 11) and human insulin levels (FIG. 12) in AAV1-mhINS treated cells compared with standard insulin construct (AAV1-hINS). These data demonstrate that mutated insulin construct is more efficient in protein production and secretion that standard insulin gene.
[0110] To provide in vivo evidences of increased insulin and C-peptide production between AAV1-mhINS vs AAV1-hINS vectors, healthy mice were injected in hindlimb muscles with a total dose of 1.4E11 vg/mouse. Glycemia and insulinemia was measured two weeks after viral injection. As shown in FIG. 13, a significant reduction in fed glycemia was observed in animals injected with AAV1-mhINS compare with AAV1-hINS. In agreement with this, insulinemia (FIG. 14A) and c-peptide (FIG. 14B) was higher in AAV1-mhINS treated mice.
[0111] The data disclosed in the present invention, clearly demonstrated a superior effect of AAV1-mhINS vectors vs AAV1-hINS and suggested that lower doses of codon mutated insulin vectors will be required to achieve same therapeutic effect than non-mutated vectors (hINS).
[0112] The use of lower doses of vectors may have several advantages for gene therapy:
[0113] a) potential immunological responses might be reduced since it has been suggested that immunological responses to AAV are dose dependent,
[0114] b) lower number of injection sites to distribute the insulin vector will be required.
[0115] c) vector manufacture demand will be lower.
EXAMPLE 7: COMBINED THERAPY AAV1-MHINS+AAV1-MHGCK
[0116] The present invention tested the efficacy of a combined gene therapy approach with AAV1 vectors carrying codon mutated human constructs in diabetic mice. To this end, we injected AAV1-mhGcK vectors, AAV1-mhINS or both (10E12 vg per vector/kg) into the hindlimbs of c57bl6 diabetic mice. Experimental T1D was induced by streptozotocin (STZ) administration as in (36) and viral vectors were injected 15 days after STZ. A control group of STZ-treated mice was injected with AAV1-null vectors (same vector capsid but without expression of any transgene).
[0117] Animals treated with a combination of AAV1-mhINS+AAV1-mhGck showed significant reduction in blood glucose levels both in fasted and fed conditions (FIGS. 15 and 16, respectively) compared with AAV1-null vector-treated mice or single treatment with AAV1-mhINS or AAV1-mhGck.
EXAMPLE 8. COMBINED THERAPY: GENE THERAPY WITH AAV1-MHGCK+EXOGENOUS INSULIN IN T1D and T2D
[0118] The present invention have also evaluated whether AAV1-mhGck gene therapy per se may have therapeutic benefit for treating diabetes.
[0119] a) Evaluation of AAV1-mhGcK in T1D.
[0120] To this end, we injected AAV1-mhGcK vectors (10E12 vg/kg) into the hindlimbs of c57bl6 diabetic mice. Experimental T1D was induced by STZ administration and viral vectors (AAV1-mhGck) were injected 15 days after STZ. A control group of STZ-treated mice was injected with AAV1-null vectors (same vector capsid but without expression of any transgene). Two-months after AAV injection an insulin tolerance test was performed using low doses of insulin (0.375 U/kg). FIG. 17 shows that AAV1-mhGck treatment dramatically increase glucose uptake and reduce glycemia in the presence of exogenous insulin. These results indicate that gene therapy with AAV1-mhGck could be combined with regular exogenous insulin injections to improve the conventional treatment of T1D diabetes.
[0121] b) AAV1-Gck Treatment in T2D.
[0122] The inventors performed experiments in high fat fed animals as a model of T2D. In these animals, AAV1-rGck vectors (2E12 vg/kg) were injected in hindlimb muscles before the induction of diabetes by the high fat diet (HFD). Three months after HFD an intraperitoneal insulin tolerance test (0.75 U/kg) was performed. Insulin sensitivity of AAV1-Gck-treated mice was similar to control healthy mice while HFD fed mice were insulin resistant (FIG. 18). These data demonstrate that AAV1-GcK gene therapy per se could be considered as a treatment for diabetic patients in which insulin production is still present, such as early phases of T2D patients.
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[0165] 43. Mendell J R, Rodino-Klapac L R, Rosales-Quintero X, Kota J, Coley B D, Galloway G, Craenen J M, Lewis S, Malik V, Shilling C, Byrne B J, Conlon T, Campbell K J, Bremer W G, Viollet L, Walker C M, Sahenk Z, Clark K R. 2009. Limb-girdle muscular dystrophy type 2D gene therapy restores alpha-sarcoglycan and associated proteins. Ann Neurol 66(3):290-297.
[0166] 44. Stroes E S, Nierman M C, Meulenberg J J, Franssen R, Twisk J, Henny C P, Maas M M, Zwinderman A H, Ross C, Aronica E, High K A, Levi M M, Hayden M R, Kastelein J J, Kuivenhoven J A. 2008. Intramuscular administration of AAV1-lipoprotein lipase S447X lowers triglycerides in lipoprotein lipase-deficient patients. Arterioscler Thromb Vasc Biol 28(12):2303-2304.
[0167] 45. Jiang H, Pierce G F, Ozelo M C, de Paula E V, Vargas J A, Smith P, Sommer J, Luk A, Manno C S, High K A, Arruda V R. 2006. Evidence of multiyear factor IX expression by AAV-mediated gene transfer to skeletal muscle in an individual with severe hemophilia B. Mol Ther 14(3):452-455.
[0168] 46. Niemeyer G P, Herzog R W, Mount J, Arruda V R, Tillson D M, Hathcock J, van Ginkel F W, High K A, Lothrop C D Jr. 2009. Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy. Blood 113(4):797-806.
[0169] 47. Simonelli F, Maguire A M, Testa F, Pierce E A, Mingozzi F, Bennicelli J L, Rossi S, Marshall K, Banfi S, Surace E M, Sun J, Redmond T M, Zhu X, Shindler K S, Ying G S, Ziviello C, Acerra C, Wright J F, McDonnell J W, High K A, Bennett J, Auricchio A. 2010. Gene therapy for Leber's congenital amaurosis is safe and effective through 1.5 years after vector administration. Mol Ther 18(3):643-650.
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Sequence CWU
1
1
412165DNAArtificial SequencepGG2humanIns from ITR to ITR (Inverted
terminal repeat)CDS(1308)..(1640) 1cagctgcgcg ctcgctcgct cactgaggcc
gcccgggcaa agcccgggcg tcgggcgacc 60tttggtcgcc cggcctcagt gagcgagcga
gcgcgcagag agggagtggc caactccatc 120actaggggtt ccttgtagtt aatgattaac
ccgccatgct acttatctac gtagccatgc 180tctagacatg gctcgacaga tctcaatatt
ggccattagc catattattc attggttata 240tagcataaat caatattggc tattggccat
tgcatacgtt gtatctatat cataatatgt 300acatttatat tggctcatgt ccaatatgac
cgccatgttg gcattgatta ttgactagtt 360attaatagta atcaattacg gggtcattag
ttcatagccc atatatggag ttccgcgtta 420cataacttac ggtaaatggc ccgcctggct
gaccgcccaa cgacccccgc ccattgacgt 480caataatgac gtatgttccc atagtaacgc
caatagggac tttccattga cgtcaatggg 540tggagtattt acggtaaact gcccacttgg
cagtacatca agtgtatcat atgccaagtc 600cgccccctat tgacgtcaat gacggtaaat
ggcccgcctg gcattatgcc cagtacatga 660ccttacggga ctttcctact tggcagtaca
tctacgtatt agtcatcgct attaccatgg 720tgatgcggtt ttggcagtac accaatgggc
gtggatagcg gtttgactca cggggatttc 780caagtctcca ccccattgac gtcaatggga
gtttgttttg gcaccaaaat caacgggact 840ttccaaaatg tcgtaacaac tgcgatcgcc
cgccccgttg acgcaaatgg gcggtaggcg 900tgtacggtgg gaggtctata taagcagagc
tcgtttagtg aaccgtcaga tcactagaag 960ctttattgcg gtagtttatc acagttaaat
tgctaacgca gtcagtgctt ctgacacaac 1020agtctcgaac ttaagctgca gtgactctct
taaggtagcc ttgcagaagt tggtcgtgag 1080gcactgggca ggtaagtatc aaggttacaa
gacaggttta aggagaccaa tagaaactgg 1140gcttgtcgag acagagaaga ctcttgcgtt
tctgataggc acctattggt cttactgaca 1200tccactttgc ctttctctcc acaggtgtcc
actcccagtt caattacagc tcttaaggct 1260agagtactta atacgactca ctataggcta
gcctcgagaa ttctgccatg gccctgtgga 1320tgcgcctcct gcccctgctg gcgctgctgg
ccctctgggg acctgaccca gccgcagcct 1380ttgtgaacca acacctgtgc ggctcagatc
tggtggaagc tctctaccta gtgtgcgggg 1440aacgaggctt cttctacaca cccaggacca
agcgggaggc agaggacctg caggtggggc 1500aggtggagct gggcgggggc cctggtgcag
gcagcctgca gcccttggcc ctggaggggt 1560cgcgacagaa gcgtggcatt gtggaacaat
gctgtaccag catctgctcc ctctaccagc 1620tggagaacta ctgcaactag acgcagctgc
aagcttatcg ataccgtcga cctcgaggaa 1680ttcacgcgtg gtacctctag agtcgacccg
ggcggccgct tccctttagt gagggttaat 1740gcttcgagca gacatgataa gatacattga
tgagtttgga caaaccacaa ctagaatgca 1800gtgaaaaaaa tgctttattt gtgaaatttg
tgatgctatt gctttatttg taaccattat 1860aagctgcaat aaacaagtta acaacaacaa
ttgcattcat tttatgtttc aggttcaggg 1920ggagatgtgg gaggtttttt aaagcaagta
aaacctctac aaatgtggta aaatccgata 1980agggactaga gcatggctac gtagataagt
agcatggcgg gttaatcatt aactacaagg 2040aacccctagt gatggagttg gccactccct
ctctgcgcgc tcgctcgctc actgaggccg 2100ggcgaccaaa ggtcgcccga cgcccgggct
ttgcccgggc ggcctcagtg agcgagcgag 2160cgcgc
216524132DNAArtificial
SequencepGG2ratGCK from ITR to ITR (Inverted terminal
repeat)CDS(1397)..(2794) 2gcagctgcgc gctcgctcgc tcactgaggc cgcccgggca
aagcccgggc gtcgggcgac 60ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga
gagggagtgg ccaactccat 120cactaggggt tccttgtagt taatgattaa cccgccatgc
tacttatcta cgtagccatg 180ctctagacat ggctcgacag atctcaatat tggccattag
ccatattatt cattggttat 240atagcataaa tcaatattgg ctattggcca ttgcatacgt
tgtatctata tcataatatg 300tacatttata ttggctcatg tccaatatga ccgccatgtt
ggcattgatt attgactagt 360tattaatagt aatcaattac ggggtcatta gttcatagcc
catatatgga gttccgcgtt 420acataactta cggtaaatgg cccgcctggc tgaccgccca
acgacccccg cccattgacg 480tcaataatga cgtatgttcc catagtaacg ccaataggga
ctttccattg acgtcaatgg 540gtggagtatt tacggtaaac tgcccacttg gcagtacatc
aagtgtatca tatgccaagt 600ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
ggcattatgc ccagtacatg 660accttacggg actttcctac ttggcagtac atctacgtat
tagtcatcgc tattaccatg 720gtgatgcggt tttggcagta caccaatggg cgtggatagc
ggtttgactc acggggattt 780ccaagtctcc accccattga cgtcaatggg agtttgtttt
ggcaccaaaa tcaacgggac 840tttccaaaat gtcgtaacaa ctgcgatcgc ccgccccgtt
gacgcaaatg ggcggtaggc 900gtgtacggtg ggaggtctat ataagcagag ctcgtttagt
gaaccgtcag atcactagaa 960gctttattgc ggtagtttat cacagttaaa ttgctaacgc
agtcagtgct tctgacacaa 1020cagtctcgaa cttaagctgc agtgactctc ttaaggtagc
cttgcagaag ttggtcgtga 1080ggcactgggc aggtaagtat caaggttaca agacaggttt
aaggagacca atagaaactg 1140ggcttgtcga gacagagaag actcttgcgt ttctgatagg
cacctattgg tcttactgac 1200atccactttg cctttctctc cacaggtgtc cactcccagt
tcaattacag ctcttaaggc 1260tagagtactt aatacgactc actataggct agcctcgaga
attccctcag ccagacagtc 1320cttacctgca acaggtggcc tcaggagtca ggaacatctc
tacttcccca acgacccctg 1380ggttgtcctc tcagagatgg ctatggatac tacaaggtgt
ggagcccagt tgttgactct 1440ggtcgagcag atcctggcag agttccagct gcaggaggaa
gacctgaaga aggtgatgag 1500ccggatgcag aaggagatgg accgtggcct gaggctggag
acccacgagg aggccagtgt 1560aaagatgtta cccacctacg tgcgttccac cccagaaggc
tcagaagtcg gagactttct 1620ctccttagac ctgggaggaa ccaacttcag agtgatgctg
gtcaaagtgg gagaggggga 1680ggcagggcag tggagcgtga agacaaaaca ccagatgtac
tccatccccg aggacgccat 1740gacgggcact gccgagatgc tctttgacta catctctgaa
tgcatctctg acttccttga 1800caagcatcag atgaagcaca agaaactgcc cctgggcttc
accttctcct tccctgtgag 1860gcacgaagac ctagacaagg gcatcctcct caattggacc
aagggcttca aggcctctgg 1920agcagaaggg aacaacatcg taggacttct ccgagatgct
atcaagagga gaggggactt 1980tgagatggat gtggtggcaa tggtgaacga cacagtggcc
acaatgatct cctgctacta 2040tgaagaccgc caatgtgagg tcggcatgat tgtgggcact
ggctgcaatg cctgctacat 2100ggaggaaatg cagaatgtgg agctggtgga aggggatgag
ggacgcatgt gcgtcaacac 2160ggagtggggc gccttcgggg actcgggcga gctggatgag
ttcctactgg agtatgaccg 2220gatggtggat gaaagctcag cgaaccccgg tcagcagctg
tacgagaaga tcatcggtgg 2280gaagtatatg ggcgagctgg tacgacttgt gctgcttaag
ctggtggacg agaaccttct 2340gttccacgga gaggcctcgg agcagctgcg cacgcgtggt
gcttttgaga cccgtttcgt 2400gtcacaagtg gagagcgact ccggggaccg aaagcagatc
cacaacatcc taagcactct 2460ggggcttcga ccctctgtca ccgactgcga cattgtgcgc
cgtgcctgtg aaagcgtgtc 2520cactcgcgcc gcccatatgt gctccgcagg actagctggg
gtcataaatc gcatgcgcga 2580aagccgcagt gaggacgtga tgcgcatcac tgtgggcgtg
gatggctccg tgtacaagct 2640gcacccgagc ttcaaggagc ggtttcacgc cagtgtgcgc
aggctgacac ccaactgcga 2700aatcaccttc atcgaatcag aggagggcag cggcagggga
gccgcactgg tctctgcggt 2760ggcctgcaag aaggcttgca tgctggccca gtgaaatcca
ggtcatatgg accgggacct 2820gggttccacg gggactccac acaccacaaa tgctcccagc
ccaccggggc aggagaccta 2880ttctgctgct acccctggaa aatggggaga ggcccctgca
agccgagtcg gccagtggga 2940cagccctagg gctctcagcc tggggcaggg ggctgggagg
aagaagagga tcagaggcgc 3000caaggccttt cttgctagaa tcaactacag aaaatggcgg
aaaatactca ggacttgcac 3060tttcacgatt cttgcttccc aagcgtgggt ctggcctccc
aagggaatgc ttcctggacc 3120ttgcaatggc ctggcttccc tgggggggac acaccttcat
ggggaggtaa cttcagcagt 3180tcggccagac cagaccccag gagagtaagg gctgctagtc
acccagacct ggctgttttc 3240ttgtctgtgg ctgaagaggc cggggagcca tgagagactg
actatccggc tacatggaga 3300ggactttcca ggcatgaaca tgccagagac tgttgccttc
atatacctcc acccgagtgg 3360cttacagttc tgggatgaac cctcccagga gatgccagag
gttagagccc cagagtcctt 3420gctctaaggg gaccagaaag gggaggcctc actctgcact
attcaagcag gaatcatctc 3480caacactcag gtccctgacc caggaggaag aagccaccct
cagtgtccct ccaagagacc 3540acccaggtcc ttctctccct cgttcccaaa tgccagcctc
tctacctggg actgtggggg 3600agtttttaat taaatattta aaactacttc aaaaaaaaaa
aaaggaattc acgcgtggta 3660cctctagagt cgacccgggc ggccgcttcc ctttagtgag
ggttaatgct tcgagcagac 3720atgataagat acattgatga gtttggacaa accacaacta
gaatgcagtg aaaaaaatgc 3780tttatttgtg aaatttgtga tgctattgct ttatttgtaa
ccattataag ctgcaataaa 3840caagttaaca acaacaattg cattcatttt atgtttcagg
ttcaggggga gatgtgggag 3900gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa
tccgataagg gactagagca 3960tggctacgta gataagtagc atggcgggtt aatcattaac
tacaaggaac ccctagtgat 4020ggagttggcc actccctctc tgcgcgctcg ctcgctcact
gaggccgggc gaccaaaggt 4080cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
gagcgagcgc gc 413232371DNAArtificial SequencepAAVmutated human
Ins from ITR to ITR (Inverted terminal repeat)CDS(1336)..(1668)
3cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
60tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
120actaggggtt cctgcggccg cacgcgtgga gctagttatt aatagtaatc aattacgggg
180tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
240cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
300gtaacgtcaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
360cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
420ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
480cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
540aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
600aatgggagtt tgttttgcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
660ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
720gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa
780gacaccggga ccgatccagc ctccgcggat tcgaatcccg gccgggaacg gtgcattgga
840acgcggattc cccgtgccaa gagtgacgta agtaccgcct atagagtcta taggcccaca
900aaaaatgctt tcttctttta atatactttt ttgtttatct tatttctaat actttcccta
960atctctttct ttcagggcaa taatgataca atgtatcatg cctctttgca ccattctaaa
1020gaataacagt gataatttct gggttaaggc aatagcaata tttctgcata taaatatttc
1080tgcatataaa ttgtaactga tgtaagaggt ttcatattgc taatagcagc tacaatccag
1140ctaccattct gcttttattt tatggttggg ataaggctgg attattctga gtccaagcta
1200ggcccttttg ctaatcatgt tcatacctct tatcttcctc ccacagctcc tgggcaacgt
1260gctggtctgt gtgctggccc atcactttgg caaagaattg ggattcgaac atcgattgaa
1320ttcctcgagg ccaccatggc cctgtggatg agactgctgc ctctgctggc cctgctggct
1380ctgtggggcc ctgaccctgc cgccgctttc gtgaaccagc acctgtgcgg cagcgatctg
1440gtggaggccc tgtacctggt ctgcggcgag agaggcttct tctacacccc taggaccaag
1500agagaggccg aggacctcca ggtcggacag gtggaactgg gcggaggacc tggcgctgga
1560tctctgcagc ctctggccct ggaaggcagc agacagaaaa ggggcatcgt ggagcagtgc
1620tgcaccagca tctgcagcct gtaccagctg gaaaactact gcaactgagg atccgtcgac
1680ctgcagaagc ttgcctcgag cagcgctgct cgagagatct acgggtggca tccctgtgac
1740ccctccccag tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc
1800ctaataaaat taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg
1860tggagggggg tggtatggag caaggggcaa gttgggaaga caacctgtag ggcctgcggg
1920gtctattggg aaccaagctg gagtgcagtg gcacaatctt ggctcactgc aatctccgcc
1980tcctgggttc aagcgattct cctgcctcag cctcccgagt tgttgggatt ccaggcatgc
2040atgaccaggc tcagctaatt tttgtttttt tggtagagac ggggtttcac catattggcc
2100aggctggtct ccaactccta atctcaggtg atctacccac cttggcctcc caaattgctg
2160ggattacagg cgtgaaccac tgctcccttc cctgtccttc tgattttgta ggtaaccacg
2220tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
2280ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
2340cggcctcagt gagcgagcga gcgcgcagct g
237143454DNAArtificial SequencepAAVmutated human GCK from ITR to ITR
(Inverted terminal repeat)CDS(1344)..(2744) 4cctgcaggca gctgcgcgct
cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60gggcgacctt tggtcgcccg
gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120actccatcac taggggttcc
tgcggccgca cgcgtggagc tagttattaa tagtaatcaa 180ttacggggtc attagttcat
agcccatata tggagttccg cgttacataa cttacggtaa 240atggcccgcc tggctgaccg
cccaacgacc cccgcccatt gacgtcaata atgacgtatg 300ttcccatagt aacgtcaata
gggactttcc attgacgtca atgggtggag tatttacggt 360aaactgccca cttggcagta
catcaagtgt atcatatgcc aagtacgccc cctattgacg 420tcaatgacgg taaatggccc
gcctggcatt atgcccagta catgacctta tgggactttc 480ctacttggca gtacatctac
gtattagtca tcgctattac catggtgatg cggttttggc 540agtacatcaa tgggcgtgga
tagcggtttg actcacgggg atttccaagt ctccacccca 600ttgacgtcaa tgggagtttg
ttttgcacca aaatcaacgg gactttccaa aatgtcgtaa 660caactccgcc ccattgacgc
aaatgggcgg taggcgtgta cggtgggagg tctatataag 720cagagctcgt ttagtgaacc
gtcagatcgc ctggagacgc catccacgct gttttgacct 780ccatagaaga caccgggacc
gatccagcct ccgcggattc gaatcccggc cgggaacggt 840gcattggaac gcggattccc
cgtgccaaga gtgacgtaag taccgcctat agagtctata 900ggcccacaaa aaatgctttc
ttcttttaat atactttttt gtttatctta tttctaatac 960tttccctaat ctctttcttt
cagggcaata atgatacaat gtatcatgcc tctttgcacc 1020attctaaaga ataacagtga
taatttctgg gttaaggcaa tagcaatatt tctgcatata 1080aatatttctg catataaatt
gtaactgatg taagaggttt catattgcta atagcagcta 1140caatccagct accattctgc
ttttatttta tggttgggat aaggctggat tattctgagt 1200ccaagctagg cccttttgct
aatcatgttc atacctctta tcttcctccc acagctcctg 1260ggcaacgtgc tggtctgtgt
gctggcccat cactttggca aagaattggg attcgaacat 1320cgattgaatt cctcgaggcc
accatggcta tggacgtgac cagaagccag gcccagaccg 1380ccctgacact ggtggagcag
atcctggccg agttccagct gcaagaagag gacctgaaga 1440aagtgatgcg gcggatgcag
aaagagatgg acagaggcct gagactggaa acccacgaag 1500aggccagcgt gaagatgctg
cccacctacg tgcggagcac ccctgagggc agcgaagtgg 1560gcgacttcct gagcctggac
ctgggcggca ccaacttcag agtgatgctg gtcaaagtgg 1620gcgagggcga agagggacag
tggagcgtga aaacaaagca ccagatgtac agcatccccg 1680aggacgccat gacaggcacc
gccgagatgc tgttcgacta catcagcgag tgtatctccg 1740acttcctgga caaacatcag
atgaagcaca agaagctgcc cctgggcttc accttcagct 1800tccccgtgcg gcacgaggac
atcgacaagg gcatcctgct gaactggacc aagggcttca 1860aggccagcgg cgctgagggc
aacaacgtgg tcggcctgct gagggacgcc atcaagagaa 1920gaggcgactt cgagatggac
gtggtggcca tggtcaacga taccgtggct accatgatca 1980gctgctacta cgaggaccac
cagtgtgaag tgggcatgat cgtgggcacc ggctgcaacg 2040cctgctacat ggaagagatg
cagaacgtgg aactcgtgga gggagatgag ggcagaatgt 2100gcgtgaacac cgagtggggc
gccttcggag actctggcga gctggacgag ttcctgctgg 2160aatacgacag actggtggac
gagagcagcg ctaaccccgg ccagcagctg tacgagaagc 2220tgatcggcgg caagtacatg
ggagagctgg tccggctggt gctgctgagg ctggtggatg 2280agaacctgct gttccacggc
gaggcctccg agcagctgag aaccagaggc gccttcgaaa 2340ccagattcgt gagccaggtg
gagagcgaca ccggcgacag aaagcagatc tacaacatcc 2400tgagcaccct gggcctgagg
cctagcacca ccgactgcga catcgtgcgg agagcctgcg 2460agagcgtgtc caccagagcc
gcccacatgt gttctgccgg actggcaggc gtgatcaaca 2520gaatgcggga gagcagatcc
gaggacgtga tgagaatcac cgtgggcgtg gacggcagcg 2580tgtacaagct gcaccccagc
ttcaaagagc ggttccacgc ctccgtgaga aggctgaccc 2640ccagctgcga gatcaccttc
atcgagagcg aggaaggctc tggcagaggc gccgctctgg 2700tgtctgccgt ggcctgcaag
aaagcctgca tgctgggcca gtgaggatcc gtcgacctgc 2760agaagcttgc ctcgagcagc
gctgctcgag agatctacgg gtggcatccc tgtgacccct 2820ccccagtgcc tctcctggcc
ctggaagttg ccactccagt gcccaccagc cttgtcctaa 2880taaaattaag ttgcatcatt
ttgtctgact aggtgtcctt ctataatatt atggggtgga 2940ggggggtggt atggagcaag
gggcaagttg ggaagacaac ctgtagggcc tgcggggtct 3000attgggaacc aagctggagt
gcagtggcac aatcttggct cactgcaatc tccgcctcct 3060gggttcaagc gattctcctg
cctcagcctc ccgagttgtt gggattccag gcatgcatga 3120ccaggctcag ctaatttttg
tttttttggt agagacgggg tttcaccata ttggccaggc 3180tggtctccaa ctcctaatct
caggtgatct acccaccttg gcctcccaaa ttgctgggat 3240tacaggcgtg aaccactgct
cccttccctg tccttctgat tttgtaggta accacgtgcg 3300gaccgagcgg ccgcaggaac
ccctagtgat ggagttggcc actccctctc tgcgcgctcg 3360ctcgctcact gaggccgggc
gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc 3420ctcagtgagc gagcgagcgc
gcagctgcct gcag 3454
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