Patent application title: High Transgene Expression Of A Pseudotyped Adeno-Associated Virus Type
Yuhong Liu (Cherry Hill, NJ, US)
Jia Luo (Philadelphia, PA, US)
Matthew During (Columbus, OH, US)
IPC8 Class: AC12N701FI
Class name: Chemistry: molecular biology and microbiology virus or bacteriophage, except for viral vector or bacteriophage vector; composition thereof; preparation or purification thereof; production of viral subunits; media for propagating
Publication date: 2010-04-15
Patent application number: 20100093058
Patent application title: High Transgene Expression Of A Pseudotyped Adeno-Associated Virus Type
NUTTER MCCLENNEN & FISH LLP
Origin: BOSTON, MA US
IPC8 Class: AC12N701FI
Patent application number: 20100093058
The present invention related to methods and compositions comprising
recombinant vectors comprising chimeric capsids and recombinant
pseudotyped vectors with non-native capsid protein(s). The recombinant
vectors of the invention confer an altered tropism that permits selective
targeting of desired cells.
32. A recombinant pseudotyped adeno-associated virion for use in neural cells comprising:a transgene flanked 5' and 3' by inverted terminal repeat (ITR) sequences, wherein at least one ITR sequence is derived from a first adeno-associated virus (AAV);a Rep expression product from the rep coding region of a second adeno-associated virus (AAV), wherein the second AAV is different from the first AAV; anda non-native capsid derived from the first adeno-associated virus (AAV), such that the transgene is packaged within the non-native capsid, and wherein the non-native capsid provides a modified tropism and can bind to an attachment site present on a cell surface of a neural cell with a higher affinity than a corresponding adeno-associated virion with a wild type capsid.
33. The recombinant virion of claim 32, wherein at least one of the ITR sequences is derived from AAV2 and the other ITR sequence is derived from an adeno-associated virus (AAV) selected from the group consisting of AAV1, AAV3, AAV4, AAV5, and AAV6.
34. The recombinant virion of claim 32, wherein the first adeno-associated virus type is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6.
35. The recombinant virion of claim 32, wherein the second adeno-associated virus type is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6.
36. The recombinant virion of claim 32, wherein the first AAV is AAV5.
37. The recombinant virion of claim 32, wherein second AAV is AAV2.
38. The recombinant virion of claim 32, wherein the Rep expression product and capsid are encoded by the nucleic acid of SEQ ID NO:14.
39. The recombinant virion of claim 32, wherein the transgene is operably linked to a promoter in a transgene expression cassette.
40. The recombinant virion of claim 39, wherein the promoter is a tissue-specific promoter.
41. The recombinant virion of claim 39, wherein the promoter is operable in a brain or spinal cord neural cell.
42. The recombinant virion of claim 39, wherein the promoter is operable in a stratium neural cell.
43. The recombinant virion of claim 39, wherein the promoter is operable in a hippocampal neural cell.
44. The recombinant virion of claim 32, wherein the recombinant virion has a cell transduction rate that is about 2-fold higher than the transduction rate of the corresponding wild type adeno-associated virion.
45. A recombinant pseudotyped adeno-associated virus type-5 virion for use in a neural cell comprising:a transgene flanked 5' and 3' by inverted terminal repeat sequences derived from adeno-associated virus-5 (AAV5);a Rep expression product from the rep coding region of adeno-associated virus-2 (AAV2); anda non-native capsid derived from adeno-associated virus-5 (AAV5), such that the transgene is packaged within the AAV5 capsid, wherein the AAV5 capsid has a higher affinity to an attachment site present on a cell surface of the neural cell than a corresponding adeno-associated virion with a wild type capsid.
46. The recombinant virion of claim 45, wherein the Rep expression product and capsid are encoded by the nucleic acid of SEQ ID NO:14.
47. The recombinant virion of claim 45, wherein the transgene is operably linked to a promoter in a transgene expression cassette.
48. The recombinant virion of claim 47, wherein the promoter is operable in a brain or spinal cord neural cell.
49. The recombinant virion of claim 47, wherein the promoter is operable in a stratium neural cell.
50. The recombinant virion of claim 47, wherein the promoter is operable in a hippocampal neural cell.
51. A method of making a recombinant pseudotyped adeno-associated virion with a modified tropism comprising:providing a first construct comprising a transgene flanked 5' and 3' with inverted terminal repeat sequences derived from a first adeno-associated virus type, wherein at least one inverted terminal repeat sequence comprises a packaging signal, and a second helper construct comprising a rep coding region derived from a second adeno-associated virus type and a cap coding region derived from the first adeno-associated virus type, wherein the cap coding region encodes for a non-native capsid, and wherein the first adeno-associated virus type is different from the second adeno-associated virus type; andcontacting a population of cells with the first and second constructs, such that the population of cells allows assembly of a recombinant virion comprising a non-native capsid, to thereby produce a recombinant pseudotyped virion with a modified tropism, wherein the recombinant pseudotyped virion can bind to an attachment site present on a cell surface of a neural cell, with a higher affinity than a corresponding adeno-associated virion with a wild type capsid.
52. The method of claim 51, wherein the first adeno-associated virus type is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6.
53. The method of claim 51, wherein the second adeno-associated virus type is adeno-associated virus type-2 (AAV2).
54. The method of claim 51, wherein the step of contacting the population of cells further comprises contacting a population of 293 cells.
55. The method of claim 51, wherein the step of contacting the population of cells comprises the recombinant pseudotyped virion capable of transducing the population of cells at about 2-fold to about 30-fold higher transduction rate than the corresponding wild type adeno-associated virion.
RELATED CASE INFORMATION
This application claims priority to U.S. Provisional Patent Application No. 60/189,110, filed Mar. 14, 2000, is a continuation-in-part of U.S. patent application Ser. No. 09/804,898 filed: Mar. 13, 2001, and is a continuation of U.S. patent application Ser. No. 10/427,129 filed on May 1, 2003.
BACKGROUND OF THE INVENTION
The technical field of this invention is recombinant viral vectors and, in particular, recombinant pseudotyped viral vectors, especially recombinant pseudotyped adeno-associated viral (AAV) vectors.
Parvoviridae are small non-enveloped viruses containing single-stranded linear DNA genomes of 4 to 6 kb in length. Adeno-associated virus (AAV) is a member of the parvoviridae family. The AAV genome contains major open reading frames coding for the Rep (replication) and Cap (capsid) proteins. Flanking the AAV coding regions are two nucleotide inverted terminal repeat (ITR) sequences which contain palindromic sequences that can fold over to form hairpin structures that function as primers during initiation of DNA replication. In addition to their role in DNA replication, the ITR sequences have been shown to be necessary for viral integration, rescue from the host genome and encapsidation of viral nucleic acid into mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158:97-129).
The capsids of parvoviridae have icosahedral symmetry and are about 20-24 nm in diameter. They are composed of three viral proteins (VP1, VP2, and VP3, which are approximately 87, 73 and 61 Kd, respectively) (Muzyczka supra). VP3 represents 90% of the total virion protein; VP2 and VP1 account for approximately 5% each.
AAV can assume two pathways upon infection of a host cell. In the presence of helper virus, AAV will enter the lytic pathway where the viral genome is transcribed, replicated, and encapsidated into newly formed viral particles. In the absence of helper virus function, the AAV genome becomes integrated as a provirus into a specific region of the host cell genome, through recombination between the AAV ITRs and host cell sequences. Specific targeting of AAV viral DNA occurs at the long arm of human chromosome 19 (Kotin et al., (1990) Proc. Natl. Acad. Sci. USA 87:2211-2215; Samulski et al., (1991) EMBO J. 10:3941-3950). This particular feature of AAV reduces the likelihood of insertional mutagenesis resulting from random integration of viral vector DNA into the coding region of a host gene.
The AAV vector has properties that make it unique for gene therapy, for example, AAV is not associated with any known diseases and is generally non-pathogenic. In addition, AAV integrates into the host chromosome in a site-specific manner (See e.g., Kotin et al., (1990) Proc. Natl. Acad. Sci. 87: 2211-2215 and Samulski et al., (1991) EMBO J. 10: 3941-3950). However, clinical trials have indicated that the low transduction rate and low titer of the virus often may limit its use as a therapy in the central nervous system (CNS).
The AAV viral vector uses cellular receptors to attach to and infect a cell. Recently identified receptors include a heparan sulfate proteoglycan receptor as the primary receptor, and either the fibroblast growth factor (FGF), or the integrin aVb5, as secondary receptors (Qing et al. (1999) Nat. Med. 5:71-77 and Summerford et al. (1999) Nat. Med. 5:78-82). Following attachment to the cell, the viral particle undergoes receptor-mediated internalization into clathrin-coated endocytic vesicles of the cell.
Although the AAV viral vectors provide a suitable means for gene delivery to a target cell, they may often display a limited tropism (i.e., the binding and entry of the virus into a cell) for particular cell types. To date, attempts to alter the tropism of AAV vectors have involved introducing a peptide ligand into the capsid coat. For example, Girod et al. introduced a 14 amino acid peptide containing the RDG motif of the laminin fragment P1 into a capsid region of the AAV2 serotype to alter tropism (Girod et al. (1999) Nature Med. 5: 1052-1056). Zavada et al. altered the tropism of an AAV vector by the addition of viral glycoproteins (Zavada et al. (1982) J. Gen. Virol. 63: 15-24). Others have added single chain fragments of variable regions of a monoclonal antibody against CD34 to the N-terminus of the VP2 capsid (Yang et al. (1998) Hum. Gene. Ther. 9: 1929-1937). The major limitation with these approaches is that they require additional steps that covalently link large molecules, such as receptor ligands and antibodies to the virus. This adds to the size of the virus as well as the cost of production. Furthermore, the targeted particles are not homogenous in structure, which may effect the efficiency of gene delivery and transfer. Therefore, a need exists to generate viral vectors with a modified tropism that interact more efficiently with a cell surface. A need also exists for viral vectors with a modified tropism to target cell types that the corresponding wild type virus does not typically target.
SUMMARY OF THE INVENTION
The invention is based on the discovery that a recombinant parvovirus vectors can be pseudotyped such that the recombinant vector is derived from a different virus than the capsid, e.g., a first parvovirus, is packaged with a capsid from a second parvovirus that is different from the first parvovirus. Such recombinant pseudotyped vectors have a modified tropism which allows them to interact with a cell surface molecule on a neural cell with an altered affinity. The recombinant pseudotyped vector is produced by packaging the wild type parvovirus vector in the capsid of a parvovirus other than the wild type. This can be accomplished, for example by using helper functions that comprise a rep coding region derived from the wild type parvovirus and a cap coding region derived from a parvovirus other than the wild type parvovirus. The resulting recombinant pseudotyped vector has a modified tropism that allows the recombinant pseudotyped vector to interact with a cell surface molecule on a neural cell with an altered affinity, e.g., a higher affinity, than a recombinant vector with a wild type capsid. Thus, the pseudotyped vector allows targeting of cells that a vector with a wild type capsid would not normally target, and permits targeting and infecting a broader range of cells and hosts compared with the wild type parvovirus. The pseudotyped vectors are particularly suitable for transduction into neural cells, for example, those present in regions of the brain.
More specifically, the invention pertains to recombinant pseudotyped adeno-associated vectors that carry the core genetic information of a first adeno-associated virus (AAV) type (i.e., the wild type AAV), and in addition the surface proteins of a second adeno-associated virus type that is different from the first adeno-associated virus type. For example, a recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, and the like, genome may be encapsidated within any one of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, and the like, capsid, provided that the AAV capsid and genome are of different types (or "serotypes").
In particularly preferred embodiments, the recombinant pseudotyped AAV virions comprise a wild type AAV2 type genome packaged within an AAV1, AAV3, AAV4, AAV5, AAV6 type capsid. In particular preferred embodiments, the recombinant pseudotyped AAV virion comprises a wild type AAV2 type genome packaged with an AAV1, or AAV5 type capsid. Most preferably, a wild type AAV2 genome packaged with an AAV1 type capsid. The present invention provides AAV helper function vectors that allow the genome to be packaged. These helper functions express rep gene products from the wild type AAV, and cap gene products from an AAV that is different from the wild type AAV to produce the recombinant pseudotyped virions of the invention.
Accordingly, in one aspect, the invention features a recombinant pseudotyped adeno-associated virion for use in neural cells comprising a transgene flanked 5' and 3' by inverted terminal repeat sequences, where the inverted terminal repeat sequences are derived from a first adeno-associated virus; and a non-native capsid derived from a second adeno-associated virus that is different from the first adeno-associated virus, such that the transgene is packaged within the non-native capsid, and where the non-native capsid provides a modified tropism and can bind to an attachment site present on a cell surface of a neural cell, with a higher affinity than a corresponding adeno-associated virion with a wild type capsid, and upon entering a cell has a transduction rate that is about 2-fold to about 30-fold higher than the transduction rate of the corresponding wild type adeno-associated virion. The transduction rate can be determined by densitometry analysis.
The first adeno-associated virus type can be selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 and the like, while the second adeno-associated virus type can also be selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 and the like, as long as the second adeno-associated virus type is different from the first AAV. In one embodiment, the modified tropism permits attachment of the virion to an attachment site present on a neural cell, for example, a neural cell in a region of a brain. The modified tropism also permits binding and entry of the adeno-associated virus into the neural cell.
In another aspect, the invention features a recombinant pseudotyped adeno-associated virus type-1 virion comprising a transgene flanked 5' and 3' by inverted terminal repeat sequences, where the inverted terminal repeat sequences are derived from adeno-associated virus type-2 (AAV2); and a non-native capsid derived from adeno-associated virus type-1 (AAV1), such that the transgene is packaged within the AAV1 capsid, wherein the AAV1 capsid provides a modified tropism and can bind to an attachment site present on a cell surface of a neural cell with a higher affinity than a corresponding adeno-associated virion with a wild type capsid, and upon entry into a cell has a transduction rate that is about 8-fold higher than the transduction rate of the corresponding wild type adeno-associated virion with a wild type capsid.
In yet another aspect, the invention features a recombinant pseudotyped adeno-associated virus type-5 virion comprising a transgene flanked 5' and 3' by inverted terminal repeat sequences, where the inverted terminal repeat sequences are derived from adeno-associated virus-2 (AAV2); and a non-native capsid derived from adeno-associated virus-5 (AAV5), such that the transgene is packaged within the AAV5 capsid, wherein the AAV5 capsid provides a modified tropism and can bind to an attachment site present on a cell surface of a neural cell with a higher affinity than a corresponding adeno-associated virion with a wild type capsid, and upon entry into a cell has a transduction rate that is about 2-fold higher than the transduction rate of the corresponding wild type adeno-associated virion with a wild type capsid.
In another aspect, the invention features a method of making a recombinant pseudotyped adeno-associated virions by providing a first construct comprising a transgene flanked 5' and 3' with inverted terminal repeat sequences derived from a first adeno-associated virus type, where at least one inverted terminal repeat sequence comprises a packaging signal, and a second helper construct comprising a rep coding region derived from the first adeno-associated virus type and a cap coding region derived from a second adeno-associated virus type, wherein the cap coding region encodes a non-native capsid. A population of cells is contacted with the first and second constructs, such that the population of cells allows assembly of a recombinant virion, to thereby produce a recombinant pseudotyped virion, wherein the recombinant pseudotyped virion has a modified tropism and can bind to an attachment site present on a cells surface of a neural cell, and has a transduction rate that is about 2-fold to about 30-fold higher than the transduction rate of the corresponding wild type adeno-associated virion with a wild type capsid.
The inverted terminal repeat sequences of the first construct can be derived from an adeno-associated virus type selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 and the like. In a preferred embodiment, the inverted terminal repeat sequences are derived from the wild type AAV, e.g., AAV2. The second construct can comprise a nucleic acid sequence encoding a capsid derived from an adeno-associated virus type that is different from the first adeno-associated virus type. The recombinant pseudotyped adeno-associated virion can be made by contacting a population of 293 cells.
In yet another aspect, the invention features a method for modifying the tropism of a recombinant adeno-associated viral vector comprising replacing a native capsid of a first adeno-associated virus type with a non-native capsid derived from a second adeno-associated virus type; and combining the non-native capsid under conditions for assembly, to thereby modify the tropism of an adeno-associated viral vector.
In one embodiment, the non-native capsid can be derived from an adeno-associated virus type selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 and the like. In a preferred embodiment, the non-native capsid is derived from AAV1. In another preferred embodiment, the non-native capsid is derived from AAV5.
The method for modifying the tropism of a recombinant adeno-associated viral vector can further comprise increasing the efficiency of entry into a cell using a recombinant pseudotyped adeno-associated viral vector by providing a transgene flanked 5' and 3' by inverted terminal repeat sequences, where the inverted terminal repeat sequences are derived from a first adeno-associated virus type, and a non-native capsid derived from a second adeno-associated virus, where the capsid has a modified tropism; and contacting a cell with the recombinant pseudotyped adeno-associated viral vector having a modified capsid tropism such that the non-native capsid binds to an attachment site on the cell surface of a neural cell, and permits the vector to enter the neural cell more efficiently that a corresponding viral vector comprising a wild type capsid. In a preferred embodiment, the inverted terminal repeat sequences are derived from AAV2 and the non-native capsid is derived from AAV1. In another embodiment, the inverted terminal repeat sequences are derived from AAV2 and the non-native capsid is derived from AAV5. The attachment site can be a site on a neural cell, e.g., a neuronal cell in a region of a brain.
In another aspect, the invention features an isolated nucleic acid molecule encoding an AAV helper function. The nucleic acid molecule comprises a rep coding region derived from a first adeno-associated virus type, where the first adeno-associated virus is the wild type virus, and a cap coding region derived from a second adeno-associated virus type that is different from the first virus. The non-native capsid provides a modified tropism which permits binding to an attachment site present on a cell surface of a neural cells with a higher affinity than a corresponding adeno-associated virion with a wild type capsid, and which upon entry into a cell has a transduction rate that is about 2-fold to about 30-fold higher than the transduction rate of the corresponding wild type adeno-associated virion.
BRIEF DESCRIPTION OF FIGURES
FIG. 1 is a schematic showing the pseudotyped helper function constructs;
FIG. 2 is a bar graph showing the yields of AAV pseudotyped vectors; and
FIG. 3 is a bar graph showing the densitometric analysis of EGFP expression in the hippocampus.
The present invention is based on the discovery that a recombinant pseudotyped adeno-associated virus (AAV) vector can be packaged efficiently producing a recombinant vector with a capsid that confers a modified tropism to the vector. The modified tropism allows the recombinant pseudotyped vector to bind to attachment sites on neural cells, with an altered affinity, e.g., a higher affinity, than a recombinant vector with wild type capsid. Alternatively, the modified tropism allows targeting of cells that would not typically be targeted by an AAV vector with a wild type capsid. These recombinant pseudotyped vectors with the modified tropism are produced by using a non-native capsid derived from an adeno-associated virus that is different from the wild type adeno-associated virus, and the genome of the wild type AAV is packaged within the non-native capsid. Alternatively, the altered tropism can be the result of recombinant vectors comprising chimeric capsids. The recombinant vector with chimeric capsids has at least one non-native amino acid sequence derived from a capsid protein from another member of the parvovirus family, and also contains a packaging sequence in the genome that can be derived from the wild type parvovirus or can be derived from another family member.
So that the invention is more clearly understood, the following terms are defined:
The term "parvoviruses" as used herein refers to any member of the subfamily Parvovirinae. It includes both autonomous parvovirus and dependovirus. The present invention include, but are not limited to, LuIII parvovirus (LuIII), minute virus of mice (MVM; e.g., MVMi and MVMp), hamster parvovirus (e.g., H1), feline panleukopenia virus, canine parvovirus, porcine parvovirus, latent rat virus, mink enteritis virus, human parvovirus (e.g., B19), bovine parvovirus, Aleutian mink disease parvovirus, adeno-associated viruses (e.g., AAV1, AAV2).
The term "pseudotyped" as used herein refers to mixed viral particles (or virions). These "pseudotyped" viral particles carry the core and genetic information of first virus, and in addition the surface capsid protein(s) of second different virus. The term "pseudotyped" is also intended to encompass surface capsid proteins of other viruses with point mutations (additions, substitutions, and deletions).
The term "pseudotyped adeno-associated virion" as used herein refers to mixed adeno-associated viral particles. These "pseudotyped" adeno-associated particles carry the core and genetic information of a first adeno-associated virus, and in addition the surface capsid protein(s) of a second different adeno-associated virus.
The term "chimeric capsid" as used herein refers to a viral protein coat with one or more non-native amino acid sequences. The chimeric capsid can comprise a combination of amino acid sequences from the same family. For example, a chimeric capsid comprising the VP1 domain of AAV2, in combination with the VP2 and VP3 domains of AAV5. The skilled artisan will appreciate that the chimeric capsid can be any combination of viral protein domains from the parvovirus family member such as, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 and the like. The term "chimeric capsid" also refers to a viral protein coat with at least one non-native amino acid sequence from a virus, such as herpesvirus, adenovirus, lentivirus, retrovirus, Epstein-Barr virus and vaccinia virus, and the like.
The term "non-native capsid" as used herein refers to an entire capsid protein that is not present in the wild type parvovirus. For example, the non-native capsid protein can be the entire capsid derived from an AAV, such as AAV1 that replaces the entire capsid of a wild type AAV, such as AAV2. The term "non-native capsid" is also intended to a include nucleic acid molecule encoding the non-native capsid protein.
The term "tropism" as used herein refers to the binding (or attachment) and entry (or internalization) of the virus into the cell, optionally and preferably, followed by expression of sequences carried by the viral genome in the cell.
The term "modified tropism" as used herein refers to a recombinant parvovirus that has an altered tropism, which allows the parvovirus to target cells that the wild type virus with a wild type capsid was unable to target. The term "modified tropism", includes reductions or enhancements in infectivity with respect to a particular cell type(s) as compared with the wild type parvovirus. These reductions or enhancements can arise due to a change in the binding or attachment of the virus to a target cell, that the wild type virus is unable to target. Alternatively, these reductions or enhancements can arise due to a change in the entry or internalization of the virus into the target cell. The term "modified tropism" also encompasses the creation of a new tropism i.e., creating a parvovirus that infects a particular cell type(s) to a significant or a detectable extent that the wild type parvovirus was unable to infect. Preferred cell types are those of the central nervous system, e.g., neural cells of the brain. As a further alternative, a "modified tropism" also refers to a more efficient delivery of a targeted parvovirus as compared with the native parvovirus (e.g., a reduced Multiplicity of Infection, "MOI").
The term "affinity" as used herein refers to the art recognized use of the term for the attraction between a ligand and receptor. An "altered affinity" is one that has an increased (i.e., a stronger attraction) or decreased (i.e., a weaker attraction) between ligand and receptor.
The term "transduction rate" as used herein refers to its the introduction of a nucleic acid sequence contained in a pseudotyped viral vector into a number of cells. Transduction by a method of the invention involves contacting cells with a viral vector such that the viral nucleic acid enters the cell and can be expressed therein. The rate of transduction can be determined by measuring the number of the cells that are transduced and by examining the extent of expression of a protein at a target site by certain amount of virus (by particle). If a vector is administered, the extent of transduction of cells can be determined by examining the distance away from an injection site that protein expression occurs. The more further away from the point of injection that expression can occurs, the greater number of cells that are transduced. The transduction rate for the pseudotyped vectors can be determined by standard techniques. For example, the rate of transduction can be assessed sterologically by counting the number of cells expressing a marker protein, such as Green Fluorescent Protein (GFP) in a region of a brain into which the recombinant vectors of the invention have been delivered. Serial continuous brain sections can be made around the injection site, e.g., approximately 50 sections, and the number of cells expressing the GFP in each brain section counted using a microscope. The sum total number of transduced cells from each brain section can be counted to provide an evaluation of the transduction rate for each of the recombinant pseudotyped vectors, or recombinant chimeric capsid vectors.
Under circumstances where sterological counting is impractical, for example where there is strong expression of the marker protein that makes it difficult to assess the number of cells expressing the marker protein, fluorescent microscopic densitometry can be used to determine the fluorescence intensity of marker protein, e.g., GFP in the target nuclei. The fluorescent images of each brain section can be captured by a digital camera under the fluorescent microscope and the relative fluorescent intensity in the transduced nuclei of each image can be analyzed by using the NIH image software. With densitometry analysis, color images of a marker protein such as GFP, are analyzed as a black and white images. The area that appears as bright white indicates highest expression of the marker protein, a grey scale indicates a lower expression, and a black scale indicates no expression of the marker protein. The rate of transduction can be determined by examining the area of "white" in a region of the brain. The greater the area of white in an image, the greater the expression of the marker protein in that region.
Another method of determining transduction rates can be by measuring luciferase activity of transduced cells using the Luciferase Assay Reagent Kit (Promega, Madison, Wis., U.S.A) according to the manufacturer's recommendations to measure the luciferase activity of cell lysates using a luminometer. Preferably the transduction rate is about 2 fold to about 30 fold higher in neural cells using the recombinant pseudotyped vectors or the recombinant chimeric capsid vectors, compared with the wild type vector having a wild type capsid.
The term "attachment site" as used herein refers to a site on a target cell to which the recombinant pseudotyped parvovirus binds to, or interacts with. The recombinant pseudotyped vectors have a modified tropism to facilitate binding of AAV to the cellular receptor, or to inhibit the binding to the receptor. In particular, the attachment site is one that is not typically targeted by a wild type virion, but is one that is targeted by the pseudotyped vector of the invention. For example, by binding to an attachment site on neural cells, e.g., an attachment site on a neural cell in a region of the brain or spinal cord.
The term "gene transfer" or "gene delivery" as used herein refers to methods or systems for reliably inserting foreign nucleic acids sequences, e.g., DNA into host cells. Such methods can result in transient expression of non-integrated transferred DNA, extra-chromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells. Gene transfer provides a unique approach for the treatment of acquired and inherited diseases. A number of systems have been developed for gene transfer into mammalian cells (See, e.g., U.S. Pat. No. 5,399,346.)
The term "transgene", as used herein refers to a nucleic acid sequence of interest. Such transgenes, or gene sequences, may be derived form a variety of sources including DNA, cDNA, synthetic DNA, and RNA. Such transgenes may comprise genomic DNA which may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with promoter regions or poly A sequences. The transgenes of the present invention are preferably cDNA. Genomic or cDNA may be obtained by means well known in the art. A transgene which may be any gene sequence whose expression produces a gene product that is to be expressed in a cell. The gene product may affect the physiology of the host cell. Alternatively the transgene may be a selectable marker gene or reporter gene.
The term "transgene expression cassette" refers to a transgene that is operably linked to a promoter or other regulatory sequence sufficient to direct transcription of the transgene. Suitable promoters include, for example, tissue specific promoters. Other regulatory sequences include post-regulatory sequences such as the woodchuck post-regulatory sequence.
The term "tissue-specific promoter" as used herein refers to a promoter that is operable in cells of the central nervous system (CNS), such as neural cells. Examples of promoters for the CNS include but are not limited to, neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477) and glial specific promoters (Morii et al. (1991) Biochem. Biophys Res. Commun. 175: 185-191). Preferably, the promoter is tissue specific and is essentially not active outside the central nervous system, or the activity of the promoter is higher in the central nervous system that in other systems. For example, a promoter specific for the spinal cord, brainstem, (medulla, pons, and midbrain), cerebellum, diencephalon (thalamus, hypothalamus), telencephalon (corpus stratium, cerebral cortex, or within the cortex, the occipital, temporal, parietal or frontal lobes), subthalamic nucleus (STN), substantia nigra (SN), or combinations, thereof. Preferred promoters are the Chicken Beta Actin (CBA) promoter and the neuron-specific enolase (NSE) promoter. The promoter may also be one that can be used in combination with an AAV to result in higher expression. For example, a cytomegalovirus enhancer/Chicken-Beta-Actin (CBA) hybrid promoter that functions in cells of the CNS (Xu et al. (2001) Hum Gene Ther. 12:563-73).
A "nucleic acid sequence" refers to a DNA or RNA sequence. The term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, 2-thiocytosine, and 2,6-diaminopurine.
The term "vector" as used herein refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, adeno-associated virus, parvovirus, virion, and the like, which is capable of replication when associated with the proper control elements and which can transfer gene sequences into cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.
The term "AAV vector" as used herein refers to a vector derived from an adeno-associated virus serotype, including but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, and the like. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking Inverted Terminal Repeat (ITR) sequences. Functional ITR sequences permit the rescue, replication and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences required for replication and packaging (e.g., functional ITRs) of the virus. The ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging.
The term "regulatory sequence" is art-recognized and intended to include control elements such as promoters, enhancers and other expression control elements (e.g., polyadenylation signals), transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, enhancer sequences, post-regulatory sequences and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these regulatory sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. Such regulatory sequences are known to those skilled in the art and are described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). It should be understood that the design of the viral vector may depend on such factors as the choice of the host cell to be transfected and/or the amount of protein to be expressed.
The term "operably linked" as used herein refers to an arrangement of elements wherein the components are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression of the coding sequence. For example, intervening untranslated yet transcribed can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.
The term "AAV rep coding region" as used herein refers to the art-recognized region of the AAV genome which encodes the replication proteins Rep 78, Rep 68, Rep 52 and Rep 40. These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAV origin of DNA replication, DNA helicase activity and modulation of transcription from AAV (or other exogenous) promoters. The Rep expression products are collectively required for replicating the AAV genome. For a description of the AAV rep coding region, see, e.g., Muzyczka (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin (1994) Human Gene Therapy 5:793-801. Suitable homologues of the AAV rep coding region include the human herpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV2 DNA replication (Thomson et al. (1994) Virology 204:304-311). In certain embodiment of the invention, the rep coding region can be derived from any AAV serotype including, but limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, etc. In a preferred embodiments, the rep coding region is derived from AAV2.
The term "AAV cap coding region" as used herein refers to the art-recognized region of the AAV genome which encodes the capsid proteins VP1, VP2, and VP3, or functional homologues thereof. These cap expression products supply the packaging functions which are collectively required for packaging the viral genome. For a description of the AAV cap coding region, See, e.g., Muzyczka (Supra). In certain embodiment, the AAV cap coding region can be derived from any AAV serotype, including, but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, etc. In a preferred embodiment, the entire cap coding region is derived from AAV1. In certain embodiment, the entire cap coding region is derived from AAV5.
The term "AAV helper functions" or "helpers" as used herein refer to AAV-derived coding sequences that can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication. Thus, AAV helper functions include the rep and cap regions. The rep expression products have been shown to possess many functions, including, among others: recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and modulation of transcription from AAV (or other heterologous) promoters. The cap expression products supply necessary packaging functions. AAV helper functions are used herein to complement AAV functions in trans that are missing from AAV vectors.
The term "pseudotyped AAV helper functions" as used herein refers to rep and cap regions where the cap region is derived from an AAV that is different from the wild type genome. In preferred embodiment, the helper functions comprise a rep coding region derived from a first adeno-associated virus, that is the wild type adeno-associated virus, and the non-native cap coding region is derived from a adeno-associated virus that is different from the wild type adeno-associated virus. In one embodiment, the AAV helper function comprises a wild type rep region derived from AAV2, and a non-native cap coding region derived from AAV1. In another embodiment, the AAV helper function comprises a wild type rep region from AAV2, and a non-native cap coding region from AAV5.
The term "accessory functions" as used herein refers to non-AAV derived viral and/or cellular functions upon which AAV is dependent for its replication (Carter, (1990) "Adeno-Associated Virus Helper Functions," in CRC Handbook of Parvoviruses, vol. I (P. Tijssen, ed.)). Thus, the term captures DNAs, RNAs and protein that are required for AAV replication, including those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1) and vaccinia virus.
The term "accessory function vector" as used herein refers generally to a nucleic acid molecule that includes nucleotide sequences providing accessory functions. An accessory function vector can be transfected into a suitable host cell, wherein the vector is then capable of supporting AAV virion production in the host cell. Thus, accessory function vectors can be in the form of a plasmid, phage, transposon, cosmid or virus that has been modified from its naturally occurring form.
The term "recombinant virus" as used herein refers to a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle.
The term "recombinant virion" as used herein refers to a complete infectious, replication-defective virus particle composed of a viral coat, encapsidating a transgene which is flanked on both sides by viral ITRs. The term "recombinant virion" is used synonymously with the term "recombinant particle."
The term "recombinant AAV virion," or "recombinant AAV particle" as used herein refers to an infectious, replication-defective virus composed of an AAV protein shell encapsidating a heterologous nucleotide sequence of interest that is flanked on both sides by AAV ITRs. A recombinant AAV virion is produced in a suitable host cell comprising an AAV vector, AAV helper functions, and/or accessory functions. In this manner, the host cell is rendered capable of encoding AAV polypeptides that are required for packaging the AAV vector (comprising a recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.
The terms "5'", "3'", "upstream" or "downstream" are art recognized terms that describe the relative position of nucleotide sequences in a particular nucleic acid molecule relative to another sequence.
The term "transfection" is used herein refers to the uptake of an exogenous nucleic acid molecule by a cell. A cell has been "transfected" when exogenous nucleic acid has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acid molecules into suitable host cells. The term refers to both stable and transient uptake of the nucleic acid molecule, and is intended to capture captures chemical, electrical, and viral-mediated transfection procedures.
The term "coding sequence" or a sequence which "encodes" a particular protein, as used herein refers to a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence.
A "fragment" or "portion" of a nucleic acid encoding a capsid protein is defined as a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the entire amino acid sequence of the capsid protein, such as VP1, VP2 or VP3. A fragment or portion of a nucleic acid molecule is about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, and about 50 nucleotides in length. Also within the scope of the invention are nucleic acid fragments which are about 60, 70, 80, 90, 100 or more nucleotides in length. Preferred fragments or portions include nucleotide sequences encode a polypeptide that alters the tropism of the chimeric capsid. The term fragment or portion also refers to an amino acid sequence of the capsid protein that has fewer amino acids than the entire sequence of the viral protein domains VP1, VP2 and VP3. The fragment is about 10 amino acids, more preferably about 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 and 200 or more amino acids in length.
The terms "polypeptide" and "protein" are used interchangeably herein and refer to a polymer of amino acids and includes full-length proteins and fragments thereof. As will be appreciated by those skilled in the art, the invention also includes nucleic acids that encode those polypeptides having slight variations in amino acid sequences or other properties from a known amino acid sequence Amino acid substitutions can be selected by known parameters to be neutral and can be introduced into the nucleic acid sequence encoding it by standard methods such as induced point, deletion, insertion and substitution mutants. Minor changes in amino acid sequence are generally preferred, such as conservative amino acid replacements, small internal deletions or insertions, and additions or deletions at the ends of the molecules. These modifications can result in changes in the amino acid sequence, provide silent mutations, modify a restriction site, or provide other specific mutations. Additionally, they can result in a beneficial change to the encoded protein.
The term "homology" or "identity" as used herein refers to the percentage of likeness between nucleic acid molecules. To determine the homology or percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. (48): 444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In another example, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another example, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty.
The term "host cell" as used herein refers to, for example microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of an AAV helper construct, an AAV vector plasmid, an accessory function vector, or other transfer DNA. The term includes the progeny of the original cell which has been transfected. Thus, a "host cell" as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to natural, accidental, or deliberate mutation.
The term "cell line" as used herein refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.
The term "central nervous system" or "CNS" as used herein refers to the art recognized use of the term. The CNS pertains to the brain, cranial nerves and spinal cord. The CNS also comprises the cerebrospinal fluid, which fills the ventricles of the brain and the central canal of the spinal cord. Regions of the brain include, but are not limited to, the striatum, hippocampus, cortex, basal ganglia, subthalamic nucleus (STN), pedunculopontine nucleus (PPN), substantia nigra (SN), thalamus, putamen, or caudate regions of the brain, as well as the spinal cord or combinations thereof.
The term "neural cells" as used herein refers to cells that have been isolated from the brain, spinal cord or cells from any region of the central nervous system, as well as any cell present in the brain, spinal cord, or central nervous system of a subject, to which the recombinant pseudotype vectors, or the recombinant chimeric capsid vectors with a modified tropism, attach to, enter, and express a transgene, with a higher affinity or transduction rate than the corresponding vector with a vectors with a wild type capsid. Examples of neural cells include neuronal cells, such as nerve cells that transmit nerve or chemical signals to and from the brain, such as sensory neurons or bipolar neurons that carry messages from the body's sense receptors (eyes, ears, etc.) to the CNS; motoneurons or multipolar neurons cells that carry signals from the muscles and glands to the CNS (e.g., spinal motor neurons, pyramidal neurons, Purkinje cells.); interneurons or pseudopolare cells which form the neural wiring within the CNS. These have two axons (instead of an axon and a dendrite).
The term neural cells is also intended to include glial cells, which make up 90 percent of the brain's cells. Glial cells are nerve cells that do not carry nerve impulses. Types of glial cells include, but are not limited to, Schwann's cells, satellite cells, microglia, oligodendroglia, and astroglia.
The term "subject" as used herein refers to any living organism in which an immune response is elicited. The term subject includes, but is not limited to, humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
Further details of the invention are described in the following sections:
I Recombinant Vectors
The invention features a method of producing recombinant vectors comprising a chimeric capsid or recombinant pseudotyped vectors that are particularly suitable for targeting cells in the central nervous system, e.g., cells in a region of the brain. Recombinant vectors can be constructed using known techniques to provide operatively linked components of control elements including a transcriptional initiation region, a transgene, and a transcriptional termination region. The control elements are selected to be functional in the targeted cell. The resulting construct which contains the operatively linked components can be flanked at the 5' and 3' region with functional parvoviral ITR sequences.
In one aspect, the invention features a recombinant pseudotyped parvovirus vector that comprises a wild type parvovirus genome, and a non-native capsid derived from a parvovirus that is different from the wild type parvovirus. In one embodiment, the recombinant pseudotyped parvovirus vector is a recombinant pseudotyped AAV vector.
The parvovirus family includes adeno-associated viruses. Examples of adeno-associated virus serotypes include, but are not limited to, AAV1 (Xiao et al. (1999), J. Virol., 73: 3994-4003, GenBank Accession No. AF063497; gi:9632547), AAV2 (Ruffing et al. (1994) J. Gen. Virol., 75: 3385-3392, GenBank Accession No. gi:9626146), AAV3 (Muramatsu et al. (1996) Virology 221: 208-217, GenBank Accession No. U48704; Rutledge et al. (1998) J. Virol., 72: 309-319, GenBank Accession No. AF028705), AAV4 (Chiorini et al. (1997), J. Virol., 71: 6823-6833, GenBank Accession No. U89790), AAV5 (Bantel et al., (1999), J. Virol. 73: 939-947 GenBank Accession No. gi:4249656) and AAV6 (Rutledge et al. (1998), J. Virol., 72: 309-319, GenBank Accession No. AF028704). The sequences of the capsid genes for such serotypes is reported in Srivastava et al., (1983) J. Virol. 45:555-564; Muzyczka (1992) Curr. Top. Micro Immunol. 158:97-129, and Ruffing et al. (1992) J. Virol. 66:6922-6930. Each serotype of AAV has a different cellular tropism and bind to different cell surface proteins. Some parvovirus family members are useful for transduction of particular cell types, but less useful for transduction of other cells.
Several serotypes of adeno-associated viruses AAV have been reported (Bantel-Schaal et al. (1999) J. Virol. 73:939-947 and Chiorini (1999) J. Virol. 73:1309-1319 (AAV5); Chiorini, et al. (1997) J. Virol. 71:6823-6833 (AAV4); Muramatsu et al. (1996) Virology 221:208-217 (AAV3); Rutledge et al. (1998) J. Virol. 72:309-319; and Xiao et al. (1999) J. Virol. 73:3994-4003 (AAV2)). Cloning and sequence characterization of these serotypes indicate that they share a similar genomic organization, which consists of two large open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs). The ITR structure is the minimal sequence required for AAV DNA replication, provirus integration, and packaging of progeny AAV DNA into virus particles. The left ORF encodes four nonstructural Rep proteins. These proteins not only are the regulators of AAV transcription (Labow et al. (1986) J. Virol. 60:251-258) but also are involved in AAV replication (Snyder et al. (1990) J. Virol. 64:6204-6213) and virus assembly (King et al. (2001) EMBO J. 20:3282-3291) and play a role in site-specific integration of the viral genome into the host chromosome during latent infection (Linden et al. (1996) Proc. Natl. Acad. Sci. USA 93:11288-11294). The sequences of the Rep ORFs of AAV2, AAV3, AAV4, and AAV6 are approximately 85% identical, but AAV5 has only 54.5% homology with the other AAV serotypes (Chiorini et al. (1999) J. Virol. 73:1309-1319). The right half of the AAV genome encodes three viral capsid proteins referred to as VP1, VP2, and VP3 and is less conserved than the Rep ORF. AAV2, AAV3, and AAV6 share about 80% homology in the amino acid sequences of the capsid proteins. However, an alignment of the capsid protein ORFs of all six serotypes results in a reduction of the overall amino acid identity to less than 45% (Bantel-Schaal et al. (1999) J. Virol. 73:939-947). This diversity in the capsid protein sequences is likely the basis for differences in the serological characteristics and altered tissue tropism among the six AAV serotypes.
A particularly preferred parvovirus is the adeno-associated virus-2 (AAV2). The AAV2 sequence is available through Genbank under accession no. gi:9626146. AAV2 has a broad host range and until recently, all human cells were thought to be infectable. However, certain cells of the central nervous system are inaccessible with AAV2. For example, AAV2 has poor tropism for human myeloid stem cells, or cells from the lymphocyte lineage. AAV2 is not associated with any disease, therefore making it safe for gene transfer applications (Cukor et al. (1984), The Parvoviruses, Ed. K. I. Bems, Plenum, N. Y., 33-36; Ostrove et al. (1981), Virology 113: 521). AAV2 integrates into the host genome upon infection so that transgene can be expressed indefinitely (Kotin et al. (1990), Proc. Natl. Acad. Sci. USA 87: 221; Samulski et al. (1991), EMBO J. 10: 3941). Integration of AAV2 into the cellular genome is independent of cell replication which is particularly important since AAV can thus transfer genes into quiescent cells (Lebkowski et al. (1988), Mol. Cell. Biol. 8: 3988).
Another particularly preferred parvovirus is the adeno-associated virus-1 (AAV1). The AAV1 sequence is available through GenBank under accession no. gi:9632547. The AAV1 genome shows more than 80% identity with other known AAV and contains the characteristic structural features. There is approximately 80% homology in the nucleotide sequence between AAV1 and AAV2. The ITRs of AAV1 are predicted to form T-shaped hairpin structures. The right and left ITRs of AAV1 are identical and virtually the same as the right ITR of AAV6, except for 1 nucleotide in the A and A' sequences and the last nucleotide in the D sequence. The AAV2 Rep binding motif found in the AAV2 preintegration region in human chromosome 19 is well conserved in AAV1. The terminal repeats of AAV1 are 143 nucleotides long, while those of AAV2, AAV3, and AAV4 are 145 or 146 nucleotides long. The p5 promoter region of AAV1 shows some divergence from homologous regions of other AAV serotypes but maintains critical regulatory elements; the repeated YY1 sites are present throughout all known AAV serotypes, including AAV1. The p19 promoter, the p40 promoter, and poly(A) can also be identified in the AAV1 genome by homology to those in known AAV serotypes, which are also highly conserved (See Xiao et al. (1999) J Virol 73:3994-4003).
Examples of a suitable transgene used in the recombinant vector of the invention include gene sequences for the disease or transgene that confers a therapeutic effect to diseases such as amyloid polyneuropathy, Alzheimer's Disease, Duchenne's muscular dystrophy, ALS, Parkinson's Disease (e.g., glutamic acid decarboxylase gene for therapeutic effect) and brain tumors. The transgene may also be a selectable marker gene which is any gene sequence capable of expressing a protein whose presence permits selective propagation of a cell which contains it. Examples of selectable markers include gene sequence capable of conferring host resistance to antibiotics (such as ampicillin, tetracycline, kanamycin, etc.), amino acid analogs, or permitting growth of bacteria on additional carbon sources or under otherwise impermissible culturing conditions.
The skilled artisan can appreciate that regulatory sequences to control expression of the transgene can often be provided from commonly used promoters derived from viruses such as, polyoma, Adenovirus 2, lentivirus, retrovirus, and Simian Virus 40. Use of viral regulatory elements to direct expression of the transgene can allow for high level constitutive expression of the protein in a variety of host cells. Ubiquitously expressing promoters can also be used include, for example, the early lentivirus, retrovirus, promoter Boshart et al. (1985) Cell 41:521-530, herpesvirus thymidine kinase (HSV-TK) promoter (McKnight et al. (1984) Cell 37: 253-262), β-actin promoters (e.g., the human β-actin promoter as described by Ng et al. (1985) Mol. Cell Biol. 5: 2720-2732) and colony stimulating factor-1 (CSF-1) promoter (Ladner et al., (1987) EMBO J. 6: 2693-2698).
Alternatively, the regulatory sequences can direct expression of the transgene preferentially in a particular cell type, i.e., tissue-specific regulatory elements can be used. Preferred promoters are those functional in the central nervous system. Particularly preferred promoters are Chicken beta Active (CBA) and neuron-specific elonase (NSE). The promoter can be any desired promoter, selected based on the level of expression required of the transgene operably linked to the promoter and the cell type in which the vector is used. The promoter may also be an AAV2 promoter selected from the group consisting of p5, p19 and p40. In a preferred embodiment, the promoter is an AAV2 p5 promoter.
The recombinant vectors can be packaged into a particle using a transgene flanked by the same parvovirus ITR sequences e.g., AAV2 ITR sequences. In another embodiment, the transgene can be flanked by inverted terminal repeat sequences from two different parvoviruses. For example, the 5' ITR can be derived from AAV2 and the 3' ITR can be derived from AAV5, as long as at least one ITR comprises a packaging sequence required to package the chimeric capsid. In one embodiment, the chimeric capsid is produced with one ITR sequence from a AAV2 and the second ITR from a parvovirus selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, and the like. In a preferred embodiment, the ITR sequences are from AAV2. In another embodiment, the transgene may also be flanked with an ITR sequence from a parvovirus and an ITR sequence from a virus. For example, the 5' ITR can be derived from AAV2 and the 3' ITR can be derived from an adenovirus as long as at least one ITR comprises a packaging sequence and functions as intended to package the virus.
The ITR sequences for AAV2 are described, for example by Kotin et al. (1994) Human Gene Therapy 5:793-801; Berns "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds.) The skilled artisan will appreciate that AAV ITR's can be modified using standard molecular biology techniques. Accordingly, AAV ITRs used in the vectors of the invention need not have a wild-type nucleotide sequence, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides. The ITR's flanking the transgene need not necessarily be identical or derived from the same AAV serotype or isolate, so long as the ITR's function as intended, i.e., to allow for excision and replication of the bounded nucleotide sequence of interest when AAV rep gene products are present in the cell. Modified ITR's have been generated and have been shown to function for their intended purpose. The modified terminal repeat sequences were competent for AAV DNA replication, encapsidation, infection, integration, and subsequent rescue from the chromosome when superinfected with Ad and wild-type AAV (See e.g., Xiao et al. (1997) J Virol 71:941-948, and U.S. Pat. No. 6,346,415).
Wild-type AAV ITRs provide a functional origin of replication (ori) and function in cis for AAV DNA replication and for rescue or excision from prokaryotic plasmids. An ITR comprises two regions, the hairpin (HP) region and the D sequence. The HP sequence comprises the terminal 125 nucleotides of the AAV2 ITR, while the D sequence comprises the adjoining 20 nucleotides. In addition, the terminal resolution site (trs) lies between the HP region and the D sequence.
The HP region contains palindromic sequence elements in the order A, C', C, B', B, A', and thus can fold back on itself to form a T-shaped hairpin structure (Muzyczka, (1992) Curr. Top. Microbiol. Immunol. 158:97-129). The terminal HP structure is apparently used as a primer for initiation of viral DNA replication, converting the single-stranded genome into a double-stranded template with a covalently closed hairpin at one end (Berns and Bohenzky, (1987) Adv. Vir. Res. 32: 243-306; and Lusby et al., (1980) J. Virol. 34: 402409).
The D sequence, which is not involved in forming the T-shaped structure of the ITR, appears to play a crucial role in high-efficiency rescue, selective replication and encapsidation of the AAV genome (Wang et al., (1997) J. Virol. 71: 3077-3082). Analysis of several D sequence mutants has shown that, when the 10 nucleotides of the D sequence distal to the HP were removed, the AAV genome could undergo efficient rescue, replication and encapsidation. However, when the deletion was extended to 15 nucleotides, rescue, replication and packaging were severely compromised.
The trs lies at the junction of the D sequence and HP sequences. The trs appears to be specifically bound and cleaved by Rep78 and Rep68 (Im and Muzyczka, (1990), Cell 61: 447-457; Im and Muzyczka, (1992), J. Virol. 66: 1119-1128; Snyder et al., (1990) Cell 60: 105-113).
Not all of the ITR appears to be essential for its various functions. For example, the 10 nucleotides of the D sequence distal to the HP region can apparently be deleted without impairing rescue, replication and encapsidation. However, much of the terminal 125 nucleotides of the HP region appears to be needed for DNA replication and terminal resolution (Bohenzky et al., (1988) Virology 166:316-327).
The recombinant vector can be constructed by directly inserting the transgene into an AAV genome which has had the major AAV open reading frames ("ORFs") excised therefrom. Other portions of the AAV genome can also be deleted, as long as a sufficient portion of the ITRs remain to allow for replication and packaging functions. These constructs can be designed using techniques well known in the art. (See, e.g., Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka (1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin (1994) Human Gene Therapy 5:793-801; Shelling et al. (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875).
Deletion or replacement of the AAV genome, e.g., the capsid region of the AAV2, results in an AAV2 nucleic acid which is incapable of encapsidating itself. The chimeric capsid proteins can be provided using a nucleic acid construct that encodes the chimeric capsid proteins. The chimeric capsid proteins are provided in one or more expression vector(s) which are introduced into a host cell along with the AAV2 nucleic acid.
Plasmid expression vectors can typically be designed and constructed such that they contain a transgene encoding a protein or a portion of a protein necessary for encapsidation of the recombinant AAV2 nucleic acid i.e., the chimeric capsid proteins, or capsids that modify the tropism of the vector. Generally, construction of such plasmids can be performed using standard methods, such as those described in Sambrook, J. et al. Molecular Cloning: A Laboratory Manual, 2nd edition (CSHL Press, Cold Spring Harbor, N.Y. 1989).
The conditions under which plasmid expression vectors are introduced into a host cell vary depending on certain factors. These factors include, for example, the size of the nucleic acid of the plasmid, the type of host cell, and the desired efficiency of transfection. There are several methods of introducing the recombinant nucleic acid into the host cells which are well-known and commonly employed by those of ordinary skill in the art. These transfection methods include, for example, calcium phosphate-mediated uptake of nucleic acids by a host cell and DEAE-dextran facilitated uptake of nucleic acid by a host cell. The methods that are most efficient in each case are typically determined empirically upon consideration of the above factors.
As with plasmid expression vectors, viral expression vectors can be designed and constructed such that they contain a foreign gene encoding a foreign protein or fragment thereof and the regulatory elements necessary for expressing the foreign protein. Examples of such viruses include retroviruses, adenoviruses and herpesvirus.
Vectors without the rep gene appear to replicate and integrate at random sites in the host cell genome, while expression of Rep proteins Rep 68 and Rep 78, can mediate genomic integration into a well-defined locus on human chromosome 19 (Kotin, et al., Proc. Natl. Acad. Sci. USA 87:2211-2215 (1990); Samulski, et al., (1991) EMBO J 10:3941-3950; Giraud, et al., (1994) Proc. Natl. Acad. Sci. USA 91:10039-10043; Weitzman et al., (1994) Proc. Natl. Acad. Sci. USA 91:5808-5812). The plasmid bearing the cap genes can encode a chimeric capsid comprising a cap gene from a parvovirus, e.g., AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6, and the like, or a portion thereof, or a virus, e.g., herpesvirus, adenovirus, lentivirus, retrovirus, Epstein-Barr virus and vaccinia virus. Non-native cap genes can be derived from a parvovirus that is different from the wild type parvovirus. For example, by encapsulating a wildtype AAV vector with a capsid protein from an AAV that is different than the wild type AAV. In one embodiment, the wildtype AAV2 type vector is encapsulated with a capsid protein derived from an AAV1 type virus.
Suitable host cells for producing particles comprising the chimeric capsids or non-native capsids include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a exogenous nucleic acid molecule.
Cells from the stable human cell line, 293 (readily available through, e.g., the ATCC under Accession No. ATCC CRL1573) are preferred in the practice of the present invention. Particularly, the human cell line 293 is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), and expresses the adenoviral E1a and E1b genes (Aiello et al. (1979) Virology 94:460). The 293 cell line is readily transfected, and provides a particularly convenient platform in which to produce virions.
The entry of viral expression vectors into host cells generally requires addition of the virus to the host cell media followed by an incubation period during which the virus enters the cell. Incubation conditions, such as the length of incubation and the temperature under which the incubation is carried out, vary depending on the type of host cell and the type of viral expression vector used. Determination of these parameters is well known to those having ordinary skill in the art. In most cases, the incubation conditions for the infection of cells with viruses typically involves the incubation of the virus in serum-free medium (minimal volume) with the tissue culture cells at 30° C. for a minimum of thirty minutes. For some viruses, such as retroviruses, a compound to facilitate the interaction of the virus with the host cell is added.
Recombinant AAV vectors can be packaged into particles by co-transfection of cells with a plasmid bearing the AAV replication and/or cap genes (e.g., chimeric cap genes or non-native cap genes). The replication and cap genes encode replication proteins or capsid proteins (e.g., chimeric capsids or non-native capsids), respectively and mediate replication and genomic integration of AAV sequence, as well as packaging and formation of AAV particles (Samulski (1993) Current Opinion in Genetics and Development 3:74-80; Muzyczka, (1992) Curr. Top. Microbiol. Immunol. 158:97-129).
Generally, AAV helper function vectors can be engineered using conventional recombinant techniques. Particularly, nucleic acid molecules can be readily assembled in any desired order by inserting one or more accessory function nucleotide sequences into a construct, such as by ligating restriction fragments or PCR-generated products into a cloning vector using polylinker oligonucleotides or the like. The newly formed nucleic acid molecule can then be excised from the vector and placed in an appropriate expression construct using restriction enzymes or other techniques that are well known in the art.
The AAV helper function vectors can be used in a variety of systems for recombinant AAV virion production. For example, suitable host cells that have been transfected with an AAV helper function vector are capable of producing recombinant AAV virions when co-transfected with an AAV vector. One or more accessory function vectors capable of being expressed in the cell may also be co-transfected to provide accessory functions. The AAV vector, AAV helper construct and the accessory function vector(s) can be introduced into the host cell, either simultaneously or serially, using transfection techniques described above.
The chimeric capsid or non-native capsid can also be produced in a suitable host cell and can be used as a delivery vehicle for an operatively linked transgene.
Standard methods of infectivity known to the skilled artisan can be used to test for the altered tropism (See e.g., Grimm et al. (1998) Hum Gene Ther 10: 2745-60). For example, efficiency of entry can be quantitated by introducing a recombinant vector with a chimeric capsid or non-native capsid into the wild type AAV vector and monitoring transduction as a function of multiplicity of infection (MOI). A reduced MOI of the recombinant vector comprising chimeric capsid, or recombinant pseudotyped vector comprising a non-native capsid, compared to a recombinant vector with a wild type capsid indicates a more efficient vector. For example, fewer AAV5 particles than AAV2 are required to get transduction into a cell in a target organ, e.g., brain.
Examples of attachment sites present on a surface cell types that can be targeted by the recombinant vector with the chimeric capsid or a recombinant pseudotyped vector with native capsid include, but are not limited to heparin and chondroitin sulfate moities found on glycosaminoglycans, sialic acid moieties found on mucins, glycoproteins, gangliosides, MHC class I glycoproteins, common carbohydrate components found in the cell membrane glycoproteins including mannose, N-acetyl-galactosamine, N-acetyl-glucosamine, fucose, galactose and the like. Particularly preferred attachments sites are those present on neural cells.
II Recombinant Pseudotyped Vectors
In one aspect the invention features recombinant pseudotyped vectors comprising a non-native capsid that is derived form a parvovirus other than the wild type parvovirus. In a preferred embodiment, the recombinant pseudotyped parvovirus are recombinant pseudotyped AAV virion vectors. The recombinant pseudotyped adeno-associated virion comprises a transgene flanked 5' and 3' by inverted terminal repeat sequences that can be derived from a first adeno-associated virus, where the first adeno-associated virus is a wild type adeno-associated virus. The transgene is encapsulated in a non-native capsid derived from a second adeno-associated virus that is different from the first adeno-associated virus, such that the transgene is packaged within the non-native capsid. Once packaged, the non-native capsid provides a modified tropism and can bind to an attachment site present on a cell surface in the central nervous system of a subject with a higher affinity than a corresponding adeno-associated virion with a wild type capsid. Upon entry into the cell the transduction rate of the pseudotyped virion is at least about 2 fold to about 100 fold higher than the corresponding wild type virion, preferably about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, and 100-fold higher than the corresponding wild type virion.
With the recombinant pseudotyped vectors, the AAV capsid packages an AAV genome of a different AAV type. For example, a recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 and the like type genome may be encapsidated within an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 capsid, provided that the AAV capsid and genome are of different types.
In particularly preferred embodiments, the recombinant pseudotyped AAV of vector has an AAV-2 genome encapsulated within an AAV1, AAV3, AAV4, AAV5, AAV6, and the like capsid. In particular preferred embodiment, the recombinant pseudotyped AAV vector comprises an AAV2 type genome encapsulated with an AAV1 capsid. In another embodiment, the recombinant pseudotyped AAV virions comprises an AAV2 type genome encapsulated with an AAV5 capsid.
The recombinant pseudotyped vectors can be prepared by using the same methodology described above using the pseudotyped helper functions. The pseudotyped AAV helper function vectors can be engineered using conventional recombinant techniques. These pseudotyped AAV helper functions comprise a capsid region that can be derived from any AAV serotype that is different from the wild type AAV. The cap coding region can be derived from AAV serotypes that include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, and the like. In one embodiment, the entire capsid region of the wild type AAV vector is replaced with the entire capsid region of a different AAV vector. In another embodiment, the entire capsid region of a different AAV vector can be altered by mutations (point, additions, substitutions). Other natural variants of the capsid region are also within the scope of the invention. Preferred capsids are derived from the AAV1, or the AAV5 serotype.
The pseudotyped AAV helper function vectors also comprise a rep coding region. The rep coding region can be derived from any AAV serotypes that include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, and the like, as long as it functions for its intended purpose. In one embodiment, the rep coding region is derived from the wild type AAV vector. For example, if the wild type AAV is AAV2, then the rep coding region can be derived from AAV2. In another embodiment, the rep coding region is derived from a AAV vector that is different from the wild type AAV. Examples of pseudotyped helper constructs are described in detail in Example 1 and shown in FIG. 1.
The pseudotyped AAV helper function vectors can be used in a variety of systems for recombinant pseudotyped AAV virion production. For example, suitable host cells that have been transfected with a pseudotyped AAV helper function vector are capable of producing recombinant pseudotyped AAV virions when cotransfected with an AAV vector comprising a transgene or transgene expression cassette. One or more accessory function vectors capable of being expressed in the cell may also be cotransfected to provide accessory functions. The AAV vector comprising a transgene or transgene expression cassette, the pseudotyped AAV helper construct and the accessory function vector(s) can be introduced into the host cell, either simultaneously or serially, using transfection techniques described above.
Suitable host cells for producing particles comprising the pseudotyped AAV helper constructs include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a exogenous nucleic acid molecule. Cells from the stable human cell line, 293 (readily available through, e.g., the ATCC under Accession No. ATCC CRL1573) are preferred in the practice of the present invention. Particularly, the human cell line 293 is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), and expresses the adenoviral E1a and E1b genes (Aiello et al. (1979) Virology 94:460). The 293 cell line is readily transfected, and provides a particularly convenient platform in which to produce virions. Example 2 describes how to produce recombinant pseudotyped virions by cotransfection into 293 kidney cells.
Standard methods of infectivity known to the skilled artisan can be used to test for the alter tropism (See e.g., Grimm et al. (1998) Hum Gene Ther 10: 2745-60). For example, efficiency of entry can be quantitated by introducing a recombinant pseudo typed into a cell and monitoring transduction as a function of multiplicity of infection (MOI). A reduced MOI of the recombinant pseudotyped vector comprising a non-native capsid, compared to a recombinant vector with a wild type capsid indicates a more efficient vector. For example, fewer AAV1-AAV2 particles are required than wild type AAV2, to get transduction into a cell in a target organ, e.g., brain.
The Examples show that infectious recombinant pseudotyped vectors can be produced using the pseudotyped helper constructs of the invention. The yields of the different serotypes are shown in the FIG. 2. The yield of pseudotyped AAV1-AAV2 was about 5 to 10 times higher than the yield of AAV2-AAV2 (wild type). These higher yields are important particularly when the vectors are to be delivered to the central nervous system, e.g., regions of the brain. Due to the high yields of genomic particles which makes the vectors highly concentrated, the pseudotyped vector can be delivered to the central nervous system, e.g., a region of the brain, in smaller volume of a suitable carrier.
The pseudotyped vectors also transfected different regions of the brain, such as the as the stratium and hippocampus. The transduction results demonstrate that the AAV1-AAV2 pseudotype vector transduced almost all the hippocampus area and stratium. The AAV1-AAV2 pseudotyped vector diffused a greater distance from the injection site than the wild type vector, and transduces a more extensive cell number and volume in the central nervous system, than the other pseuodotyped vectors.
The pseudotyped AAV vectors of the invention can be used to escape pre-existing immune responses in a subject. For example, the AAV1-AAV2 pseudotyped vector containing the AAV1 capsid can be used is to escape the pre-existing immune responses to the AAV2 vector. The AAV1-AAV2 pseudotyped vector can be used in the patients who already has AAV2 neutralizing antibodies by natural infection or by previous administration of AAV2 vectors. The pseudotyped vectors of the invention can also be co-administration with one or more other pseudotyped vectors. For example, if two or more genes are required to be transduced into same cells, separating one gene from the other and placing them in different pseudotyped vectors, may increase the co-transduction rate because each pseudotyped vector uses a different receptor to bind to, and enter the cell.
III Recombinant Vectors Comprising Chimeric Capsids
The invention also features a method of producing recombinant vectors comprising a chimeric capsid. Recombinant vectors can be constructed using known techniques to provide operatively linked components of control elements including a transcriptional initiation region, a transgene, and a transcriptional termination region, as described above.
The recombinant viral vectors comprising a chimeric capsid have at least one non-native amino acid sequence, where the non-native amino acid sequence is derived from a capsid protein domain of a parvovirus, a virus, or a combination thereof, and where the chimeric capsid is capable of binding to an attachment site present on a cell surface of a neural cell; and a transgene flanked 5' and 3' by inverted terminal repeat sequences. The inverted terminal repeat sequences can be derived from a parvovirus, a virus, or a combination thereof, as long as at least one inverted terminal repeat sequence comprises a packaging signal that allows assembly of the chimeric capsid.
In one embodiment, the invention features a recombinant AAV2 vector comprising a chimeric capsid having at least one native AAV2 amino acid sequence and at least one non-native amino acid sequence derived from a parvovirus other than AAV2, wherein the chimeric capsid is capable of binding to an attachment site present on a cell surface; and a transgene flanked 5' and 3' by a first inverted terminal repeat sequences derived from AAV2 and a second inverted terminal repeat sequence derived from a parvovirus.
The chimeric capsids can be constructed in which the capsid region comprising the capsid viral protein subunits, VP1, VP2 and VP3 of a first AAV, can be replaced entirely with the VP1, VP2 and VP3 subunits of a second AAV. Alternatively, the chimeric capsid can be constructed so that a portion of a capsid subunits can be replaced. The nucleotide sequences for the various AAV serotypes are available from Genbank using the accession numbers provided above, as well as from a number of references such as Chiorini et al. (1999) J. Virol. 73: 1309-1319; Xiao et al. (1999) J. Virol. 73: 3994-4003 and Rutledge et al. (1998) J. Virol. 72: 309-319.
The entire capsid region of the AAV, or the individual VP regions, can be generated using standard molecular biology techniques such as PCR amplification, as described, for example, Sambrook J., Fritsch E. F., Maniatis T.: Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory, 1989) and the Examples section.
The entire capsid coding region (i.e. VP1, VP2, and VP3) for AAV1 spans from nucleotide positions 2222 (ATG) through 4433 (TAA) of SEQ ID NO: 1. More specifically, the VP1 region begins at nucleotide position 2222 (ATG) and ends at nucleotide position 2827. The VP2 region begins at nucleotide position 2828 (ACG) and ends at nucleotide position 2806. The VP3 region begins at nucleotide position 2807 (ATG) and ends at nucleotide position 4410.
The entire capsid coding region (i.e. VP1, VP2, and VP3) for AAV2 spans from nucleotide positions 2203 (ATG) through 4410 (TAA) of SEQ ID NO: 2. More specifically, the VP1 region begins at nucleotide position 2203 (ATG) and ends at nucleotide position 2613. The VP2 region begins at nucleotide position 2614 (ACG) and ends at nucleotide position 2808. The VP3 region begins at nucleotide position 2809 (ATG) and ends at nucleotide position 4410.
The entire capsid coding region (i.e. VP1, VP2, and VP3) for AAV3 spans from nucleotide positions 2209 (ATG) through 4410 (TAA) of SEQ ID NO: 3. More specifically, the VP1 region begins at nucleotide position 2209 (ATG) and ends at nucleotide position 2619. The VP2 region begins at nucleotide position 2620 (ACG) and ends at nucleotide position 2814. The VP3 region begins at nucleotide position 2815 (ATG) and ends at nucleotide position 4419.
The entire capsid coding region (i.e. VP1, VP2, and VP3) for AAV4 spans from nucleotide positions 2260 (ATG) through 4464 (TAA) of SEQ ID NO: 4. More specifically, the VP1 region begins at nucleotide position 2260 (ATG) and ends at nucleotide position 2667. The VP2 region begins at nucleotide position 2668 (ACG) and ends at nucleotide position 2853. The VP3 region begins at nucleotide position 2854 (ATG) and ends at nucleotide position 4464.
The entire capsid coding region (i.e. VP1, VP2, and VP3) for AAV5 spans from nucleotide positions 2207 (ATG) through 4381 (TAA) of SEQ ID NO: 5. More specifically, the VP1 region begins at nucleotide position 2207 (ATG) and ends at nucleotide position 2614. The VP2 region begins at nucleotide position 2615 (ACG) and ends at nucleotide position 2782. The VP3 region begins at nucleotide position 2783 (ATG) and ends at nucleotide position 4381.
The entire capsid coding region (i.e. VP1, VP2, and VP3) for AAV6 spans from nucleotide positions 2208 (ATG) through 4418 (TAA) of SEQ ID NO: 6. More specifically, the VP1 region begins at nucleotide position 2208 (ATG) and ends at nucleotide position 2618. The VP2 region begins at nucleotide position 2619 (ACG) and ends at nucleotide position 2813. The VP3 region begins at nucleotide position 2814 (ATG) and ends at nucleotide position 4418.
In one embodiment, the chimeric capsids of the recombinant vectors are produced by "complete substitutions", this term as used herein refers to replacing the entire capsid viral protein domain of the host with a non-native amino acid sequence. For example, a recombinant AAV2 vector in which the amino acid sequence of the VP1 domain of AAV2 is retained, but the entire amino acid sequence of the VP2 and VP3 domain of AAV2 is replaced with the entire amino acid sequence of the VP2 domain from another parvovirus, such as AAV5.
In another embodiment, the chimeric capsids of the recombinant vectors are produced by "patch substitution" this term as used herein refers to replacing a fragment of the capsid viral protein domain of the host with a fragment of non-native amino acid sequence from another parvovirus. For example, a recombinant AAV2 vector in which a fragment of the amino acid sequence of the VP1 domain of AAV2 is replaced with a corresponding fragment of a non-native amino acid sequence from AAV5. The non-native amino acid sequence preferably comprises a determinant that alters the tropism of the capsid. The altered tropism can allow the chimeric capsid to bind to an attachment site on cell surface with a higher affinity than a wild type capsid. The modified tropism of the chimeric capsid allows a wider range of host cells to be targeted. The infective properties of such a particle can be improved above those of a recombinant vector containing a wild type capsid. Alternatively, the altered tropism can prevent the chimeric capsid from binding to an attachment site on a cell surface. This provides for a method of selecting cell types for specific targeting of a transgene, while excluding expression of the transgene where it is not wanted. Other embodiments include mutations (single amino acid substitution or deletion mutations) within the capsid viral protein domain that alter the tropism.
In one embodiment, the invention features recombinant vectors with a chimeric capsid where the chimeric capsid comprises fragments of the entire AAV2 capsid protein, VP1, VP2, or VP3 sequences. The fragments can be an amino acid sequence comprising about 10 amino acids, more preferably about 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 and 200 or more amino acids in length.
Additionally, modifications can be made to the nucleic acid molecule encoding the capsid protein or fragment thereof, such that modifications to the nucleotide sequences that encode a capsid protein produce a capsid protein with a modified amino acid sequence. Such means of generating modification to a sequence are standard in the art (See e.g., Sambrook J., Fritsch E. F., Maniatis T.: Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory, 1989) and can be performed.
Also within the scope of the invention are AAV2 recombinant vectors with a chimeric capsid comprising VP1, VP2, VP3 proteins that can have at least 60% homology to the polypeptide encoded by nucleotides at position 2202 to nucleotide at position 4412 of AAV2 (Genbank accession no. gi:9626146). The capsid protein can have about 70% homology, about 75% homology, about 80% homology, about 85% homology, about 90% homology, about 95% homology, about 99% homology to the polypeptide encoded by nucleotides at position 2202 to nucleotide at position 4412.
In another aspect, the invention features a recombinant pseudotyped parvovirus vector that comprises a wild type parvovirus genome, and a non-native capsid derived from a parvovirus that is different from the wild type parvovirus. In one embodiment, the recombinant pseudotyped parvovirus vector is a recombinant pseudotyped AAV vector.
It is also preferred that the wild type AAV genome comprises one or more AAV inverted terminal repeat(s). Typically, a recombinant AAV genome will retain only those elements required in cis (e.g., one or more AAV ITRs), with the rest of the genome (e.g., the rep/cap genes) being provided in trans.
In another embodiment, the recombinant vector of the invention can be a vector comprising a chimeric capsid containing amino acid sequences from a parvovirus, and a non-native amino acid sequence from a virus that can be used to target a neural cell. Examples of a suitable virus include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6. Examples of a suitable virus include, but are not limited to, herpesvirus, adenovirus, lentivirus, retrovirus, Epstein-Barr virus and vaccinia virus. The recombinant vector with a chimeric capsid can have an altered tropism that allows the capsid coat to bind to the surface of cell types with a higher affinity than a recombinant vector with a wild type capsid. Alternatively, the modified tropism prevents the capsid from targeting particular cell types.
The skilled artisan can appreciate there are numerous viruses that can comprise capsid proteins which can be used to construct the recombinant vector with the chimeric capsid. For example, the herpesviruses is a large double stranded DNA viruses consisting of an icosahedral capsid surrounded by an envelope. The group has been classified as alpha, beta and gamma herpesviruses on the basis of genome structure and biological properties (See e.g., Roizman et al. (1981) Int. virology 16, 201-217). The herpes particle constitutes over 30 different proteins which are assembled within the host cell. About 6-8 are used in the capsid.
The herpes simplex virus 1 (HSV-1) genome specifies an abundant capsid protein complex which in denaturing gels forms multiple bands due to different molecular weights of the component proteins. Details of the HSV-1 capsid have been well documented, see for example, Davison et al. (1992) J. Gen. Virol. 73:2709-2713; Gibson et al. (1972) J. Virol. 10:1044-1052; and Newcomb et al., (1991) J. Virol., 65:613-620). Several herpesvirus sequences are available from GenBank.
The human adenovirus is comprised of a linear 36 kilobase double-stranded DNA genome, which is divided into 100 map units, each of which is 360 base pair in length. The DNA contains short inverted terminal repeats (ITR) at each end of the genome that are required for viral DNA replication. The gene products are organized into early (E1 through E4) and late (L1 through L5) regions, based on expression before or after the initiation of viral DNA synthesis (See, e.g., Horwitz, Virology, 2d edit., ed. B. N. Fields, Raven Press, Ltd. New York (1990)).
The adenovirus capsid has been well characterized and nucleic acid molecules of various adenoviruses are available in GenBank. Adenovirus interacts with eukaryotic cells by virtue of specific receptor recognition by domains in the knob portion of the fiber protein which protrude from each of the twelve vertices of the icosahedral capsid (See e.g., Henry et al. (1994) J. Virol. 68:5239-5246; Stevenson et al. (1995) J. Virol. 69:2850-2857; and Louis et al. (1994) J. Virol. 68:4104-4106). These or other regions of the adenovirus capsid may be used to construct the chimeric capsid of the invention. Nucleic acid sequences of many lentivirus, retrovirus types are available from GenBank.
IV Administration of Recombinant Vectors
Administration of the recombinant vectors of the invention (i.e., recombinant vectors comprising a chimeric capsid or recombinant pseudotyped vectors comprising a non-native capsid) to a cell (e.g., a neural cell) can be accomplished by standard methods in the art. Preferably, the vector is packaged into a particle and the particle is added to the cells at the appropriate multiplicity of infection. The modified tropism of the recombinant vector allows the capsid to interact with an attachment site on a cell surface of a neural cell that a wild type capsid fails to interact with, for example, the AAV2 has a poor tropism for human myeloid stem cells. However, a recombinant vector with a chimeric capsid comprising non-native capsid proteins from different member of the parvovirus family, or the recombinant pseudotyped vectors may confer the ability to AAV2 to interact with human myeloid stem cells. Alternatively, the modified tropism can prevent the capsid from interacting with a particular cell type, to thereby selectively target desired cell types.
Administration of the recombinant vectors of the invention to the cell can be by any means, including contacting the recombinant vector with the cell. For such in vitro method, the vector can be administered to the cell by standard transduction methods. (See e.g., Sambrook, Supra.) The cells being transduced can be derived from a human, and other mammals such as primates, horse, sheep, goat, pig, dog, rat, and mouse. Cell types and tissues that can be targeted include, but are not limited to, adipocytes, adenocyte, adrenal cortex, amnion, aorta, ascites, astrocytes, bladder, bone, bone marrow, brain, breast, bronchus, cells of the central nervous system (CNS), cardiac muscle, cecum, cervix, chorion, colon, conjunctiva, connective tissue, cornea, dermis, duodenum, endometrium, endothelium, epithelial tissue, epidermis, ependymal, esophagus, eye, fascia, fibroblasts, foreskin, gastric, glial cells, glioblast, gonad, hepatic cells, histocyte, ileum, intestine, small intestine, jejumim, keratinocytes, kidney, larynx, leukocytes, lipocyte, liver, lung, lymph node, lymphoblast, lymphocytes, macrophages, mammary alveolar nodule, mammary gland, mastocyte, maxilla, melanocytes, monocytes, mouth, microglia, myelin, nervous tissue, neural cells, neuroblast, neurons, neuroglia, oligodendrocytes, osteoblasts, osteogenic cells, ovary, palate, pancreas, papilloma, cells of the peripheral nervous system, peritoneum, pituicytes, pharynx, placenta, plasma cells, pleura, prostate, rectum, salivary gland, skeletal muscle, skin, smooth muscle, somatic, spleen, squamous, stomach, submandibular gland, submaxillary gland, synoviocytes, testis, thymus, thyroid, trabeculae, trachea, turbinate, umbilical cord, ureter, and uterus. In a preferred embodiment, the cells are neural cells.
The recombinant vectors of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises the recombinant vectors of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion.
The recombinant vectors of the invention can be incorporated into a pharmaceutical composition suitable for parenteral administration. Other suitable buffers include but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride can be used to modify the toxicity of the solution at a concentration of 0-300 mM (optimally 150 mM for a liquid dosage form). Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Bulking agents can be included for a lyophilized dosage form, principally 1-10% mannitol (optimally 2-4%). Stabilizers can be used in both liquid and lyophilized dosage forms, principally 1-50 mM L-Methionine (optimally 5-10 mM). Other suitable bulking agents include glycine, arginine, can be included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants.
The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antigen, antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and spray-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
The pharmaceutical compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of the recombinant vector. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the recombinant vector may vary according to factors such as the disease state, age, sex, and weight of the individual and the ability of the vector to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the recombinant vector is outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
For in vitro administration of the vectors of the invention into a neural cells, standard procedures such as transduction can be performed. (See e.g., Sambrook, id.). For in vivo administration of the vectors of the invention, broad distribution of the vectors into the CNS can be accomplished by injecting the vector into the cerebrospinal fluid, e.g., by lumbar puncture (See e.g., Kapadia et al. (1996) Neurosurg 10: 585-587). Alternatively, precise delivery of the vector into specific sites of the brain to target a neural cell, can be conducted using stereotactic microinjection techniques. For example, the subject being treated can be placed within a stereotactic frame base (MRI-compatible) and then imaged using high resolution MRI to determine the three-dimensional positioning of the particular region to be treated. The MRI images can then be transferred to a computer having the appropriate stereotactic software, and a number of images are used to determine a target site and trajectory for antibody microinjection. The software translates the trajectory into three-dimensional coordinates that are precisely registered for the stereotactic frame. In the case of intracranial delivery, the skull will be exposed, burr holes will be drilled above the entry site, and the stereotactic apparatus used to position the needle and ensure implantation at a predetermined depth. The vector can be delivered to regions, such as the cells of the stratium, hippocampus, spinal cord, brainstem, (medulla, pons, and midbrain), cerebellum, diencephalon (thalamus, hypothalamus), telencephalon (corpus stratium, cerebral cortex, or within the cortex, the occipital, temporal, parietal or frontal lobes), or combinations, thereof. In another preferred embodiment, the vector is delivered using other delivery methods suitable for localized delivery, such as localized permeation of the blood-brain barrier. Particularly preferred delivery methods are those that deliver the vector to regions of the brain that require modification.
Modification as used herein refers to a change in the cellular activity in the region of the brain injected with the vector. The change in cellular activity can result from changing the expression, or production of genes responsible for stimulating, activating, or inhibiting, a cell. For example, delivery of a vector comprising a nucleotide sequence encoding GAD, to a region of the brain that is overstimulated, such as the basal ganglia. In particular, delivery of the vector to the STN which are overactive in diseases such as Parkinson's, will result in expression of GAD in this region.
V. Therapeutic Uses of Recombinant Vectors
The recombinant vectors with the chimeric capsids, or the recombinant pseudotyped vectors of the invention offer the advantage over current vector systems for delivery into cells, in particular into neural cells. Due to their modified tropism, the recombinant vectors can efficiently and safely deliver transgenes to cells that are not normally targeted by vectors with a wild type capsid. The recombinant vectors of the invention may also be used to selectively target desired cell types, while excluded of the cell types based on the modified tropism. The pseudotyped vectors of the invention are particularly suitable for delivering transgenes to cells of the central nervous system. In particular to the brain or different regions of the brain.
The recombinant vector with a chimeric capsid, or the recombinant pseudotyped vectors can comprise a transgene sequence that is associated with a disease or a disorder such that expression of the transgene would result in amelioration of the disease or disorder. There are a number of neurological and neurodegenerative diseases that can benefit from such a therapy, which include, but are not limited to, Parkinson's disease, Huntington disease, Alzheimer disease, ALS, epilepsy, stroke and central nervous system tumors. These include astrocytomas, oligodendrogliomas, meningiomas, neurofibromas, ependymomas, Schwannomas, neurofibrosarcomas, glioblastomas, and the like.
The term "neurodegenerative disorder" "neurological disorder" as used herein refers to a disorder which causes morphological and/or functional abnormality of a neural cell or a population of neural cells. The neurodegenerative disorder can result in an impairment or absence of a normal neurological function or presence of an abnormal neurological function in a subject. For example, neurodegenerative disorders can be the result of disease, injury, and/or aging. Non-limiting examples of morphological and functional abnormalities include physical deterioration and/or death of neural cells, abnormal growth patterns of neural cells, abnormalities in the physical connection between neural cells, under- or over production of a substance or substances, e.g., a neurotransmitter, by neural cells, failure of neural cells to produce a substance or substances which it normally produces, production of substances, e.g., neurotransmitters, and/or transmission of electrical impulses in abnormal patterns or at abnormal times. Neurodegeneration can occur in any area of the brain of a subject and is seen with many disorders including, for example, head trauma, stroke, ALS, multiple sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease.
The recombinant vectors of the invention are particularly useful for diseases such as Parkinson's disease which is associated with a disturbances of posture, locomotion, facial expression or speech. Symptoms of Parkinson's disease are caused by loss of nerve cells in the pigmented substantia nigra pars compacta (SNPC) and the locus coeruleus in the midbrain. The stratium or corpus stratium is a structure in the cerebral hemispheres consisting of two basal ganglia (the caudate nucleus and the putnam) and the fibre of the internal capsule that separate them. Parkinson's disease in humans primarily effects the subcortical structures, especially the substantai nigra and the locus ceruleus. It is characterized by the loss of dopamine neurons in the substanta nigra, which have the basal ganglia as their major target organ. Cell loss also occurs in the globus pallidus and putamen.
Parkinson's disease is also associated with eosinophilic intraneural inclusion granules (Lewy bodies) which are present in the basal ganglia, brainstem, spinal cord, and sympathetic ganglia. The pars compacta neurons of the substantia nigra (SN) provide dopaminergic input into the stratium, which is part of the basal ganglia. These dopaminergic neurons modulate a monosynaptic gamma-aminobutyric acid (GABA) inhibitory output in the globus pallidus interna and pars reticulata of the substantia nigra. In Parkinson's disease, loss of dopaminergic cells in the substantia nigra leads to stratial dopamine depletion. This loss of dopamine alters the activity of neurons within the basal ganglia circuitry, including excessive firing and activity of these cells. Accordingly, the recombinant vectors of the invention can be used to deliver a therapeutic gene to the site of domaminergic cell loss or other regions of the basal ganglia and output nuclei.
Several animal models of Parkinson's disease have been generated in which effective therapies are indicative of therapeutic efficacy in humans. These animal models include three rat models (the rats having lesions in substantia nigral dopaminergic cells caused by treatment with 6-hydroxydopamine, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), or surgical transection of the nigral striatal pathway) (See, e.g. Bjorklund et al. (1982) Nature 298:652-654), a rhesus monkey model (the monkeys having lesions in substantia nigral dopaminergic cells caused by treatment with MPTP) (See, e.g., Smith, et al. (1993) Neuroscience 52):7-16; Bakay et al. (1985) Appl. Neurophysiol. 48:358-361; Zamir. et al. (1984) Brain Res. 322:356-360), and a sheep model (the sheep having lesions in substantia nigral dopaminergic cells caused by treatment with MPTP) (Baskin, et al. (1994) Life Sci. 54:471-479). In another embodiment, the antigen, antibody or antibody portion of the invention can be used to treat a subject with Parkinson's disease. To assess therapeutic strategies, morphological and immunohistochemical studies can be performed by conventional techniques and behavioral tests can also be performed to determine the efficacy of the therapy, such as the Barnes Circular Maze test, or the lisne crossing mobility test, as described previously (Barnes et al. (1979) J. Comp. Physiol. Psychol. 93: 74-104; and (Carlsson et al. (1990) Life Sci. 47: 1729).
The recombinant vectors of the invention can also be used to ameliorate the symptoms of Huntington's disease. Models of Huntington's diseases have been developed in several different animals. For example, rat (Isacson et al. (1985) Neuroscience 16:799-817), monkey (Kanazawa, et al. (1986) Neurosci. Lett. 71:241-246), and baboon (Hantraye. et al. (1992) Proc. Natl. Acad. Sci. USA 89:4187-4191; Hantraye., et al. (1990) Exp. Neurol. 108:91-014; Isacson, et al. (1989) Exp. Brain Res. 75(1):213-220). Neurodegeneration in Huntington's disease typically involves degeneration in one or both nuclei forming the stratium or corpus stratium, the caudate nucleus and putamen. To assess therapeutic strategies, morphological and immunohistochemical studies can be performed by conventional techniques and behavioral tests can also be performed to determine the efficacy of the therapy, such as the Barnes Circular Maze test, or the lisne crossing mobility test, as described previously (Barnes et al. (1979) J. Comp. Physiol. Psychol. 93: 74-104; and (Carlsson et al. (1990) Life Sci. 47: 1729).
The recombinant vectors of the invention can also be used to ameliorate the symptoms of Amyloid Lateral Sclerosis (ALS). Several models of ALS are available. Mutations in the superoxide dismutase gene 1 (SOD-1) are found in patients with familial amyotrophic lateral sclerosis (FALS). Overexpression of a mutated human SOD-1 gene in mice results in neurodegenerative disease as result of motor neuron loss in lumbar spinal cord, providing a suitable model for FALS (See e.g., Mohajeri et al. (1998) Exp Neurol 150:329-336). Transgenic models of ALS are also described (See e.g., Gurney (1997) J Neurol Sci 152:S67-73). Expression of mutant SOD1 genes in transgenic mice causes a progressive paralytic disease whose general features resemble ALS in humans. These models can be used to examine the effect of an antigen, antibody or antibody portion that can be used to modify the function of receptors or transporter proteins associated with ALS (e.g., EAAT2 transporter protein). A gain-of-function in these models can monitored, for example, improvement in motor impairments of the animal's limbs.
One skilled in the art will appreciate further features and advantages of the invention based on the above-described embodiments. Accordingly, the invention is not to be limited by what has been particularly shown and described, except as indicated by the appended claims. All publications and references cited herein are expressly incorporated herein by reference in their entirety.
Construction of Pseudotyped Adeno Associated Helper Plasmids
The pseudotyped adeno-associated helper plasmids for AAV1 and AAV5 are shown in FIG. 1. These pseudotyped helper plasmids were constructed using an AAV2 helper plasmid, referred to as p5E18 (Xiao, et al (1998). J. Virol. 72:10222-10226 & Xiao, et al, (1999) J. Virol. 73:3994-4003), as the backbone, and replacing its AAV2 capsid gene with either an AAV1 or AAV5 capsid gene. The primer nucleotide sequences used to generate the psueodtyped AAV vectors are based on AAV1 (Genbank accession no:gi:9632547), AAV2 (Genbank accession no: gi:9626146), and AAV5 (Genbank accession no: gi:4249656).
The AAV2 rep region (partial) was amplified using forward primer 5'-CGAGTCAGTTGCGCAGCCATCGACGTCAGA-3' (SEQ ID NO: 7) which corresponded with nucleotide positions 1847-1876 in the AAV2 genome and reverse primer 5'-CTGGAAGATAACCATCGGCAGCCATACCTGATTTAAATCATTTATTGTTC-3' (SEQ ID NO: 8) which correspond with nucleotide positions 2178-2202 in AAV2 genome using plasmid P5E18 as template. The 5' 25 nucleotides of the reverse primer (SEQ ID NO. 8) corresponded with the AAV1 genome at positions 2223-2247.
The AAV1 capsid gene was amplified using primers that corresponded with nucleotide positions 2223-2247 in AAV1 genome using forward primer 5'-GAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAG-3' (SEQ ID NO: 9) and nucleotide positions 4550-4579 using the reverse primer 5'GGACTCTAGAGTAACCCGATGACGTAAGTCTTTTATCGCG-3' (SEQ ID NO: 10).
The subsequent PCR products were linked together by PCR amplification using the rep forward primer (SEQ ID NO: 7) and the cap reverse primer (SEQ ID NO: 10). After the PCR reaction, the PCR product was digested with HindIII and XbaI and the fragment subcloned into p5E18 at the HindIII and XbaI cloning sites as described by Xiao et al. (1999) J. Virol. 73:3994-4003. The resulting plasmid is designated pHyb21, a recombinant pseudotyped adeno-associated virus with an AAV1 capsid and AAV2 rep sequences, and is shown in SEQ ID NO: 11.
The same procedure was used to generate a recombinant pseudotyped adeno-associated virus with an AAV5 capsid and AAV2 rep sequences. The AAV2 rep region was amplified using primers that corresponded with nucleotide positions 1847-1876 in AAV2 genome with forward primer 5'-CGAGTCAGTTGCGCAGCCATCGACGTCAGA-3' (SEQ ID NO: 7) and nucleotide positions 2178-2202 in AAV2 genome with reverse primer 5'-CTGGAGGGTGATCAACAAAAGACATACCTGATTTAAATCATTTATTGTTC-3' (SEQ ID NO: 8) to amplify the rep sequence using plasmid P5E18 as template (The 5' 25 nucleotides of this primer corresponded with positions 2207-2231 of AAV5 genome). The AAV5 capsid gene was amplified using primers that corresponded with nucleotide positions 2207-2231 in AAV5 genome using forward primer 5'-GAACAATAAATGATTTAAATCAGGTATGTCTTTTGTTGATCACCCTCCAG-3' (SEQ ID NO: 12). The 5' 25 nucleotides of this primer corresponded with nucleotide positions 2178-2202 of AAV2 genome). The reverse primer corresponded with nucleotide positions 4419-4448 and has a sequence of 5'-GGACTCTAGAGACCACAAGAGGCAGTATTTTACTGACACG-3' (SEQ ID NO: 13).
The subsequent PCR products were linked together by PCR amplification using the rep forward primer (SEQ ID NO: 7) and the cap reverse primer (SEQ ID NO: 13). After the PCR reaction, the PCR product was digested with HindIII and XbaI and the fragment subcloned into p5E18 at the HindIII and XbaI cloning sites as described by Xiao et al. (1999) J. Virol. 73:3994-4003. The resulting plasmid is designated pHyb25, a recombinant pseudotyped adeno-associated virus with an AAV5 capsid and AAV2 rep sequences, and is shown in SEQ ID NO: 14. The helper plasmid with AAV-2 rep and AAV-2 capsid is shown in SEQ ID NO: 15.
Construction of Pseudotyped Vectors
To test the packaging of the virus using the pseudotyped helper plasmids, expression cassettes containing detectable markers were generated. Enhanced green fluorescent protein (EGFP) and luciferase were used as markers. The CBA-EGFP-WPRE-BGH poly A vector was constructed by cloning the enhanced green fluorescent protein (EGFP) into an AAVs genome plasmid under the control of a chicken beta actin (CBA) promoter followed by a woodchuck post-regulatory element (WPRE), with a bovine growth hormone (bGH) polyadenylation site between the two AAV2 inverted terminal repeat sequences, as previously described (During et al. (1998) Nature Med. 4:1131-1135). The CBA-luciferase-WPRE-BGH poly A vector was constructed by cloning the luciferase gene into the AAV plasmid under the control of a chicken beta actin (CBA) promoter followed by a woodchuck post-regulatory element (WPRE), with a bovine growth hormone (bGH) polyadenylation site between the two AAV2 inverted terminal repeat sequences.
To determine whether the these expression cassettes could be packaged into recombinant virions using the pseudotyped helper plasmids, triple plasmid experiments were performed. Recombinant AAV EGFP or AAV luciferase viruses were generated by cotransfecting into 293 cells, the pseudotyped helper constructs described in Example 1, along with the CBA-EGFP-WPRE-BGH poly A and CBA-luciferase-WPRE-BGH poly A vectors described in Example 2, and an adeno helper plasmid at a ratio of 1:1:2, using calcium phosphate precipitation methods. Cells were incubated at 37° C. for 72-96 hours after transfection. After incubation, the cells were harvested and the viruses purified by double CsCl gradient. The viral titer was determined by Quantitative PCR.
In-Vitro Infectivity of the Pseudotyped Vectors
To test the in-vitro infectivity of the recombinant pseudotyped vectors, and to determine whether the AAV helpers supported recombinant AAV production, the pseudotyped helper constructs described in Example 1 were cotransfected into 293 cells, along with the CBA-EGFP-WPRE-BGH poly A and CBA-luciferase-WPRE-BGH poly A vectors described in Example 2, and an adeno helper plasmid at a ratio of 1:1:2, using calcium phosphate precipitation methods in a triple transfection. Cells were incubated at 37° C. for 72-96 hours post transfection. After incubation, the cells were harvested and the viruses purified by double CsCl gradient. The vector yield was determined by measuring the GFP and luciferase expression in 293 cells using cell lysate from the above preparation. At MOIs of 10-1000, robust expression was seen with the recombinant pseudotyped viruses.
The yields of the different serotypes are shown in the FIG. 2. From 15 dishes of 293 cell transfections (15 cm diameter), the yield for recombinant pseudotype AAV1-AAV2 was 1.17×1013 genomic particles encoding luciferase and 5.06×1012 genomic particles encoding EGFP. For the recombinant AAV2-AAV2 (wild type) virus, the yield was 5.60×1011 genomic particles encoding luciferase and 1.03×1012 genomic particles encoding EGFP. For recombinant pseudotype AAV5-AAV2, the yield was 1.80×1012 genomic particles luciferase, and 1.62×1012 genomic particles encoding EGFP. The results showed that the yield of AAV1-AAV2 was about 5 to 10 times higher than the yield of AAV2-AAV2 or AAV5-AAV2. These higher yields are important particularly when the vectors are to be delivered to the central nervous system, e.g., regions of the brain. Due to the high yields of genomic particles which makes the vectors highly concentrated, the pseudotyped vector can be delivered to the brain in smaller volume of a suitable carrier.
In Vivo Effect of the Pseudotyped Vector
To test the in vivo effect of the pseudotyped virions, the pseudotyped virions were prepared as described in Example 2, purified by CsCl gradient, and delivered to the brain. The pseudotyped vector were delivered specifically to the mid-stratium and hippocampus regions of the brain.
For delivery into the stratium, 3 μl (1.5×1010 genomic particles) of CBA-EGFP-WPRE-BGH vector plus 1.5 μl of mannitol were stereotaxically injected into the left mid-striatum of male Sprawl Dawley rats (275-325 g) (n=3) that had been anaesthetized with a mixture of Ketamine (67 mg/kg)/Xylazine (6.7 mg/kg) given interperitonially (ip). The experiment was not repeated with the CBA-luciferase-WPRE-BGH poly A vector.
For delivery into the hippocampus, 20 (1×1010 genomic particles) of each vector (recombinant CBA-EGFP-WPRE-BGH poly A or CBA-luciferase-WPRE-BGH poly A, packaged in AAV1, AAV2 or AAV5 capsids) plus 1 μl of mannitol were injected into the right hippocampus. The intracerebral infusion was administered at the rate of 0.2 μl/min The needle was left in situ for additional 5 min before removal.
The brain tissue of the animals were examined using standard histology methods. Four weeks after in vivo administration of the pseudotyped vectors, the animals were perfused intracardially with phosphate buffered saline followed by 4% paraformaldehyde. The brain was removed and postfixed by 4% paraformaldehyde for about 4 hours and then transferred to 20% and 30% sucrose solution until the brain sank. The brain was cut coronally into 20 μm sections using a freezing cryostat (Leica, Germany). The section was then examined by fluorescent and confocal microscopy. Transgene expression was assessed by using stereology for cell counting. Individual brain slices (e.g., 50 brain slices) were examined to determine the region and number of cells that were transduced, the total number of cells from each slice was then added together from each one of the slides to provide a 3-dimensional configuration of the total area of the brain that had been transduced. The sum total number of transduced cells from each brain section was counted to provide an evaluation of the transduction rate for each of the recombinant pseudotyped vectors, or recombinant chimeric capsid vectors.
Under circumstances where sterological counting was impractical, for example where there is strong expression of the marker protein, fluorescent microscopic densitometry was used to determine the fluorescence intensity of marker protein in the target nuclei. The fluorescent images of each brain section were captured by a digital camera under the fluorescent microscope and the relative fluorescent intensity in the transduced nuclei of each image was analyzed by using the NIH image software. With densitometry analysis, color images of a marker protein were analyzed as a black and white images. The area that appeared as bright white indicated a region of the highest expression of the marker protein, a grey scale indicated a lower expression, and a black scale indicated no expression of the marker protein. The rate of transduction was determined by examining the area of "white" in a region of the brain. The greater the area of white in an image, the greater the expression of the marker protein in that region.
The transduction results of the difference pseudotyped vectors in different regions of the brain were by examining the fluorescence of GFP in the hippocampus (data not shown), four weeks after transduction. The data showed that in the hippocampus and in the hippocampus the AAV1-AAV2 pseudotype vector transduced almost all the hippocampus area. The neuron like cells in the CA1, CA2, CA3, dentate gyrus regions all strongly expressed GFP florescence, with the entire region turning green. Moreover, numerous fibers also turned green. This data demonstrates that the AAV1-AAV2 pseudotype vector transduced well in the brain, and transduced a greater number of cells further away from the needle tract than the other pseudotyped vectors. For the AAV2-AAV2 vector and AAV5-AAV2 pseudotyped vector, the transduced area was limited to the area near the needle tract. The AAV5-AAV2 pseudotyped vector showed more green fluorescence than the AAV2-AAV2 (wild type vector).
A similar result was observed in the stratium with the pseudotyped vectors. Again, in the striatum, more cells and fiber were transduced by the AAV1-AAV2 pseudotyped vector compared with other two stereotypes vectors. The AAV1-AAV2 pseudotyped vector also diffused further away from the needle tract, thereby transducing a greater number of cells which appeared green.
In order to quantitate the transduction difference of different pseudotyped vectors in the hippocampus, NIH imagine analysis was used to quantify the relative densitometry of the hippocampus, and the results depicted in FIG. 3. The results show that the GFP expression using the pseudotyped AAV1-AAV2 vector was significantly stronger than the GFP expression observed with the AAV2-AAV2 and the AAV2-AAV5 pseudotyped vectors (P<0.05, one way ANOVA, post hoc). The data shows that based on densitometric analysis, the rate of transduction and expression of the AAV1-AAV2 pseudotyped vector is about 8-10 fold higher than the AAV2-AAV2 vector. The rate of transduction and expression of the AAV5-AAV2 pseudotyped vector is about 2-fold higher than the AAV2-AAV2 vector.
Analysis of Markers for Different Cell Types in the Brain
To further examine the types of cells that were transduced with the pseudotyped vectors, antibodies to specific markers present on different cell types were used. In particular, primary antibodies against the neuronal specific marker, NeuN, and glial cell specific marker, GFAP, were used. The EGFP transduced cells in the brain were examined by incubating sections of the brain with primary antibodies NeuN diluted in buffer (1:200, Chemicon), and GFAP (1:1000, Chemicon). The secondary fluorescent antibody, Cy3 (1:100, Jackson) was used to bind to the primary antibody. The sections of the brain were observed under the Confocal microscope.
The results demonstrate that the major cells infected by both the AAV2-AAV2 and AAV5-AAV2 pseudotyped vectors were NeuN positive cells, i.e., hippocampus pyramidal cells, granule cells, inter neurons, and striatum neurons of different shapes and sizes. These cells appeared red in both the ipsilateral hippocampus and collateral hippocampus examined at ×40 magnification. Similarly, the AAV1-AAV2 pseudotyped vector also transduced almost exclusively to the neurons.
In the hippocampus, AAV1-AAV2 pseudotyped vector infected pyramidal neurons in the region CA1, CA2 and CA3, granule cell layer in the dentate gyrus. The fibers in the contralateral sites of the hippocampus also turned green, suggesting that these fibers arise from the transduced neurons from injected sites. Moreover, some of the neuron cell bodies in the contralateral sites were also GFP positive, although much weaker compared with ipsilateral sites. These contralateral transduced cells were located in the CA2, CA3 and halius area. The CA1 region and granule cells in the dentate gyrus remained uninfected at the same time. One reasonable explanation as to why the neurons were transduced, was that these cells had terminals in the injected sites and virus entered into the cells from their terminal and slowed traveled to the soma.
These results also demonstrate that the transduction rate with the AAV1-AAV2 pseudotyped vector was much higher than the transduction rate with AAV2-AAV2. When the same amounts of CBA-EGFP, AAV1-AAV2 and AAV2-AAV2 vectors were delivered into the hippocampus, and striatum of the brain, the EGFP expression level with the AAV1-AAV2 pseudotyped vector was 10 fold higher than that of AAV2-AAV2. The AAV1-AAV2 pseudotyped vector also diffused a greater distance from the injection site than the AAV2-AAV2 vector. When AAV1-AAV2 was delivered into the hippocampus, the whole hippocampus was transduced, while only small area around needle tract was transduced by AAV2-AAV2 vector. Collectively, these results demonstrate that the AAV1-AAV2 pseudotype vector is a suitable vector for gene delivery in the CNS. The AAV1-AAV2 pseudotype vector shows a higher yield, efficient transduction rate, and transduces a more extensive cell number and volume in the CNS, than the other pseuodotyped vectors. For all these reasons, the AAV1-AAV2 pseudotype vector is suitable for delivering genes to a target site in the CNS.
1514718DNAadeno-associated virus 2 1ttgcccactc cctctctgcg cgctcgctcg ctcggtgggg cctgcggacc aaaggtccgc 60agacggcaga gctctgctct gccggcccca ccgagcgagc gagcgcgcag agagggagtg 120ggcaactcca tcactagggg taatcgcgaa gcgcctccca cgctgccgcg tcagcgctga 180cgtaaattac gtcatagggg agtggtcctg tattagctgt cacgtgagtg cttttgcgac 240attttgcgac accacgtggc catttagggt atatatggcc gagtgagcga gcaggatctc 300cattttgacc gcgaaatttg aacgagcagc agccatgccg ggcttctacg agatcgtgat 360caaggtgccg agcgacctgg acgagcacct gccgggcatt tctgactcgt ttgtgagctg 420ggtggccgag aaggaatggg agctgccccc ggattctgac atggatctga atctgattga 480gcaggcaccc ctgaccgtgg ccgagaagct gcagcgcgac ttcctggtcc aatggcgccg 540cgtgagtaag gccccggagg ccctcttctt tgttcagttc gagaagggcg agtcctactt 600ccacctccat attctggtgg agaccacggg ggtcaaatcc atggtgctgg gccgcttcct 660gagtcagatt agggacaagc tggtgcagac catctaccgc gggatcgagc cgaccctgcc 720caactggttc gcggtgacca agacgcgtaa tggcgccgga ggggggaaca aggtggtgga 780cgagtgctac atccccaact acctcctgcc caagactcag cccgagctgc agtgggcgtg 840gactaacatg gaggagtata taagcgcctg tttgaacctg gccgagcgca aacggctcgt 900ggcgcagcac ctgacccacg tcagccagac ccaggagcag aacaaggaga atctgaaccc 960caattctgac gcgcctgtca tccggtcaaa aacctccgcg cgctacatgg agctggtcgg 1020gtggctggtg gaccggggca tcacctccga gaagcagtgg atccaggagg accaggcctc 1080gtacatctcc ttcaacgccg cttccaactc gcggtcccag atcaaggccg ctctggacaa 1140tgccggcaag atcatggcgc tgaccaaatc cgcgcccgac tacctggtag gccccgctcc 1200gcccgcggac attaaaacca accgcatcta ccgcatcctg gagctgaacg gctacgaacc 1260tgcctacgcc ggctccgtct ttctcggctg ggcccagaaa aggttcggga agcgcaacac 1320catctggctg tttgggccgg ccaccacggg caagaccaac atcgcggaag ccatcgccca 1380cgccgtgccc ttctacggct gcgtcaactg gaccaatgag aactttccct tcaatgattg 1440cgtcgacaag atggtgatct ggtgggagga gggcaagatg acggccaagg tcgtggagtc 1500cgccaaggcc attctcggcg gcagcaaggt gcgcgtggac caaaagtgca agtcgtccgc 1560ccagatcgac cccacccccg tgatcgtcac ctccaacacc aacatgtgcg ccgtgattga 1620cgggaacagc accaccttcg agcaccagca gccgttgcag gaccggatgt tcaaatttga 1680actcacccgc cgtctggagc atgactttgg caaggtgaca aagcaggaag tcaaagagtt 1740cttccgctgg gcgcaggatc acgtgaccga ggtggcgcat gagttctacg tcagaaaggg 1800tggagccaac aaaagacccg cccccgatga cgcggataaa agcgagccca agcgggcctg 1860cccctcagtc gcggatccat cgacgtcaga cgcggaagga gctccggtgg actttgccga 1920caggtaccaa aacaaatgtt ctcgtcacgc gggcatgctt cagatgctgt ttccctgcaa 1980gacatgcgag agaatgaatc agaatttcaa catttgcttc acgcacggga cgagagactg 2040ttcagagtgc ttccccggcg tgtcagaatc tcaaccggtc gtcagaaaga ggacgtatcg 2100gaaactctgt gccattcatc atctgctggg gcgggctccc gagattgctt gctcggcctg 2160cgatctggtc aacgtggacc tggatgactg tgtttctgag caataaatga cttaaaccag 2220gtatggctgc cgatggttat cttccagatt ggctcgagga caacctctct gagggcattc 2280gcgagtggtg ggacttgaaa cctggagccc cgaagcccaa agccaaccag caaaagcagg 2340acgacggccg gggtctggtg cttcctggct acaagtacct cggacccttc aacggactcg 2400acaaggggga gcccgtcaac gcggcggacg cagcggccct cgagcacgac aaggcctacg 2460accagcagct caaagcgggt gacaatccgt acctgcggta taaccacgcc gacgccgagt 2520ttcaggagcg tctgcaagaa gatacgtctt ttgggggcaa cctcgggcga gcagtcttcc 2580aggccaagaa gcgggttctc gaacctctcg gtctggttga ggaaggcgct aagacggctc 2640ctggaaagaa acgtccggta gagcagtcgc cacaagagcc agactcctcc tcgggcatcg 2700gcaagacagg ccagcagccc gctaaaaaga gactcaattt tggtcagact ggcgactcag 2760agtcagtccc cgatccacaa cctctcggag aacctccagc aacccccgct gctgtgggac 2820ctactacaat ggcttcaggc ggtggcgcac caatggcaga caataacgaa ggcgccgacg 2880gagtgggtaa tgcctcagga aattggcatt gcgattccac atggctgggc gacagagtca 2940tcaccaccag cacccgcacc tgggccttgc ccacctacaa taaccacctc tacaagcaaa 3000tctccagtgc ttcaacgggg gccagcaacg acaaccacta cttcggctac agcaccccct 3060gggggtattt tgatttcaac agattccact gccacttttc accacgtgac tggcagcgac 3120tcatcaacaa caattgggga ttccggccca agagactcaa cttcaaactc ttcaacatcc 3180aagtcaagga ggtcacgacg aatgatggcg tcacaaccat cgctaataac cttaccagca 3240cggttcaagt cttctcggac tcggagtacc agcttccgta cgtcctcggc tctgcgcacc 3300agggctgcct ccctccgttc ccggcggacg tgttcatgat tccgcaatac ggctacctga 3360cgctcaacaa tggcagccaa gccgtgggac gttcatcctt ttactgcctg gaatatttcc 3420cttctcagat gctgagaacg ggcaacaact ttaccttcag ctacaccttt gaggaagtgc 3480ctttccacag cagctacgcg cacagccaga gcctggaccg gctgatgaat cctctcatcg 3540accaatacct gtattacctg aacagaactc aaaatcagtc cggaagtgcc caaaacaagg 3600acttgctgtt tagccgtggg tctccagctg gcatgtctgt tcagcccaaa aactggctac 3660ctggaccctg ttatcggcag cagcgcgttt ctaaaacaaa aacagacaac aacaacagca 3720attttacctg gactggtgct tcaaaatata acctcaatgg gcgtgaatcc atcatcaacc 3780ctggcactgc tatggcctca cacaaagacg acgaagacaa gttctttccc atgagcggtg 3840tcatgatttt tggaaaagag agcgccggag cttcaaacac tgcattggac aatgtcatga 3900ttacagacga agaggaaatt aaagccacta accctgtggc caccgaaaga tttgggaccg 3960tggcagtcaa tttccagagc agcagcacag accctgcgac cggagatgtg catgctatgg 4020gagcattacc tggcatggtg tggcaagata gagacgtgta cctgcagggt cccatttggg 4080ccaaaattcc tcacacagat ggacactttc acccgtctcc tcttatgggc ggctttggac 4140tcaagaaccc gcctcctcag atcctcatca aaaacacgcc tgttcctgcg aatcctccgg 4200cggagttttc agctacaaag tttgcttcat tcatcaccca atactccaca ggacaagtga 4260gtgtggaaat tgaatgggag ctgcagaaag aaaacagcaa gcgctggaat cccgaagtgc 4320agtacacatc caattatgca aaatctgcca acgttgattt tactgtggac aacaatggac 4380tttatactga gcctcgcccc attggcaccc gttaccttac ccgtcccctg taattacgtg 4440ttaatcaata aaccggttga ttcgtttcag ttgaactttg gtctcctgtc cttcttatct 4500tatcggttac catggttata gcttacacat taactgcttg gttgcgcttc gcgataaaag 4560acttacgtca tcgggttacc cctagtgatg gagttgccca ctccctctct gcgcgctcgc 4620tcgctcggtg gggcctgcgg accaaaggtc cgcagacggc agagctctgc tctgccggcc 4680ccaccgagcg agcgagcgcg cagagaggga gtgggcaa 471824675DNAadeno-associated virus 2 2ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120gccaactcca tcactagggg ttcctggagg ggtggagtcg tgacgtgaat tacgtcatag 180ggttagggag gtcctgtatt agaggtcacg tgagtgtttt gcgacatttt gcgacaccat 240gtggtcacgc tgggtattta agcccgagtg agcacgcagg gtctccattt tgaagcggga 300ggtttgaacg cgcagccgcc atgccggggt tttacgagat tgtgattaag gtccccagcg 360accttgacgg gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg 420aatgggagtt gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga 480ccgtggccga gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc 540cggaggccct tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc 600tcgtggaaac caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg 660aaaaactgat tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg 720tcacaaagac cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc 780ccaattactt gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac 840agtatttaag cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga 900cgcacgtgtc gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc 960cggtgatcag atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca 1020aggggattac ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca 1080atgcggcctc caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta 1140tgagcctgac taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacattt 1200ccagcaatcg gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt 1260ccgtctttct gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg 1320ggcctgcaac taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct 1380acgggtgcgt aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg 1440tgatctggtg ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc 1500tcggaggaag caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga 1560ctcccgtgat cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga 1620ccttcgaaca ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc 1680tggatcatga ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa 1740aggatcacgt ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa 1800gacccgcccc cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc 1860agccatcgac gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat 1920gttctcgtca cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga 1980atcagaattc aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg 2040tgtcagaatc tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc 2100atcatatcat gggaaaggtg ccagacgctt gcactgcctg cgatctggtc aatgtggatt 2160tggatgactg catctttgaa caataaatga tttaaatcag gtatggctgc cgatggttat 2220cttccagatt ggctcgagga cactctctct gaaggaataa gacagtggtg gaagctcaaa 2280cctggcccac caccaccaaa gcccgcagag cggcataagg acgacagcag gggtcttgtg 2340cttcctgggt acaagtacct cggacccttc aacggactcg acaagggaga gccggtcaac 2400gaggcagacg ccgcggccct cgagcacgta caaagcctac gaccggcagc tcgacagcgg 2460agacaacccg tacctcaagt acaaccacgc cgacgcggag tttcaggagc gccttaaaga 2520agatacgtct tttgggggca acctcggacg agcagtcttc caggcgaaaa agagggttct 2580tgaacctctg ggcctggttg aggaacctgt taagacggct ccgggaaaaa agaggccggt 2640agagcactct cctgtggagc cagactcctc ctcgggaacc ggaaaggcgg gccagcagcc 2700tgcaagaaaa agattgaatt ttggtcagac tggagacgca gactcagtac ctgaccccca 2760gcctctcgga cagccaccag cagccccctc tggtctggga actaatacga tggctacagg 2820cagtggcgca ccaatggcag acaataacga gggcgccgac ggagtgggta attcctccgg 2880aaattggcat tgcgattcca catggatggg cgacagagtc atcaccacca gcacccgaac 2940ctgggccctg cccacctaca acaaccacct ctacaaacaa atttccagcc aatcaggagc 3000ctcgaacgac aatcactact ttggctacag caccccttgg gggtattttg acttcaacag 3060attccactgc cacttttcac cacgtgactg gcaaagactc atcaacaaca actggggatt 3120ccgacccaag agactcaact tcaagctctt taacattcaa gtcaaagagg tcacgcagaa 3180tgacggtacg acgacgattg ccaataacct taccagcacg gttcaggtgt ttactgactc 3240ggagtaccag ctcccgtacg tcctcggctc ggcgcatcaa ggatgcctcc cgccgttccc 3300agcagacgtc ttcatggtgc cacagtatgg atacctcacc ctgaacaacg ggagtcaggc 3360agtaggacgc tcttcatttt actgcctgga gtactttcct tctcagatgc tgcgtaccgg 3420aaacaacttt accttcagct acacttttga ggacgttcct ttccacagca gctacgctca 3480cagccagagt ctggaccgtc tcatgaatcc tctcatcgac cagtacctgt attacttgag 3540cagaacaaac actccaagtg gaaccaccac gcagtcaagg cttcagtttt ctcaggccgg 3600agcgagtgac attcgggacc agtctaggaa ctggcttcct ggaccctgtt accgccagca 3660gcgagtatca aagacatctg cggataacaa caacagtgaa tactcgtgga ctggagctac 3720caagtaccac ctcaatggca gagactctct ggtgaatccg gccatggcaa gccacaagga 3780cgatgaagaa aagttttttc ctcagagcgg ggttctcatc tttgggaagc aaggctcaga 3840gaaaacaaat gtgaacattg aaaaggtcat gattacagac gaagaggaaa tcggaacaac 3900caatcccgtg gctacggagc agtatggttc tgtatctacc aacctccaga gaggcaacag 3960acaagcagct accgcagatg tcaacacaca aggcgttctt ccaggcatgg tctggcagga 4020cagagatgtg taccttcagg ggcccatctg ggcaaagatt ccacacacgg acggacattt 4080tcacccctct cccctcatgg gtggattcgg acttaaacac cctcctccac agattctcat 4140caagaacacc ccggtacctg cgaatccttc gaccaccttc agtgcggcaa agtttgcttc 4200cttcatcaca cagtactcca cgggacacgg tcagcgtgga gatcgagtgg gagctgcaga 4260aggaaaacag caaacgctgg aatcccgaaa ttcagtacac ttccaactac aacaagtctg 4320ttaatcgtgg acttaccgtg gatactaatg gcgtgtattc agagcctcgc cccattggca 4380ccagatacct gactcgtaat ctgtaattgc ttgttaatca ataaaccgtt taattcgttt 4440cagttgaact ttggtctctg cgtatttctt tcttatctag tttccatggc tacgtagata 4500agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag ttggccactc 4560cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg 4620gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg gccaa 467534722DNAadeno-associated virus 2 3tggccactcc ctctatgcgc actcgctcgc tcggtggggc ctggcgacca aaggtcgcca 60gacggacgtg ctttgcacgt ccggccccac cgagcgagcg agtgcgcata gagggagtgg 120ccaactccat cactagaggt atggcagtga cgtaacgcga agcgcgcgaa gcgagaccac 180gcctaccagc tgcgtcagca gtcaggtgac ccttttgcga cagtttgcga caccacgtgg 240ccgctgaggg tatatattct cgagtgagcg aaccaggagc tccattttga ccgcgaaatt 300tgaacgagca gcagccatgc cggggttcta cgagattgtc ctgaaggtcc cgagtgacct 360ggacgagcac ctgccgggca tttctaactc gtttgttaac tgggtggccg agaaggaatg 420ggagctgccg ccggattctg acatggatcc gaatctgatt gagcaggcac ccctgaccgt 480ggccgaaaag cttcagcgcg agttcctggt ggagtggcgc cgcgtgagta aggccccgga 540ggccctcttt tttgtccagt tcgaaaaggg ggagacctac ttccacctgc acgtgctgat 600tgagaccatc ggggtcaaat ccatggtggt cggccgctac gtgagccaga ttaaagagaa 660gctggtgacc cgcatctacc gcggggtcga gccgcagctt ccgaactggt tcgcggtgac 720caaaacgcga aatggcgccg ggggcgggaa caaggtggtg gacgactgct acatccccaa 780ctacctgctc cccaagaccc agcccgagct ccagtgggcg tggactaaca tggaccagta 840tttaagcgcc tgtttgaatc tcgcggagcg taaacggctg gtggcgcagc atctgacgca 900cgtgtcgcag acgcaggagc agaacaaaga gaatcagaac cccaattctg acgcgccggt 960catcaggtca aaaacctcag ccaggtacat ggagctggtc gggtggctgg tggaccgcgg 1020gatcacgtca gaaaagcaat ggattcagga ggaccaggcc tcgtacatct ccttcaacgc 1080cgcctccaac tcgcggtccc agatcaaggc cgcgctggac aatgcctcca agatcatgag 1140cctgacaaag acggctccgg actacctggt gggcagcaac ccgccggagg acattaccaa 1200aaatcggatc taccaaatcc tggagctgaa cgggtacgat ccgcagtacg cggcctccgt 1260cttcctgggc tgggcgcaaa agaagttcgg gaagaggaac accatctggc tctttgggcc 1320ggccacgacg ggtaaaacca acatcgcgga agccatcgcc cacgccgtgc ccttctacgg 1380ctgcgtaaac tggaccaatg agaactttcc cttcaacgat tgcgtcgaca agatggtgat 1440ctggtgggag gagggcaaga tgacggccaa ggtcgtggag agcgccaagg ccattctggg 1500cggaagcaag gtgcgcgtgg accaaaagtg caagtcatcg gcccagatcg aacccactcc 1560cgtgatcgtc acctccaaca ccaacatgtg cgccgtgatt gacgggaaca gcaccacctt 1620cgagcatcag cagccgctgc aggaccggat gtttaaattt gaacttaccc gccgtttgga 1680ccatgacttt gggaaggtca ccaaacagga agtaaaggac tttttccggt gggcttccga 1740tcacgtgact gacgtggctc atgagttcta cgtcagaaag ggtggagcta agaaacgccc 1800cgcctccaat gacgcggatg taagcgagcc aaaacggcag tgcacgtcac ttgcgcagcc 1860gacaacgtca gacgcggaag caccggcgga ctacgcggac aggtaccaaa acaaatgttc 1920tcgtcacgtg ggcatgaatc tgatgctttt tccctgtaaa acatgcgaga gaatgaatca 1980aatttccaat gtctgtttta cgcatggtca aagagactgt ggggaatgct tccctggaat 2040gtcagaatct caacccgttt ctgtcgtcaa aaagaagact tatcagaaac tgtgtccaat 2100tcatcatatc ctgggaaggg cacccgagat tgcctgttcg gcctgcgatt tggccaatgt 2160ggacttggat gactgtgttt ctgagcaata aatgacttaa accaggtatg gctgctgacg 2220gttatcttcc agattggctc gaggacaacc tttctgaagg cattcgtgag tggtgggctc 2280tgaaacctgg agtccctcaa cccaaagcga accaacaaca ccaggacaac cgtcggggtc 2340ttgtgcttcc gggttacaaa tacctcggac ccggtaacgg actcgacaaa ggagagccgg 2400tcaacgaggc ggacgcggca gccctcgaac acgacaaagc ttacgaccag cagctcaagg 2460ccggtgacaa cccgtacctc aagtacaacc acgccgacgc cgagtttcag gagcgtcttc 2520aagaagatac gtcttttggg ggcaaccttg gcagagcagt cttccaggcc aaaaagagga 2580tccttgagcc tcttggtctg gttgaggaag cagctaaaac ggctcctgga aagaagaggc 2640ctgtagatca gtctcctcag gaaccggact catcatctgg tgttggcaaa tcgggcaaac 2700agcctgccag aaaaagacta aatttcggtc agactggcga ctcagagtca gtcccagacc 2760ctcaacctct cggagaacca ccagcagccc ccacaagttt gggatctaat acaatggctt 2820caggcggtgg cgcaccaatg gcagacaata acgagggtgc cgatggagtg ggtaattcct 2880caggaaattg gcattgcgat tcccaatggc tgggcgacag agtcatcacc accagcacca 2940gaacctgggc cctgcccact tacaacaacc atctctacaa gcaaatctcc agccaatcag 3000gagcttcaaa cgacaaccac tactttggct acagcacccc ttgggggtat tttgacttta 3060acagattcca ctgccacttc tcaccacgtg actggcagcg actcattaac aacaactggg 3120gattccggcc caagaaactc agcttcaagc tcttcaacat ccaagttaaa gaggtcacgc 3180agaacgatgg cacgacgact attgccaata accttaccag cacggttcaa gtgtttacgg 3240actcggagta tcagctcccg tacgtgctcg ggtcggcgca ccaaggctgt ctcccgccgt 3300ttccagcgga cgtcttcatg gtccctcagt atggatacct caccctgaac aacggaagtc 3360aagcggtggg acgctcatcc ttttactgcc tggagtactt cccttcgcag atgctaagga 3420ctggaaataa cttccaattc agctatacct tcgaggatgt accttttcac agcagctacg 3480ctcacagcca gagtttggat cgcttgatga atcctcttat tgatcagtat ctgtactacc 3540tgaacagaac gcaaggaaca acctctggaa caaccaacca atcacggctg ctttttagcc 3600aggctgggcc tcagtctatg tctttgcagg ccagaaattg gctacctggg ccctgctacc 3660ggcaacagag actttcaaag actgctaacg acaacaacaa cagtaacttt ccttggacag 3720cggccagcaa atatcatctc aatggccgcg actcgctggt gaatccagga ccagctatgg 3780ccagtcacaa ggacgatgaa gaaaaatttt tccctatgca cggcaatcta atatttggca 3840aagaagggac aacggcaagt aacgcagaat tagataatgt aatgattacg gatgaagaag 3900agattcgtac caccaatcct gtggcaacag agcagtatgg aactgtggca aataacttgc 3960agagctcaaa tacagctccc acgactagaa ctgtcaatga tcagggggcc ttacctggca 4020tggtgtggca agatcgtgac gtgtaccttc aaggacctat ctgggcaaag attcctcaca 4080cggatggaca ctttcatcct tctcctctga tgggaggctt tggactgaaa catccgcctc 4140ctcaaatcat gatcaaaaat actccggtac cggcaaatcc tccgacgact ttcagcccgg 4200ccaagtttgc ttcatttatc actcagtact ccactggaca ggtcagcgtg gaaattgagt 4260gggagctaca gaaagaaaac agcaaacgtt ggaatccaga gattcagtac acttccaact 4320acaacaagtc tgttaatgtg gactttactg tagacactaa tggtgtttat agtgaacctc 4380gccctattgg aacccggtat ctcacacgaa acttgtaatc ctggttaatc aataaaccgt 4440ttaattcgtt tcagttgaac tttggctctt gtgcacttct tatcttatct tgtttccatg 4500gctactgcgt agataagcag cggcctgcgg cgcttgcgct tcgcggttta caactgctgg 4560ttaatattta actctcgcca tacctctagt gatggagttg gccactccct ctatgcgcac 4620tcgctcgctc ggtggggccg gacgtgcaaa gcacgtccgt ctggcgacct ttggtcgcca 4680ggccccaccg agcgagcgag tgcgcataga gggagtggcc aa 472244767DNAadeno-associated virus 2 4ttggccactc cctctatgcg cgctcgctca ctcactcggc cctggagacc aaaggtctcc 60agactgccgg cctctggccg gcagggccga gtgagtgagc gagcgcgcat agagggagtg 120gccaactcca tcatctaggt ttgcccactg acgtcaatgt gacgtcctag ggttagggag 180gtccctgtat tagcagtcac gtgagtgtcg tatttcgcgg agcgtagcgg agcgcatacc 240aagctgccac gtcacagcca cgtggtccgt ttgcgacagt ttgcgacacc atgtggtcag 300gagggtatat aaccgcgagt gagccagcga ggagctccat tttgcccgcg aattttgaac 360gagcagcagc catgccgggg ttctacgaga tcgtgctgaa ggtgcccagc gacctggacg 420agcacctgcc cggcatttct gactcttttg tgagctgggt ggccgagaag gaatgggagc 480tgccgccgga ttctgacatg gacttgaatc tgattgagca ggcacccctg accgtggccg 540aaaagctgca acgcgagttc ctggtcgagt ggcgccgcgt gagtaaggcc ccggaggccc 600tcttctttgt ccagttcgag aagggggaca gctacttcca cctgcacatc ctggtggaga 660ccgtgggcgt caaatccatg gtggtgggcc gctacgtgag ccagattaaa gagaagctgg 720tgacccgcat ctaccgcggg gtcgagccgc agcttccgaa ctggttcgcg gtgaccaaga
780cgcgtaatgg cgccggaggc gggaacaagg tggtggacga ctgctacatc cccaactacc 840tgctccccaa gacccagccc gagctccagt gggcgtggac taacatggac cagtatataa 900gcgcctgttt gaatctcgcg gagcgtaaac ggctggtggc gcagcatctg acgcacgtgt 960cgcagacgca ggagcagaac aaggaaaacc agaaccccaa ttctgacgcg ccggtcatca 1020ggtcaaaaac ctccgccagg tacatggagc tggtcgggtg gctggtggac cgcgggatca 1080cgtcagaaaa gcaatggatc caggaggacc aggcgtccta catctccttc aacgccgcct 1140ccaactcgcg gtcacaaatc aaggccgcgc tggacaatgc ctccaaaatc atgagcctga 1200caaagacggc tccggactac ctggtgggcc agaacccgcc ggaggacatt tccagcaacc 1260gcatctaccg aatcctcgag atgaacgggt acgatccgca gtacgcggcc tccgtcttcc 1320tgggctgggc gcaaaagaag ttcgggaaga ggaacaccat ctggctcttt gggccggcca 1380cgacgggtaa aaccaacatc gcggaagcca tcgcccacgc cgtgcccttc tacggctgcg 1440tgaactggac caatgagaac tttccgttca acgattgcgt cgacaagatg gtgatctggt 1500gggaggaggg caagatgacg gccaaggtcg tagagagcgc caaggccatc ctgggcggaa 1560gcaaggtgcg cgtggaccaa aagtgcaagt catcggccca gatcgaccca actcccgtga 1620tcgtcacctc caacaccaac atgtgcgcgg tcatcgacgg aaactcgacc accttcgagc 1680accaacaacc actccaggac cggatgttca agttcgagct caccaagcgc ctggagcacg 1740actttggcaa ggtcaccaag caggaagtca aagacttttt ccggtgggcg tcagatcacg 1800tgaccgaggt gactcacgag ttttacgtca gaaagggtgg agctagaaag aggcccgccc 1860ccaatgacgc agatataagt gagcccaagc gggcctgtcc gtcagttgcg cagccatcga 1920cgtcagacgc ggaagctccg gtggactacg cggacaggta ccaaaacaaa tgttctcgtc 1980acgtgggtat gaatctgatg ctttttccct gccggcaatg cgagagaatg aatcagaatg 2040tggacatttg cttcacgcac ggggtcatgg actgtgccga gtgcttcccc gtgtcagaat 2100ctcaacccgt gtctgtcgtc agaaagcgga cgtatcagaa actgtgtccg attcatcaca 2160tcatggggag ggcgcccgag gtggcctgct cggcctgcga actggccaat gtggacttgg 2220atgactgtga catggaacaa taaatgactc aaaccagata tgactgacgg ttaccttcca 2280gattggctag aggacaacct ctctgaaggc gttcgagagt ggtgggcgct gcaacctgga 2340gcccctaaac ccaaggcaaa tcaacaacat caggacaacg ctcggggtct tgtgcttccg 2400ggttacaaat acctcggacc cggcaacgga ctcgacaagg gggaacccgt caacgcagcg 2460gacgcggcag ccctcgagca cgacaaggcc tacgaccagc agctcaaggc cggtgacaac 2520ccctacctca agtacaacca cgccgacgcg gagttccagc agcggcttca gggcgacaca 2580tcgtttgggg gcaacctcgg cagagcagtc ttccaggcca aaaagagggt tcttgaacct 2640cttggtctgg ttgagcaagc gggtgagacg gctcctggaa agaagagacc gttgattgaa 2700tccccccagc agcccgactc ctccacgggt atcggcaaaa aaggcaagca gccggctaaa 2760aagaagctcg ttttcgaaga cgaaactgga gcaggcgacg gaccccctga gggatcaact 2820tccggagcca tgtctgatga cagtgagatg cgtgcagcag ctggcggagc tgcagtcgag 2880ggcggacaag gtgccgatgg agtgggtaat gcctcgggtg attggcattg cgattccacc 2940tggtctgagg gccacgtcac gaccaccagc accagaacct gggtcttgcc cacctacaac 3000aaccacctct acaagcgact cggagagagc ctgcagtcca acacctacaa cggattctcc 3060accccctggg gatactttga cttcaaccgc ttccactgcc acttctcacc acgtgactgg 3120cagcgactca tcaacaacaa ctggggcatg cgacccaaag ccatgcgggt caaaatcttc 3180aacatccagg tcaaggaggt cacgacgtcg aacggcgaga caacggtggc taataacctt 3240accagcacgg ttcagatctt tgcggactcg tcgtacgaac tgccgtacgt gatggatgcg 3300ggtcaagagg gcagcctgcc tccttttccc aacgacgtct ttatggtgcc ccagtacggc 3360tactgtggac tggtgaccgg caacacttcg cagcaacaga ctgacagaaa tgccttctac 3420tgcctggagt actttccttc gcagatgctg cggactggca acaactttga aattacgtac 3480agttttgaga aggtgccttt ccactcgatg tacgcgcaca gccagagcct ggaccggctg 3540atgaaccctc tcatcgacca gtacctgtgg ggactgcaat cgaccaccac cggaaccacc 3600ctgaatgccg ggactgccac caccaacttt accaagctgc ggcctaccaa cttttccaac 3660tttaaaaaga actggctgcc cgggccttca atcaagcagc agggcttctc aaagactgcc 3720aatcaaaact acaagatccc tgccaccggg tcagacagtc tcatcaaata cgagacgcac 3780agcactctgg acggaagatg gagtgccctg acccccggac ctccaatggc cacggctgga 3840cctgcggaca gcaagttcag caacagccag ctcatctttg cggggcctaa acagaacggc 3900aacacggcca ccgtacccgg gactctgatc ttcacctctg aggaggagct ggcagccacc 3960aacgccaccg atacggacat gtggggcaac ctacctggcg gtgaccagag caacagcaac 4020ctgccgaccg tggacagact gacagccttg ggagccgtgc ctggaatggt ctggcaaaac 4080agagacattt actaccaggg tcccatttgg gccaagattc ctcataccga tggacacttt 4140cacccctcac cgctgattgg tgggtttggg ctgaaacacc cgcctcctca aatttttatc 4200aagaacaccc cggtacctgc gaatcctgca acgaccttca gctctactcc ggtaaactcc 4260ttcattactc agtacagcac tggccaggtg tcggtgcaga ttgactggga gatccagaag 4320gagcggtcca aacgctggaa ccccgaggtc cagtttacct ccaactacgg acagcaaaac 4380tctctgttgt gggctcccga tgcggctggg aaatacactg agcctagggc tatcggtacc 4440cgctacctca cccaccacct gtaataacct gttaatcaat aaaccggttt attcgtttca 4500gttgaacttt ggtctccgtg tccttcttat cttatctcgt ttccatggct actgcgtaca 4560taagcagcgg cctgcggcgc ttgcgcttcg cggtttacaa ctgccggtta atcagtaact 4620tctggcaaac cagatgatgg agttggccac attagctatg cgcgctcgct cactcactcg 4680gccctggaga ccaaaggtct ccagactgcc ggcctctggc cggcagggcc gagtgagtga 4740gcgagcgcgc atagagggag tggccaa 476754652DNAadeno-associated virus 2 5tggcactctc ccccctgtcg cgttcgctcg ctcgctggct cgtttggggg ggtggcagct 60caaagagctg ccagacgacg gccctctggc cgtcgccccc ccaaacgagc cagcgagcga 120gcgaacgcga caggggggag agtgccacac tctcaagcaa gggggttttg taagcagtga 180tgtcataatg atgtaatgct tattgtcacg cgatagttaa tgattaacag tcatgtgatg 240tgttttatcc aataggaaga aagcgcgcgt atgagttctc gcgagacttc cggggtataa 300aagaccgagt gaacgagccc gccgccattc tttgctctgg actgctagag gaccctcgct 360gccatggcta ccttctatga agtcattgtt cgcgtcccat ttgacgtgga ggaacatctg 420cctggaattt ctgacagctt tgtggactgg gtaactggtc aaatttggga gctgcctcca 480gagtcagatt taaatttgac tctggttgaa cagcctcagt tgacggtggc tgatagaatt 540cgccgcgtgt tcctgtacga gtggaacaaa ttttccaagc aggagtccaa attctttgtg 600cagtttgaaa agggatctga atattttcat ctgcacacgc ttgtggagac ctccggcatc 660tcttccatgg tcctcggccg ctacgtgagt cagattcgcg cccagctggt gaaagtggtc 720ttccagggaa ttgaacccca gatcaacgac tgggtcgcca tcaccaaggt aaagaagggc 780ggagccaata aggtggtgga ttctgggtat attcccgcct acctgctgcc gaaggtccaa 840ccggagcttc agtgggcgtg gacaaacctg gacgagtata aattggccgc cctgaatctg 900gaggagcgca aacggctcgt cgcgcagttt ctggcagaat cctcgcagcg ctcgcaggag 960gcggcttcgc agcgtgagtt ctcggctgac ccggtcatca aaagcaagac ttcccagaaa 1020tacatggcgc tcgtcaactg gctcgtggag cacggcatca cttccgagaa gcagtggatc 1080caggaaaatc aggagagcta cctctccttc aactccaccg gcaactctcg gagccagatc 1140aaggccgcgc tcgacaacgc gaccaaaatt atgagtctga caaaaagcgc ggtggactac 1200ctcgtgggga gctccgttcc cgaggacatt tcaaaaaaca gaatctggca aatttttgag 1260atgaatggct acgacccggc ctacgcggga tccatcctct acggctggtg tcagcgctcc 1320ttcaacaaga ggaacaccgt ctggctctac ggacccgcca cgaccggcaa gaccaacatc 1380gcggaggcca tcgcccacac tgtgcccttt tacggctgcg tgaactggac caatgaaaac 1440tttcccttta atgactgtgt ggacaaaatg ctcatttggt gggaggaggg aaagatgacc 1500aacaaggtgg ttgaatccgc caaggccatc ctggggggct caaaggtgcg ggtcgatcag 1560aaatgtaaat cctctgttca aattgattct acccctgtca ttgtaacttc caatacaaac 1620atgtgtgtgg tggtggatgg gaattccacg acctttgaac accagcagcc gctggaggac 1680cgcatgttca aatttgaact gactaagcgg ctcccgccag attttggcaa gattactaag 1740caggaagtca aggacttttt tgcttgggca aaggtcaatc aggtgccggt gactcacgag 1800tttaaagttc ccagggaatt ggcgggaact aaaggggcgg agaaatctct aaaacgccca 1860ctgggtgacg tcaccaatac tagctataaa agtctggaga agcgggccag gctctcattt 1920gttcccgaga cgcctcgcag ttcagacgtg actgttgatc ccgctcctct gcgaccgctc 1980aattggaatt caaggtatga ttgcaaatgt gactatcatg ctcaatttga caacatttct 2040aacaaatgtg atgaatgtga atatttgaat cggggcaaaa atggatgtat ctgtcacaat 2100gtaactcact gtcaaatttg tcatgggatt cccccctggg aaaaggaaaa cttgtcagat 2160tttggggatt ttgacgatgc caataaagaa cagtaaataa agcgagtagt catgtctttt 2220gttgatcacc ctccagattg gttggaagaa gttggtgaag gtcttcgcga gtttttgggc 2280cttgaagcgg gcccaccgaa accaaaaccc aatcagcagc atcaagatca agcccgtggt 2340cttgtgctgc ctggttataa ctatctcgga cccggaaacg gtctcgatcg aggagagcct 2400gtcaacaggg cagacgaggt cgcgcgagag cacgacatct cgtacaacga gcagcttgag 2460gcgggagaca acccctacct caagtacaac cacgcggacg ccgagtttca ggagaagctc 2520gccgacgaca catccttcgg gggaaacctc ggaaaggcag tctttcaggc caagaaaagg 2580gttctcgaac cttttggcct ggttgaagag ggtgctaaga cggcccctac cggaaagcgg 2640atagacgacc actttccaaa aagaaagaag gctcggaccg aagaggactc caagccttcc 2700acctcgtcag acgccgaagc tggacccagc ggatcccagc agctgcaaat cccagcccaa 2760ccagcctcaa gtttgggagc tgatacaatg tctgcgggag gtggcggccc attgggcgac 2820aataaccaag gtgccgatgg agtgggcaat gcctcgggag attggcattg cgattccacg 2880tggatggggg acagagtcgt caccaagtcc acccgaacct gggtgctgcc cagctacaac 2940aaccaccagt accgagagat caaaagcggc tccgtcgacg gaagcaacgc caacgcctac 3000tttggataca gcaccccctg ggggtacttt gactttaacc gcttccacag ccactggagc 3060ccccgagact ggcaaagact catcaacaac tactggggct tcagaccccg gtccctcaga 3120gtcaaaatct tcaacattca agtcaaagag gtcacggtgc aggactccac caccaccatc 3180gccaacaacc tcacctccac cgtccaagtg tttacggacg acgactacca gctgccctac 3240gtcgtcggca acgggaccga gggatgcctg ccggccttcc ctccgcaggt ctttacgctg 3300ccgcagtacg gttacgcgac gctgaaccgc gacaacacag aaaatcccac cgagaggagc 3360agcttcttct gcctagagta ctttcccagc aagatgctga gaacgggcaa caactttgag 3420tttacctaca actttgagga ggtgcccttc cactccagct tcgctcccag tcagaacctg 3480ttcaagctgg ccaacccgct ggtggaccag tacttgtacc gcttcgtgag cacaaataac 3540actggcggag tccagttcaa caagaacctg gccgggagat acgccaacac ctacaaaaac 3600tggttcccgg ggcccatggg ccgaacccag ggctggaacc tgggctccgg ggtcaaccgc 3660gccagtgtca gcgccttcgc cacgaccaat aggatggagc tcgagggcgc gagttaccag 3720gtgcccccgc agccgaacgg catgaccaac aacctccagg gcagcaacac ctatgccctg 3780gagaacacta tgatcttcaa cagccagccg gcgaacccgg gcaccaccgc cacgtacctc 3840gagggcaaca tgctcatcac cagcgagagc gagacgcagc cggtgaaccg cgtggcgtac 3900aacgtcggcg ggcagatggc caccaacaac cagagctcca ccactgcccc cgcgaccggc 3960acgtacaacc tccaggaaat cgtgcccggc agcgtgtgga tggagaggga cgtgtacctc 4020caaggaccca tctgggccaa gatcccagag acgggggcgc actttcaccc ctctccggcc 4080atgggcggat tcggactcaa acacccaccg cccatgatgc tcatcaagaa cacgcctgtg 4140cccggaaata tcaccagctt ctcggacgtg cccgtcagca gcttcatcac ccagtacagc 4200accgggcagg tcaccgtgga gatggagtgg gagctcaaga aggaaaactc caagaggtgg 4260aacccagaga tccagtacac aaacaactac aacgaccccc agtttgtgga ctttgccccg 4320gacagcaccg gggaatacag aaccaccaga cctatcggaa cccgatacct tacccgaccc 4380ctttaaccca ttcatgtcgc ataccctcaa taaaccgtgt attcgtgtca gtaaaatact 4440gcctcttgtg gtcattcaat gaataacagc ttacaacatc tacaaaacct ccttgcttga 4500gagtgtggca ctctcccccc tgtcgcgttc gctcgctcgc tggctcgttt gggggggtgg 4560cagctcaaag agctgccaga cgacggccct ctggccgtcg cccccccaaa cgagccagcg 4620agcgagcgaa cgcgacaggg gggagagtgc ca 465264683DNAadeno-associated virus 2 6ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120gccaactcca tcactagggg ttcctggagg ggtggagtcg tgacgtgaat tacgtcatag 180ggttagggag gtcctgtatt agaggtcacg tgagtgtttt gcgacatttt gcgacaccat 240gtggtcacgc tgggtattta agcccgagtg agcacgcagg gtctccattt tgaagcggga 300ggtttgaacg cgcagcgcca tgccggggtt ttacgagatt gtgattaagg tccccagcga 360ccttgacgag catctgcccg gcatttctga cagctttgtg aactgggtgg ccgagaagga 420atgggagttg ccgccagatt ctgacatgga tctgaatctg attgagcagg cacccctgac 480cgtggccgag aagctgcagc gcgacttcct ggtccagtgg cgccgcgtga gtaaggcccc 540ggaggccctc ttctttgttc agttcgagaa gggcgagtcc tacttccacc tccatattct 600ggtggagacc acgggggtca aatccatggt gctgggccgc ttcctgagtc agattaggga 660caagctggtg cagaccatct accgcgggat cgagccgacc ctgcccaact ggttcgcggt 720gaccaagacg cgtaatggcg ccggaggggg gaacaaggtg gtggacgagt gctacatccc 780caactacctc ctgcccaaga ctcagcccga gctgcagtgg gcgtggacta acatggagga 840gtatataagc gcgtgtttaa acctggccga gcgcaaacgg ctcgtggcgc acgacctgac 900ccacgtcagc cagacccagg agcagaacaa ggagaatctg aaccccaatt ctgacgcgcc 960tgtcatccgg tcaaaaacct ccgcacgcta catggagctg gtcgggtggc tggtggaccg 1020gggcatcacc tccgagaagc agtggatcca ggaggaccag gcctcgtaca tctccttcaa 1080cgccgcctcc aactcgcggt cccagatcaa ggccgctctg gacaatgccg gcaagatcat 1140ggcgctgacc aaatccgcgc ccgactacct ggtaggcccc gctccgcccg ccgacattaa 1200aaccaaccgc atttaccgca tcctggagct gaacggctac gaccctgcct acgccggctc 1260cgtctttctc ggctgggccc agaaaaggtt cggaaaacgc aacaccatct ggctgtttgg 1320gccggccacc acgggcaaga ccaacatcgc ggaagccatc gcccacgccg tgcccttcta 1380cggctgcgtc aactggacca atgagaactt tcccttcaac gattgcgtcg acaagatggt 1440gatctggtgg gaggagggca agatgacggc caaggtcgtg gagtccgcca aggccattct 1500cggcggcagc aaggtgcgcg tggaccaaaa gtgcaagtcg tccgcccaga tcgatcccac 1560ccccgtgatc gtcacctcca acaccaacat gtgcgccgtg attgacggga acagcaccac 1620cttcgagcac cagcagccgt tgcaggaccg gatgttcaaa tttgaactca cccgccgtct 1680ggagcatgac tttggcaagg tgacaaagca ggaagtcaaa gagttcttcc gctgggcgca 1740ggatcacgtg accgaggtgg cgcatgagtt ctacgtcaga aagggtggag ccaacaagag 1800acccgccccc gatgacgcgg ataaaagcga gcccaagcgg gcctgcccct cagtcgcgga 1860tccatcgacg tcagacgcgg aaggagctcc ggtggacttt gccgacaggt accaaaacaa 1920atgttctcgt cacgcgggca tgcttcagat gctgtttccc tgcaaaacat gcgagagaat 1980gaatcagaat ttcaacattt gcttcacgca cgggaccaga gactgttcag aatgtttccc 2040cggcgtgtca gaatctcaac cggtcgtcag aaagaggacg tatcggaaac tctgtgccat 2100tcatcatctg ctggggcggg ctcccgagat tgcttgctcg gcctgcgatc tggtcaacgt 2160ggatctggat gactgtgttt ctgagcaata aatgacttaa accaggtatg gctgccgatg 2220gttatcttcc agattggctc gaggacaacc tctctgaggg cattcgcgag tggtgggact 2280tgaaacctgg agccccgaaa cccaaagcca accagcaaaa gcaggacgac ggccggggtc 2340tggtgcttcc tggctacaag tacctcggac ccttcaacgg actcgacaag ggggagcccg 2400tcaacgcggc ggatgcagcg gccctcgagc acgacaaggc ctacgaccag cagctcaaag 2460cgggtgacaa tccgtacctg cggtataacc acgccgacgc cgagtttcag gagcgtctgc 2520aagaagatac gtcttttggg ggcaacctcg ggcgagcagt cttccaggcc aagaagaggg 2580ttctcgaacc ttttggtctg gttgaggaag gtgctaagac ggctcctgga aagaaacgtc 2640cggtagagca gtcgccacaa gagccagact cctcctcggg cattggcaag acaggccagc 2700agcccgctaa aaagagactc aattttggtc agactggcga ctcagagtca gtccccgacc 2760cacaacctct cggagaacct ccagcaaccc ccgctgctgt gggacctact acaatggctt 2820caggcggtgg cgcaccaatg gcagacaata acgaaggcgc cgacggagtg ggtaatgcct 2880caggaaattg gcattgcgat tccacatggc tgggcgacag agtcatcacc accagcaccc 2940gaacatgggc cttgcccacc tataacaacc acctctacaa gcaaatctcc agtgcttcaa 3000cgggggccag caacgacaac cactacttcg gctacagcac cccctggggg tattttgatt 3060tcaacagatt ccactgccat ttctcaccac gtgactggca gcgactcatc aacaacaatt 3120ggggattccg gcccaagaga ctcaacttca agctcttcaa catccaagtc aaggaggtca 3180cgacgaatga tggcgtcacg accatcgcta ataaccttac cagcacggtt caagtcttct 3240cggactcgga gtaccagttg ccgtacgtcc tcggctctgc gcaccagggc tgcctccctc 3300cgttcccggc ggacgtgttc atgattccgc agtacggcta cctaacgctc aacaatggca 3360gccaggcagt gggacggtca tccttttact gcctggaata tttcccatcg cagatgctga 3420gaacgggcaa taactttacc ttcagctaca ccttcgagga cgtgcctttc cacagcagct 3480acgcgcacag ccagagcctg gaccggctga tgaatcctct catcgaccag tacctgtatt 3540acctgaacag aactcagaat cagtccggaa gtgcccaaaa caaggacttg ctgtttagcc 3600gggggtctcc agctggcatg tctgttcagc ccaaaaactg gctacctgga ccctgttacc 3660ggcagcagcg cgtttctaaa acaaaaacag acaacaacaa cagcaacttt acctggactg 3720gtgcttcaaa atataacctt aatgggcgtg aatctataat caaccctggc actgctatgg 3780cctcacacaa agacgacaaa gacaagttct ttcccatgag cggtgtcatg atttttggaa 3840aggagagcgc cggagcttca aacactgcat tggacaatgt catgatcaca gacgaagagg 3900aaatcaaagc cactaacccc gtggccaccg aaagatttgg gactgtggca gtcaatctcc 3960agagcagcag cacagaccct gcgaccggag atgtgcatgt tatgggagcc ttacctggaa 4020tggtgtggca agacagagac gtatacctgc agggtcctat ttgggccaaa attcctcaca 4080cggatggaca ctttcacccg tctcctctca tgggcggctt tggacttaag cacccgcctc 4140ctcagatcct catcaaaaac acgcctgttc ctgcgaatcc tccggcagag ttttcggcta 4200caaagtttgc ttcattcatc acccagtatt ccacaggaca agtgagcgtg gagattgaat 4260gggagctgca gaaagaaaac agcaaacgct ggaatcccga agtgcagtat acatctaact 4320atgcaaaatc tgccaacgtt gatttcactg tggacaacaa tggactttat actgagcctc 4380gccccattgg cacccgttac ctcacccgtc ccctgtaatt gtgtgttaat caataaaccg 4440gttaattcgt gtcagttgaa ctttggtctc atgtcgttat tatcttatct ggtcaccata 4500gcaaccggtt acacattaac tgcttagttg cgcttcgcga atacccctag tgatggagtt 4560gcccactccc tctatgcgcg ctcgctcgct cggtggggcc ggcagagcag agctctgccg 4620tctgcggacc tttggtccgc aggccccacc gagcgagcga gcgcgcatag agggagtggg 4680caa 4683730DNAadeno-associated virus 2 7cgagtcagtt gcgcagccat cgacgtcaga 30850DNAadeno-associated virus 2 8ctggaagata accatcggca gccatacctg atttaaatca tttattgttc 50950DNAadeno-associated virus 2 9gaacaataaa tgatttaaat caggtatggc tgccgatggt tatcttccag 501040DNAadeno-associated virus 2 10ggactctaga gtaacccgat gacgtaagtc ttttatcgcg 401113804DNAadeno-associated virus 2 11cgggcccccc ctcgaggtcg acggtatcgg gggagctcgc agggtctcca ttttgaagcg 60ggaggtttga acgcgcagcc gccatgccgg ggttttacga gattgtgatt aaggtcccca 120gcgaccttga cgggcatctg cccggcattt ctgacagctt tgtgaactgg gtggccgaga 180aggaatggga gttgccgcca gattctgaca tggatctgaa tctgattgag caggcacccc 240tgaccgtggc cgagaagctg cagcgcgact ttctgacgga atggcgccgt gtgagtaagg 300ccccggaggc ccttttcttt gtgcaatttg agaagggaga gagctacttc cacatgcacg 360tgctcgtgga aaccaccggg gtgaaatcca tggttttggg acgtttcctg agtcagattc 420gcgaaaaact gattcagaga atttaccgcg ggatcgagcc gactttgcca aactggttcg 480cggtcacaaa gaccagaaat ggcgccggag gcgggaacaa ggtggtggat gagtgctaca 540tccccaatta cttgctcccc aaaacccagc ctgagctcca gtgggcgtgg actaatatgg 600aacagtattt aagcgcctgt ttgaatctca cggagcgtaa acggttggtg gcgcagcatc 660tgacgcacgt gtcgcagacg caggagcaga acaaagagaa tcagaatccc aattctgatg 720cgccggtgat cagatcaaaa acttcagcca ggtacatgga gctggtcggg tggctcgtgg 780acaaggggat tacctcggag aagcagtgga tccaggagga ccaggcctca tacatctcct 840tcaatgcggc ctccaactcg cggtcccaaa tcaaggctgc cttggacaat gcgggaaaga 900ttatgagcct gactaaaacc gcccccgact acctggtggg ccagcagccc gtggaggaca 960tttccagcaa tcggatttat aaaattttgg aactaaacgg gtacgatccc caatatgcgg 1020cttccgtctt tctgggatgg gccacgaaaa agttcggcaa gaggaacacc atctggctgt 1080ttgggcctgc aactaccggg aagaccaaca tcgcggaggc catagcccac actgtgccct 1140tctacgggtg cgtaaactgg accaatgaga
actttccctt caacgactgt gtcgacaaga 1200tggtgatctg gtgggaggag gggaagatga ccgccaaggt cgtggagtcg gccaaagcca 1260ttctcggagg aagcaaggtg cgcgtggacc agaaatgcaa gtcctcggcc cagatagacc 1320cgactcccgt gatcgtcacc tccaacacca acatgtgcgc cgtgattgac gggaactcaa 1380cgaccttcga acaccagcag ccgttgcaag accggatgtt caaatttgaa ctcacccgcc 1440gtctggatca tgactttggg aaggtcacca agcaggaagt caaagacttt ttccggtggg 1500caaaggatca cgtggttgag gtggagcatg aattctacgt caaaaagggt ggagccaaga 1560aaagacccgc ccccagtgac gcagatataa gtgagcccaa acgggtgcgc gagtcagttg 1620cgcagccatc gacgtcagac gcggaagctt cgatcaacta cgcagacagg taccaaaaca 1680aatgttctcg tcacgtgggc atgaatctga tgctgtttcc ctgcagacaa tgcgagagaa 1740tgaatcagaa ttcaaatatc tgcttcactc acggacagaa agactgttta gagtgctttc 1800ccgtgtcaga atctcaaccc gtttctgtcg tcaaaaaggc gtatcagaaa ctgtgctaca 1860ttcatcatat catgggaaag gtgccagacg cttgcactgc ctgcgatctg gtcaatgtgg 1920atttggatga ctgcatcttt gaacaataaa tgatttaaat caggtatggc tgccgatggt 1980tatcttccag attggctcga ggacaacctc tctgagggca ttcgcgagtg gtgggacttg 2040aaacctggag ccccgaagcc caaagccaac cagcaaaagc aggacgacgg ccggggtctg 2100gtgcttcctg gctacaagta cctcggaccc ttcaacggac tcgacaaggg ggagcccgtc 2160aacgcggcgg acgcagcggc cctcgagcac gacaaggcct acgaccagca gctcaaagcg 2220ggtgacaatc cgtacctgcg gtataaccac gccgacgccg agtttcagga gcgtctgcaa 2280gaagatacgt cttttggggg caacctcggg cgagcagtct tccaggccaa gaagcgggtt 2340ctcgaacctc tcggtctggt tgaggaaggc gctaagacgg ctcctggaaa gaaacgtccg 2400gtagagcagt cgccacaaga gccagactcc tcctcgggca tcggcaagac aggccagcag 2460cccgctaaaa agagactcaa ttttggtcag actggcgact cagagtcagt ccccgatcca 2520caacctctcg gagaacctcc agcaaccccc gctgctgtgg gacctactac aatggcttca 2580ggcggtggcg caccaatggc agacaataac gaaggcgccg acggagtggg taatgcctca 2640ggaaattggc attgcgattc cacatggctg ggcgacagag tcatcaccac cagcacccgc 2700acctgggcct tgcccaccta caataaccac ctctacaagc aaatctccag tgcttcaacg 2760ggggccagca acgacaacca ctacttcggc tacagcaccc cctgggggta ttttgatttc 2820aacagattcc actgccactt ttcaccacgt gactggcagc gactcatcaa caacaattgg 2880ggattccggc ccaagagact caacttcaaa ctcttcaaca tccaagtcaa ggaggtcacg 2940acgaatgatg gcgtcacaac catcgctaat aaccttacca gcacggttca agtcttctcg 3000gactcggagt accagcttcc gtacgtcctc ggctctgcgc accagggctg cctccctccg 3060ttcccggcgg acgtgttcat gattccgcaa tacggctacc tgacgctcaa caatggcagc 3120caagccgtgg gacgttcatc cttttactgc ctggaatatt tcccttctca gatgctgaga 3180acgggcaaca actttacctt cagctacacc tttgaggaag tgcctttcca cagcagctac 3240gcgcacagcc agagcctgga ccggctgatg aatcctctca tcgaccaata cctgtattac 3300ctgaacagaa ctcaaaatca gtccggaagt gcccaaaaca aggacttgct gtttagccgt 3360gggtctccag ctggcatgtc tgttcagccc aaaaactggc tacctggacc ctgttatcgg 3420cagcagcgcg tttctaaaac aaaaacagac aacaacaaca gcaattttac ctggactggt 3480gcttcaaaat ataacctcaa tgggcgtgaa tccatcatca accctggcac tgctatggcc 3540tcacacaaag acgacgaaga caagttcttt cccatgagcg gtgtcatgat ttttggaaaa 3600gagagcgccg gagcttcaaa cactgcattg gacaatgtca tgattacaga cgaagaggaa 3660attaaagcca ctaaccctgt ggccaccgaa agatttggga ccgtggcagt caatttccag 3720agcagcagca cagaccctgc gaccggagat gtgcatgcta tgggagcatt acctggcatg 3780gtgtggcaag atagagacgt gtacctgcag ggtcccattt gggccaaaat tcctcacaca 3840gatggacact ttcacccgtc tcctcttatg ggcggctttg gactcaagaa cccgcctcct 3900cagatcctca tcaaaaacac gcctgttcct gcgaatcctc cggcggagtt ttcagctaca 3960aagtttgctt cattcatcac ccaatactcc acaggacaag tgagtgtgga aattgaatgg 4020gagctgcaga aagaaaacag caagcgctgg aatcccgaag tgcagtacac atccaattat 4080gcaaaatctg ccaacgttga ttttactgtg gacaacaatg gactttatac tgagcctcgc 4140cccattggca cccgttacct tacccgtccc ctgtaattac gtgttaatca ataaaccggt 4200tgattcgttt cagttgaact ttggtctcct gtccttctta tcttatcggt taccatggtt 4260atagcttaca cattaactgc ttggttgcgc ttcgcgataa aagacttacg tcgtagccat 4320gctctagagg tcctgtatta gaggtcacgt gagtgttttg cgacattttg cgacaccatg 4380tggtcacgct gggtatttaa gcccgagtga gcacgcaggg tctccatttt gaagcgggag 4440gtttgaacgc gcagccgcca agccgaattc tgcagatatc catcacactg gcggccgctc 4500gactagagcg gccgccaccg cggtggagct ccagcttttg ttccctttag tgagggttaa 4560ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 4620caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 4680tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 4740cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 4800gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 4860tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 4920agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 4980cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 5040ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 5100tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5160gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 5220gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5280gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5340ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5400ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 5460ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 5520gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 5580ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 5640tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 5700ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 5760gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 5820tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 5880cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 5940ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 6000gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 6060caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 6120gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 6180ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 6240tgcataattc tcttactgtc atgccatccg taagatgctg cccggcgtca atacgggata 6300ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 6360gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 6420ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 6480ggcacgggcc ccccctcgag gtcgacggta tcgggggagc tcgcagggtc tccattttga 6540agcgggaggt ttgaacgcgc agccgccatg ccggggtttt acgagattgt gattaaggtc 6600cccagcgacc ttgacgggca tctgcccggc atttctgaca gctttgtgaa ctgggtggcc 6660gagaaggaat gggagttgcc gccagattct gacatggatc tgaatctgat tgagcaggca 6720cccctgaccg tggccgagaa gctgcagcgc gactttctga cggaatggcg ccgtgtgagt 6780aaggccccgg aggccctttt ctttgtgcaa tttgagaagg gagagagcta cttccacatg 6840cacgtgctcg tggaaaccac cggggtgaaa tccatggttt tgggacgttt cctgagtcag 6900attcgcgaaa aactgattca gagaatttac cgcgggatcg agccgacttt gccaaactgg 6960ttcgcggtca caaagaccag aaatggcgcc ggaggcggga acaaggtggt ggatgagtgc 7020tacatcccca attacttgct ccccaaaacc cagcctgagc tccagtgggc gtggactaat 7080atggaacagt atttaagcgc ctgtttgaat ctcacggagc gtaaacggtt ggtggcgcag 7140catctgacgc acgtgtcgca gacgcaggag cagaacaaag agaatcagaa tcccaattct 7200gatgcgccgg tgatcagatc aaaaacttca gccaggtaca tggagctggt cgggtggctc 7260gtggacaagg ggattacctc ggagaagcag tggatccagg aggaccaggc ctcatacatc 7320tccttcaatg cggcctccaa ctcgcggtcc caaatcaagg ctgccttgga caatgcggga 7380aagattatga gcctgactaa aaccgccccc gactacctgg tgggccagca gcccgtggag 7440gacatttcca gcaatcggat ttataaaatt ttggaactaa acgggtacga tccccaatat 7500gcggcttccg tctttctggg atgggccacg aaaaagttcg gcaagaggaa caccatctgg 7560ctgtttgggc ctgcaactac cgggaagacc aacatcgcgg aggccatagc ccacactgtg 7620cccttctacg ggtgcgtaaa ctggaccaat gagaactttc ccttcaacga ctgtgtcgac 7680aagatggtga tctggtggga ggaggggaag atgaccgcca aggtcgtgga gtcggccaaa 7740gccattctcg gaggaagcaa ggtgcgcgtg gaccagaaat gcaagtcctc ggcccagata 7800gacccgactc ccgtgatcgt cacctccaac accaacatgt gcgccgtgat tgacgggaac 7860tcaacgacct tcgaacacca gcagccgttg caagaccgga tgttcaaatt tgaactcacc 7920cgccgtctgg atcatgactt tgggaaggtc accaagcagg aagtcaaaga ctttttccgg 7980tgggcaaagg atcacgtggt tgaggtggag catgaattct acgtcaaaaa gggtggagcc 8040aagaaaagac ccgcccccag tgacgcagat ataagtgagc ccaaacgggt gcgcgagtca 8100gttgcgcagc catcgacgtc agacgcggaa gcttcgatca actacgcaga caggtaccaa 8160aacaaatgtt ctcgtcacgt gggcatgaat ctgatgctgt ttccctgcag acaatgcgag 8220agaatgaatc agaattcaaa tatctgcttc actcacggac agaaagactg tttagagtgc 8280tttcccgtgt cagaatctca acccgtttct gtcgtcaaaa aggcgtatca gaaactgtgc 8340tacattcatc atatcatggg aaaggtgcca gacgcttgca ctgcctgcga tctggtcaat 8400gtggatttgg atgactgcat ctttgaacaa taaatgattt aaatcaggta tggctgccga 8460tggttatctt ccagattggc tcgaggacaa cctctctgag ggcattcgcg agtggtggga 8520cttgaaacct ggagccccga agcccaaagc caaccagcaa aagcaggacg acggccgggg 8580tctggtgctt cctggctaca agtacctcgg acccttcaac ggactcgaca agggggagcc 8640cgtcaacgcg gcggacgcag cggccctcga gcacgacaag gcctacgacc agcagctcaa 8700agcgggtgac aatccgtacc tgcggtataa ccacgccgac gccgagtttc aggagcgtct 8760gcaagaagat acgtcttttg ggggcaacct cgggcgagca gtcttccagg ccaagaagcg 8820ggttctcgaa cctctcggtc tggttgagga aggcgctaag acggctcctg gaaagaaacg 8880tccggtagag cagtcgccac aagagccaga ctcctcctcg ggcatcggca agacaggcca 8940gcagcccgct aaaaagagac tcaattttgg tcagactggc gactcagagt cagtccccga 9000tccacaacct ctcggagaac ctccagcaac ccccgctgct gtgggaccta ctacaatggc 9060ttcaggcggt ggcgcaccaa tggcagacaa taacgaaggc gccgacggag tgggtaatgc 9120ctcaggaaat tggcattgcg attccacatg gctgggcgac agagtcatca ccaccagcac 9180ccgcacctgg gccttgccca cctacaataa ccacctctac aagcaaatct ccagtgcttc 9240aacgggggcc agcaacgaca accactactt cggctacagc accccctggg ggtattttga 9300tttcaacaga ttccactgcc acttttcacc acgtgactgg cagcgactca tcaacaacaa 9360ttggggattc cggcccaaga gactcaactt caaactcttc aacatccaag tcaaggaggt 9420cacgacgaat gatggcgtca caaccatcgc taataacctt accagcacgg ttcaagtctt 9480ctcggactcg gagtaccagc ttccgtacgt cctcggctct gcgcaccagg gctgcctccc 9540tccgttcccg gcggacgtgt tcatgattcc gcaatacggc tacctgacgc tcaacaatgg 9600cagccaagcc gtgggacgtt catcctttta ctgcctggaa tatttccctt ctcagatgct 9660gagaacgggc aacaacttta ccttcagcta cacctttgag gaagtgcctt tccacagcag 9720ctacgcgcac agccagagcc tggaccggct gatgaatcct ctcatcgacc aatacctgta 9780ttacctgaac agaactcaaa atcagtccgg aagtgcccaa aacaaggact tgctgtttag 9840ccgtgggtct ccagctggca tgtctgttca gcccaaaaac tggctacctg gaccctgtta 9900tcggcagcag cgcgtttcta aaacaaaaac agacaacaac aacagcaatt ttacctggac 9960tggtgcttca aaatataacc tcaatgggcg tgaatccatc atcaaccctg gcactgctat 10020ggcctcacac aaagacgacg aagacaagtt ctttcccatg agcggtgtca tgatttttgg 10080aaaagagagc gccggagctt caaacactgc attggacaat gtcatgatta cagacgaaga 10140ggaaattaaa gccactaacc ctgtggccac cgaaagattt gggaccgtgg cagtcaattt 10200ccagagcagc agcacagacc ctgcgaccgg agatgtgcat gctatgggag cattacctgg 10260catggtgtgg caagatagag acgtgtacct gcagggtccc atttgggcca aaattcctca 10320cacagatgga cactttcacc cgtctcctct tatgggcggc tttggactca agaacccgcc 10380tcctcagatc ctcatcaaaa acacgcctgt tcctgcgaat cctccggcgg agttttcagc 10440tacaaagttt gcttcattca tcacccaata ctccacagga caagtgagtg tggaaattga 10500atgggagctg cagaaagaaa acagcaagcg ctggaatccc gaagtgcagt acacatccaa 10560ttatgcaaaa tctgccaacg ttgattttac tgtggacaac aatggacttt atactgagcc 10620tcgccccatt ggcacccgtt accttacccg tcccctgtaa ttacgtgtta atcaataaac 10680cggttgattc gtttcagttg aactttggtc tcctgtcctt cttatcttat cggttaccat 10740ggttatagct tacacattaa ctgcttggtt gcgcttcgcg ataaaagact tacgtcgtag 10800ccatgctcta gaggtcctgt attagaggtc acgtgagtgt tttgcgacat tttgcgacac 10860catgtggtca cgctgggtat ttaagcccga gtgagcacgc agggtctcca ttttgaagcg 10920ggaggtttga acgcgcagcc gccaagccga attctgcaga tatccatcac actggcggcc 10980gctcgactag agcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg 11040ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 11100ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 11160tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 11220ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 11280gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 11340gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 11400ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 11460ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 11520cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 11580ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 11640tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 11700gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 11760tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 11820gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 11880tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 11940ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 12000agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 12060gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 12120attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 12180agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 12240atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 12300cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 12360ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 12420agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 12480tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 12540gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 12600caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 12660ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 12720gcactgcata attctcttac tgtcatgcca tccgtaagat gctgcccggc gtcaatacgg 12780gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 12840gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 12900gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 12960ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 13020ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 13080atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 13140gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa 13200tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat 13260agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg 13320tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac 13380catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta 13440aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag 13500ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg 13560taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca 13620ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg 13680cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac 13740gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg 13800gtac 138041250DNAadeno-associated virus 2 12gaacaataaa tgatttaaat caggtatgtc ttttgttgat caccctccag 501340DNAadeno-associated virus 2 13ggactctaga gaccacaaga ggcagtattt tactgacacg 401413676DNAadeno-associated virus 2 14cgggcccccc ctcgaggtcg acggtatcgg gggagctcgc agggtctcca ttttgaagcg 60ggaggtttga acgcgcagcc gccatgccgg ggttttacga gattgtgatt aaggtcccca 120gcgaccttga cgggcatctg cccggcattt ctgacagctt tgtgaactgg gtggccgaga 180aggaatggga gttgccgcca gattctgaca tggatctgaa tctgattgag caggcacccc 240tgaccgtggc cgagaagctg cagcgcgact ttctgacgga atggcgccgt gtgagtaagg 300ccccggaggc ccttttcttt gtgcaatttg agaagggaga gagctacttc cacatgcacg 360tgctcgtgga aaccaccggg gtgaaatcca tggttttggg acgtttcctg agtcagattc 420gcgaaaaact gattcagaga atttaccgcg ggatcgagcc gactttgcca aactggttcg 480cggtcacaaa gaccagaaat ggcgccggag gcgggaacaa ggtggtggat gagtgctaca 540tccccaatta cttgctcccc aaaacccagc ctgagctcca gtgggcgtgg actaatatgg 600aacagtattt aagcgcctgt ttgaatctca cggagcgtaa acggttggtg gcgcagcatc 660tgacgcacgt gtcgcagacg caggagcaga acaaagagaa tcagaatccc aattctgatg 720cgccggtgat cagatcaaaa acttcagcca ggtacatgga gctggtcggg tggctcgtgg 780acaaggggat tacctcggag aagcagtgga tccaggagga ccaggcctca tacatctcct 840tcaatgcggc ctccaactcg cggtcccaaa tcaaggctgc cttggacaat gcgggaaaga 900ttatgagcct gactaaaacc gcccccgact acctggtggg ccagcagccc gtggaggaca 960tttccagcaa tcggatttat aaaattttgg aactaaacgg gtacgatccc caatatgcgg 1020cttccgtctt tctgggatgg gccacgaaaa agttcggcaa gaggaacacc atctggctgt 1080ttgggcctgc aactaccggg aagaccaaca tcgcggaggc catagcccac actgtgccct 1140tctacgggtg cgtaaactgg accaatgaga actttccctt caacgactgt gtcgacaaga 1200tggtgatctg gtgggaggag gggaagatga ccgccaaggt cgtggagtcg gccaaagcca 1260ttctcggagg aagcaaggtg cgcgtggacc agaaatgcaa gtcctcggcc cagatagacc 1320cgactcccgt gatcgtcacc tccaacacca acatgtgcgc cgtgattgac gggaactcaa 1380cgaccttcga acaccagcag ccgttgcaag accggatgtt caaatttgaa ctcacccgcc 1440gtctggatca tgactttggg aaggtcacca agcaggaagt caaagacttt ttccggtggg 1500caaaggatca cgtggttgag gtggagcatg aattctacgt caaaaagggt ggagccaaga 1560aaagacccgc ccccagtgac gcagatataa gtgagcccaa acgggtgcgc gagtcagttg 1620cgcagccatc gacgtcagac gcggaagctt cgatcaacta cgcagacagg taccaaaaca 1680aatgttctcg tcacgtgggc atgaatctga tgctgtttcc ctgcagacaa tgcgagagaa 1740tgaatcagaa ttcaaatatc tgcttcactc acggacagaa agactgttta gagtgctttc 1800ccgtgtcaga atctcaaccc gtttctgtcg tcaaaaaggc gtatcagaaa ctgtgctaca 1860ttcatcatat catgggaaag gtgccagacg cttgcactgc ctgcgatctg gtcaatgtgg 1920atttggatga ctgcatcttt gaacaataaa tgatttaaat caggtatgtc ttttgttgat 1980caccctccag attggttgga agaagttggt gaaggtcttc gcgagttttt gggccttgaa 2040gcgggcccac cgaaaccaaa acccaatcag cagcatcaag atcaagcccg tggtcttgtg 2100ctgcctggtt ataactatct cggacccgga aacggtctcg atcgaggaga gcctgtcaac
2160agggcagacg aggtcgcgcg agagcacgac atctcgtaca acgagcagct tgaggcggga 2220gacaacccct acctcaagta caaccacgcg gacgccgagt ttcaggagaa gctcgccgac 2280gacacatcct tcgggggaaa cctcggaaag gcagtctttc aggccaagaa aagggttctc 2340gaaccttttg gcctggttga agagggtgct aagacggccc ctaccggaaa gcggatagac 2400gaccactttc caaaaagaaa gaaggctcgg accgaagagg actccaagcc ttccacctcg 2460tcagacgccg aagctggacc cagcggatcc cagcagctgc aaatcccagc ccaaccagcc 2520tcaagtttgg gagctgatac aatgtctgcg ggaggtggcg gcccattggg cgacaataac 2580caaggtgccg atggagtggg caatgcctcg ggagattggc attgcgattc cacgtggatg 2640ggggacagag tcgtcaccaa gtccacccga acctgggtgc tgcccagcta caacaaccac 2700cagtaccgag agatcaaaag cggctccgtc gacggaagca acgccaacgc ctactttgga 2760tacagcaccc cctgggggta ctttgacttt aaccgcttcc acagccactg gagcccccga 2820gactggcaaa gactcatcaa caactactgg ggcttcagac cccggtccct cagagtcaaa 2880atcttcaaca ttcaagtcaa agaggtcacg gtgcaggact ccaccaccac catcgccaac 2940aacctcacct ccaccgtcca agtgtttacg gacgacgact accagctgcc ctacgtcgtc 3000ggcaacggga ccgagggatg cctgccggcc ttccctccgc aggtctttac gctgccgcag 3060tacggttacg cgacgctgaa ccgcgacaac acagaaaatc ccaccgagag gagcagcttc 3120ttctgcctag agtactttcc cagcaagatg ctgagaacgg gcaacaactt tgagtttacc 3180tacaactttg aggaggtgcc cttccactcc agcttcgctc ccagtcagaa cctgttcaag 3240ctggccaacc cgctggtgga ccagtacttg taccgcttcg tgagcacaaa taacactggc 3300ggagtccagt tcaacaagaa cctggccggg agatacgcca acacctacaa aaactggttc 3360ccggggccca tgggccgaac ccagggctgg aacctgggct ccggggtcaa ccgcgccagt 3420gtcagcgcct tcgccacgac caataggatg gagctcgagg gcgcgagtta ccaggtgccc 3480ccgcagccga acggcatgac caacaacctc cagggcagca acacctatgc cctggagaac 3540actatgatct tcaacagcca gccggcgaac ccgggcacca ccgccacgta cctcgagggc 3600aacatgctca tcaccagcga gagcgagacg cagccggtga accgcgtggc gtacaacgtc 3660ggcgggcaga tggccaccaa caaccagagc tccaccactg cccccgcgac cggcacgtac 3720aacctccagg aaatcgtgcc cggcagcgtg tggatggaga gggacgtgta cctccaagga 3780cccatctggg ccaagatccc agagacgggg gcgcactttc acccctctcc ggccatgggc 3840ggattcggac tcaaacaccc accgcccatg atgctcatca agaacacgcc tgtgcccgga 3900aatatcacca gcttctcgga cgtgcccgtc agcagcttca tcacccagta cagcaccggg 3960caggtcaccg tggagatgga gtgggagctc aagaaggaaa actccaagag gtggaaccca 4020gagatccagt acacaaacaa ctacaacgac ccccagtttg tggactttgc cccggacagc 4080accggggaat acagaaccac cagacctatc ggaacccgat accttacccg acccctttaa 4140cccattcatg tcgcataccc tcaataaacc gtgtattcgt gtcagtaaaa tactgcctct 4200tgtggtcatt caatgaataa cagcttacaa catcgtagcc atgctctaga ggtcctgtat 4260tagaggtcac gtgagtgttt tgcgacattt tgcgacacca tgtggtcacg ctgggtattt 4320aagcccgagt gagcacgcag ggtctccatt ttgaagcggg aggtttgaac gcgcagccgc 4380caagccgaat tctgcagata tccatcacac tggcggccgc tcgactagag cggccgccac 4440cgcggtggag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat 4500catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 4560gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 4620ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 4680gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 4740tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 4800cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 4860gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 4920gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 4980gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 5040ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 5100atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 5160tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 5220ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 5280gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 5340ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 5400ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 5460agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 5520ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 5580aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 5640tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 5700cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 5760tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 5820cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 5880ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 5940gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 6000gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 6060gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 6120gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 6180tcatgccatc cgtaagatgc tgcccggcgt caatacggga taataccgcg ccacatagca 6240gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 6300taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 6360cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 6420agggaataag ggcgcgggcc ccccctcgag gtcgacggta tcgggggagc tcgcagggtc 6480tccattttga agcgggaggt ttgaacgcgc agccgccatg ccggggtttt acgagattgt 6540gattaaggtc cccagcgacc ttgacgggca tctgcccggc atttctgaca gctttgtgaa 6600ctgggtggcc gagaaggaat gggagttgcc gccagattct gacatggatc tgaatctgat 6660tgagcaggca cccctgaccg tggccgagaa gctgcagcgc gactttctga cggaatggcg 6720ccgtgtgagt aaggccccgg aggccctttt ctttgtgcaa tttgagaagg gagagagcta 6780cttccacatg cacgtgctcg tggaaaccac cggggtgaaa tccatggttt tgggacgttt 6840cctgagtcag attcgcgaaa aactgattca gagaatttac cgcgggatcg agccgacttt 6900gccaaactgg ttcgcggtca caaagaccag aaatggcgcc ggaggcggga acaaggtggt 6960ggatgagtgc tacatcccca attacttgct ccccaaaacc cagcctgagc tccagtgggc 7020gtggactaat atggaacagt atttaagcgc ctgtttgaat ctcacggagc gtaaacggtt 7080ggtggcgcag catctgacgc acgtgtcgca gacgcaggag cagaacaaag agaatcagaa 7140tcccaattct gatgcgccgg tgatcagatc aaaaacttca gccaggtaca tggagctggt 7200cgggtggctc gtggacaagg ggattacctc ggagaagcag tggatccagg aggaccaggc 7260ctcatacatc tccttcaatg cggcctccaa ctcgcggtcc caaatcaagg ctgccttgga 7320caatgcggga aagattatga gcctgactaa aaccgccccc gactacctgg tgggccagca 7380gcccgtggag gacatttcca gcaatcggat ttataaaatt ttggaactaa acgggtacga 7440tccccaatat gcggcttccg tctttctggg atgggccacg aaaaagttcg gcaagaggaa 7500caccatctgg ctgtttgggc ctgcaactac cgggaagacc aacatcgcgg aggccatagc 7560ccacactgtg cccttctacg ggtgcgtaaa ctggaccaat gagaactttc ccttcaacga 7620ctgtgtcgac aagatggtga tctggtggga ggaggggaag atgaccgcca aggtcgtgga 7680gtcggccaaa gccattctcg gaggaagcaa ggtgcgcgtg gaccagaaat gcaagtcctc 7740ggcccagata gacccgactc ccgtgatcgt cacctccaac accaacatgt gcgccgtgat 7800tgacgggaac tcaacgacct tcgaacacca gcagccgttg caagaccgga tgttcaaatt 7860tgaactcacc cgccgtctgg atcatgactt tgggaaggtc accaagcagg aagtcaaaga 7920ctttttccgg tgggcaaagg atcacgtggt tgaggtggag catgaattct acgtcaaaaa 7980gggtggagcc aagaaaagac ccgcccccag tgacgcagat ataagtgagc ccaaacgggt 8040gcgcgagtca gttgcgcagc catcgacgtc agacgcggaa gcttcgatca actacgcaga 8100caggtaccaa aacaaatgtt ctcgtcacgt gggcatgaat ctgatgctgt ttccctgcag 8160acaatgcgag agaatgaatc agaattcaaa tatctgcttc actcacggac agaaagactg 8220tttagagtgc tttcccgtgt cagaatctca acccgtttct gtcgtcaaaa aggcgtatca 8280gaaactgtgc tacattcatc atatcatggg aaaggtgcca gacgcttgca ctgcctgcga 8340tctggtcaat gtggatttgg atgactgcat ctttgaacaa taaatgattt aaatcaggta 8400tgtcttttgt tgatcaccct ccagattggt tggaagaagt tggtgaaggt cttcgcgagt 8460ttttgggcct tgaagcgggc ccaccgaaac caaaacccaa tcagcagcat caagatcaag 8520cccgtggtct tgtgctgcct ggttataact atctcggacc cggaaacggt ctcgatcgag 8580gagagcctgt caacagggca gacgaggtcg cgcgagagca cgacatctcg tacaacgagc 8640agcttgaggc gggagacaac ccctacctca agtacaacca cgcggacgcc gagtttcagg 8700agaagctcgc cgacgacaca tccttcgggg gaaacctcgg aaaggcagtc tttcaggcca 8760agaaaagggt tctcgaacct tttggcctgg ttgaagaggg tgctaagacg gcccctaccg 8820gaaagcggat agacgaccac tttccaaaaa gaaagaaggc tcggaccgaa gaggactcca 8880agccttccac ctcgtcagac gccgaagctg gacccagcgg atcccagcag ctgcaaatcc 8940cagcccaacc agcctcaagt ttgggagctg atacaatgtc tgcgggaggt ggcggcccat 9000tgggcgacaa taaccaaggt gccgatggag tgggcaatgc ctcgggagat tggcattgcg 9060attccacgtg gatgggggac agagtcgtca ccaagtccac ccgaacctgg gtgctgccca 9120gctacaacaa ccaccagtac cgagagatca aaagcggctc cgtcgacgga agcaacgcca 9180acgcctactt tggatacagc accccctggg ggtactttga ctttaaccgc ttccacagcc 9240actggagccc ccgagactgg caaagactca tcaacaacta ctggggcttc agaccccggt 9300ccctcagagt caaaatcttc aacattcaag tcaaagaggt cacggtgcag gactccacca 9360ccaccatcgc caacaacctc acctccaccg tccaagtgtt tacggacgac gactaccagc 9420tgccctacgt cgtcggcaac gggaccgagg gatgcctgcc ggccttccct ccgcaggtct 9480ttacgctgcc gcagtacggt tacgcgacgc tgaaccgcga caacacagaa aatcccaccg 9540agaggagcag cttcttctgc ctagagtact ttcccagcaa gatgctgaga acgggcaaca 9600actttgagtt tacctacaac tttgaggagg tgcccttcca ctccagcttc gctcccagtc 9660agaacctgtt caagctggcc aacccgctgg tggaccagta cttgtaccgc ttcgtgagca 9720caaataacac tggcggagtc cagttcaaca agaacctggc cgggagatac gccaacacct 9780acaaaaactg gttcccgggg cccatgggcc gaacccaggg ctggaacctg ggctccgggg 9840tcaaccgcgc cagtgtcagc gccttcgcca cgaccaatag gatggagctc gagggcgcga 9900gttaccaggt gcccccgcag ccgaacggca tgaccaacaa cctccagggc agcaacacct 9960atgccctgga gaacactatg atcttcaaca gccagccggc gaacccgggc accaccgcca 10020cgtacctcga gggcaacatg ctcatcacca gcgagagcga gacgcagccg gtgaaccgcg 10080tggcgtacaa cgtcggcggg cagatggcca ccaacaacca gagctccacc actgcccccg 10140cgaccggcac gtacaacctc caggaaatcg tgcccggcag cgtgtggatg gagagggacg 10200tgtacctcca aggacccatc tgggccaaga tcccagagac gggggcgcac tttcacccct 10260ctccggccat gggcggattc ggactcaaac acccaccgcc catgatgctc atcaagaaca 10320cgcctgtgcc cggaaatatc accagcttct cggacgtgcc cgtcagcagc ttcatcaccc 10380agtacagcac cgggcaggtc accgtggaga tggagtggga gctcaagaag gaaaactcca 10440agaggtggaa cccagagatc cagtacacaa acaactacaa cgacccccag tttgtggact 10500ttgccccgga cagcaccggg gaatacagaa ccaccagacc tatcggaacc cgatacctta 10560cccgacccct ttaacccatt catgtcgcat accctcaata aaccgtgtat tcgtgtcagt 10620aaaatactgc ctcttgtggt cattcaatga ataacagctt acaacatcgt agccatgctc 10680tagaggtcct gtattagagg tcacgtgagt gttttgcgac attttgcgac accatgtggt 10740cacgctgggt atttaagccc gagtgagcac gcagggtctc cattttgaag cgggaggttt 10800gaacgcgcag ccgccaagcc gaattctgca gatatccatc acactggcgg ccgctcgact 10860agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc 10920gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 10980tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 11040ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 11100ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 11160ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 11220agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 11280catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 11340tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 11400gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 11460ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 11520cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 11580caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 11640ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 11700taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 11760taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 11820cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 11880tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 11940gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 12000catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 12060atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 12120ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 12180gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 12240agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 12300gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 12360agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 12420catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 12480aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 12540gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 12600taattctctt actgtcatgc catccgtaag atgctgcccg gcgtcaatac gggataatac 12660cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 12720actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 12780ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 12840aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 12900ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 12960atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 13020taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca 13080ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag 13140atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc 13200aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc 13260taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc 13320ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa 13380gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc 13440acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc 13500aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg 13560ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt 13620aaaacgacgg ccagtgagcg cgcgtaatac gactcactat agggcgaatt gggtac 136761512136DNAadeno-associated virus 2 15cgggcccccc ctcgaggtcg acggtatcgg gggagctcgc agggtctcca ttttgaagcg 60ggaggtttga acgcgcagcc gccatgccgg ggttttacga gattgtgatt aaggtcccca 120gcgaccttga cgggcatctg cccggcattt ctgacagctt tgtgaactgg gtggccgaga 180aggaatggga gttgccgcca gattctgaca tggatctgaa tctgattgag caggcacccc 240tgaccgtggc cgagaagctg cagcgcgact ttctgacgga atggcgccgt gtgagtaagg 300ccccggaggc ccttttcttt gtgcaatttg agaagggaga gagctacttc cacatgcacg 360tgctcgtgga aaccaccggg gtgaaatcca tggttttggg acgtttcctg agtcagattc 420gcgaaaaact gattcagaga atttaccgcg ggatcgagcc gactttgcca aactggttcg 480cggtcacaaa gaccagaaat ggcgccggag gcgggaacaa ggtggtggat gagtgctaca 540tccccaatta cttgctcccc aaaacccagc ctgagctcca gtgggcgtgg actaatatgg 600aacagtattt aagcgcctgt ttgaatctca cggagcgtaa acggttggtg gcgcagcatc 660tgacgcacgt gtcgcagacg caggagcaga acaaagagaa tcagaatccc aattctgatg 720cgccggtgat cagatcaaaa acttcagcca ggtacatgga gctggtcggg tggctcgtgg 780acaaggggat tacctcggag aagcagtgga tccaggagga ccaggcctca tacatctcct 840tcaatgcggc ctccaactcg cggtcccaaa tcaaggctgc cttggacaat gcgggaaaga 900ttatgagcct gactaaaacc gcccccgact acctggtggg ccagcagccc gtggaggaca 960tttccagcaa tcggatttat aaaattttgg aactaaacgg gtacgatccc caatatgcgg 1020cttccgtctt tctgggatgg gccacgaaaa agttcggcaa gaggaacacc atctggctgt 1080ttgggcctgc aactaccggg aagaccaaca tcgcggaggc catagcccac actgtgccct 1140tctacgggtg cgtaaactgg accaatgaga actttccctt caacgactgt gtcgacaaga 1200tggtgatctg gtgggaggag gggaagatga ccgccaaggt cgtggagtcg gccaaagcca 1260ttctcggagg aagcaaggtg cgcgtggacc agaaatgcaa gtcctcggcc cagatagacc 1320cgactcccgt gatcgtcacc tccaacacca acatgtgcgc cgtgattgac gggaactcaa 1380cgaccttcga acaccagcag ccgttgcaag accggatgtt caaatttgaa ctcacccgcc 1440gtctggatca tgactttggg aaggtcacca agcaggaagt caaagacttt ttccggtggg 1500caaaggatca cgtggttgag gtggagcatg aattctacgt caaaaagggt ggagccaaga 1560aaagacccgc ccccagtgac gcagatataa gtgagcccaa acgggtgcgc gagtcagttg 1620cgcagccatc gacgtcagac gcggaagctt cgatcaacta cgcagacagg taccaaaaca 1680aatgttctcg tcacgtgggc atgaatctga tgctgtttcc ctgcagacaa tgcgagagaa 1740tgaatcagaa ttcaaatatc tgcttcactc acggacagaa agactgttta gagtgctttc 1800ccgtgtcaga atctcaaccc gtttctgtcg tcaaaaaggc gtatcagaaa ctgtgctaca 1860ttcatcatat catgggaaag gtgccagacg cttgcactgc ctgcgatctg gtcaatgtgg 1920atttggatga ctgcatcttt gaacaataaa tgatttaaat caggtatggc tgccgatggt 1980tatcttccag attggctcga ggacactctc tctgaaggaa taagacagtg gtggaagctc 2040aaacctggcc caccaccacc aaagcccgca gagcggcata aggacgacag caggggtctt 2100gtgcttcctg ggtacaagta cctcggaccc ttcaacggac tcgacaaggg agagccggtc 2160aacgaggcag acgccgcggc cctcgagcac gacaaagcct acgaccggca gctcgacagc 2220ggagacaacc cgtacctcaa gtacaaccac gccgacgcgg agtttcagga gcgccttaaa 2280gaagatacgt cttttggggg caacctcgga cgagcagtct tccaggcgaa aaagagggtt 2340cttgaacctc tgggcctggt tgaggaacct gttaagacgg ctccgggaaa aaagaggccg 2400gtagagcact ctcctgtgga gccagactcc tcctcgggaa ccggaaaggc gggccagcag 2460cctgcaagaa aaagattgaa ttttggtcag actggagacg cagactcagt acctgacccc 2520cagcctctcg gacagccacc agcagccccc tctggtctgg gaactaatac gatggctaca 2580ggcagtggcg caccaatggc agacaataac gagggcgccg acggagtggg taattcctcg 2640ggaaattggc attgcgattc cacatggatg ggcgacagag tcatcaccac cagcacccga 2700acctgggccc tgcccaccta caacaaccac ctctacaaac aaatttccag ccaatcagga 2760gcctcgaacg acaatcacta ctttggctac agcacccctt gggggtattt tgacttcaac 2820agattccact gccacttttc accacgtgac tggcaaagac tcatcaacaa caactgggga 2880ttccgaccca agagactcaa cttcaagctc tttaacattc aagtcaaaga ggtcacgcag 2940aatgacggta cgacgacgat tgccaataac cttaccagca cggttcaggt gtttactgac 3000tcggagtacc agctcccgta cgtcctcggc tcggcgcatc aaggatgcct cccgccgttc 3060ccagcagacg tcttcatggt gccacagtat ggatacctca ccctgaacaa cgggagtcag 3120gcagtaggac gctcttcatt ttactgcctg gagtactttc cttctcagat gctgcgtacc 3180ggaaacaact ttaccttcag ctacactttt gaggacgttc ctttccacag cagctacgct 3240cacagccaga gtctggaccg tctcatgaat cctctcatcg accagtacct gtattacttg 3300agcagaacaa acactccaag tggaaccacc acgcagtcaa ggcttcagtt ttctcaggcc 3360ggagcgagtg acattcggga ccagtctagg aactggcttc ctggaccctg ttaccgccag 3420cagcgagtat caaagacatc tgcggataac aacaacagtg aatactcgtg gactggagct 3480accaagtacc acctcaatgg
cagagactct ctggtgaatc cgggcccggc catggcaagc 3540cacaaggacg atgaagaaaa gttttttcct cagagcgggg ttctcatctt tgggaagcaa 3600ggctcagaga aaacaaatgt ggacattgaa aaggtcatga ttacagacga agaggaaatc 3660aggacaacca atcccgtggc tacggagcag tatggttctg tatctaccaa cctccagaga 3720ggcaacagac aagcagctac cgcagatgtc aacacacaag gcgttcttcc aggcatggtc 3780tggcaggaca gagatgtgta ccttcagggg cccatctggg caaagattcc acacacggac 3840ggacattttc acccctctcc cctcatgggt ggattcggac ttaaacaccc tcctccacag 3900attctcatca agaacacccc ggtacctgcg aatccttcga ccaccttcag tgcggcaaag 3960tttgcttcct tcatcacaca gtactccacg ggacaggtca gcgtggagat cgagtgggag 4020ctgcagaagg aaaacagcaa acgctggaat cccgaaattc agtacacttc caactacaac 4080aagtctgtta atgtggactt tactgtggac actaatggcg tgtattcaga gcctcgcccc 4140attggcacca gatacctgac tcgtaatctg taattgcttg ttaatcaata aaccgtttaa 4200ttcgtttcag ttgaactttg gtctctgcgt atttctttct tatctagttt ccatgctcta 4260ggatccacta gtaacggccg ccagtgtgct ggaattcggc tttgtagtta atgattaacc 4320cgccatgcta cttatctacg tagccatgca ttcaatgaat aacagcttac aacatcgtag 4380ccatgctcta gaggtcctgt attagaggtc acgtgagtgt tttgcgacat tttgcgacac 4440catgtggtca cgctgggtat ttaagcccga gtgagcacgc agggtctcca ttttgaagcg 4500ggaggtttga acgcgcagcc gccaagccga attctgcaga tatccatcac actggcggcc 4560gctcgactag agcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg 4620ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 4680ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 4740tgagtgagct aacgggcccc ccctcgaggt cgacggtatc gggggagctc gcagggtctc 4800cattttgaag cgggaggttt gaacgcgcag ccgccatgcc ggggttttac gagattgtga 4860ttaaggtccc cagcgacctt gacgggcatc tgcccggcat ttctgacagc tttgtgaact 4920gggtggccga gaaggaatgg gagttgccgc cagattctga catggatctg aatctgattg 4980agcaggcacc cctgaccgtg gccgagaagc tgcagcgcga ctttctgacg gaatggcgcc 5040gtgtgagtaa ggccccggag gcccttttct ttgtgcaatt tgagaaggga gagagctact 5100tccacatgca cgtgctcgtg gaaaccaccg gggtgaaatc catggttttg ggacgtttcc 5160tgagtcagat tcgcgaaaaa ctgattcaga gaatttaccg cgggatcgag ccgactttgc 5220caaactggtt cgcggtcaca aagaccagaa atggcgccgg aggcgggaac aaggtggtgg 5280atgagtgcta catccccaat tacttgctcc ccaaaaccca gcctgagctc cagtgggcgt 5340ggactaatat ggaacagtat ttaagcgcct gtttgaatct cacggagcgt aaacggttgg 5400tggcgcagca tctgacgcac gtgtcgcaga cgcaggagca gaacaaagag aatcagaatc 5460ccaattctga tgcgccggtg atcagatcaa aaacttcagc caggtacatg gagctggtcg 5520ggtggctcgt ggacaagggg attacctcgg agaagcagtg gatccaggag gaccaggcct 5580catacatctc cttcaatgcg gcctccaact cgcggtccca aatcaaggct gccttggaca 5640atgcgggaaa gattatgagc ctgactaaaa ccgcccccga ctacctggtg ggccagcagc 5700ccgtggagga catttccagc aatcggattt ataaaatttt ggaactaaac gggtacgatc 5760cccaatatgc ggcttccgtc tttctgggat gggccacgaa aaagttcggc aagaggaaca 5820ccatctggct gtttgggcct gcaactaccg ggaagaccaa catcgcggag gccatagccc 5880acactgtgcc cttctacggg tgcgtaaact ggaccaatga gaactttccc ttcaacgact 5940gtgtcgacaa gatggtgatc tggtgggagg aggggaagat gaccgccaag gtcgtggagt 6000cggccaaagc cattctcgga ggaagcaagg tgcgcgtgga ccagaaatgc aagtcctcgg 6060cccagataga cccgactccc gtgatcgtca cctccaacac caacatgtgc gccgtgattg 6120acgggaactc aacgaccttc gaacaccagc agccgttgca agaccggatg ttcaaatttg 6180aactcacccg ccgtctggat catgactttg ggaaggtcac caagcaggaa gtcaaagact 6240ttttccggtg ggcaaaggat cacgtggttg aggtggagca tgaattctac gtcaaaaagg 6300gtggagccaa gaaaagaccc gcccccagtg acgcagatat aagtgagccc aaacgggtgc 6360gcgagtcagt tgcgcagcca tcgacgtcag acgcggaagc ttcgatcaac tacgcagaca 6420ggtaccaaaa caaatgttct cgtcacgtgg gcatgaatct gatgctgttt ccctgcagac 6480aatgcgagag aatgaatcag aattcaaata tctgcttcac tcacggacag aaagactgtt 6540tagagtgctt tcccgtgtca gaatctcaac ccgtttctgt cgtcaaaaag gcgtatcaga 6600aactgtgcta cattcatcat atcatgggaa aggtgccaga cgcttgcact gcctgcgatc 6660tggtcaatgt ggatttggat gactgcatct ttgaacaata aatgatttaa atcaggtatg 6720gctgccgatg gttatcttcc agattggctc gaggacactc tctctgaagg aataagacag 6780tggtggaagc tcaaacctgg cccaccacca ccaaagcccg cagagcggca taaggacgac 6840agcaggggtc ttgtgcttcc tgggtacaag tacctcggac ccttcaacgg actcgacaag 6900ggagagccgg tcaacgaggc agacgccgcg gccctcgagc acgacaaagc ctacgaccgg 6960cagctcgaca gcggagacaa cccgtacctc aagtacaacc acgccgacgc ggagtttcag 7020gagcgcctta aagaagatac gtcttttggg ggcaacctcg gacgagcagt cttccaggcg 7080aaaaagaggg ttcttgaacc tctgggcctg gttgaggaac ctgttaagac ggctccggga 7140aaaaagaggc cggtagagca ctctcctgtg gagccagact cctcctcggg aaccggaaag 7200gcgggccagc agcctgcaag aaaaagattg aattttggtc agactggaga cgcagactca 7260gtacctgacc cccagcctct cggacagcca ccagcagccc cctctggtct gggaactaat 7320acgatggcta caggcagtgg cgcaccaatg gcagacaata acgagggcgc cgacggagtg 7380ggtaattcct cgggaaattg gcattgcgat tccacatgga tgggcgacag agtcatcacc 7440accagcaccc gaacctgggc cctgcccacc tacaacaacc acctctacaa acaaatttcc 7500agccaatcag gagcctcgaa cgacaatcac tactttggct acagcacccc ttgggggtat 7560tttgacttca acagattcca ctgccacttt tcaccacgtg actggcaaag actcatcaac 7620aacaactggg gattccgacc caagagactc aacttcaagc tctttaacat tcaagtcaaa 7680gaggtcacgc agaatgacgg tacgacgacg attgccaata accttaccag cacggttcag 7740gtgtttactg actcggagta ccagctcccg tacgtcctcg gctcggcgca tcaaggatgc 7800ctcccgccgt tcccagcaga cgtcttcatg gtgccacagt atggatacct caccctgaac 7860aacgggagtc aggcagtagg acgctcttca ttttactgcc tggagtactt tccttctcag 7920atgctgcgta ccggaaacaa ctttaccttc agctacactt ttgaggacgt tcctttccac 7980agcagctacg ctcacagcca gagtctggac cgtctcatga atcctctcat cgaccagtac 8040ctgtattact tgagcagaac aaacactcca agtggaacca ccacgcagtc aaggcttcag 8100ttttctcagg ccggagcgag tgacattcgg gaccagtcta ggaactggct tcctggaccc 8160tgttaccgcc agcagcgagt atcaaagaca tctgcggata acaacaacag tgaatactcg 8220tggactggag ctaccaagta ccacctcaat ggcagagact ctctggtgaa tccgggcccg 8280gccatggcaa gccacaagga cgatgaagaa aagttttttc ctcagagcgg ggttctcatc 8340tttgggaagc aaggctcaga gaaaacaaat gtggacattg aaaaggtcat gattacagac 8400gaagaggaaa tcaggacaac caatcccgtg gctacggagc agtatggttc tgtatctacc 8460aacctccaga gaggcaacag acaagcagct accgcagatg tcaacacaca aggcgttctt 8520ccaggcatgg tctggcagga cagagatgtg taccttcagg ggcccatctg ggcaaagatt 8580ccacacacgg acggacattt tcacccctct cccctcatgg gtggattcgg acttaaacac 8640cctcctccac agattctcat caagaacacc ccggtacctg cgaatccttc gaccaccttc 8700agtgcggcaa agtttgcttc cttcatcaca cagtactcca cgggacaggt cagcgtggag 8760atcgagtggg agctgcagaa ggaaaacagc aaacgctgga atcccgaaat tcagtacact 8820tccaactaca acaagtctgt taatgtggac tttactgtgg acactaatgg cgtgtattca 8880gagcctcgcc ccattggcac cagatacctg actcgtaatc tgtaattgct tgttaatcaa 8940taaaccgttt aattcgtttc agttgaactt tggtctctgc gtatttcttt cttatctagt 9000ttccatgctc taggatccac tagtaacggc cgccagtgtg ctggaattcg gctttgtagt 9060taatgattaa cccgccatgc tacttatcta cgtagccatg cattcaatga ataacagctt 9120acaacatcgt agccatgctc tagaggtcct gtattagagg tcacgtgagt gttttgcgac 9180attttgcgac accatgtggt cacgctgggt atttaagccc gagtgagcac gcagggtctc 9240cattttgaag cgggaggttt gaacgcgcag ccgccaagcc gaattctgca gatatccatc 9300acactggcgg ccgctcgact agagcggccg ccaccgcggt ggagctccag cttttgttcc 9360ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga 9420aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 9480tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 9540cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 9600ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 9660cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 9720ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 9780aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 9840cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 9900cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 9960gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 10020tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 10080cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 10140ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 10200gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 10260gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 10320accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 10380ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 10440tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 10500aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 10560taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 10620gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 10680agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 10740cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 10800tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 10860gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 10920agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 10980gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 11040atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgctgcccg 11100gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 11160aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 11220taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 11280tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 11340tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 11400atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 11460tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa 11520atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata 11580aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac 11640tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc 11700cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa 11760atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg 11820cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg 11880tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc 11940attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 12000tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 12060tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat 12120agggcgaatt gggtac 12136
Patent applications by Matthew During, Columbus, OH US
Patent applications by NEUROLOGIX, INC.
Patent applications in class VIRUS OR BACTERIOPHAGE, EXCEPT FOR VIRAL VECTOR OR BACTERIOPHAGE VECTOR; COMPOSITION THEREOF; PREPARATION OR PURIFICATION THEREOF; PRODUCTION OF VIRAL SUBUNITS; MEDIA FOR PROPAGATING
Patent applications in all subclasses VIRUS OR BACTERIOPHAGE, EXCEPT FOR VIRAL VECTOR OR BACTERIOPHAGE VECTOR; COMPOSITION THEREOF; PREPARATION OR PURIFICATION THEREOF; PRODUCTION OF VIRAL SUBUNITS; MEDIA FOR PROPAGATING