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Title:  Recombinant adeno-associated virus virions for the treatment of lysosomal disorders

United States Patent:  6,582,692

Issued:  June 24, 2003

Inventors:  Podsakoff; Gregory (Fullerton, CA); Watson; Gordon (El Sobrante, CA)

Assignee:  Avigen, Inc. (Alameda, CA); Children's Hospital Medical Center of Northern California (Oakland, CA)

Appl. No.:  715858

Filed:  November 17, 2000


AAV expression vectors and recombinant virions produced using these vectors, which include genes coding for enzymes defective or missing in lysosomal storage disorders, are described. These recombinant AAV virions are useful in the treatment of a variety of lysosomal storage disorders and the methods described herein provide for long-term, sustained expression of the defective or missing enzyme.


The practice of the present invention will employ, unless otherwise indicated, conventional methods of virology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (Current Edition); DNA Cloning: A Practical Approach, Vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., Current Edition); Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition); CRC Handbook of Parvoviruses, vol. I & II (P. Tijssen, ed.); Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.); Freshney Culture of Animal Cells, A Manual of Basic Technique (Wiley-Liss, Third Edition); and Ausubel et al. (1991) Current Protocols in Molecular Biology (Wiley Interscience, N.Y.).

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a recombinant AAV virion" includes a mixture of two or more virions, and the like.

General Methods

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

Although a number of compositions and methods similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.

The present invention is directed to novel AAV expression vectors and recombinant virions produced using these vectors, which include genes coding for enzymes defective or missing in lysosomal storage disorders. These recombinant AAV virions are useful in the treatment of a variety of such disorders. The methods described herein provide for long-term, sustained expression of the defective or missing enzyme. Surprisingly, methods described herein allow delivery of a gene of interest to a variety of tissues, including without limitation brain and/or liver. Delivery to desired target tissues may be by any of various methods, described further below, including by administration of recombinant AAV virions to the bloodstream, or by intrathecal, intramuscular and other routes of administration. Moreover, the methods provide for elimination or reduction of storage granules in the treated subject.

Lysosomal storage diseases that may be treated using the methods of the invention include, but are not limited to, Gaucher's disease (see, e.g., Barranger et al. Neurochemical Res. (1999) 24:601-615 and NIH Technology Assessment Conference Statement, Feb. 27, 1995-Mar. 1, 1995) including Types 1, 2 and 3, Fabry's disease (see, e.g., Takanaka et al. Exp. Hem. (1999) 27:1149-1159, Ziegler et al. Hum. Gene Ther. (1999) 10:1667-1682 and Takanaka et al. Hum. Gene Ther. (1999) 10:1931-1939), Tay-Sachs disease (see, e.g., Guidotti et al. Hum. Mol. Gen. (1999) 8:831-838 and Daly et al. Proc. Natl. Acad. Sci USA (1999) 96:2296-2300), Neimann-Pick Disease, Types A, B and C, (OAT) deficiency (see, e.g., Jensen et al. Hum. Gene Ther. (1997) 8:2125-2132, Locrazza et al. Gene Ther. (1995) 2:22-28, Rivero et al. Hum. Gene Ther. (1994) 5:701-707), hereditary homocysteinemia (see, e.g., McGill et al. Am. J. Med. Gen. (1990) 36:45-52, Eikelboom et al. Ann. Int. Med. (1999) 131:363-365, Watanabe et al. PNAS (1995) 92:1585-1589.), Mannosidoses, Fucosidoses, Sialodosis, the Mucolipidoses, such as I-cell Disease (Mucolipidoses II) and Pseudo-Hurler Polydystrophy (Mucolipidoses III), Acid Lipase Deficiency, such as Wolman Disease and Cholesterol Ester Storage Disease, Sulfatide Lipidosis including Metachromatic Dystrophy and Multiple Sulfatase Deficiency, MPS I (Hurler's disease) (see e.g., Lutzko et al. Hum. Gene Ther. (1999) 10:1521-1532, Hartung et al. Hum. Gene Ther. (1999) 10:2163-2172), MPS II (Hunter syndrome) (see e.g., Rathmann et. al. Am. J. Hum. Genet. (1996) 59:1202-1209, Stronicek et al. Transfusion (1999) 39:343-350, Li et al. J. Med. Genet. (1999) 36:21-27), MPS III (Sanfilippo syndrome) (see e.g., Scott et al. Nat. Genet. (1995) 11:465-467, Jone et al. J. Neuropath. Exp. Neur. (1997) 56(10):1158-1167), MPS IV (Morquoi's syndrome) (see e.g., Nothover et al. J. Inherit. Metab. Dis. (1996) 19:357-365), MPS V (Scheie's syndrome) (see e.g., Dekaban et al. Arch. Pathol. Lab. Med. (1976) 100:231-245), MPS VI (Maroteaux-Lamy syndrome) (see e.g., Hershovitz et al. J. Inherit. Metab. Dis. (1999) 22:50-62, Villani et al. Biochim. Biophys. Acta. (1999) 1453:185-192, Yogalingam et al. Biochim. Biophys. Acta. (1999) 1453:284-296), and MPS VII (Sly syndrome) (see, e.g. Watson et al. Gene Ther. (1998) 5:1642-1649, Elliger et al. Gene Ther. (1999) 6:1175-1178, Stein et al. J. Virol. (1999) 73 (4):3424-3429, Daly et al. PNAS (1999) 96:2296-2300, Daly et al. Hum. Gene Ther. (1999) 10:85-94); and Sandhoff disease.

The enzymes deficient in the above diseases are known to those of skill in the art. For example, for MPS VII (Sly syndrome), the deficient enzyme is .beta.-glucuronidase (GUS); for Gaucher's disease, the deficient enzyme is glucocerebrosidase (GC); for Fabry's disease, the deficient enzyme is .alpha.-galactosidase; for Tay-Sachs disease, the deficient enzyme is the .beta.-hexosaminidase .alpha.-subunit; for OAT disease, the defective enzyme is; for hereditary homocysteinemia, the defective enzyme can be any of cystathioninemia .beta.-synthase, 5,10-methylenetetrahydrofolate reductase, and methionine synthase; for MPS I (Hurler's disease), the defective enzyme is iduronidase; for MPS II (Hunter's syndrome), the defective enzyme is iduronate-2-sulfatase; for MPS III (Sanfilippo syndrome), the defective enzyme is sulphamidase; for MPS IV (Morquoi's syndrome), the defective enzyme is N-acetylgalactosamine-6-sulphate sulphatase; and for MPS VI (Maroteaux-Lamy syndrome), the defective enzyme is aryl-sulphatase B, for Neimann-Pick Disease the defective enzyme is acid sphingomyelinase, for lysosomal storage diseases caused by defects in glycoprotein degradation such as Mannosidoses, Fucosidoses and Sialodosis, the defective enzymes are exoglycosidases, such as endo-.beta.-N-acetylglycosaminidase and aspartylglucosiminidase, for the Muclolipidoses, the defective enzymes are phosphotransferases, for Sulfatide Lipidosis, the defective enzyme is arylsufatase A, for Sandhoff disease, the defective enzyme is hexosaminidase B.

The invention has been described with particular reference to AAV virions which include genes encoding GUS or GC. The nucleotide sequence and the corresponding amino acid sequence of GUS is well known and described. See, e.g., GenBank Accession No. 4504222 and Oshima et al. (1987) Proc Natl Acad Sci USA 84:685-689 (human GUS) and GenBank Accession No. 6754097 and Wawrzyniak et al. (1989) Mol Cell Biol 9:4074-4078 (mouse GUS). Additionally, representative nucleotide and amino acid sequences for GUS are depicted in SEQ ID NOS:1-2 (human GUS) and SEQ ID NOS:3-4 (mouse GUS) herein. For human GUS, the mature peptide begins at position 93 of SEQ ID NO:1 (the upstream portion representing a signal peptide). For mouse GUS, the mature peptide begins at position 79 of SEQ ID NO:3 (the upstream portion representing a signal peptide). These sequences, as well as other known GUS sequences, either with or without the sequence encoding the signal peptide, as well as fragments and variants thereof, may be used in the constructs of the present invention, as described further below.

Similarly, the GC sequences are well known. See, e.g., GenBank Accession Nos. M16328 and M11080; and Sorge et al. (1985) Proc Natl Acad Sci USA 82:7289-7293, as well as SEQ ID NOS:5-8, herein for the human GC sequences. In particular, the nucleotide sequence for GC includes two potential start codons. See, e.g., Am J Hum Genet (1987) 41:1016-1024 and Biochem J (1996) 317:81-88. One sequence, depicted in SEQ ID NOS:5-6, encodes a GC protein with 516 amino acids, the first 19 amino acids of which represent a signal peptide. The other sequence, depicted in SEQ ID NOS:7-8, begins with a start codon 60 base pairs upstream of the sequence shown in SEQ ID NO:5 and encodes an amino acid sequence with 536 amino acids, the first 39 amino acids of which represent a signal peptide. Either of these sequences for GC may be used in AAV constructs of the present invention. Moreover, these sequences, as well as other known GC sequences, either with or without the sequence encoding the signal peptide, as well as fragments and variants thereof, may be used in the constructs of the present invention, as described further below.

These genes may be used in their entireties. Alternatively, fragments and variants thereof, that retain the ability to express functional proteins that are deficient or lacking in the lysosomal storage disease of interest, may be used. The term "variant" refers to biologically active derivatives of the reference molecule, or fragments of such derivatives, that retain desired activity, as described above. In general, the term "variant" refers to a molecule, such as a polynucleotide or polypeptide, having a native sequence and structure with one or more additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy enzymatic activity. Preferably, the variant has at least the same biological activity as the parent molecule, and may even display enhanced activity over the parent molecule. Methods for making such variants are well known in the art. For example, the polypeptide produced by the polynucleotide of interest may include up to about 1-70 conservative or non-conservative amino acid substitutions, such as 5-50, 15-25, or any integer between 1-70, so long as the desired function of the molecule remains intact. One of skill in the art may readily determine regions of the molecule of interest that can be modified with a reasonable likelihood of retaining biological activity as defined herein.

By "fragment" is intended a molecule consisting of only a part of the intact, full-length sequence and structure which retains biological activity as described above. The fragment can include a C-terminal deletion and/or an N-terminal deletion of the native polypeptide and will generally include at least about 10-350, such as 25-150 contiguous amino acid residues of the full-length molecule, preferably at least about 50-250 contiguous amino acid residues of the full-length molecule, and most preferably at least about 100-150 or more contiguous amino acid residues of the full-length molecule, or any integer between 10 amino acids and the full-length sequence, provided that the fragment in question retains biological activity as described herein. These examples of fragments, of course, are merely representative and are not meant to be limiting.

Once obtained, the gene of interest is included in an AAV expression vector which is subsequently used to produce recombinant AAV virions which are delivered either directly to the affected subject, or delivered ex vivo, e.g., to cells from the subject, and reintroduced into the subject for the in vivo expression of the defective or missing enzyme. These procedures are described in detail below.

1. AAV Expression Vectors

AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest and a transcriptional termination region. The control elements are selected to be functional in the target mammalian cell or tissue. The resulting construct which contains the operatively linked components is bounded (5' and 3') with functional AAV ITR sequences.

The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence. 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. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc. Furthermore, 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the DNA molecule into the recipient cell genome when AAV Rep gene products are present in the cell.

Suitable DNA molecules for use in AAV vectors will be less than about 5 kilobases (kb) in size and will include a gene that encodes a protein that is defective or missing from a recipient subject due to a lysosomal storage disease, such as a disease described above. For example, if the targeted disease is MPS VII (Sly syndrome), the gene administered will be that encoding GUS (.beta.-glucuronidase) or a biologically active fragment or variant thereof. If the targeted disease is Gaucher Disease, the gene administered will encode GC (glucocerebrosidase), or a biologically active fragment or variant thereof. Sequences for these genes are described above.

The selected nucleotide sequence is operably linked to control elements that direct the transcription or expression thereof in the subject if the compositions will be delivered in vivo. Such control elements can comprise control sequences normally associated with the selected gene. Alternatively, heterologous control sequences can be employed. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from nonviral genes, such as the murine metallothionein gene, will also find use herein. Other inducible promoters for use in the transcriptional promoter region include the heat shock promoter (hsp); the tetracycline promoter (see, e.g., Bohl et al., (1998) Blood 92:1512-1517; Baron et al., (1999) Proc. Natl. Acad. Sci. USA 96:1013-1018; Rossi et al., (1998) Nature Gen. 20:389-393; Serguera et al., (1999) Hum. Gene Ther. 10:375-383); the rapamycin promoter (see, e.g., Ye et al., (1999) Science 283:88-91; Rivera et al., (1999) Proc. Natl. Acad. Sci. USA 96:8657-8662; Magari et al., (1997) J. Clin. Inv. 100:2865-2872; Liberles, et al., (1997) Proc. Natl. Acad. Sci. USA 94:7825-7830); and the RU486/mifepristone promoter system (Burcin et al. (1998) Proc. Natl. Acad. Sci. USA 96:355-360; Oligino et al., (1998) Gene Ther. 5:491-496; Abruzzese et al., (1999) Hum. Gene Ther. 10:1499-1507). Tissue-specific promoters, which allow for the gene of interest to be expressed when present in particular target tissues, such as muscle and liver, will also find use herein. Such promoters are well-known in the art.

These promoters need not be present in their entireties, but need only retain those elements necessary for directing transcription of a downstream sequence. In fact, due to the size constraints for packaging AAV vectors, minimal promoter sequences are often desirable. These and other promoter sequences are commercially available from, e.g., Stratagene (San Diego, Calif.) or can be obtained from commercially available plasmids, using techniques well known in the art. See, e.g., Sambrook et al., supra.

One or more enhancer sequences may also be present in the AAV constructs, such as, but not limited to the SV40 early gene enhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1 982b) 79:6777, and elements derived from human CMV, such as elements included in the CMV intron A sequence as described in Boshart et al., Cell (1985) 41:521. For example, an intron sequence may also be present, located upstream of the gene of interest, in order to enhance expression thereof. Introns are non-coding regions present in most pre-mRNA transcripts produced in the mammalian cell nucleus. Intron sequences can profoundly enhance gene expression when included in heterologous expression vectors. See, e.g., Buchman et al., Molec. Cell. Biol. (1988) 8:4395-4405. Such intron sequences are known and include those derived from, e.g., the human growth hormone sequence, a beta-globin derived intron sequence, a thymidine kinase-derived intron sequence, intron A of the human CMV IE1 enhancer/promoter, and the like.

Transcription terminator/polyadenylation signals may also be present on the various AAV vectors of the invention, located 3' to the translation stop codon for the coding sequence. Such sequences include, but are not limited to, those derived from SV40, as described in Sambrook et al., supra, as well as polyadenylation and termination sequences derived from human and bovine growth hormone. Spacer sequences are optionally present between the polyadenylation sequence and one or both of the flanking ITRs. The spacer length is variable and chosen to ensure that the final packaged vector length is smaller than 5.2 kb, and preferably 4.1 to 4.9 kb.

The AAV expression vectors can be constructed by directly inserting the selected sequence(s) 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, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,858,351; 5,962,313; 5,846,528; 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (published Mar. 4, 1993); 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 and Smith, (1994) Gene Therapy 1:165-169; and Zhou et al., (1994) J. Exp. Med. 179:1867-1875.

Alternatively, AAV ITRs can be excised from the viral genome or from an AAV vector containing the same and fused 5' and 3' of a selected nucleic acid construct that is present in another vector using standard ligation techniques, such as those described in Sambrook et al., supra. For example, ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 10 mM DTT, 33 .mu.g/ml BSA, 10 mM-50 mM NaCl, and either 40 .mu.M ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0oC. (for "sticky end" ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14oC. (for "blunt end" ligation). Intermolecular "sticky end" ligations are usually performed at 30-100 .mu.g/ml total DNA concentrations (5-100 nM total end concentration). AAV vectors which contain ITRs have been described in, e.g., U.S. Pat. No. 5,139,941. In particular, several AAV vectors are described therein which are available from the American Type Culture Collection ("ATCC") under Accession Numbers 53222, 53223, 53224, 53225 and 53226.

Additionally, chimeric genes can be produced synthetically to include AAV ITR sequences arranged 5' and 3' of one or more selected nucleic acid sequences. Preferred codons for expression of the chimeric gene sequence in the targeted mammalian cell or tissue can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem. (1984) 259:6311.

In order to produce rAAV virions, AAV expression vectors are introduced into suitable host cells using known techniques, such as by transfection. 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. Particularly suitable transfection methods include calcium phosphate co-precipitation (Graham et al., (1973) Virol. 52:456-467), direct micro-injection into cultured cells (Capecchi, M. R., (1980) Cell 22:479-488), electroporation (Shigekawa et al., (1988) BioTechniques 6:742-751), liposome mediated gene transfer (Mannino et al., (1988) BioTechniques 6:682-690), lipid-mediated transduction (Felgner et al., (1987) Proc. Natl. Acad. Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocity microprojectiles (Klein et al., (1987) Nature 327:70-73).

For the purposes of the invention, suitable host cells for producing rAAV virions include microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a heterologous DNA molecule and that are capable of growth in suspension culture. 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. Cells from the stable human cell line, 293 (readily available through, e.g., the American Type Culture Collection under Accession Number 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 rAAV virions.

2. AAV Helper Functions

Host cells containing the above-described AAV expression vectors must be rendered capable of providing AAV helper functions in order to replicate and encapsidate the nucleotide sequences flanked by the AAV ITRs to produce rAAV virions. AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication. AAV helper functions are used herein to complement necessary AAV functions that are missing from the AAV expression vectors. Thus, AAV helper functions include one, or both of the major AAV ORFs, namely the rep and cap coding regions, or functional homologues thereof.

By "AAV rep coding region" is meant 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 heterologous) 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, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, R. M. (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 AAV-2 DNA replication (Thomson et al. (1994) Virology 204:304-311).

By "AAV cap coding region" is meant 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, N. and Kotin, R. M. (supra).

AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of the AAV expression vector. AAV helper constructs are thus used to provide at least transient expression of AAV rep and/or cap genes to complement missing AAV functions that are necessary for productive AAV infection. AAV helper constructs lack AAV ITRs and can neither replicate nor package themselves.

These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the commonly used plasmids pAAV/Ad and pIM29+45 which encode both Rep and Cap expression products. See, e.g., Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. A number of other vectors have been described which encode Rep and/or Cap expression products. See, e.g., U.S. Pat. Nos. 5,139,941; 6,001,650; and 6,027,931, incorporated herein by reference in their entireties.

Both AAV expression vectors and AAV helper constructs can be constructed to contain one or more optional selectable markers. Suitable markers include genes which confer antibiotic resistance or sensitivity to, impart color to, or change the antigenic characteristics of those cells which have been transfected with a nucleic acid construct containing the selectable marker when the cells are grown in an appropriate selective medium. Several selectable marker genes that are useful in the practice of the invention include the neomycin resistance gene (encoding Aminoglycoside phosphotransferase (APH)) that allows selection in mammalian cells by conferring resistance to G418 (available from Sigma, St. Louis, Mo.). Other suitable markers are known to those of skill in the art.

3. AAV Accessory Functions

The host cell (or packaging cell) must also be rendered capable of providing nonAAV-derived functions, or "accessory functions," in order to produce rAAV virions. Accessory functions are nonAAV-derived viral and/or cellular functions upon which AAV is dependent for its replication. Thus, accessory functions include at least those nonAAV proteins and RNAs that are required in AAV replication, including those 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.

In particular, accessory functions can be introduced into and then expressed in host cells using methods known to those of skill in the art. Typically, accessory functions are provided by infection of the host cells with an unrelated helper virus. A number of suitable helper viruses are known, including adenoviruses; herpesviruses such as herpes simplex virus types 1 and 2; and vaccinia viruses. Nonviral accessory functions will also find use herein, such as those provided by cell synchronization using any of various known agents. See, e.g., Buller et al. (1981) J. Virol. 40:241-247; McPherson et al. (1985) Virology 147:217-222; Schlehofer et al. (1986) Virology 152:110-117.

Alternatively, accessory functions can be provided using an accessory function vector. Accessory function vectors include nucleotide sequences that provide one or more accessory functions. An accessory function vector is capable of being introduced into a suitable host cell in order to support efficient AAV virion production in the host cell. Accessory function vectors can be in the form of a plasmid, phage, transposon or cosmid. Accessory vectors can also be in the form of one or more linearized DNA or RNA fragments which, when associated with the appropriate control elements and enzymes, can be transcribed or expressed in a host cell to provide accessory functions.

Nucleic acid sequences providing the accessory functions can be obtained from natural sources, such as from the genome of an adenovirus particle, or constructed using recombinant or synthetic methods known in the art. In this regard, adenovirus-derived accessory functions have been widely studied, and a number of adenovirus genes involved in accessory functions have been identified and partially characterized. See, e.g., Carter (1990) "Adeno-Associated Virus Helper Functions," in CRC Handbook of Parvoviruses, vol. I (P. Tijssen, ed.), and Muzyczka, (1992) Curr. Topics. Microbiol. and Immun. 158:97-129. Specifically, early adenoviral gene regions E1A; the E1B 19 kDa protein coding region; the E2A 72 kDa protein coding region; open reading frame 6 (orf 6) or open reading frame 3 (orf 3) of the E4 coding region; and the VA RNA coding region, are thought to participate in the accessory process. See, e.g., U.S. Pat. Nos. 5,945,335 and 6,004,797, incorporated herein by reference in their entireties; and Janik et al., (1981) Proc. Natl. Acad. Sci. USA 78:1925-1929. Herpesvirus-derived accessory functions have been described. See, e.g., Young et al., (1979) Prog. Med. Virol. 25:113. Vaccinia virus-derived accessory functions have also been described. See, e.g., Carter, B. J., (1990), supra., Schlehofer et al., (1986) Virology 152:110-117.

As a consequence of the infection of the host cell with a helper virus, or transfection of the host cell with an accessory function vector, accessory functions are expressed which transactivate the AAV helper construct to produce AAV Rep and/or Cap proteins. The rep expression products excise the recombinant DNA (including the DNA of interest) from the AAV expression vector. The Rep proteins also serve to duplicate the AAV genome. The expressed Cap proteins assemble into capsids, and the recombinant AAV genome is packaged into the capsids. Thus, productive AAV replication ensues, and the DNA is packaged into rAAV virions.

Following recombinant AAV replication, rAAV virions can be purified from the host cell using a variety of conventional purification methods, such as CsCl gradients. Further, if infection is employed to express the accessory functions, residual helper virus can be inactivated, using known methods. For example, adenovirus can be inactivated by heating to temperatures of approximately 60oC. for, e.g., 20 minutes or more. This treatment effectively inactivates only the helper virus since AAV is extremely heat stable while the helper adenovirus is heat labile.

The resulting rAAV virions containing the heterologous nucleotide sequence of interest can then be used for gene delivery, such as in gene therapy applications, to deliver genes to aid in the treatment of lysosomal storage diseases.

4. In vitro and In vivo Delivery of rAAV Virions

Generally, rAAV virions are introduced into the subject using either in vivo or in vitro transduction techniques. If transduced in vitro, the desired recipient cell will be removed from the subject, transduced with rAAV virions and reintroduced into the subject. Alternatively, syngeneic or xenogeneic cells can be used where those cells will not generate an inappropriate immune response in the subject.

Suitable methods for the delivery and introduction of transduced cells into a subject have been described. For example, cells can be transduced in vitro by combining recombinant AAV virions with the subject's cells e.g., in appropriate media, and screening for those cells harboring the DNA of interest using conventional techniques such as Southern blots and/or PCR, or by using selectable markers. Transduced cells can then be formulated into pharmaceutical compositions, described more fully below, and the composition introduced into the subject by various techniques, such as by intramuscular, intravenous, intraarterial, subcutaneous and intraperitoneal injection, or by injection into smooth and cardiac muscle, using e.g., a catheter.

For in vivo delivery, the rAAV virions will be formulated into pharmaceutical compositions and can be administered parenterally in such a way that the systemic circulation is reached. For example, the pharmaceutical compositions may be delivered directly to the bloodstream using intravenous or intraarterial injection, such as into the portal vein or hepatic artery. Alternatively, the rAAV virions may be delivered to a target organ, such as directly to the liver or brain, or by a route that will deliver the rAAV virions to the desired target tissue, such as the liver, e.g., via the portal vein or hepatic artery, or the brain, e.g., by intrathecal delivery to the cerebrospinal fluid space through the spinal cord.

Pharmaceutical compositions will comprise sufficient genetic material to produce a therapeutically effective amount of the protein of interest, i.e., an amount sufficient to reduce or ameliorate symptoms of the disease state in question or an amount sufficient to confer the desired benefit. For example, a reduction in the number of storage vesicles (also termed storage granules), or elimination thereof, will provide a therapeutic benefit to the treated subject. Methods for detecting the presence of storage granules in a tissue of interest are well known in the art and described further below in the examples. Such methods entail microscopic examination of tissue sections. See, e.g., Vogler et al. (1990) Am J Pathol 136: 207-217. Moreover, reduction in the accumulation of substances due to the particular enzyme deficiency in question, will also confer a therapeutic benefit in the treated subject. Such substances may be readily detected using known assays. For example, MPS VII results in a build-up of undegraded glycosaminoglycans (GAGs). GAG levels can be readily measured using methods developed by Farndale et al. (Farndale et al. (1982) Con Tissue Res 9: 247-248) and Poorthuis et al. (Poorthuis et al. (1994) Pediatr Res 36: 187-193). See, also, the examples below.

The pharmaceutical compositions will also contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).

Appropriate doses will depend on the mammal being treated (e.g., human or nonhuman primate or other mammal), age and general condition of the subject to be treated, the severity of the condition being treated, the particular therapeutic protein in question, the mode of administration, among other factors. An appropriate effective amount can be readily determined by one of skill in the art.

Thus, a "therapeutically effective amount" will fall in a relatively broad range that can be determined through clinical trials. For example, for in vivo injection, i.e., injection directly to the bloodstream, as well as intrathecal administration, a therapeutically effective dose will be on the order of from about 106 to 1015 of the rAAV virions, more preferably 108 to 1014 rAAV virions. For in vitro transduction, an effective amount of rAAV virions to be delivered to cells will be on the order of 108 to 1013 of the rAAV virions. The amount of transduced cells in the pharmaceutical compositions will be from about 104 to 1010 cells, more preferably 105 to 108 cells. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.

Dosage treatment may be a single dose schedule or a multiple dose schedule. Moreover, the subject may be administered as many doses as appropriate. One of skill in the art can readily determine an appropriate number of doses.

Claim 1 of 8 Claims

We claim:

1. A method of delivering a recombinant adeno-associated virus (AAV) virion to a mammalian subject to treat a lysosomal storage disease, said method comprising:

(a) providing a recombinant AAV virion composition which comprises AAV virions, wherein said AAV virions comprise a nucleic acid molecule, said nucleic acid molecule comprising a gene encoding a lysosomal storage enzyme missing or defective in the subject operably linked to control elements capable of directing the in vivo transcription and translation of said gene; and

(b) delivering said recombinant AAV virion directly to the bloodstream of said subject, to result in expression of said gene for at least 13 weeks at a level which reduces lysosomal storage in said mammalian subject.



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