AAV vectors

Details AAV vectors AAV vectors

1997-09-02

2003-02-18

Wilson, Michael C. (Department: 1632)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

C424S093100

Reexamination Certificate

active

06521225

ABSTRACT:

BACKGROUND
The therapeutic treatment of diseases and disorders by gene therapy involves the transfer and stable insertion of new genetic information into cells. Although a variety of physical and chemical methods have been developed for introducing exogenous DNA into eukaryotic cells, viruses have generally been proven to be more efficient for this purpose. Several DNA-containing viruses, such as parvoviruses, adenoviruses and herpesviruses, and RNA-containing viruses, such as retroviruses, have been used to construct eukaryotic cloning and expression vectors and explored as gene therapy vehicles.
Retrovirus- and adenovirus-based vectors are associated with certain complications and disadvantages. For example, retroviruses are intimately associated with neoplastic events. See Donahue, Helper virus induced T cell lymphoma in non-human primates after retroviral mediated gene transfer,
J. Exp. Med
. 176 (1992) 1125-1135. Adenovirus induces a CTL response. See Yang, MHC class 1-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses,
Immunity
1 (1994) 433-442. It also requires a relatively large (35 kb) viral genome, making its usefulness as a vehicle to deliver large sequences limited.
Thus, an alternative vector which is neither pathogenic nor immunogenic would be advantageous. In contrast to adenoviruses, the parvovirus, adeno-associated virus (AAV), has a much smaller genome, most of which can be replaced by foreign DNA. Parvoviruses are small, icohedral viruses approximately 25 nm in diameter containing a single strand DNA genome of approximately 5 kilobases (kb). They consist of two major classes: the dependoviruses, including AAV and its subtypes (AAV1, AAV2, AAV3, AAV4 and AAV5), and the autonomous parvoviruses. The latter lytically infect permissive, proliferating cells in nonintegrating manner without helper virus assistance. On the other hand, AAV is a non-pathogenic human parvovirus that requires co-infection with a helper virus, usually adenovirus (or herpesvirus), for its optimal replication. See for example, Berns, Parvovirus replication,
Microbiol. Rev
. 54 (1990) 316-329 and Berns and Bohenzky, Adeno-associated viruses: an update,
Adv. Virus Res
. 32 (1987) 243-306.
In the absence of a helper virus, the wild-type (wt) AAV has been shown to integrate into the human chromosome 19 in a site-specific manner. See Kotin and Berns, Organization of adeno-associated virus DNA in latently infected Detroit 6 cells,
Virol
. 170 (1989) 460-467; Kotin, Mapping and direct visualization of a region-specific viral DNA integration site on chromosome 19q13-qter,
Genomics
10 (1991) 831-834; Kotin, Site-specific integration by adeno-associated virus,
Proc. Natl. Acad. Sci
. 87 (1990) 2211-2215 and Samulski, Targeted integration of adeno-associated virus (AAV) into human chromosome 19
, EMBO J
. 10 (1991) 3941-3950. Recombinant AAV vectors appear to lack this site-specificity of integration. See Ponnazhagan, Adeno-associated virus 2-mediated transduction of murine hematopoietic cells and long-term expression of a human globin gene in vivo, 6th
Parvovirus Workshop
, Montpellier, France. p29, (1995). Nevertheless, it has been suggested that the AAV-based vector system may prove to be a safer alternative to the more commonly used retrovirus- and adenovirus-based vectors. See, for example, Muzyczka, Use of adeno-associated virus as a general transduction vector for mammalian cells,
Curr. Top. Microbiol. Immunol
. 158 (1992) 97-129. Because approximately 90% of the human population is sero-positive for AAV (see, for example, Blacklow, A sero-epidemiologic study of adeno-associated virus infection in infants and children,
Am. J. Epidemiol
. 94 (1971) 359-366), accidental infection by recombinant AAV is not likely to be problematic. Furthermore, relatively higher stability, higher titers, and higher transduction efficiency of AAV have added to the desirable features of AAV vectors. See Carter, Adeno-associated virus vectors,
Curr. Opin. Biotechnol
. 3 (1993) 533-538 and Srivastava, Parvovirus-based vectors for human gene therapy,
Blood Cells
20 (1994) 531-538.
A number of studies have reported AAV-mediated successful transduction and expression of therapeutic genes in vitro. For example, see Chatterjee, Dual target inhibition of HIV-1 in vitro by means of an adeno-associated virus antisense vector,
Science
258 (1992) 1485-1488; Walsh, Regulated high level expression of a human &ggr;-globin gene introduced into erythroid cells by an adeno-associated virus vector,
Proc. Natl. Acad. Sci
. 89 (1992) 7257-7261; Walsh, Phenotypic correction of Fanconi anemia in human hematopoietic cells with a recombinant adeno-associated virus vector,
J. Clin. Invest
. 94 (1994) 1440-1448; Flotte, Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter,
J. Biol. Chem
. 268 (1993) 3781-3790; Ponnazhagan, Suppression of human &agr;-globin gene expression mediated by the recombinant adeno-associated virus 2-based antisense vectors,
J. Exp. Med
. 179 (1994) 733-738; Miller, Recombinant adeno-associated virus (rAAV)-mediated expression of human &ggr;-globin gene in human progenitor-derived erythroid cells,
Proc. Natl. Acad. Sci
. 91 (1994) 10183-10187; Einerhand, Regulated high-level human beta-globin gene expression in erythroid cells following recombinant adeno-associated virus-mediated gene transfer,
Gene Ther
. 2 (1995) 336-343; Luo, Adeno-associated virus 2-mediated gene transfer and functional expression of the human granulocyte-macrophage colony-stimulating factor,
Exp. Hematol
. 23 (1995) 1261-1267; and Zhou, Adeno-associated virus 2-mediated transduction and erythroid cell-specific expression of a human &bgr;-globin gene,
Gene Therapy
3 (1996) 223-229.
A few studies have examined the safety and efficacy of the AAV vectors in vivo (see Flotte, Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector,
Proc. Natl. Acad. Sci
. 90 (1993) 10613-10617 and Kaplitt, Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain,
Nature Genet
. 8 (1994) 148-153).
A disadvantage of AAV vectors in some clinical indications is the generalized nature of AAV infection. Previous studies have indicated that AAV possesses a wide host range that transcends the species barrier. See for example Muzyczka, Use of adeno-associated virus as a general transduction vector for mammalian cells,
Curr. Top. Microbiol. Immunol
. 158 (1992) 97-129. The autonomous parvovirus, LuIII, appears to possess a similarly wide host range, since liver specific expression has been obtained only via use of recombinants containing a liver-specific enhancer and a regulated promoter. See Maxwell, Autonomous parvovirus transduction of a gene under control of tissue-specific or inducible promoters,
Gene Therapy
3 (1996) 28036. Surprisingly, we have discovered that AAV exhibits organ tropism for the liver and is therefore uniquely adapted for the treatment of diseases or conditions of the liver, diseases or conditions characterized by involving a protein made in the liver or diseases or conditions in which systemic administration of a therapeutic via the liver is desirable or advantageous.
INVENTION SUMMARY
In one aspect, the invention provides methods for selectively expressing therapeutic molecules, such as secretory proteins, antisense molecules and ribozymes, in the liver. The methods find use in treating hepatic diseases or conditions. The methods also find use in treating any disease or condition in which systemic administration of the therapeutic substance, for example a secretory protein, is desired. The methods also find use in treating or diseases or conditions involving proteins that originate or are normally made in the liver.
The methods involve administering to a mammalian patient having a need for liver expression of a therapeutic molecule a therapeutically effective amo

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