United States Patent:  6,110,456

Assignee:  Yale University (New Haven, CT)

Filed:  June 7, 1995

A method of expressing a gene product in the gut of an animal, which comprises administering a recombinant AAV vector to the gut of the animal, wherein the vector comprises a non-AAV gene of interest ligated into an AAV vector genome.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention is quite straightforward: prior to this invention recombinant AAV vectors were well known and were known to be able to transduce a number of cells and tissues, but had not been used or suggested for use in expressing gene products in the gut of animals. The invention therefore comprises administering to the gut of a target animal a recombinant AAV vector containing a gene whose expression is desired (along with the appropriate control elements, if desired or necessary in the normal manner for vectors). No new vectors are required, as previously known AAV vectors have been shown to work well for gut expression. Thus the invention is in part a discovery that no particular adaption of AAV vectors is required for gut expression, which is surprising in view of the strict requirements for AAV reproduction (i.e., presence of a helper virus) and the normal association of AAV with the lungs and nasal passages.

A number of scientific and patent publications describe the state of the art in the AAV vector field. Since no particular adaptations of prior art vectors are required for practice of the present invention, there is no need here to detail at great length the already well-known state of the art. However, the following publications are herein incorporated by reference, as are the patent and the patent applications (and their published equivalents) identified in the Introduction section of this specification, as these materials may be useful for those less experienced in the AAV field:

1. Samulski, R. J. et al. (1982) Proc. Natl. Acad. Sci. USA. 79:2077-2081 "Cloning of Adeno-Associated Virus into pBR322: Rescue of Intact Virus from Recombinant Plasmid in Human Cells"

2. Samulski, R. J. et al. (1983) Cell 33:135-143 "Rescue of Adeno-Associated Virus from Recombinant Plasmids: Gene Correction within the Terminal Repeats of AAV"

3. Laughlin et al. (1983) Gene 23:65-73 "Cloning of Infectious Adeno-Associated Virus Genomes in Bacterial Plasmids"

4. Hermanot, P. L. and Muzycka, N. (1984) Proc. Natl. Acad. Sci. USA. 81:6466-6470 "Use of Adeno-Associated Virus as a Mammalian DNA Cloning Vector: Transduction of Neomycin Resistance into Mammalian Tissue Culture Cells"

5. Senepathy, P. et al. (1984) J. Mol. Biol. 178, 179:1-20 "Replication of Adeno-Associated Virus DNA Complementation of Naturally Occurring rep^- Mutants by a Wild-type Genome or an ori^- Mutant and Correction of Terminal Palindrome Deletions"

6. Tratschin et al. (1984) J. Virol 51:611-619 "Genetic Analysis of Adeno-Associated Virus: Properties of Deletion Mutants Constructed In Vitro and Evidence for an Adeno-Associated Virus Replication Function"

7. Tratschin et al. (1984) Mol. Cell. Biol. 4:2072-2081 "A Human Parvovirus, Adeno-Associated Virus, as a Eukaryotic Vector: Transient Expression and Encapsidation of the Prokaryotic Gene for Chloramphenicol Acetyltransferase"

8. Miller et al. (1986) Somatic Cell and Molecular Genetics 12:175-183 "Factors Involved in Production of Helper Virus-Free Retrovirus Vectors"

9. Bosselman et al. (1987) Mol. Cell. Biol. 7:1797-1806 "Replication-Defective Chimeric Helper Proviruses and Factors Affecting Generation of Competent Virus: Expression of Moloney Murine Leukemia Virus Structural Genes via the Metallothionein Promoter"

10. Ohi et al. (1988) J. Cell. Biol. 107:304A "Construction and Characterization of Recombinant Adeno-Associated Virus Genome Containing .beta.-globin cDNA"

11. McLaughlin et al. (1988) J. Virol. 62:1963-1973 "Adeno-Associated General Transduction Vectors: Analysis of Proviral Structures"

12. Lebkowski et al. (1988) Mol. Cell Biol. 8:3988-3996 "Adeno-Associated Virus: a Vector System for Efficient Introduction and Integration of DNA into a Variety of Mammalian Cell Types"

13. Samulski et al. (1989) J. Virol. 63:3822-3828 "Helper-Free Stocks of Recombinant Adeno-Associated Viruses: Normal Integration Does not Require Viral Gene Expression"

14. Srivastava et al. (October 1989) Proc. Natl. Acad. Sci. U.S.A. 86:20, 8078-82 "Construction of a recombinant human parvo virus-B19: adeno-associated virus-2 (AAV) DNA inverted terminal repeats are functional in an AAV-B19 hybrid virus--vector construction; potential application gene cloning in bone marrow cell culture and gene therapy"

15. Ohi, S. et al. (1990) J.Cell.Biochem. (Suppl.14A,D422) "Construction of recombinant adeno-associated virus that harbors human beta-globin cDNA--vector construction for potential application in hemoglobinopathy gene therapy; gene cloning and expression in 293 cell culture"

16. Ohi, S. et al. (1990) Gene 89 2:279-82 "Construction and replication of an adeno-associated virus expression vector that contains human beta-globin cDNA--plasmid PAVh-beta-GHP11 and plasmid PAVh-beta-G-psi-1 construction; potential application in gene therapy of e.g. sickle cell anemia or thalassemia"

17. Ohi, S. et al. (1990) FASEB J. 4:7, A2288) "Production and expression of recombinant adeno-associated viruses harboring human beta-globin cDNA--adeno-associated virus expression in 293 cell culture; potential gene therapy for hemoglobinopathy disease"

18. Samulski et al. (1991) Embo J. 10:3941-3950 "Targeted Integration of Adeno-associated virus AAV Into human chromosome 19"

19. Ruffing et al. (December 1992) J. Virol. 66:6922-6930 "Assembly of Viruslike Particles by Recombinant Structural Proteins of Adeno-Associated Virus Type 2 in Insect Cells"

20. Sitaric et al, (1991) FASEB 5:A1550 "Production of a Helper-free Recombinant Adeno-Associated Virus That Harbors Human .beta.-globin cDNA"

21. Walsh et al. (1991) Clin. Res. 2:325 "Gene Transfer and High-level Expression of a human .gamma.-globin Gene Mediated by a Novel Adeno-Associated Virus Promoter"

22. Carter, B. J. (October 1992) Curr. Opinion in Biotechnol. 3:533-539 "Adeno-Associated Virus Vectors"

23. Ohi et al. (1992) (Jun. 21-22, 1991) EXP Hematol 20 119 "Synthesis of a human beta globin in the recombinant adeno-associated virus-infected cells towards gene therapy of hemoglobinopathies"

24. Flotte et al. (1993) J. B. C. 268:3781-3790 "Expression of the Cystic Fibrosis Transmembrane Conductance Regulator from a Novel Adeno-Associated Virus Promoter"

25. Wong et al. (1993) Blood 82:302A. "High efficiency gene transfer into growth arrested cells utilizing an adeno-associated virus (AAV)-based vector"

26. Shaughnessey, et al. (1994) Proc. Am. Assoc. Cancer Res. 35:373 "Adeno-associated virus vectors for MDR1 gene therapy--multidrug-resistance gene cloning and gene transfer into hematopoietic stem cell culture using adeno-associated virus vector CWRSP for potential gene therapy"

27. Tenenbaum, L. et al. (1994) Gene Ther. (1, Suppl.1,S80) "Adeno-Associated Virus (AAV) as a Vector for Gene Transfer into Glial Cells of the Human Central Nervous System--Potential Gene Therapy"

28. Friedmann, T. (1994) Gene Ther. (1, Suppl. 1, S47-S48) "Gene Therapy for Disorders of the CNS--Parkinson Disease Alzheimer Disease Therapy by Gene Transfer Using Herpes Simplex Virus, Adeno Virus and Adeno-Associated Virus Vector"

29. DE 42 19626 A1 Assignee: Wehling, P. Filed: Jun. 16, 1992 Publication: Dec. 23, 1993 "Methods for Introducing Therapeutically Relevant Genes into Cells"

30. WO 91/18088 Assignee: Nat. Inst. Health-Bethesda Filed: May 17, 1991 (Priority May 23, 1990) Inventors: Chatterjee and Wong Publication: Nov. 28, 1991 "Adeno-Associated Virus (AAV)-based Eukaryotic Vectors"

31. EP 0 592 836 A1 Assignee: American Cyanamide Co. Filed: Sep. 16, 1993 (priority Sep. 17, 1992 U.S. 947127) Publication: Apr. 20, 1994 "Human Adeno-Associated Virus Integration Site DNA and use thereof"

32. WO 93/24641 Assignee: U.S. Dept. Health-Human-Serv. Filed: Jun. 2, 1993 (Priority Jun. 2, 1992) Publication: Apr. 20, 1994 "Adeno-Associated Virus with Inverted Terminal Repeat Sequences as Promoter"

33. WO 93/09239 Assignee: Res. Corp. Technol. Filed: Nov. 6, 1992 (U.S. priority Nov. 8, 1991) Publication: May 13, 1993 "Adeno-Associated Virus-2 Basal Vectors"

34. EP 0 488 528 A1 Assignee: Appl. Immune Sci. Filed: Oct. 29, 1991 (U.S. priority Oct. 30, 1990) Publication: Jun. 3, 1992 "Recombinant adeno-associated Virus Vectors"

35. U.S. Pat. No. 4,797,368 Assignee: U.S. Dept. Health-Human-Serv. Filed: Mar. 15, 1985 Issued: Jan. 10, 1989 "Adeno-associated Virus as Eukaryotic Expression Vector"

Two recent review article provide a particularly complete overview of the recent status of gene therapy using AAV virus and include a collection of additional recent scientific publications in this field.

36. Samulski, R. J. "Adeno-associated Viral Vectors"Chapter 3 in "Viruses in Human Gene Therapy" Chapman & Hall, J.-M. H. Vos., ed.

37. Samulski, R. J. "Adeno-associated Virus-based Vectors for Human Gene Therapy"Chapter 11 in "Gene Therapy: From Laboratory to the Clinic" World Scientific, K. M. Hui, ed.

Actual delivery of the viral vector for purposes of the invention is accomplished by using any physical method that will transport the AAV recombinant vector to the gut. In this discussion on administration, "AAV vector" means both a bare recombinant AAV DNA vector or AAV vector DNA packaged into viral capsids. Simply dissolving an AAV vector in phosphate buffered saline has been demonstrated to be sufficient for useful gut expression, and there are no known restrictions on the carriers or other components that can be coadministered with the vector (although compositions that degrade DNA should be avoided in the normal manner with vectors). Pharmaceutical compositions can be prepared as oral tablets, capsules, or ingestible liquids or as suppositories. The vectors can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

The AAV vector may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the AAV vector may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1 .mu.g, preferably 10-1000 .mu.g of AAV vector DNA, or 5x10³ to 5x10⁶ infectious units AAV vector per kg body weight. The amount of AAV vector in a therapeutically useful composition is that which is sufficient to produce gene expression at a therapeutically useful level. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 10 and 1000 .mu.g of AAV vector DNA or 10⁴ to 10⁶ infectious units AAV vector.

The tablets, troches, pills, capsules and the like may also contain the following: a binder such as polyvinylpyrrolidone, gum tragacanth, acacia, sucrose, corn starch or gelatin; an excipient such as calcium phosphate, sodium citrate and calcium carbonate; a disintegrating agent such as corn starch, potato starch, tapioca starch, certain complex silicates, alginic acid and the like; a lubricant such as sodium lauryl sulfate, talc and magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin; or a flavoring agent such as peppermint, oil of wintergreen or cherry flavoring. Solid compositions of a similar type are also employed as fillers in soft and hard-filled gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the AAV vector, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, flavoring such as cherry or orange flavor, emulsifying agents and/or suspending agents, as well as such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the AAV vector may be incorporated into sustained-release preparations and formulations.

Since AAV has in the past been shown to have a broad host range (for pulmonary expression) and has now been demonstrated to be operable in the gut, there are no known limits on the animals in which gut expression can take place, although expression in animals with evolutionarily developed small and large intestines is preferred, particularly in mammals, birds, fish, and reptiles, especially domesticated mammals and birds such as cattle, sheep, pigs, horses, dogs, cats, chickens, and turkeys. Both human and veterinary uses are particularly preferred.

The gene being expressed can be either a DNA segment encoding a protein, with whatever control elements (e.g., promoters, operators, ribosome binding sites) are desired by the user, or a non-coding DNA segment, the transcription of which produces all or part of some RNA-containing molecule or anti-sense molecule that is functional in cells. Since the present invention is directed to a route of delivery and to the vector rather than to the material being delivered, there are no limitations on the foreign DNA (non-AAV DNA) being delivered by the vector. While delivery of genes associated with correction of genetic deficiencies related to gut expression is preferred, expression of genes in the gut has the capability of correcting aberrant gene expression in other locations as a result of transport of expression products throughout the body.

We have demonstrated the invention by correcting lactase deficiency in the gut. We used a recombinant adeno-associated virus (AAV) expressing .beta.-galactosidase (AAVlac) and delivered the vector to the proximal intestine using a peroral route. Lactase-deficient rats that received AAVlac were able to metabolize an acute lactose load as demonstrated by a rise in plasma glucose. In contrast, phosphate-buffered saline(PBS)-treated controls demonstrated no effect of lactose on plasma glucose. Furthermore, when animals were placed on a restricted, lactose-only diet, PBS-treated rats continued to lose weight over the entire 2-week test-diet period. In contrast, AAVlac-treated animals had no weight loss during the second week. PCR and RT-PCR and histological analysis confirmed intestinal persistence of viral DNA and expression of the vector-encoded .beta.-galactosidase for the life of the animal (extending to 6 months). Moreover, when animals were re-challenged with a lactose load at 3 months after a single AAVlac or PBS treatment, AAVlac animals retained their ability to metabolize lactose and maintained body weight on a lactose diet. These data indicate that oral delivery of an AAV vector can result in long-lasting phenotypic correction of lactase deficiency.

This demonstration system was selected both to prove the principle of the invention and to demonstrate the invention in a therapeutically useful mode. Adult-type hypolactasis is genetically determined by an autosomal recessive gene (Sahi et al. Lancet 1973 2:823-828). It is the world's most common genetic disorder, afflicting over 50% of the world's population ranging from 100% in some Southeast Asian populations to less than 5% in some Northern European countries (Flatz Human Genet. 1984 36:306-310). Although the symptoms associated with lactose intolerance are relatively mild and readily controlled by omitting lactose-containing foods, there is some debate as to the potential clinical significance of the dietary restrictions which typically accompany lactose intolerance. Specifically, the reduction in calcium-intake associated with complying with a lactose-free diet may lead to an acceleration in the loss of bone mass in the elderly (Flatz 1987 Advances in Human Genet. 16:1-77 New York Plenum Press); and in adolescents and young adults, it may reduce the bone mineral mass (Mobassaleh et al. Pediatrics 75:160-166 1985).

We elected to study lactase deficiency in the rat as a model of a gastrointestinal genetic disease. We were particularly interested in determining whether we could obtain phenotypic correction using an orally delivered viral vector. We have previously shown that AAV vectors can result in long-term transgene expression in terminally differentiated cells following in vivo administration (Birge et al. NEJM 1967 276:445-448). AAV has several features which make it particularly attractive for gene therapy. It is a defective, helper-dependent virus, and the wild-type is non-pathogenic. Vectors can be generated which are completely free of helper virus (Bayless et al. 1975 NEJM 292:1156-1159). Furthermore, some recombinant AAV vectors retain just 145 base terminal repeats with the entire coding sequences removed. In other AAV vectors, non-AAV DNA is operably linked to a vector comprising a double-D AAV genomic segment consisting of 165 basepairs including an internal terminal repeat with D segments at both ends. These vectors therefore are devoid of all viral genes, minimizing any possibility of recombination and viral gene expression. Moreover, unlike adenovirus, they do not appear to elicit any immune response. Another feature of AAV which makes it potentially suitable for an orally based vector is that of hardiness--AAV is resistant to temperature, pH extremes and solvents (Sandler et al. Am. J. Clin. Nutr. 1985 42:270-274). Furthermore, during an active infection in humans, wild-type AAV is typically found in both respiratory tract and gastrointestinal secretions, the gut is therefore a normal host tissue for the virus.

Lactose intolerance is most commonly associated with a reduction in intestinal lactase activity. Lactose digestion is dependent on the enzyme, lactase-phlorizin hydrolase (LPH), a microvillar protein which has both galactosidase activity and glycosyl-N-acylsphingosine glucohydrolase activities. However, dietary administration of yeast or bacterial .beta.-galactosidase is sufficient to confer the ability to metabolize lactose (Kaplitt et al. Nature Genet. 1994 8:148-154).

Most mammalian species are relatively lactase deficient following weaning, although this developmental change in LPH expression does not appear to be simply a reduction in gene transcription. In both humans and rats, although LPH mRNA declines after weaning, it reappears during adulthood. However, this increase in mRNA is not associated with an increase in translation; and adult enzyme levels within the brush-border remain low. It appears that some LPH protein is expressed, but the enzyme is accumulated within the golgi region and is not transported to the brush-border.

Claim 1 of 8 Claims

1. A method of obtaining production of a protein in cells of the small intestine of an animal, which comprises:

orally administering an encapsidated recombinant adeno-associated virus (AAV) DNA vector to said animal, wherein said recombinant AAV DNA vector comprises a promoter operably linked to a non-AAV DNA encoding said protein, and wherein said recombinant AAV DNA vector is packaged into an AAV capsid.

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