STABLE EXPRESSION OF AAV VECTORS IN JUVENILE SUBJECTS

Abstract

The invention relates to the use of adeno-associated virus (AAV) vectors to achieve long term expression of a transgene in the liver of a juvenile subject. The invention includes the stable long-term amelioration of disease symptoms of the subjection following a single administration of an AAV vector to a juvenile subject, wherein the AAV vector delivers the transgene to the subject's liver.

Claims

1. A method of ameliorating the symptoms of a genetic disorder in a juvenile subject suffering from the genetic disorder comprising administering to the juvenile subject a therapeutically effective amount of a therapeutic AAV virus encoding a therapeutic protein, wherein the expression of the therapeutic protein ameliorates the symptoms of the genetic disorder.

2. A use of a therapeutic AAV virus for the preparation of a medicament for ameliorating symptoms of a genetic disorder in a juvenile subject suffering from the genetic disorder, wherein the medicament comprises a therapeutically effective amount of a therapeutic AAV virus encoding a therapeutic protein, wherein the expression of the therapeutic protein ameliorates the symptoms of the genetic disorder.

3. A composition comprising a therapeutically effective amount of a therapeutic AAV virus encoding a therapeutic protein for use in ameliorating symptoms of a genetic disorder in a juvenile subject suffering from the genetic disorder.

4. The method, use or composition of any one of claims 1-3, wherein the therapeutic protein is a functional copy of a non-functional endogenous protein.

5. The method, use or composition of any one of claims 1-3, wherein the therapeutic protein is a modified version of the endogenous protein.

6. The method, use or composition of any one of claims 1-3, wherein the therapeutic protein is a heterologous protein that compensates for a non-functional endogenous protein.

7. The method, use or composition of any of the preceding claims, wherein the juvenile subject is a juvenile human.

8. The method, use or composition of claim 7, wherein the juvenile human is less than 18 years old.

9. The method, use or composition of claim 7, wherein the juvenile human is less than 12 years old.

10. The method, use or composition of any of the preceding claims, wherein the therapeutic protein is expressed by the hepatocytes of the juvenile subject following administration of the therapeutic AAV virus.

11. The method of any of the preceding claims, wherein the therapeutic AAV virus is administered intravenously.

12. The use or composition of any of the preceding claims, wherein the medicament is formulated for intravenous administration.

13. The method, use or composition of any of the preceding claims, wherein the genetic disorder is a hemophilia.

14. The method, use or composition of claim 13, wherein the hemophilia is hemophilia A and the therapeutic protein is Factor VIII.

15. The method, use or composition of claim 14, wherein the Factor VIII is Factor VIII-SQ.

16. The method, use or composition of claim 14, wherein the therapeutic AAV virus is AAV5-FVIII-SQ.

17. The method, use or composition of claim 13, wherein the hemophilia is hemophilia B and the therapeutic protein is Factor IX.

18. The method, use or composition of claim 17, wherein the Factor IX is R338L Factor IX.

19. The method, use or composition of any one of claims 1 to 12, wherein the genetic disorder is phenylketonuria (PKU) and the therapeutic protein is phenylalanine hydroxylase (PAH).

20. The method, use or composition of any of the preceding claims, wherein the amount of therapeutic AAV virus administered to the juvenile subject corresponds to the same absolute number of therapeutic AAV virus that is effective in adult subjects.

21. The method, use or composition of claim 20, wherein from about 1E12 vg/kg to about 1E15 vg/kg of the therapeutic AAV virus are administered to the juvenile subject.

22. The method, use or composition of claim 20, wherein from about 6E13 vg/kg to about 6E14 vg/kg of the therapeutic AAV virus are administered to the juvenile subject.

23. The method, use or composition of any one of claims 20 to 22, wherein the AAV virus is formulated as a pharmaceutical composition comprising sodium phosphate, dibasic at a concentration of from about 0.1 mg/ml to about 3 mg/ml, sodium phosphate monobasic monohydrate at a concentration of from about 0.1 mg/ml to about 3 mg/ml, sodium chloride at a concentration of from about 1 mg/ml to about 20 mg/ml, mannitol at a concentration of from about 5 mg/ml to about 40 mg/ml, and poloxamer 188 at a concentration of from about 0.1 mg/ml to about 4 mg/ml.

24. The method, use or composition of any one of claims 20 to 23, wherein the juvenile subject is treated prophylactically with a corticosteroid at a concentration ranging from 5 mg/day to 60 mg/day.

25. The method, use or composition of any one of claims 20 to 23, wherein the juvenile subject is treated therapeutically with a corticosteroid at a concentration from 5 mg/day to 60 mg/day.

26. The method, use or composition of any one of claims 20 to 25, which results in the expression of at least about 5 IU/dl of functional Factor VIII protein in the juvenile subject.

27. The method, use or composition of any one of claims 20 to 25, which results in an increase in functional Factor VIII protein of at least about 1 IU/dl in the juvenile subject.

28. A method of reducing bleeding time of a bleeding episode in a juvenile subject suffering from hemophilia comprising administering to the juvenile subject, prior to the bleeding episode, a therapeutically effective amount of a therapeutic AAV virus.

29. A use of a therapeutically effective amount of a therapeutic AAV virus for the preparation of a medicament for reducing bleeding time of a bleeding episode in a juvenile subject suffering from hemophilia, wherein the medicament is administered to the juvenile subject prior to the bleeding episode.

30. A composition comprising a therapeutically effective amount of a therapeutic AAV virus useful for reducing bleeding time of a bleeding episode in a juvenile subject suffering from hemophilia, wherein the composition is administered to the juvenile subject prior to the bleeding episode

31. The method, composition or use of any one of claims 28-30, wherein the administering occurs at least three weeks prior to the bleeding episode.

32. The method of any one of claims 28-31, wherein the therapeutic AAV virus is administered intravenously.

33. The use or composition of any one of claims 28-31, wherein the therapeutic AAV is formulated for intravenous administration

34. The method, use or composition any one of claims 28-33, wherein the hemophilia is hemophilia A and the therapeutic AAV virus expresses Factor VIII.

35. The method, use or composition of claim 34, wherein the Factor VIII is Factor VIII-SQ.

36. The method, use or composition of claim 34, wherein the therapeutic AAV virus is AAV5-FVIII-SQ.

37. The method, use or composition of claim 28-33, wherein the hemophilia is hemophilia B and the therapeutic AAV virus expresses Factor IX.

38. The method, use or composition of claim 37, wherein the Factor IX is R338L Factor IX.

39. The method, use or composition of any one of claims 28 to 38, wherein the amount of therapeutic AAV virus administered to the juvenile subject corresponds to the same absolute number of therapeutic AAV virus that is effective in adult subjects.

40. The method, use or composition of claim 39, wherein from about 1E12 vg/kg to about 1E15 vg/kg of the therapeutic AAV virus are administered to the juvenile subject.

41. The method, use or composition of claim 39, wherein from about 6E13 vg/kg to about 6E14 vg/kg of the therapeutic AAV virus are administered to the juvenile subject.

42. The method, use or composition of any one of claims 28 to 41, wherein therapeutic AAV virus is formulated in a solution comprising sodium phosphate, dibasic at a concentration of from about 0.1 mg/ml to about 3 mg/ml, sodium phosphate monobasic monohydrate at a concentration of from about 0.1 mg/ml to about 3 mg/ml, sodium chloride at a concentration of from about 1 mg/ml to about 20 mg/ml, mannitol at a concentration of from about 5 mg/ml to about 40 mg/ml, and poloxamer 188 at a concentration of from about 0.1 mg/ml to about 4 mg/ml.

43. A method of increasing Factor VIII protein expression in a juvenile subject in need thereof comprising administering to the juvenile subject a therapeutic virus, wherein the therapeutic AAV virus is AAV5-FVIII-SQ.

44. Use of a therapeutic AAV virus for the preparation of a medicament for increasing Factor VIII protein expression in a juvenile subject in need thereof, wherein the AAV virus is AAV5-FVIII-SQ.

45. A composition comprising a therapeutic AAV virus for increasing Factor VIII protein expression in a juvenile subject in need thereof, wherein the AAV virus is AAV5-FVIII-SQ.

46. The method of claim 43, wherein the therapeutic AAV virus is administered intravenously.

47. The use or composition of claim 44 or 45, wherein the AAV virus is formulated for intravenous administration

48. The method, use or composition of any one of claims 43-47, wherein the amount of therapeutic AAV virus administered to the juvenile subject corresponds to the same absolute number of therapeutic AAV virus that is effective in adult subjects.

49. The method, use or composition of claim 48, wherein from about 1E12 vg/kg to about 1E15 vg/kg of the therapeutic AAV virus are administered to the juvenile subject.

50. The method, use or composition of claim 48, wherein from about 6E13 vg/kg to about 6E14 vg/kg of the therapeutic AAV virus are administered to the juvenile subject.

51. The method, use or composition of any one of claims 43-50 which results in expression of at least about 5 IU/dl of functional Factor VIII protein in the juvenile subject.

52. The method, use or composition of claim 51 which results in expression of at least about 1 IU/dl of functional Factor VIII protein in the juvenile subject.

53. The method, use or composition of any one of claims 43-52 which results in an increase in functional FVIII activity of at least about 1 IU/dl in the juvenile subject.

54. The method, use or composition of any one of claims 41-50, wherein the juvenile subject is treated with a corticosteroid at a concentration ranging from 5 mg/day to 60 mg/day.

55. The method, use or composition of claim 54, wherein the corticosteroid treatment is performed prophylactically.

56. The method, use or composition of claim 54, wherein the corticosteroid treatment is performed therapeutically.

57. The method, use or composition of claim 54-56, wherein the juvenile subject is treated with a corticosteroid at a concentration ranging from 5 mg/day to 60 mg/day over a continuous period of at least 3, 4, 5, 6, 7, 8, 9 or 10 weeks or greater.

58. The method of any one of claims 54-57 further comprising a step of determining the absence or presence of anti-AAV capsid antibodies in the serum of the juvenile subject after administration of the therapeutically effective amount of the AAV5-FVIII-SQ.

59. The method of claim 58 further comprising the step of administering an effective amount of a corticosteroid to the subject after a determination of the presence of anti-AAV capsid antibodies in the serum of the juvenile subject is made.

60. A method of increasing phenylalanine hydroxylase (PAH) protein expression in a juvenile subject in need thereof comprising administering to the juvenile subject a therapeutic virus, wherein the therapeutic AAV virus comprises a nucleic acid sequence encoding a functionally active PAH.

61. Use of a therapeutic AAV virus for the preparation of a medicament for increasing phenylalanine hydroxylase (PAH) protein expression in a juvenile subject in need thereof, wherein the AAV virus comprises a nucleic acid sequence encoding a functionally active PAH.

62. A composition comprising a therapeutic AAV virus for increasing phenylalanine hydroxylase (PAH) protein expression in a juvenile subject in need thereof, wherein the AAV virus comprises a nucleic acid sequence encoding a functionally active PAH.

63. The method of claim 60, wherein the therapeutic AAV virus is administered intravenously.

64. The use or composition of claim 61 or 62, wherein the AAV virus is formulated for intravenous administration

65. The method, use or composition of any one of claims 60-64, wherein about 1E12 vg/kg to about 2E16 vg/kg of the therapeutic AAV virus are administered to the juvenile subject.

65. The method, use or composition of any one of claims 60-64, wherein about 2E12 vg/kg to about 2E14 vg/kg of the therapeutic AAV virus are administered to the juvenile subject.

66. The method, use or composition of any one of claims 60-64, wherein about 6E12 vg/kg to about 2E14 vg/kg of the therapeutic AAV virus are administered to the juvenile subject.

67. The method, use or composition of any one of claim 60-66, wherein the juvenile subject is 3 weeks to 5 weeks of age.

68. The method of any one of claims 60-67 further comprising a step of determining the absence or presence of anti-AAV capsid antibodies in the serum of the juvenile subject after administration of the therapeutically effective amount of the AAV virus comprising a nucleic acid sequence encoding a functionally active PAH.

Description

DESCRIPTION OF DRAWINGS

[0065] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the United States Patent and Trademark Office upon request and payment of the necessary fee.

[0066] FIG. 1A and FIG. 1B provides an illustration and chart of the study design, respectively. In brief, control adult mice (8 weeks old) were given a single dose of AAV5-FVIII-SQ (3.5E13 vg/kg which is approximately 8.9E11 vg per mouse). Juvenile mice (2 days old) were divided into two cohorts. One cohort received a single dose of AAV5-FVIII-SQ that was the same total vg as adult mice (8.9E11 vg/mouse). The other cohort received a single dose of AAV5-FVIII-SQ that was the same vg/kg as adult mice (3.5E13 vg/kg).

[0067] FIG. 2 is a set of graphs that show that juveniles (dosed with same total vg as adults) have the same capacity as adults to take up AAV viral DNA as shown by total liver Factor VIII DNA in the left hand graph and total Factor VIII RNA in liver in the right hand graph.

[0068] FIG. 3 is a set of graphs that show that both body weight (left hand graph) and liver weight (right hand graph) of juvenile mice increased rapidly after AAV5-FVIII-SQ administration.

[0069] FIG. 4 is a graph that shows that AAV5-FVIII-SQ did not cause liver injury in juvenile mice, as determined by ALT measurement.

[0070] FIG. 5 is a set of graphs that show that juvenile mice need the same total amount of vector genome as adults to maintain therapeutic Factor VIII levels in adulthood. The left panel shows plasma Factor VIII concentration over time and the right panel shows total circulating Factor VIII in juvenile mice dosed with the same total amount of vector genome as adults (8.9E11 vg/mouse which is equivalent to approximately 4.5E14 vg/kg) or the vg per body weight as adults (3.5E13 vg/kg). In both, these values are compared to adults treated with 3.5E13 vg/kg. Total circulating FVIII was determined by multiplying plasma FVIII concentration by the estimated blood volume (in this case the estimated blood volume is 10% of body weight).

[0071] FIG. 6A and FIG. 6B. FIG. 6A is immunohistochemical analysis of AAV5 capsid in liver sections from adult mice treated with AAV5-FVIII-SQ. FIG. 6B is western blot analysis of AAV5 capsid from juvenile mice treated with AAV5-FVIII-SQ.

[0072] FIG. 7 is a table showing the overall design of a study that will determine the optimal conditions for expressing PAH in the livers of PKU subjects.

[0073] FIG. 8A-8C provide the body weight (g; meanSEM) for each group of AAV- and vehicle treated all ENU mice prior to and 8 weeks after dosing with 210.sup.14 vg/kg of AAV5-PAH or vehicle. FIG. 8A provides the body weight prior to treatment. FIG. 8B provides the body weight 4 weeks post dosing. FIG. 8C provides the body weight 8 weeks post dosing. * p<0.05, **** p, 0.0001 as determined by one-way ANOVA.

[0074] FIG. 9A-9B provide the plasma PHE concentration (M; meanSEM) for each group of AAV- and vehicle treated all ENU mice prior to and 8 weeks after dosing with 210.sup.14 vg/kg of AAV5-PAH or vehicle. FIG. 9A provides the plasma PHE concentration 4 weeks after dosing. FIG. 9B provides the plasma PHE concentration 8 weeks post dosing. * p<0.05, **** p, 0.0001 as determined by one-way ANOVA.

DETAILED DESCRIPTION

[0075] The present invention provides for AAV vectors encoding functionally active therapeutic proteins (e.g., completely packaged AAV Factor VIII vectors, AAV Factor IX vectors, and AAV PAH vectors). The recombinant AAV therapeutic protein vectors of the invention have improved transgene expression, as well as improved AAV virus production yield and simplified purification. Introducing one or more introns into the therapeutic protein-coding region enhances expression. Reconfiguring the number and positioning of enhancers also enhances expression.

AAV Vectors

[0076] As used herein, the term AAV is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. There are currently thirteen serotypes of AAV that have been characterized. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228; and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, it is fully expected that these same principles will be applicable to additional AAV serotypes since it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to inverted terminal repeat sequences (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0077] An AAV vector as used herein refers to a vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs) and operably linked to one or more expression control elements. Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products.

[0078] An AAV virion or AAV viral particle or AAV vector particle refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an AAV vector particle or simply an AAV vector. Thus, production of AAV vector particle necessarily includes production of AAV vector, as such a vector is contained within an AAV vector particle.

[0079] AAV rep and cap genes are genes encoding replication and encapsidation proteins, respectively. AAV rep and cap genes have been found in all AAV serotypes examined to date, and are described herein and in the references cited. In wild-type AAV, the rep and cap genes are generally found adjacent to each other in the viral genome (i.e., they are coupled together as adjoining or overlapping transcriptional units), and they are generally conserved among AAV serotypes. AAV rep and cap genes are also individually and collectively referred to as AAV packaging genes. The AAV cap genes in accordance with the present invention encode Cap proteins which are capable of packaging AAV vectors in the presence of rep and adeno helper function and are capable of binding target cellular receptors. In some embodiments, the AAV cap gene encodes a capsid protein having an amino acid sequence derived from a particular AAV serotype.

[0080] The AAV sequences employed for the production of AAV can be derived from the genome of any AAV serotype. Generally, the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels, provide a similar set of genetic functions, produce virions which are essentially physically and functionally equivalent, and replicate and assemble by practically identical mechanisms. For the genomic sequence of AAV serotypes and a discussion of the genomic similarities. (See, e.g., GenBank Accession number U89790; GenBank Accession number J01901; GenBank Accession number AF043303; GenBank Accession number AF085716; Chiorini et al., J. Vir. (1997) vol. 71, pp. 6823-6833; Srivastava et al., J. Vir. (1983) vol. 45, pp. 555-564; Chiorini et al., J. Vir. (1999) vol. 73, pp. 1309-1319; Rutledge et al., J. Vir. (1998) vol. 72, pp. 309-319; and Wu et al., J. Vir. (2000) vol. 74, pp. 8635-8647).

[0081] The genomic organization of all known AAV serotypes is very similar. The genome of AAV is a linear, single-stranded DNA molecule that is less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the non-structural replication (Rep) proteins and the structural (VP) proteins. The VP proteins form the capsid. The terminal 145 nt are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. The Rep genes encode the Rep proteins, Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, and Rep 52 and Rep40 are transcribed from the p19 promoter. The cap genes encode the VP proteins, VP1, VP2, and VP3. The cap genes are transcribed from the p40 promoter. The ITRs employed in the vectors of the present invention may correspond to the same serotype as the associated cap genes, or may differ. In a particularly preferred embodiment, the ITRs employed in the vectors of the present invention correspond to an AAV2 serotype and the cap genes correspond to an AAV5 serotype.

[0082] In some embodiments, a nucleic acid sequence encoding an AAV capsid protein is operably linked to expression control sequences for expression in a specific cell type, such as Sf9 or HEK cells. Techniques known to one skilled in the art for expressing foreign genes in insect host cells or mammalian host cells can be used to practice the invention. Methodology for molecular engineering and expression of polypeptides in insect cells is described, for example, in Summers and Smith (1986) A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex.; Luckow (1991) In Prokop et al., Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DNA Technology and Applications, 97-152; King, L. A. and R. D. Possee (1992) The baculovirus expression system, Chapman and Hall, United Kingdom; O'Reilly, D. R., L. K. Miller, V. A. Luckow (1992) Baculovirus Expression Vectors: A Laboratory Manual, New York; W.H. Freeman and Richardson, C. D. (1995) Baculovirus Expression Protocols, Methods in Molecular Biology, volume 39; U.S. Pat. No. 4,745,051; US2003148506; and WO 03/074714, all of which are incorporated by reference in their entireties. A particularly suitable promoter for transcription of a nucleotide sequence encoding an AAV capsid protein is e.g. the polyhedron promoter. However, other promoters that are active in insect cells are known in the art, e.g. the p10, p35 or IE-1 promoters and further promoters described in the above references are also contemplated.

[0083] Use of insect cells for expression of heterologous proteins is well documented, as are methods of introducing nucleic acids, such as vectors, e.g., insect-cell compatible vectors, into such cells and methods of maintaining such cells in culture. (See, e.g., METHODS IN MOLECULAR BIOLOGY, ed. Richard, Humana Press, N J (1995); O'Reilly et al., BACULOVIRUS EXPRESSION VECTORS, A LABORATORY MANUAL, Oxford Univ. Press (1994); Samulski et al., J. Vir. (1989) vol. 63, pp. 3822-3828; Kajigaya et al., Proc. Nat'l. Acad. Sci. USA (1991) vol. 88, pp. 4646-4650; Ruffing et al., J. Vir. (1992) vol. 66, pp. 6922-6930; Kirnbauer et al., Vir. (1996) vol. 219, pp. 37-44; Zhao et al., Vir. (2000) vol. 272, pp. 382-393; and U.S. Pat. No. 6,204,059). In some embodiments, the nucleic acid construct encoding AAV in insect cells is an insect cell-compatible vector. An insect cell-compatible vector or vector as used herein refers to a nucleic acid molecule capable of productive transformation or transfection of an insect or insect cell. Exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector can be employed as long as it is insect cell-compatible. The vector may integrate into the insect cells genome but the presence of the vector in the insect cell need not be permanent and transient episomal vectors are also included. The vectors can be introduced by any means known, for example by chemical treatment of the cells, electroporation, or infection. In some embodiments, the vector is a baculovirus, a viral vector, or a plasmid. In a more preferred embodiment, the vector is a baculovirus, i.e. the construct is a baculoviral vector. Baculoviral vectors and methods for their use are described in the above cited references on molecular engineering of insect cells.

[0084] Baculoviruses are enveloped DNA viruses of arthropods, two members of which are well known expression vectors for producing recombinant proteins in cell cultures. Baculoviruses have circular double-stranded genomes (80-200 kbp) which can be engineered to allow the delivery of large genomic content to specific cells. The viruses used as a vector are generally Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) or Bombyx mori (Bm)NPV).

[0085] Baculoviruses are commonly used for the infection of insect cells for the expression of recombinant proteins. In particular, expression of heterologous genes in insects can be accomplished as described in for instance U.S. Pat. No. 4,745,051; Friesen et al (1986); EP 127,839; EP 155,476; Vlak et al (1988); Miller et al (1988); Carbonell et al (1988); Maeda et al (1985); Lebacq-Verheyden et al (1988); Smith et al (1985); Miyajima et al (1987); and Martin et al (1988). Numerous baculovirus strains and variants and corresponding permissive insect host cells that can be used for protein production are described in Luckow et al (1988), Miller et al (1986); Maeda et al (1985) and McKenna (1989).

Methods for Producing Recombinant AAVs

[0086] The present disclosure provides materials and methods for producing recombinant AAVs in insect or mammalian cells. In some embodiments, the viral construct further comprises a promoter and a restriction site downstream of the promoter to allow insertion of a polynucleotide encoding one or more proteins of interest, wherein the promoter and the restriction site are located downstream of the 5 AAV ITR and upstream of the 3 AAV ITR. In some embodiments, the viral construct further comprises a posttranscriptional regulatory element downstream of the restriction site and upstream of the 3 AAV ITR. In some embodiments, the viral construct further comprises a polynucleotide inserted at the restriction site and operably linked with the promoter, where the polynucleotide comprises the coding region of a protein of interest. As a skilled artisan will appreciate, any one of the AAV vector disclosed in the present application can be used in the method as the viral construct to produce the recombinant AAV.

[0087] In some embodiments, the helper functions are provided by one or more helper plasmids or helper viruses comprising adenoviral or baculoviral helper genes. Non-limiting examples of the adenoviral or baculoviral helper genes include, but are not limited to, E1A, E1B, E2A, E4 and VA, which can provide helper functions to AAV packaging.

[0088] Helper viruses of AAV are known in the art and include, for example, viruses from the family Adenoviridae and the family Herpesviridae. Examples of helper viruses of AAV include, but are not limited to, SAdV-13 helper virus and SAdV-13-like helper virus described in US Publication No. 20110201088 (the disclosure of which is incorporated herein by reference), helper vectors pHELP (Applied Viromics). A skilled artisan will appreciate that any helper virus or helper plasmid of AAV that can provide adequate helper function to AAV can be used herein.

[0089] In some embodiments, the AAV cap genes are present in a plasmid. The plasmid can further comprise an AAV rep gene which may or may not correspond to the same serotype as the cap genes. The cap genes and/or rep gene from any AAV serotype (including, but not limited to, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 and any variants thereof) can be used herein to produce the recombinant AAV. In some embodiments, the AAV cap genes encode a capsid from serotype 1, serotype 2, serotype 4, serotype 5, serotype 6, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11, serotype 12, serotype 13 or a variant thereof.

[0090] In some embodiments, the insect or mammalian cell can be transfected with the helper plasmid or helper virus, the viral construct and the plasmid encoding the AAV cap genes; and the recombinant AAV virus can be collected at various time points after co-transfection. For example, the recombinant AAV virus can be collected at about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, or a time between any of these two time points after the co-transfection.

[0091] Recombinant AAV can also be produced using any conventional methods known in the art suitable for producing infectious recombinant AAV. In some instances, a recombinant AAV can be produced by using an insect or mammalian cell that stably expresses some of the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) comprising AAV rep and cap genes, and a selectable marker, such as a neomycin resistance gene, can be integrated into the genome of the cell. The insect or mammalian cell can then be co-infected with a helper virus (e.g., adenovirus or baculovirus providing the helper functions) and the viral vector comprising the 5 and 3 AAV ITR (and the nucleotide sequence encoding the heterologous protein, if desired). The advantages of this method are that the cells are selectable and are suitable for large-scale production of the recombinant AAV. As another non-limiting example, adenovirus or baculovirus rather than plasmids can be used to introduce rep and cap genes into packaging cells. As yet another non-limiting example, both the viral vector containing the 5 and 3 AAV LTRs and the rep-cap genes can be stably integrated into the DNA of producer cells, and the helper functions can be provided by a wild-type adenovirus to produce the recombinant AAV.

Cell Types Used in AAV Production

[0092] The viral particles comprising the AAV vectors of the invention may be produced using any invertebrate cell type which allows for production of AAV or biologic products and which can be maintained in culture. For example, the insect cell line used can be from Spodoptera frugiperda, such as SF9, SF21, SF900+, drosophila cell lines, mosquito cell lines, e.g., Aedes albopictus derived cell lines, domestic silkworm cell lines, e.g. Bombyx mori cell lines, Trichoplusia ni cell lines such as High Five cells or Lepidoptera cell lines such as Ascalapha odorata cell lines. Preferred insect cells are cells from the insect species which are susceptible to baculovirus infection, including High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAml, BM-N, Ha2302, Hz2E5 and Ao38.

[0093] Baculoviruses are enveloped DNA viruses of arthropods, two members of which are well known expression vectors for producing recombinant proteins in cell cultures. Baculoviruses have circular double-stranded genomes (80-200 kbp) which can be engineered to allow the delivery of large genomic content to specific cells. The viruses used as a vector are generally Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) or Bombyx mori (Bm-NPV) (Kato et al., 2010).

[0094] Baculoviruses are commonly used for the infection of insect cells for the expression of recombinant proteins. In particular, expression of heterologous genes in insects can be accomplished as described in for instance U.S. Pat. No. 4,745,051; Friesen et al (1986); EP 127,839; EP 155,476; Vlak et al (1988); Miller et al (1988); Carbonell et al (1988); Maeda et al (1985); Lebacq-Verheyden et al (1988); Smith et al (1985); Miyajima et al (1987); and Martin et al (1988). Numerous baculovirus strains and variants and corresponding permissive insect host cells that can be used for protein production are described in Luckow et al (1988), Miller et al (1986); Maeda et al (1985) and McKenna (1989).

[0095] In another aspect of the invention, the methods of the invention are also carried out with any mammalian cell type which allows for replication of AAV or production of biologic products, and which can be maintained in culture. Preferred mammalian cells used can be HEK293, HeLa, CHO, NSO, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5 cells.

Pharmaceutical Formulations

[0096] In other embodiments, the present invention is directed to pharmaceutical formulations of therapeutic protein expressing AAV vectors/virions useful for administration to subjects suffering from a genetic disorder. In certain aspects, the pharmaceutical formulations of the present invention are liquid formulations that comprise recombinant therapeutic protein expressing AAV virions produced from the vectors disclosed herein, wherein the concentration of recombinant AAV virions in the formulation may vary widely. In certain embodiments, the concentration of recombinant AAV virion in the formulation may range from 1E12 vg/ml to 2E16 vg/ml. In a particularly preferred embodiment, the concentration of recombinant AAV virion in the formulation is about 2E13 vg/ml. In a preferred embodiment, the recombinant AAV virion present in the formulation is derived from AAV5-FVIII-SQ. In other preferred embodiments of the invention the recombinant AAV virion present in the formulation is derived from AAV vectors expressing Factor IX or expressing PAH.

[0097] In other aspects, the AAV pharmaceutical formulation of the invention comprises one or more pharmaceutically acceptable excipients to provide the formulation with advantageous properties for storage and/or administration to subjects for the treatment of the genetic disorder. In certain embodiments, the pharmaceutical formulations of the present invention are capable of being stored at 65 C. for a period of at least 2 weeks, preferably at least 4 weeks, more preferably at least 6 weeks and yet more preferably at least about 8 weeks, without detectable change in stability. In this regard, the term stable means that the recombinant AAV virus present in the formulation essentially retains its physical stability, chemical stability and/or biological activity during storage. In certain embodiments of the present invention, the recombinant AAV virus present in the pharmaceutical formulation retains at least about 80% of its biological activity in a human patient during storage for a determined period of time at 65 C., more preferably at least about 85%, 90%, 95%, 98% or 99% of its biological activity in a juvenile human subject.

[0098] In certain aspects, the formulation comprising recombinant AAV virions further comprises one or more buffering agents. For example, in various aspects, the formulation of the present invention comprises sodium phosphate dibasic at a concentration of about 0.1 mg/ml to about 3 mg/ml, about 0.5 mg/ml to about 2.5 mg/ml, about 1 mg/ml to about 2 mg/ml, or about 1.4 mg/ml to about 1.6 mg/ml. In a particularly preferred embodiment, the AAV formulation of the present invention comprises about 1.42 mg/ml of sodium phosphate, dibasic (dried). Another buffering agent that may find use in the recombinant AAV formulations of the present invention is sodium phosphate, monobasic monohydrate which, in some embodiments, finds use at a concentration of from about 0.1 mg/ml to about 3 mg/ml, about 0.5 mg/ml to about 2.5 mg/ml, about 1 mg/ml to about 2 mg/ml, or about 1.3 mg/ml to about 1.5 mg/ml. In a particularly preferred embodiment, the AAV formulation of the present invention comprises about 1.38 mg/ml of sodium phosphate, monobasic monohydrate. In a yet more particularly preferred embodiment of the present invention, the recombinant AAV formulation of the present invention comprises about 1.42 mg/ml of sodium phosphate, dibasic and about 1.38 mg/ml of sodium phosphate, monobasic monohydrate.

[0099] In another aspect, the recombinant AAV formulation of the present invention may comprise one or more isotonicity agents, such as sodium chloride, preferably at a concentration of about 1 mg/ml to about 20 mg/ml, for example, about 1 mg/ml to about 10 mg/ml, about 5 mg/ml to about 15 mg/ml, or about 8 mg/ml to about 20 mg/ml. In a particularly preferred embodiment, the formulation of the present invention comprises about 8.18 mg/ml sodium chloride. Other buffering agents and isotonicity agents known in the art are suitable and may be routinely employed for use in the formulations of the present disclosure.

[0100] In another aspect, the recombinant AAV formulations of the present invention may comprise one or more bulking agents. Exemplary bulking agents include without limitation mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24). In certain preferred embodiments, the formulations of the present invention comprise mannitol, which may be present in an amount from about 5 mg/ml to about 40 mg/ml, or from about 10 mg/ml to about 30 mg/ml, or from about 15 mg/ml to about 25 mg/ml. In a particularly preferred embodiment, mannitol is present at a concentration of about 20 mg/ml.

[0101] In yet another aspect, the recombinant AAV formulations of the present invention may comprise one or more surfactants, which may be non-ionic surfactants. Exemplary surfactants include ionic surfactants, non-ionic surfactants, and combinations thereof. For example, the surfactant can be, without limitation, TWEEN 80 (also known as polysorbate 80, or its chemical name polyoxyethylene sorbitan monooleate), sodium dodecylsulfate, sodium stearate, ammonium lauryl sulfate, TRITON AG 98 (Rhone-Poulenc), poloxamer 407, poloxamer 188 and the like, and combinations thereof. In a particularly preferred embodiment, the formulation of the present invention comprises poloxamer 188, which may be present at a concentration of from about 0.1 mg/ml to about 4 mg/ml, or from about 0.5 mg/ml to about 3 mg/ml, from about 1 mg/ml to about 3 mg/ml, about 1.5 mg/ml to about 2.5 mg/ml, or from about 1.8 mg/ml to about 2.2 mg/ml. In a particularly preferred embodiment, poloxamer 188 is present at a concentration of about 2.0 mg/ml.

[0102] In a particular preferred embodiment of the present invention, the pharmaceutical formulation of the present invention comprises AAV5-FVIII-SQ formulated in a liquid solution that comprises about 1.42 mg/ml of sodium phosphate, dibasic, about 1.38 mg/ml of sodium phosphate, monobasic monohydrate, about 8.18 mg/ml sodium chloride, about 20 mg/ml mannitol and about 2 mg/ml poloxamer 188.

[0103] The recombinant therapeutic protein expressing AAV virus-containing formulations of the present disclosure are stable and can be stored for extended periods of time without an unacceptable change in quality, potency, or purity. In one aspect, the formulation is stable at a temperature of about 5 C. (e.g., 2 C. to 8 C.) for at least 1 month, for example, at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or more. In another aspect, the formulation is stable at a temperature of less than or equal to about 20 C. for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more. In another aspect, the formulation is stable at a temperature of less than or equal to about 40 C. for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more. In another aspect, the formulation is stable at a temperature of less than or equal to about 60 C. for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more.

Methods of Treatment

[0104] In certain embodiments, the present invention is directed to methods for treating a subject suffering from a genetic disorder comprising administering to that subject a therapeutically effective amount of an AAV vector expressing a therapeutic protein or a pharmaceutical composition comprising the same. In this instance, a therapeutically effective amount is an amount of AAV vector that after administration results in the expression of the therapeutic protein in a level sufficient to at least partially and preferably fully ameliorate the symptoms of the genetic disorder.

[0105] For example, the present invention is directed to methods of treating diseases or disorders including cancer such as carcinoma, sarcoma, leukemia, lymphoma; and autoimmune diseases such as multiple sclerosis. Non-limiting examples of carcinomas include esophageal carcinoma; hepatocellular carcinoma; basal cell carcinoma, squamous cell carcinoma (various tissues); bladder carcinoma, including transitional cell carcinoma; bronchogenic carcinoma; colon carcinoma; colorectal carcinoma; gastric carcinoma; lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung; adrenocortical carcinoma; thyroid carcinoma; pancreatic carcinoma; breast carcinoma; ovarian carcinoma; prostate carcinoma; adenocarcinoma; sweat gland carcinoma; sebaceous gland carcinoma; papillary carcinoma; papillary adenocarcinoma; cystadenocarcinoma; medullary carcinoma; renal cell carcinoma; ductal carcinoma in situ or bile duct carcinoma; choriocarcinoma; seminoma; embryonal carcinoma; Wilm's tumor; cervical carcinoma; uterine carcinoma; testicular carcinoma; osteogenic carcinoma; epithelieal carcinoma; and nasopharyngeal carcinoma. Non-limiting examples of sarcomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas. Non-limiting examples of solid tumors include glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma. Non-limiting examples of leukemias include chronic myeloproliferative syndromes; acute myelogenous leukemias; chronic lymphocytic leukemias, including B-cell CLL, T-cell CLL prolymphocytic leukemia, and hairy cell leukemia; and acute lymphoblastic leukemias. Examples of lymphomas include, but are not limited to, B-cell lymphomas, such as Burkitt's lymphoma; Hodgkin's lymphoma; and the like. Other non-liming examples of the diseases that can be treated using the AAV vectors, recombinant viruses and methods disclosed herein include genetic disorders including sickle cell anemia, cystic fibrosis, lysosomal acid lipase (LAL) deficiency 1, Tay-Sachs disease, Phenylketonuria, Mucopolysaccharidoses, Glycogen storage diseases (GSD, e.g., GSD types I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, and XIV), Galactosemia, muscular dystrophy (e.g., Duchenne muscular dystrophy), Wilson's disease, hereditary angioedema (HAE), alpha 1 antitrypsin deficiency, Fabry Disease, Gaucher Disease and hemophilia such as hemophilia A (classic hemophilia) and hemophilia B (Christmas Disease). In addition, the AAV vectors, recombinant viruses and methods disclosed herein can be used to other disorders that can be treated by local expression of a transgene in the liver or by expression of a secreted protein from the liver or a hepatocyte.

[0106] In a preferred embodiment, the present invention is directed to methods for reducing bleeding time during a bleeding episode in a juvenile subject suffering from hemophilia A comprising administering to that juvenile subject a therapeutically effective amount of an AAV FVIII vector, recombinant AAV FVIII virus or a pharmaceutical composition comprising the same. In this regard, a therapeutically effective amount, in reference to the treatment of hemophilia A or for use in a method for reducing bleeding time during a bleeding episode in a subject suffering from hemophilia A, refers to an amount capable of invoking one or more of the following effects: (1) reduction, inhibition, or prevention, to some extent, of one or more of the physiological symptoms of hemophilia A including, for example, bruising, joint pain or swelling, prolonged headache, vomiting or fatigue, (2) improvement in the capability to clot blood, (3) reduction of overall bleeding time during a bleeding episode, (4) administration resulting in a measurable increase in the concentration or activity of functional FVIII protein in the plasma of a subject, and/or (5) relief, to some extent, of one or more symptoms associated with the disorder.

[0107] A therapeutically effective amount of an AAV vector or virus or a pharmaceutical composition comprising the same for purposes of treatment as described herein may be determined empirically and in a routine manner. In a particularly preferred embodiment, a therapeutically effective amount of a therapeutic AAV virus for treatment of a juvenile subject is the same absolute number of viral particles that has been determined, calculated, or estimated to produce a therapeutic response in adult subjects. Accordingly, the invention provides administering AAV vectors to juvenile subjects at higher doses compared to adults when measured as vg/kg body weight. In some embodiments this corresponds to 2 to 15 times the amount of AAV vector given to an adult when expressed as vg/kg. In certain embodiments, however, a therapeutically effective amount of recombinant AAV virus ranges from about 1E12 vg/kg body weight to about 1E14 vg/kg body weight, preferably from about 6E12 vg/kg body weight to about 6E13 vg/kg body weight. In a preferred embodiment, a therapeutically effective amount of recombinant AAV virus is about 2E13 vg/kg body weight. In another preferred embodiment, a therapeutically effective amount of recombinant AAV virus is about 6E13 vg/kg body weight.

[0108] Recombinant AAV vectors/virus of the present invention may be administered to a juvenile subject, preferably a juvenile mammalian subject, more preferably a juvenile human subject, through a variety of known administration techniques. In a preferred embodiment, the recombinant AAV gene therapy virus is administered by intravenous injection either as a single bolus or over a prolonged time period, which may be at least about 1, 5, 10, 15, 30, 45, 60, 75, 90, 120, 150, 180, 210 or 240 minutes, or more. In preferred embodiments the recombinant AAV virus administered expresses Factor VIII, Factor IX, or PAH. In a particularly preferred embodiment of the present invention, the recombinant AAV virus administered is AAV5-FVIII-SQ.

[0109] Administration of a recombinant AAV FVIII vector/virus, or pharmaceutical formulation comprising the same, of the present invention preferably results in an increase in functional FVIII protein activity in the plasma of the subject of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more IU/dl as compared to the amount of functional FVIII protein activity present in the plasma in the subject prior to administration. In certain embodiments, administration of a recombinant AAV FVIII vector/virus, or pharmaceutical formulation comprising the same, of the present invention results in the expression of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more IU/dl of functional FVIII protein activity in the plasma of the subject. In this regard, the term IU or international unit in regards to FVIII activity is a well understood and accepted term, wherein 1 IU of FVIII activity is equivalent to the quantity of FVIII in one ml of normal human plasma. FVIII activity in the plasma may be quantitatively determined by a number of well-known and accepted assays including, for example, the activated partial thromboplastin time (APPT) method (see, e.g., Miletich J P: Activated partial thromboplastin time. In Williams Hematology. Fifth edition. Edited by E Beutler, M A Lichtman, B A Coller, T J Kipps. New York, McGraw-Hill, 1995, pp L85-86, Greaves and Preston, Approach to the bleeding patient. In Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Fourth edition. Edited by R W Colman, J Hirsh, V J Marder, et al. Philadelphia, JB Lippincott Co, 2001, pp 1197-1234 and Olson et al, (1998) Arch. Pathol. Lab. Med., vol. 122, pp. 782-798) or chromogenic FXa assay (Harris et al., (2011) Thromb. Res., vol. 128, pp. 125-129).

[0110] In other embodiments of the present invention, bleeding time in a subject may be measured by well-known and accepted techniques including, for example, the Ivy method (see, e.g., Ivy et al., (1935) Surg. Gynec. Obstet., vol. 60, page 781 (1935) and Ivy et al., (1941) J. Lab. Clin. Med., vol. 26, page 1812) or the Duke method (see, e.g., Duke et al., (1910) JAMA, vol. 55, page 1185). A bleeding episode in a subject refers to an injury that results in bleeding in the subject, either externally or internally, and generally comprises the time period from injury to formation of a blood clot.

[0111] In aspects of the invention involving an AAV vector expressing PAH to treat PKU in juvenile subjects, the effectiveness of the AAV vector can be monitored by measuring levels of phenylalanine in the blood of the treated juvenile subject. Precise quantitate assays for determining circulating levels of phenylalanine are well known in the art and include fluorometric assays (see, McCaman, M. W. and Robins, E., (1962) J. Lab. Clin. Med., vol. 59, pp. 885-890); thin layer chromatography based assays (see, Tsukerman, G. L. (1985) Laboratornoe delo, vol. 6, pp. 326-327); enzymatic assays (see, La Du, B. N., et al. (1963) Pediatrics, vol. 31, pp. 39-46; and Peterson, K., et al. (1988) Biochem. Med. Metab. Biol., vol. 39, pp. 98-104); methods employing high pressure liquid chromatography (HPLC) (see, Rudy, J. L., et al. (1987) Clin. Chem., vol. 33, pp. 1152-1154); and high-throughput automation (see, Hill, J. B., et al. (1985) Clin. Chem., vol. 5, pp. 541-546).

[0112] Administration of an AAV virus of the present invention may, in some cases, result in an observable degree of hepatotoxicity. Hepatotoxicity may be measured by a variety of well-known and routinely used techniques for example, measuring concentrations of certain liver-associated enzyme(s) (e.g., alanine transaminase, ALT) in the bloodstream of a subject both prior to AAV administration (i.e., baseline) and after AAV administration. An observable increase in ALT concentration after AAV administration (as compared to prior to administration) is indicative of drug-induced hepatotoxicity. In certain embodiments of the present invention, in addition to administration of a therapeutically effective amount of AAV virus, the subject may be treated either prophylactically, therapeutically, or both with a corticosteroid to prevent and/or treat any hepatotoxicity associated with administration of the AAV virus.

[0113] Prophylactic corticosteroid treatment refers to the administration of a corticosteroid to prevent hepatotoxicity and/or to prevent an increase in measured ALT levels in the subject. Therapeutic corticosteroid treatment refers to the administration of a corticosteroid to reduce hepatotoxicity caused by administration of an AVV virus and/or to reduce an elevated ALT concentration in the bloodstream of the subject caused by administration of an AAV virus. In certain embodiments, prophylactic or therapeutic corticosteroid treatment may comprise administration of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more mg/day of the corticosteroid to the subject. In certain embodiments, prophylactic or therapeutic corticosteroid treatment of a subject may occur over a continuous period of at least about 3, 4, 5, 6, 7, 8, 9, 10 weeks, or more. Corticosteroids that find use in the methods described herein include any known or routinely-employed corticosteroid including, for example, dexamethasone, prednisone, fludrocortisone, hydrocortisone, and the like.

Detection of Anti-AAV Antibodies

[0114] To maximize the likelihood of successful liver transduction with systemic AAV-mediated therapeutic gene transfer, prior to administration of an AAV vector in a therapeutic regimen to a human patient as described above, the prospective patient may be assessed for the presence of anti-AAV capsid antibodies that are capable of blocking cell transduction or otherwise reduce the overall efficiency of the therapeutic regimen. Such antibodies may be present in the serum of the prospective patient and may be directed against an AAV capsid of any serotype. In one embodiment, the serotype against which pre-existing antibodies are directed is AAV5.

[0115] Methods to detect pre-existing AAV immunity are well known and routinely employed in the art and include cell-based in vitro transduction inhibition (TI) assays, in vivo (e.g., in mice) TI assays, and ELISA-based detection of total anti-capsid antibodies (TAb) (see, e.g., Masat et al., Discov. Med., vol. 15, pp. 379-389 and Boutin et al., (2010) Hum. Gene Ther., vol. 21, pp. 704-712). TI assays may employ host cells into which an AAV-inducible reporter vector has been previously introduced. The reporter vector may comprise an inducible reporter gene such as GFP, etc. whose expression is induced upon transduction of the host cell by an AAV virus. Anti-AAV capsid antibodies present in human serum that are capable of preventing/reducing host cell transduction would thereby reduce overall expression of the reporter gene in the system. Therefore, such assays may be employed to detect the presence of anti-AAV capsid antibodies in human serum that are capable of preventing/reducing cell transduction by the therapeutic FVIII AAV virus.

[0116] TAb assays to detect anti-AAV capsid antibodies may employ solid-phase-bound AAV capsid as a capture agent over which human serum is passed, thereby allowing anti-capsid antibodies present in the serum to bind to the solid-phase-bound capsid capture agent. Once washed to remove non-specific binding, a detection agent may be employed to detect the presence of anti-capsid antibodies bound to the capture agent. The detection agent may be an antibody, an AAV capsid, or the like, and may be detectably-labeled to aid in detection and quantitation of bound anti-capsid antibody. In one embodiment, the detection agent is labeled with ruthenium or a ruthenium-complex that may be detected using electrochemiluminescence techniques and equipment.

[0117] The same above-described methodology may be employed to assess and detect the generation of an anti-AAV capsid immune response in a patient previously treated with a therapeutic AAV virus of interest. As such, not only may these techniques be employed to assess the presence of anti-AAV capsid antibodies prior to treatment with a therapeutic AAV virus, they may also be employed to assess and measure the induction of an immune response against the administered therapeutic AAV virus after administration. As such, the present invention contemplates methods that combine techniques for detecting anti-AAV capsid antibodies in human serum and administration of a therapeutic AAV virus for the treatment of hemophilia A, wherein the techniques for detecting anti-AAV capsid antibodies in human serum may be performed either prior to or after administration of the therapeutic AAV virus.

[0118] Other aspects and advantages of the present invention will be understood upon consideration of the following illustrative examples.

EXAMPLES

Example 1

AAV Age Comparison Study

[0119] To examine the ability of juvenile mice to respond to AAV mediated gene therapy, two doses of an AAV expressing human Factor VIII (AAV5-FVIII-SQ) were administered to two cohorts of juvenile (2 day old) Rag2/FVIII double knock-out mice (DKO). All doses were administered intravenously via tail veins. Adult (8 week old) Rag2/FVIII DKO mice were used as controls and were treated with 3.5E13 vg/kg which in the adult corresponds to 8.9E11 vg per mouse. Cohort 1 of the juvenile mice (2 days old) were treated with the same dose per body weight as adults (i.e. 3.5E13 vg/kg) whereas cohort 2 of the juvenile mice (2 days old) were treated with the same absolute vg per mouse as adults (i.e. 8.9E11 vg per mouse which corresponds to 4.5E14 vg/kg in 2 day old mice). FIG. 1A and FIG. 1B provide the study design and the sample collection time points. Groups of juvenile mice were studied well into adulthood (beyond 8 weeks of age) following AAV administration.

[0120] To determine the capacity of juvenile hepatocytes to take-up AAV5-FVIII-SQ, the amounts of FVIII DNA and FVIII RNA in the livers of treated juvenile and adult mice were measured. As shown in FIG. 2, juvenile mice receiving the same total vg as adult mice (8.9 E11 vg per mouse; 4.5E14 vg/kg body weight juvenile mice) had similar levels of FVIII DNA as adult mice demonstrating that there was no additional loss of DNA due to hepatocyte division in juvenile mice. This result was surprising in light of the literature on AAV vectors, which teaches that hepatocytes of juveniles treated with AAV lose viral genomes due to cell division and growth of the animal. In addition, as shown in FIG. 2 (right hand graph) Factor VIII RNA levels increased over time in adult mice dosed with AAV5-FVIII-SQ. Surprisingly, juvenile mice dosed with the same absolute dose as the adult mice demonstrated a similar increase in Factor VIII RNA over time. These data demonstrate that juvenile hepatocytes have the same ability to take up AAV genomes and express AAV delivered transgenes as do adult hepatocytes and that juvenile hepatocytes, contrary to the teachings of the art, do not lose viral genomes during tissue growth and cell division.

[0121] The effect of AAV treatment on the overall health and development of the juvenile mice was assessed by measuring both body weight and liver weight over time after AAV administration. As shown in FIG. 3, both the liver weight and body weight of AAV treated juvenile mice increased over time and reached normal adult levels by 8 weeks post-administration. Further, to evaluate whether AAV treatment that targets transgene expression in liver resulted in liver injury, blood levels of alanine aminotransferase (ALT) were measured. As shown in FIG. 4, ALT levels did not increase in response to AAV5-FVIII-SQ treatment despite the juvenile mice being given the same absolute amount of vector genome as adults, i.e. higher levels of vg per body weight than the control adult animals. Taken together, these data demonstrate that AAV treatment did not have a negative effect on overall health or development of juvenile mice and did not cause liver injury despite the relatively high levels of viral genomes being administered.

[0122] The therapeutic effectiveness of AAV delivered transgenes in juvenile mice were assessed by measuring the plasma Factor VIII concentration and estimating total circulating Factor VIII protein. As shown in FIG. 5, juveniles treated with the same dose on a per body weight basis as adults (3.5E13 vg/kg) did not produce therapeutic levels of Factor VIII when they reached adulthood. This result is consistent with prior studies demonstrating that juvenile hepatocytes stopped producing detectable levels of AAV administered transgenes several weeks after AAV administration. Surprisingly, juvenile mice dosed with the adult dose of absolute number of viral genomes (8.9E11 vg), initially express high levels of plasma Factor VIII. These levels decreased between weeks 1 and 3 due to blood volume expansion. However, the total amount of circulating Factor VIII remained stable from week 1 to the end of the study at week 16. This maintained level of Factor VIII protein in juvenile mice, while five to six fold lower than levels seen in treated adult mice, remained within the therapeutically effective window. Accordingly, these data demonstrate that AAV are effective in delivering a therapeutic dose of a transgene when the AAV are administered at an adult dose of absolute viral genome amount per subject. Moreover, these data demonstrate that the transgene expression stabilizes and remains constant for long durations.

Example 2

Delivery and Expression of PAH in the Livers of Juvenile PKU Subjects

[0123] In mammals, the liver enzyme phenylalanine hydroxylase (PAH) converts excess phenylalanine (Phe) in the body to tyrosine (Tyr). In humans, mutations in the gene coding for PAH can result in diminished or lack of production or activity of the enzyme, resulting in an accumulation of Phe and decrease of Tyr levels in the body, with phenotypic consequences, including growth failure, light skin and hair coloration, cognitive deficits, sleep disturbance, and seizures. In humans this disease state is called phenylketonuria (PKU). The ENU2 mouse model of PKU (Sheldovsky 1993) was created by chemical mutagenesis, using N-ethyl-N-nitrosourea (ENU), in exon 7 of the gene coding for PAH. Phe263 is replaced by Ser263, resulting in a mild reduction of PAH protein levels, but no detectable PAH catalytic activity. This is analogous to a mutation found in a large subset of human PKU patients where Phe263 has been mutated to Leu263. ENU2 mice have phenotypes which recapitulate several of those seen in PKU patients, including high plasma and tissue levels of Phe, low levels of Tyr, small size/body weight, a light brown coat color (while wild-type counterparts are black), and seizures.

[0124] Male ENU2 mice were enrolled into individual age groups (n=10/group) as listed below and injected intravenously with the AAV5-PAH at 2e14 vg/kg at the approximate age: Group One, AAV administered at 2 days of age; Group Two, AAV administered at 1 week of age; Group Three, AAV administered at 2 weeks of age; Group Four, AAV administered at 3 weeks of age; Group Five, AAV administered at 5 weeks of age; and Group Six, AAV administered at 8 weeks of age (see FIG. 7 for overall study design).

[0125] Body weights were measured prior to study start, and at four and eight weeks post-dose. Blood samples were collected 4 and 8 weeks post-dose, processed to plasma, and analyzed for Phe by liquid chromatography/mass spectrometry. Plasma proteins were precipitated using acetonitrile containing a stable isotope internal standards (13C9,15N (PheIS)). The supernatant was derivatized by reaction with Benzoyl Chloride and diluted prior to LC-MS/MS injection.

[0126] Body weights of animals in each age group were similar prior to dosing (FIG. 8A). Four weeks post dosing (FIG. 8B), animals treated with AAV5-PAH at either 3 or 8 weeks of age, but not those treated at 2 days, or 1, 2, or 5 weeks of age gained significantly more weight than their vehicle-treated counterparts. By 8 weeks post-dosing (FIG. 8C), mice treated at 5 weeks of age also achieved significantly more weight gain than controls.

[0127] At both the 4 (FIG. 9A) and 8-week (FIG. 9B) time points, Phe levels were reduced to WT range in mice treated at 5 or 8 weeks of age. Mice treated at 3 weeks of age had more variable phe levels, suggesting a partial response. Mice treated at 2 days, 1 week, or 2 weeks of age did not have appreciable reduction in plasma Phe.

[0128] Maximal effect of treatment with AAV5PAH, 2E14 vg/kg, on the ENU2 phenotypes of low body weight and high plasma Phe was achieved when mice were at least 5 weeks old at time of treatment. A significant effect on body weight, but only a partial effect on Phe reduction, was achieved when mice were treated at 3 weeks of age.