Gene therapy for treating galactosemia

Abstract

To provide a gene therapy for treating galactosemia.SOLUTION: Provided is a recombinant AAV vector encoding GALT protein. Also provided are a composition and a method for treating galactosemia. The present invention provides an isolated polynucleotide including a sequence identical to any one of sequence numbers 1 to 3 by at least 85% or an equivalent of each thereof, where, as needed, at least one or more of nucleotides modified from a wild type sequence are not modified from corresponding wild type sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62 / 725,225, filed August 30, 2018, the contents of which are incorporated herein by reference.

[0002] background The present disclosure relates generally to the field of gene therapy, and in particular to the field of gene therapy for the treatment of galactosemia. [Background technology]

[0003] Currently, there are no treatment options for galactosemia patients other than dietary restriction of galactose-containing foods. Despite strict adherence to a galactose-free diet, many patients suffer from long-term mental and physical impairments due to the small amount of galactose in food and endogenously produced galactose. Thus, there is a need in the art for an effective treatment. The present disclosure meets this need and provides related advantages. Summary of the Invention [Means for solving the problem]

[0004] The disclosed gene therapy provides galactosemia patients with a sustainable, long-term treatment option by efficiently delivering the GALT gene to their cells, including the brain, providing sustained expression of functional GALT protein and alleviation of disease symptoms.

[0005] To achieve this treatment, provided herein is a polynucleotide or recombinant adeno-associated virus ("AAV") vector comprising, consisting essentially of, or consisting of a polynucleotide sequence encoding galactose-1-phosphate uridyl transferase ("GALT"). In one aspect, the polynucleotide sequence encoding GALT comprises, consists essentially of, or consists of a nucleotide sequence at least 85% identical to SEQ ID NO:1. In one aspect, the sequence is at least 85% identical to SEQ ID NO:1, provided that at least one or more of the nucleotides modified from the wild-type sequence are not modified from SEQ ID NO:1 (see FIG. 4). In another aspect, the polynucleotide sequence encoding GALT comprises, consists essentially of, or consists of the nucleotide sequence shown in SEQ ID NO:1. In another embodiment, the polynucleotide sequence encodes the amino acid sequence of SEQ ID NO:4 or its equivalent.

[0006] In one embodiment, the vector is a self-complementary vector or a single-stranded DNA (ssDNA) vector.

[0007] In one embodiment, recombinant adeno-associated virus ("AAV") comprises, consists essentially of, or consists of AAV, such as scAAV or ssAAV, flanked by two inverted terminal repeats (ITRs). These ITRs form hairpins at the end of the sequence to act as a primer to initiate second strand synthesis before the subsequent steps of infection can begin. Second strand synthesis is believed to be one of several obstacles to efficient infection. Additional advantages of scAAV include increased and extended transgene expression in vitro and in vivo. Thus, in one embodiment, AAV further comprises two ITRs.

[0008] Non-limiting examples of recombinant AAV backbones for making vectors include AAV vector serotypes from the group of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B or AAV rh74. In further embodiments, the vector backbone is an AAV9 serotype, rh74 serotype or a modified AAVrh74 serotype. Also provided are polynucleotides encoding a modified AAVrh74 VP1 capsid protein or equivalent thereof comprising one or more modifications selected from the group of a substitution of asparagine with isoleucine at amino acid position 502, an optional substitution of tryptophan with arginine at amino acid position 505 of VP1 of AAVrh74, and an insertion of the peptides YIG or YIGSR at amino acid position 591 of VP1 of AAVrh74, or polynucleotides which are at least 85% identical, provided that one or more, or two or more, or three or more, or four or more modifications are identical to an amino acid in the modified AAVrh74 VP1 capsid protein, and complements of these polynucleotides. Also provided is a modified AAVrh74 VP1 capsid protein or equivalent thereof comprising one or more modifications selected from the group of a substitution of asparagine with isoleucine at amino acid position 502, an optional substitution of tryptophan with arginine at amino acid position 505 of VP1 of AAVrh74, and an insertion of the peptide YIG at amino acid position 591 of VP1 of AAVrh74, or a polynucleotide which is at least 85% identical, provided that one or more, or two or more, or three or more, or four or more modifications are identical to an amino acid in the modified AAVrh74 VP1 capsid protein.

[0009] The polynucleotide sequence encoding GALT is optionally functionally linked to promoter, tissue-specific regulatory element or constitutive promoter.Non-limiting examples of constitutive promoter include, for example, Rous sarcoma virus (RSV) LTR promoter (optionally with RSV enhancer), cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter or EF1 promoter.In a specific embodiment, β-actin promoter is chicken β-actin ("CBA") promoter. In another embodiment, the promoter is selected from a CMV promoter, an EF1a promoter, an SV40 promoter, a PGK1 (human or mouse) promoter, a P5 promoter, a Ubc promoter, a human beta-actin promoter, a CAG promoter, a TRE promoter, a UAS promoter, an Ac5 promoter, a polyhedrin promoter, a CaMKIIa promoter, a Gal1 promoter, a TEF1, a GDS promoter, an ADH1 promoter, a CaMV35S promoter, a Ubi promoter, an H1 promoter, a U6 promoter, or an alpha-1-antitrypsin promoter.

[0010] In further embodiments, the polynucleotide or recombinant AAV further comprises a polynucleotide encoding an enhancer element. Non-limiting examples include the CMV enhancer, the WPRE, and the RSV enhancer.

[0011] In certain embodiments, the recombinant polynucleotide or AAV vector comprises, consists essentially of, or consists of a nucleotide sequence at least 85% identical to SEQ ID NO:2 or SEQ ID NO:3. In one embodiment, the sequence is at least 85% identical to SEQ ID NO:2 or 3, with the proviso that at least one or more of the nucleotides modified from the wild-type GALT (see FIG. 4) sequence are not modified from SEQ ID NO:2 or 3. In a further embodiment, the recombinant AAV vector comprises, consists essentially of, or consists of the nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3.

[0012] The recombinant polynucleotide or AAV vector described herein can further comprise a detectable or purification marker. The polynucleotide or vector can be contained in a composition comprising the polynucleotide or vector and a carrier, such as a preservative or a pharma- ceutically acceptable carrier.

[0013] Also provided are cells or viral particles that contain the polynucleotide and / or AAV vector described herein.The cells can be prokaryotic or eukaryotic, and can include packaging cell lines.The cells and viral particles can be contained in a composition that includes the vector and a carrier, such as a preservative or a pharmaceutically acceptable carrier.

[0014] The polynucleotides and vectors are useful in therapeutic or other methods, and are contained within wild-type or modified capsid proteins.Therefore, provided herein are AAV viral particles that contain the recombinant polynucleotides and / or AAV vectors disclosed herein, and compositions that contain polynucleotides and / or viral particles for use in the methods disclosed herein.Furthermore, methods are provided for preparing vectors, polynucleotides, and viral particles that contain them.In one embodiment, a method is provided for introducing functional GALT enzyme into cells, comprising contacting cells with recombinant polynucleotides and / or AAV vectors, or capsids or viral particles that contain them, or compositions described herein.Contacting can be ex vivo or in vivo.

[0015] Also provided is a method for treating galactosemia in a subject in need thereof, comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a recombinant polynucleotide and / or AAV vector, a viral particle comprising a recombinant polynucleotide and / or AAV vector, or a composition described herein.

[0016] In one embodiment, there is also provided a method of increasing galactose metabolism in a subject possibly suffering from galactosemia, comprising, consisting essentially of, or consisting of administering to the subject a recombinant polynucleotide and / or AAV vector, viral particle, or composition described herein. In one embodiment, the amount delivered is an effective amount as determined by the treating professional.

[0017] Further provided is a method for reducing disease symptoms or symptoms associated with galactosemia in a subject suffering from galactosemia, comprising, consisting essentially of, or consisting of administering recombinant polynucleotide and / or AAV vector, viral particle, or composition described herein to the subject, wherein the disease symptoms include one or more of jaundice, hepatosplenomegaly, hepatocellular dysfunction, hypoglycemia, renal tubular dysfunction, hypotonia, sepsis, cataracts, ataxia, tremor, reduced bone mineral density, fertility in adult females, motor dysfunction and growth retardation in GalT deficiency or primary ovarian insufficiency.In one embodiment, the method further comprises identifying the subject as suffering from galactosemia, and then treating the subject identified by the method.In one embodiment, the amount delivered is an effective amount as determined by the treating professional.

[0018] In each of these methods, the galactosemia to be treated is galactosemia type 1, galactosemia type 2, or galactosemia type 3. In certain embodiments, the galactosemia is type 1 galactosemia.

[0019] The recombinant polynucleotide and / or AAV vector, viral particle, or composition is administered by intramuscular or intravenous injection. Alternatively, the recombinant polynucleotide and / or AAV vector, polynucleotide and / or viral particle, or composition is administered systemically. In addition, the recombinant polynucleotide and / or AAV vector, viral particle, or composition is administered parenterally by injection, infusion, or implantation. In a further embodiment, the polynucleotide, recombinant AAV vector, viral particle, or composition is administered systemically after intramuscular or intravenous injection.

[0020] Kits comprising the recombinant polynucleotides and / or AAV vectors, viral particles, and / or compositions described herein are also provided by the present disclosure. The kits can optionally contain instructions for making and using the polynucleotides, vectors, viral particles, or compositions. In certain embodiments, for example, the following items are provided: (Item 1) An isolated polynucleotide comprising a sequence at least 85% identical to any one of SEQ ID NOs: 1-3, or their respective equivalents, and optionally wherein at least one or more of the nucleotides modified from the wild-type sequence are not modified from their corresponding wild-type sequence. (Item 2) An isolated polynucleotide encoding the amino acid sequence of SEQ ID NO:4 or an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:4, or an equivalent thereof. (Item 3) A polynucleotide comprising the complement of the polynucleotide of items 1 or 2. (Item 4) A polynucleotide comprising the polynucleotide of SEQ ID NO:1 or an equivalent thereof, optionally wherein at least one or more of the nucleotides modified from the wild-type sequence are not modified from SEQ ID NO:1. (Item 5) A vector comprising the polynucleotide according to any one of items 2 to 4. (Item 6) 6. The vector according to item 5, further comprising a promoter and / or enhancer element. (Item 7) 7. The isolated polynucleotide according to any one of items 1 to 4 or the vector according to item 5 or 6, further comprising a label. (Item 8) 6. The vector according to item 5, wherein the vector is an AAV vector. (Item 9) 9. The vector of item 8, further comprising a polynucleotide encoding a capsid protein. (Item 10) 10. The vector of item 9, wherein the capsid protein is a wild-type capsid protein or a mutated capsid protein. (Item 11) 11. An isolated cell comprising the isolated polynucleotide according to any one of items 1 to 4 or the vector according to any one of items 5 to 10. (Item 12) A recombinant AAV vector comprising a polynucleotide sequence encoding galactose-1-phosphate uridyl transferase ("GALT"). (Item 13) 13. The recombinant AAV vector of item 12, wherein the polynucleotide sequence encoding GALT comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:1, and optionally, at least one or more of the nucleotides modified from the wild-type sequence are unmodified from SEQ ID NO:1. (Item 14) Item 13. The recombinant AAV vector of item 12, wherein the polynucleotide sequence encoding GALT comprises the nucleotide sequence set forth in SEQ ID NO:1. (Item 15) 15. The recombinant AAV vector of any one of items 12 to 14, wherein the vector is of the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B or AAV rh74. (Item 16) 15. The recombinant AAV vector of any one of items 12 to 14, wherein the vector is AAV9, rh74, mutated rh74 or AAV PHP.B. (Item 17) 17. The recombinant AAV vector of any one of items 12 to 16, wherein the vector is operably linked to a promoter, a tissue-specific regulatory element, or a constitutive promoter. (Item 18) 18. The recombinant AAV vector of item 17, wherein the constitutive promoter comprises a Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a beta-actin promoter, a phosphoglycerol kinase (PGK) promoter, a U6 promoter, or an EF1 promoter. (Item 19) 19. The recombinant AAV vector of item 18, wherein the β-actin promoter is a chicken β-actin ("CBA") promoter. (Item 20) 13. The recombinant AAV vector of item 12, comprising a nucleotide sequence that is at least 95% identical to SEQ ID NO:2, and optionally wherein at least one or more of the nucleotides modified from the wild-type sequence are unmodified from SEQ ID NO:2. (Item 21) Item 13. The recombinant AAV vector of item 12, comprising the nucleotide sequence set forth in SEQ ID NO:2. (Item 22) 13. The recombinant AAV vector of claim 12, comprising a nucleotide sequence that is at least 85% identical to SEQ ID NO:3, and optionally wherein at least one or more of the nucleotides modified from the wild-type sequence are unmodified from SEQ ID NO:3. (Item 23) Item 13. The recombinant AAV vector of item 12, comprising the nucleotide sequence set forth in SEQ ID NO:3. (Item 24) 18. The recombinant AAV vector according to any one of items 12 to 17, wherein the polynucleotide sequence encoding the GALT enzyme encodes the amino acid sequence of SEQ ID NO: 4. (Item 25) 25. The recombinant AAV vector of any one of items 12 to 24, further comprising a detectable or purification marker. (Item 26) 25. A viral particle comprising the AAV vector of any one of items 12 to 24 and one or more capsid proteins. (Item 27) 27. The viral particle of item 26, wherein the capsid protein comprises at least one wild-type capsid protein. (Item 28) 27. The viral particle according to item 26, wherein the capsid protein comprises at least one non-naturally occurring or mutated protein. (Item 29) 27. The virus particle of item 26, wherein the capsid protein comprises at least one wild-type capsid protein and at least one mutated capsid protein. (Item 30) 27. The viral particle of item 26, wherein the capsid protein comprises a modified AAVrh74 VP1 capsid protein comprising one or more modifications selected from the group of: a substitution of asparagine at amino acid position 502 with isoleucine, and an optional substitution of tryptophan at amino acid position 505 of VP1 of AAVrh74 with arginine, and an insertion of peptide YIG at amino acid position 591 of VP1 of AAVrh74. (Item 31) A polynucleotide encoding a modified AAVrh74 VP1 capsid protein or its equivalent comprising one or more modifications selected from the group consisting of a substitution of asparagine with isoleucine at amino acid position 502, a substitution of tryptophan with arginine at amino acid 505 of VP1 of AAVrh74, and an insertion of the peptide YIG at amino acid position 591 of VP1 of AAVrh74, or a polynucleotide which is at least 85% identical thereto, provided that the one or more modifications are identical to an amino acid in the modified AAVrh74 VP1 capsid protein. (Item 32) A modified AAVrh74 VP1 capsid protein or an equivalent thereof comprising one or more modifications selected from the group consisting of a substitution of asparagine with isoleucine at amino acid position 502, a substitution of tryptophan with arginine at amino acid 505 of VP1 of AAVrh74, and an insertion of the peptide YIG at amino acid position 591 of VP1 of AAVrh74, or a polynucleotide which is at least 85% identical thereto, provided that the one or more modifications are identical to an amino acid in the modified AAVrh74 VP1 capsid protein. (Item 33) The complement of the polynucleotide of item 31. (Item 34) A composition comprising a polynucleotide according to any one of items 1 to 14, 31 or 33, a capsid protein according to item 32, and / or a viral particle according to any one of items 15 to 19, and a carrier, optionally a pharma- ceutically acceptable carrier. (Item 35) 20. A method for introducing a functional GALT enzyme into a cell, comprising contacting said cell with the polynucleotide according to any one of items 1 to 13, 31 or 33, the capsid protein according to item 32, the composition according to item 14 or the viral particle according to any one of items 15 to 19. (Item 36) 36. The method of item 35, wherein the contacting is ex vivo or in vivo. (Item 37) 20. A method for treating galactosemia in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide according to any one of items 1 to 13, or the composition according to item 14, or the viral particle according to any one of items 15 to 19. (Item 38) 20. A method for increasing galactose metabolism in a subject suffering from galactosemia, comprising administering to the subject a polynucleotide according to any one of items 1 to 13, or a composition according to item 14, or a viral particle according to any one of items 15 to 19. (Item 39) 20. A method for reducing a disease symptom in a subject suffering from galactosemia, comprising administering to the subject the polynucleotide according to any one of items 1 to 13, the composition according to item 14, or the viral particle according to any one of items 15 to 19, wherein the disease symptom is selected from jaundice, hepatosplenomegaly, hepatocellular dysfunction, hypoglycemia, renal tubular dysfunction, hypotonia, sepsis, cataracts, ataxia, tremor, reduced bone density or primary ovarian insufficiency. (Item 40) 40. The method according to any one of items 37 to 39, wherein the galactosemia is type 1 galactosemia. (Item 41) 41. The method according to any one of items 37 to 40, wherein the subject is a mammal. (Item 42) 42. The method of claim 41, wherein the subject is a human. (Item 43) 43. The method of claim 42, wherein the subject is a human newborn. (Item 44) 44. The method of claim 43, wherein the human newborn is selected for administration by genetic testing for lack of expression of a functional GALT gene. (Item 45) 45. The method of any one of items 37 to 44, wherein the administration is by intramuscular injection, local delivery to the liver or intravenous injection. (Item 46) 45. The method of any one of items 37 to 44, wherein administration comprises systemic administration. (Item 47) 45. The method of any one of items 37 to 44, wherein the administration comprises localized delivery to the liver. (Item 48) A kit comprising one or more of the polynucleotide according to any one of items 1 to 13, 31 or 33, the capsid according to item 32, the composition according to item 14 or the viral particle according to any one of items 15 to 19, and instructions for use. (Item 49) A host cell comprising the recombinant AAV vector according to any one of items 1 to 13. (Item 50) 50. The host cell according to item 49, wherein the cell is a prokaryotic or eukaryotic cell. (Item 51) 51. The host cell of item 50, wherein the cell is a packaging cell. (Item 52) 34. An AAV packaging system comprising the polynucleotide of any one of items 1 to 13, 31 or 33 and a helper cell line. [Brief description of the drawings]

[0021] [Figure 1A] Figures 1A-1C show maps of three exemplary vectors: pAAV-CB-GALT-WPRE-kan (Figure 1A), pAAV-CB-hGALT (Figure 1B), and pAAV-CB-hGALT-WPREv2 (Figure 1C). [Figure 1B] Figures 1A-1C show maps of three exemplary vectors: pAAV-CB-GALT-WPRE-kan (Figure 1A), pAAV-CB-hGALT (Figure 1B), and pAAV-CB-hGALT-WPREv2 (Figure 1C). [Figure 1C] Figures 1A-1C show maps of three exemplary vectors: pAAV-CB-GALT-WPRE-kan (Figure 1A), pAAV-CB-hGALT (Figure 1B), and pAAV-CB-hGALT-WPREv2 (Figure 1C).

[0022] [Diagram 2] FIG. 2 shows the biodistribution of the pAAV-CB-GALT-WPRE-kan vector (FIG. 1A).

[0023] [Diagram 3] FIG. 3 shows the expression of human GALT protein expressed using the pAAV-CB-GALT-WPRE-Kan vector (FIG. 1A).

[0024] [Figure 4] FIG. 4 is a sequence alignment of the codon-optimized recombinant polynucleotide encoding GALT described herein compared to the corresponding wild-type sequence.

[0025] [Diagram 5] Figure 5 shows the biodistribution of AAV9 vectors. C57Bl / 6J (WT) or GalT- / - (CG) female mice were intravenously injected with AAV9 / lucEYFP reporter virus with 1E+12 viral genome (Vg), and tissues were harvested 1 week later and assayed for vector genome copies by qPCR.

[0026] [Figure 6] Figure 6 shows protein expression. Plasmid pAAV-CB-GALT-WPRE or pAAV-CB-lucEYFP reporter plasmid was transfected into HEK293 cells and GALT expression was assayed by Western blot analysis 2 days later. GALT transfected cell lysate (30 or 3 μg), LucEYFP cell lysate (138 μg), HepG2 endogenous GALT protein control (135 μg), purified bacterially expressed GALT protein standard with a slightly higher molecular weight due to the purification tag. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description The embodiments according to the present disclosure will be described more completely below. However, aspects of the present disclosure can be embodied in different forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided so that the present disclosure will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art. The terms used in the description herein are merely intended to describe specific embodiments, and are not intended to be limiting.

[0028] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art. Terms as defined in commonly used dictionaries should be interpreted to have a meaning consistent with their meaning in the context of this application and related art, and it is further understood that they should not be interpreted in an idealized or overly formal sense unless expressly defined as such herein. Although not expressly defined below, such terms should be interpreted according to their general meaning.

[0029] The terms used in the description herein are for the purpose of describing particular embodiments only and are not intended to limit the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0030] Unless otherwise indicated, the practice of the present technique employs conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art.

[0031] Unless the context dictates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.Furthermore, the present disclosure also contemplates that in some embodiments, any feature or combination of features described herein can be excluded or omitted.To illustrate, if the specification states that a complex comprises components A, B, and C, it is specifically intended that any or any combination of A, B, or C can be omitted and can be excluded from the scope, either alone or in any combination.

[0032] Unless otherwise stated, all specified embodiments, features and terms are intended to include both the recited embodiment, feature or term and their biological equivalents.

[0033] All numerical designations, including ranges, such as pH, temperature, time, concentration and molecular weight, are approximate values ​​that are varied (+) or (-) by increments of 1.0 or 0.1, or by variations of + / -15%, or + / -10%, or + / -5%, or + / -2%, as appropriate. It should be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about". It should also be understood, although not always explicitly stated, that the reagents described herein are merely exemplary, and that equivalent reagents to such are known in the art.

[0034] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation or by Arabic numerals. Full citations for publications identified by Arabic numerals are set forth immediately before the claims. The disclosures of these publications, patents and published patent specifications are incorporated by reference in their entireties into this disclosure in order to more fully describe the state of the art to which this invention pertains. definition

[0035] Unless otherwise indicated, the practice of the present technology employs conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols In Molecular Biology (FM Ausubel et al., eds., (1987)); the series Methods in Enzymology (Academic Press, 1999); Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)); Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (R. I. Freshney, ed. (1987)).

[0036] As used in the description of this invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0037] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude other elements. As used herein, the transitional phrase (and grammatical variations) consisting essentially of should be interpreted to include the recited materials or steps, as well as materials or steps that do not substantially affect the basic and novel characteristics of the recited embodiment. Thus, the term "consisting essentially of" as used herein should not be interpreted as equivalent to "comprising." "Consisting of" is intended to mean excluding more than trace amounts of other ingredients and substantial method steps for administering the compositions disclosed herein. The embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0038] The term "about" as used herein when referring to a measurable value, such as an amount or concentration, is intended to encompass a variation of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0039] The terms "acceptable," "effective," or "sufficient" when used to describe the selection of any ingredient, range, dosage form, etc. disclosed herein intend that said ingredient, range, dosage form, etc. is appropriate for the purpose disclosed.

[0040] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or").

[0041] The term "adeno-associated virus" or "AAV" as used herein refers to a member of a class of viruses related to the name and belonging to the Dependoparvovirus genus of the Parvoviridae family. Multiple serotypes of this virus are known to be suitable for gene delivery, and all known serotypes can infect cells from various tissue types. At least eleven consecutively numbered AAV serotypes are known in the art. Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the eleven serotypes, such as AAV2, AAV8, AAV9, or variant serotypes, such as AAV-DJ and AAV PHP.B. AAV particles contain three major viral proteins: VP1, VP2, and VP3. In one embodiment, AAV refers to serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, or AAV rh74.

[0042] The term "galactosemia" refers to a genetic disorder that affects a subject's ability to metabolize galactose. Lactose in foods (such as dairy products) is broken down into glucose and galactose by the enzyme lactase. In individuals with galactosemia, the enzymes required for further metabolism of galactose (galactokinase and galactose-1-phosphate uridyltransferase) are greatly reduced or completely missing, causing toxic levels of galactose or galactose 1-phosphate (depending on which enzyme is missing) in various tissues, resulting in disease symptoms including, but not limited to, jaundice, hepatomegaly (enlarged liver), renal tubular dysfunction, hypotonia, sepsis, hepatocellular dysfunction, cirrhosis, renal failure, cataracts, vomiting, seizures, hypoglycemia, lethargy, brain damage, and ovarian failure, as in classical galactosemia. Thus, these symptoms, when associated with galactosemia in a subject, can also be appropriately treated by the methods of the present disclosure.

[0043] Based on the enzyme that is deficient in the subject, galactosemia can be classified as type 1 galactosemia (galactose-1-phosphate uridyl transferase), type 2 galactosemia (galactokinase) and type 3 galactosemia (UDP galactose epimerase). In one embodiment, the recombinant vector or method disclosed herein can be used to treat type 1, type 2 or type 3 galactosemia. In another embodiment, the recombinant or method can be used to treat type 1 galactosemia. Since galactosemia is associated with many disease symptoms, in another embodiment, the present disclosure provides a method of reducing disease symptoms in a subject suffering from galactosemia, wherein the disease symptoms are selected from jaundice, hepatomegaly (enlarged liver), renal tubular dysfunction, hypotonia, sepsis, hepatocellular dysfunction, cirrhosis, renal failure, cataracts, vomiting, seizures, hypoglycemia, lethargy, brain damage or ovarian failure.

[0044] Other disease symptoms associated with galactosemia include, but are not limited to, speech deficit, ataxia, Friedreich's ataxia, dysmetria, reduced bone density, or premature ovarian failure. These symptoms can also be treated by the methods of the present disclosure.

[0045] As used herein, the term "cell" can refer to either prokaryotic or eukaryotic cells, obtained from the subject or from a commercially available source, as appropriate.

[0046] "Eukaryotic cells" includes all kingdoms of life except Monera. Eukaryotic cells can be easily identified through their membrane-bound nuclei. Animals, plants, fungi and protists are eukaryotes, i.e. organisms whose cells are organized into complex structures by internal membranes and cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless specifically stated, the term "host" includes eukaryotic hosts, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or eukaryotic hosts include monkey, cow, pig, mouse, rat, bird, reptile and human, e.g., HEK293 and 293T cells.

[0047] "Prokaryotic cells", which usually lack a nucleus or any other membrane-bound organelles, are divided into two domains: bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in circular loops called episomes. Bacterial cells are very small, roughly the size of an animal mitochondrion (approximately 1-2 μm in diameter and 10 μm in length). Prokaryotic cells are characterized by three main shapes: rod-shaped, spherical and spiral. Instead of undergoing an elaborate replication process like eukaryotes, bacterial cells divide by binary fission. Examples include, but are not limited to, the bacteria Bacillus, Escherichia coli and Salmonella.

[0048] The term "encoding" when applied to a nucleic acid sequence refers to a polynucleotide that is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those of skill in the art, it can be transcribed and / or translated to produce an mRNA for a polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced from the antisense strand.

[0049] The terms "equivalent" or "biological equivalent" are used interchangeably when referring to a particular molecule, biological or cellular material, and are intended to have minimal homology while still maintaining a desired structure or function. Non-limiting examples of equivalent polypeptides include polypeptides or polypeptide sequences having at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% identity, or polypeptides encoded by polynucleotides or their complements that hybridize to polynucleotides encoding such polypeptide sequences under high stringency conditions. High stringency conditions are described herein and are incorporated by reference. Alternatively, the equivalent is a polypeptide encoded by a polynucleotide or its complement that has at least 70%, or at least 75%, or 80%, or at least 85%, or at least 90%, or at least 95% identity, or at least 97% sequence identity to a reference polynucleotide, such as a wild-type polynucleotide.

[0050] Non-limiting examples of equivalent polypeptides include polynucleotides having at least 60%, or at least 65%, or at least 70%, or at least 75%, or 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97% identity to a reference polynucleotide. Equivalent also contemplates polynucleotides or their complements that hybridize to a reference polynucleotide under conditions of high stringency.

[0051] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are identical when comparing the two sequences. Alignment and percent homology or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. In certain embodiments, default parameters are used for alignment. A non-limiting exemplary alignment program is BLAST, using default parameters. In particular, exemplary programs include BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. Sequence identity and percent identity can be determined by incorporating them into clustalW (available at the Web address: genome.jp / tools / clustalw / , last accessed on January 13, 2017).

[0052] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that can be aligned for comparison. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or less than 25% identity, with one of the sequences of the present disclosure.

[0053] "Homology" or "identity" or "similarity" can also refer to two nucleic acid molecules that hybridize under stringent conditions.

[0054] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogsteen binding, or in any other sequence-specific manner. The complex may contain two strands forming a double-stranded structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a larger process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0055] Examples of stringent hybridization conditions include an incubation temperature of about 25°C to about 37°C; a hybridization buffer concentration of about 6xSSC to about 10xSSC; a formamide concentration of about 0% to about 25%; and a wash solution of about 4xSSC to about 8xSSC. Examples of mild hybridization conditions include an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9xSSC to about 2xSSC; a formamide concentration of about 30% to about 50%; and a wash solution of about 5xSSC to about 2xSSC. Examples of high stringency conditions include an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1xSSC to about 0.1xSSC; a formamide concentration of about 55% to about 75%; and a wash solution of about 1xSSC, 0.1xSSC, or deionized water. Generally, hybridization incubation times are 5 minutes to 24 hours, with one, two or more washing steps, with wash incubation times of about 1, 2 or 15 minutes. SSC is a 0.15M NaCl and 15mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be used.

[0056] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0057] "Gene" refers to a polynucleotide containing at least one open reading frame (ORF) that can encode a particular polypeptide or protein after being transcribed and translated. "Gene product" or "gene expression product" refers to the amino acid (e.g., peptide or polypeptide) produced when a gene is transcribed and translated.

[0058] "Under transcriptional control" is a term well understood in the art and indicates that transcription of a polynucleotide sequence, typically a DNA sequence, is dependent on being operably linked to elements that contribute to or facilitate the initiation of transcription. "Operatively linked" refers to a polynucleotide positioned such that it can function within a cell. In one embodiment, the present invention provides a method for the synthesis of a downstream sequence, e.g., a suicide gene, VEGF, 165A, or a nucleotide sequence that is operably linked to a downstream ... Promoters operably linked to VEGF, tet activators, and the like are provided.

[0059] The term "encoding" as applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those of skill in the art, it can be transcribed and / or translated to produce an mRNA for a polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced from the antisense strand.

[0060] As used herein, the term "isolated" refers to a molecule or biological or cellular material that is substantially free of other materials.

[0061] As used herein, the term "functional" may be used to modify any molecule, biologic, or cellular material with the intent of achieving a particular, specified effect.

[0062] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.Thus, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or polymers that contain purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases.

[0063] As used herein, the term "promoter" refers to any sequence that controls the expression of a coding sequence, such as a gene. A promoter can be, for example, constitutive, inducible, repressible, or tissue-specific. A "promoter" is a regulatory sequence that is a region of a polynucleotide sequence at which the initiation and rate of transcription are regulated. A promoter can contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. Non-limiting exemplary promoters include the Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, the U6 promoter, or the EF1 promoter. In some embodiments, the promoter is a chicken β-actin ("CBA") promoter (e.g., base pairs numbered 439-708 of SEQ ID NO:2 or base pairs numbered 644-921 of SEQ ID NO:3, or equivalents of each of these).

[0064] Further non-limiting exemplary promoters with particular target specificity are described herein below and include, but are not limited to, CMV, EF1a, SV40, PGK1 (human or mouse), P5, Ubc, human β-actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIa, Gal1, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6 and alpha-1-antitrypsin promoters. Synthetic derived promoters can be used for ubiquitous or tissue-specific expression. Additionally, viral derived promoters, some of which are described above, such as CMV, HIV, adenovirus and AAV promoters, can be useful in the methods disclosed herein. In some embodiments, the promoter is coupled to an enhancer to increase transcription efficiency. Non-limiting examples of enhancers include the RSV enhancer or the CMV enhancer (e.g., base pairs numbered 153-432 of SEQ ID NO:2).

[0065] Enhancers are regulatory elements that increase the expression of a target sequence. A "promoter / enhancer" is a polynucleotide that contains a sequence that can provide both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. Enhancers / promoters can be "endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer / promoter is one that is naturally linked to a particular gene in a genome. An "exogenous" or "heterologous" enhancer / promoter is one that has been juxtaposed to a gene using genetic engineering (i.e., molecular biology techniques) such that transcription of that gene is induced by the linked enhancer / promoter.

[0066] The terms "protein", "peptide" and "polypeptide" are used interchangeably and in their broadest sense refer to a compound of two or more subunits of amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein's or peptide's sequence. As used herein, the term "amino acid" refers to any of the natural and / or unnatural or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs and peptidomimetics.

[0067] As used herein, the term "vector" refers to a non-chromosomal nucleic acid that contains an intact replicon such that the vector can replicate when placed in a cell, for example, by the process of transformation. Vectors can be viral or non-viral. Viral vectors include retroviruses, adenoviruses, herpes viruses, baculoviruses, modified baculoviruses, papoviruses, or other modified naturally occurring viruses. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; particles containing DNA-protein complexes and DNA condensed with cationic polymers, such as heterologous polylysine, oligopeptides of defined length, and polyethyleneimine, optionally contained in liposomes; and ternary complexes containing viruses and polylysine-DNA.

[0068] "Viral vector" is defined as a recombinantly produced virus or virus particle that contains a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, AAV vectors, lentiviral vectors, adenoviral vectors, alphavirus vectors, and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827.

[0069] In another embodiment, the promoter is an inducible promoter. In certain related embodiments, the promoter is an inducible tetracycline promoter. The Tet-Off and Tet-On gene expression systems provide researchers with ready access to the controlled, high-level gene expression systems described by Gossen & Bujard (1992; Tet-Off) and Gossen et al. (1995; Tet-On). In the Tet-Off system, gene expression is turned on when tetracycline (Tc) or doxycycline (Dox; a Tc derivative) is removed from the medium. In contrast, in the Tet-On system, expression is turned on by adding Dox. Both systems allow for tight control of gene expression in response to changes in the concentration of Tc or Dox. The maximum expression levels in the Tet system are very high and compare favorably with the maximum levels obtained from strong constitutive mammalian promoters such as CMV (Yin et al., 1996). Unlike other inducible mammalian expression systems, gene regulation in the Tet system is highly specific, so that pleiotropic effects or nonspecific induction do not complicate the interpretation of results. In E. coli, the Tet repressor protein (TetR) negatively regulates the genes of the tetracycline resistance operon on the Tn10 transposon. TetR blocks transcription of these genes by binding to the tet operator sequence (tetO) in the absence of Tc. TetR and tetO provide the basis for regulation and induction for use in mammalian experimental systems. In the Tet-On system, the regulatory protein is based on the "reverse" Tet repressor (rTetR), created by four amino acid changes in TetR (Hillen & Berens, 1994; Gossen et al., 1995). The resulting protein, rtTA (reverse tTA is also called tetracycline-activating protein), is encoded by the pTet-On regulatory plasmid. This gene may be present in a separate vector from the GALT gene or may be encoded on the same gene.

[0070] In a related embodiment, the vector further comprises, consists essentially of, or consists of a nucleic acid encoding a tetracycline activating protein and a promoter regulating expression of the tetracycline activating protein.

[0071] Other inducible systems useful in the vectors, isolated cells, viral packaging systems and methods described herein include regulation by ecdysone, estrogen, progesterone, chemical inducers of dimerization and isopropyl-β-D1-thiogalactopyranoside (EPTG).

[0072] As used herein, the term "recombinant expression system" or "recombinant vector" refers to one or more genetic constructs for the expression of specific genetic material formed by recombination.

[0073] "Gene delivery vehicle" is defined as any molecule that can carry inserted polynucleotide into host cell.Examples of gene delivery vehicles are biocompatible polymers, including liposomes, micelles, natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial virus envelopes; metal particles; and bacteria or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophages, cosmids, plasmids, fungal vectors and other recombinant vehicles typically used in the art that have been described for expression in various eukaryotic and prokaryotic hosts and can be used for gene therapy, in addition to simple protein expression.

[0074] The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vehicles. As used herein, "gene delivery", "gene transfer", "transduction" and the like are terms that refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for the transfer. Such methods include various well-known techniques, such as vector-mediated gene transfer (e.g., by viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes), as well as techniques that facilitate the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery, and various other techniques used for the transfer of polynucleotides). The transferred polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the transferred polynucleotide contains an origin of replication compatible with the host cell, or is integrated into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid), or into the nuclear or mitochondrial chromosome. As known in the art and described herein, many vectors are known to be capable of mediating the transfer of genes into mammalian cells.

[0075] A "plasmid" is an extrachromosomal DNA molecule that can replicate independently of chromosomal DNA and is separate from chromosomal DNA. Plasmids are often circular and double stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms and usually provide a selective advantage under given environmental conditions. Plasmids may carry genes that confer resistance to naturally occurring antibiotics in competitive niches, or the proteins produced may act as toxins under similar circumstances.

[0076] "Plasmids" used in genetic engineering are called "plasmid vectors". Many plasmids are commercially available for such use. The gene to be replicated is inserted into a copy of the plasmid that contains a gene that makes the cell resistant to a particular antibiotic and a multiple cloning site (MCS, or polylinker), a short region that contains several commonly used restriction sites that make it easy to insert a DNA fragment into this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria that contain a plasmid with the gene of interest. Just as bacteria produce proteins that confer antibiotic resistance, they can also be induced to produce large amounts of proteins from an inserted gene.

[0077] In the embodiment where gene transfer is mediated by DNA virus vectors such as adenovirus (Ad) or adeno-associated virus (AAV), vector construct refers to the polynucleotide and transgene that comprises the viral genome or a part thereof. Adenovirus (Ad) is a relatively well-characterized homogeneous group of viruses that includes more than 50 serotypes. Adenovirus does not require integration into the host cell genome. Recombinant Ad-derived vectors have also been constructed, especially those that reduce the possibility of recombination and the generation of wild-type virus. Such vectors are commercially available from sources such as Takara Bio USA (Mountain View, CA), Vector Biolabs (Philadelphia, PA) and Creative Biogene (Shirley, NY). Wild-type AAV has high infectivity and specificity to integrate into the genome of host cells. See Wold and Toth (2013) Curr. Gene. Ther. 13(6):421-433, Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0078] Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operably linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are available from Agilent. These vectors are commercially available from sources such as Biosciences, Inc. Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the clone to remove extra potentially inappropriate alternative translation initiation codons or other sequences that may disrupt or reduce expression at either the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be inserted immediately 5' to the initiation codon to enhance expression.

[0079] Gene delivery vehicles also include DNA / liposome complexes, micelles, and targeted viral protein-DNA complexes. Liposomes that also contain targeting antibodies or fragments thereof can be used in the methods disclosed herein. In addition to the delivery of polynucleotides to cells or cell populations, the proteins described herein can be directly introduced into cells or cell populations by non-limiting techniques of protein transfection, or culture conditions that can enhance the expression and / or increase the activity of the proteins disclosed herein are other non-limiting techniques.

[0080] As used herein, the term "signal peptide" or "signal polypeptide" refers to an amino acid sequence that is usually present at the N-terminal end of newly synthesized secretory or membrane polypeptides or proteins. "Signal peptide" or "signal polypeptide" acts to guide a polypeptide to a specific cellular location, for example, across the cell membrane, into the cell membrane, or into the nucleus. In some embodiments, the signal peptide is removed after localization. Examples of signal peptides are well known in the art. Non-limiting examples are those described in U.S. Patent Nos. 8,853,381, 5,958,736, and 8,795,965.

[0081] As used herein, the term "viral capsid" or "capsid" refers to the proteinaceous shell or coat of a viral particle. The capsid functions to encapsidate, protect, transport, and release the viral genome into the host cell. Capsids are generally composed of oligomeric structural subunits of proteins ("capsid proteins"). As used herein, the term "encapsidated" means enclosed within a viral capsid.

[0082] As used herein, the term "helper" with respect to a virus or plasmid refers to a virus or plasmid that is used to provide additional components required for the replication and packaging of a viral particle or recombinant viral particle, such as the modified AAV disclosed herein. The components encoded by the helper virus may include any genes required for virion assembly, encapsidation, genome replication and / or packaging. For example, the helper virus may encode an enzyme required for the replication of the viral genome. Non-limiting examples of helper viruses and plasmids suitable for use with AAV constructs include pHELP (plasmid), adenovirus (virus) or herpesvirus (virus).

[0083] As used herein, the term "AAV" is the standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells that are provided with specific functions by a co-infecting helper virus. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol.1, pp.169-228 and Berns, 1990, Virology, pp.1743-1764, Raven Press, (New York). It is well known that various serotypes are very closely related, both structurally and functionally, even at the genetic level, so it is fully expected that the same principles described in these reviews can be applied to additional AAV serotypes that have been characterized after the publication date of these reviews. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R. Puttison, 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 possess three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis revealing extensive cross-hybridization between serotypes along the length of the genome, and the presence at the ends of similar self-annealing segments that correspond to "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0084] As used herein, "AAV vector" refers to a vector that contains one or more polynucleotides of interest (or transgenes) flanked by AAV terminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell transfected with a vector that encodes and expresses the rep and cap gene products.

[0085] "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a virus particle that is composed of at least one AAV capsid protein and a polynucleotide AAV vector enclosed in the capsid.When the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to mammalian cells), it is usually referred to as "AAV vector particle" or simply "AAV vector".Therefore, since such vector is contained within the AAV vector particle, the production of the AAV vector particle necessarily includes the production of the AAV vector.

[0086] In some embodiments, AAV is a replication-deficient parvovirus, whose single-stranded DNA genome is about 4.7 kb long, including two 145-nucleotide inverted terminal repeats (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided under GenBank Accession No. NC_002077. The complete genome of AAV-2 is provided under GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983). The complete genome of AAV-3 is provided under GenBank Accession No. NC_1829. The complete genome of AAV-4 is provided under GenBank Accession No. NC_001829. The AAV-5 genome is provided under GenBank Accession No. AF085716. The complete genome of AAV-6 is provided under GenBank Accession No. NC_00 1862. At least a portion of the AAV-7 and AAV-8 genomes are provided under GenBank accession numbers AX753246 and AX753249, respectively. The AAV-9 genome is provided in Gao et al., J. Virol., 78:6381-6388 (2004). The AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006). The AAV-11 genome is provided in Virology, 330(2):375-383 (2004). The sequence of the AAV rh.74 genome is provided in U.S. Patent No. 9,434,928, which is incorporated herein by reference. U.S. Patent No. 9,434,928 also provides the sequences of capsid proteins and self-complementary genomes. In one embodiment, the genome is a self-complementary genome. Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the AAV ITRs. Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes.Two rep promoters (p5 and pi9), coupled with alternative splicing of a single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52 and rep40) from the rep gene. The Rep proteins have multiple enzymatic properties that are ultimately involved in the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2 and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0087] AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example in gene therapy. AAV infection of cells in culture is noncytotoxic, and natural infection of humans and other animals is asymptomatic and symptomless. In addition, AAV can infect many mammalian cells and target many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and persist as transcriptionally active nuclear episomes (extrachromosomal elements) for essentially the lifetime of these cells. The AAV proviral genome is inserted as cloned DNA in a plasmid, making the construction of recombinant genomes feasible. Furthermore, because signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding the replication and structural capsid protein, rep-cap) can be replaced with foreign DNA. To generate AAV vectors, the rep and cap proteins can be provided in trans. Another important feature of AAV is that it is a highly stable and robust virus. AAV readily survives the conditions used to inactivate adenovirus (56°-65°C for several hours), making cryopreservation of AAV less important. AAV can even be lyophilized. Finally, cells infected with AAV are not resistant to superinfection.

[0088] Several studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8:659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93:14082-14087 (1996); and Xiao et al., J Virol, 70:8098-8108 (1996). See also Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). Furthermore, since muscle is highly vascularized, recombinant AAV transduction resulted in the appearance of transgene products in the systemic circulation after intramuscular injection, as described in Herzog et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94:13921-13926 (1997). Furthermore, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the cellular factors necessary for correct antibody glycosylation, folding and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics. The recombinant AAV (rAAV) genome of the present invention comprises a nucleic acid molecule (e.g., SEQ ID NO: 1) encoding GALT and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in the rAAV genome can be derived from any AAV serotype from which recombinant virus can be derived, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV PHP.B and AAV rh74. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV mutants are also contemplated, such as rAAV with capsid mutations. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0089] As used herein, the term "external" with respect to a viral capsid protein refers to a surface, domain, region or end of the capsid protein that faces outward in an assembled viral capsid. The term "internal" with respect to a viral capsid protein refers to a surface, domain, region or end (amino or carboxyl) of the capsid protein that faces inward in an assembled viral capsid. When used with respect to an assembled viral capsid, the term "internal" refers to the capsid-enclosed space inside the viral capsid and the capsid's inward-facing surface that is exposed to the enclosed space. The internal space is enclosed in the capsid by the viral capsid protein and may contain nucleic acid such as the viral genome, viral proteins, host or packaging cell proteins, and any other components or factors that are packaged or encapsidated during replication, virion assembly, encapsidation and / or packaging.

[0090] As used herein, the term "conjugated" refers to any method of attaching, binding, fusing and / or linking a viral capsid protein to a GALT protein or its equivalent.Non-limiting examples of conjugation include recombinant fusion proteins in which the GALT protein or its equivalent and the viral capsid protein are encoded by a single polynucleotide that contains genes for both the GALT protein or its equivalent and the viral capsid protein, modular intein-based assembly of GALT-intein protein and viral capsid-intein protein, post-translational modification that forms a chemical bond between the GALT protein or its equivalent and the viral capsid protein, and linking the GALT or its equivalent to the viral capsid protein via one or more linkers.In some embodiments, conjugation can be a temporary or transient association state between the viral capsid protein and its equivalent. For example, GALT or its equivalent can be transiently linked to a viral capsid protein via a polymer that is sensitive to changes in pH or ionic gradients at later stages of infection or within specific cellular microenvironments, such as an oxime bond (see, e.g., Jin et al., Biomacromolecules, 2011, 12(10), pp 3460-3468 and Yoshida et al., Expert Opin Drug Deliv. 2013 Nov;10(11):1497-1513).

[0091] As used herein, the term "label" refers to a directly or indirectly detectable compound or composition that is directly or indirectly conjugated to a composition to be detected, e.g., a protein such as a polynucleotide or an antibody, to generate a "labeled" composition. The term also includes sequences conjugated to a polynucleotide that provide a signal upon expression of the inserted sequence, such as green fluorescent protein (GFP). The label may be detectable alone (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze a chemical change in a substrate compound or composition that is detectable. The label may be suitable for small-scale detection or may be more suitable for high-throughput screening. Thus, suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or quantified. A response that is simply detected generally includes a response whose presence is merely confirmed, whereas a response that is quantified generally includes a response that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other property. In luminescent or fluorescent assays, the detectable response can be generated directly, using a luminophore or fluorophore associated with the assay component actually involved in binding, or indirectly, using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0092] Examples of luminescent labels that generate signals include, but are not limited to, bioluminescence and chemiluminescence. Detectable luminescent responses generally include the change or generation of a luminescent signal. Suitable methods and luminophores for luminescent labeling of assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0093] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, Malachite Green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).

[0094] In another embodiment, the fluorescent label is functionalized to facilitate covalent binding to cellular components present in or on the surface of cells or tissues, such as cell surface markers.Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which can be used to attach the fluorescent label to a second molecule.The choice of functional group of the fluorescent label depends on the site of attachment to either linker, drug, marker, or second labeling agent.

[0095] Fluorescent labels can be attached directly to the cellular component or compound or via a linker. Binding pairs suitable for use in indirectly linking fluorescent labels to intermediates include, but are not limited to, antigen / antibody, e.g., rhodamine / anti-rhodamine, biotin / avidin and biotin / streptavidin.

[0096] The term "solid support" refers to a non-aqueous surface such as a "culture plate", "gene chip" or "microarray". Such gene chips or microarrays can be used for diagnostic and therapeutic purposes by many techniques known to those skilled in the art. In one technique, oligonucleotides are attached and arranged on the gene chip to determine DNA sequences by hybridization approaches, such as those outlined in U.S. Pat. Nos. 6,025,136 and 6,018,041. The polynucleotides of the invention can be modified into probes, which can then be used to detect gene sequences. Such techniques are described, for example, in U.S. Pat. Nos. 5,968,740 and 5,858,659. Probes can also be attached or affixed to electrode surfaces for electrochemical detection of nucleic acid sequences, as described by Kayem et al., U.S. Pat. No. 5,952,172 and Kelley et al., (1999) Nucleic Acids Res. 27:4830-4837.

[0097] A "composition" is intended to mean a combination of an active polypeptide, polynucleotide or antibody with another compound or composition, inert (e.g., a detectable label) or active (e.g., a gene delivery vehicle).

[0098] A "pharmaceutical composition" is intended to include a combination of an active polynucleotide, polynucleotide or antibody with an inert or active carrier, such as a solid support, making the composition suitable for in vitro, in vivo or ex vivo diagnostic or therapeutic use.

[0099] As used herein, the term "pharmaceutical acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, and emulsions, such as oil / water or water / oil emulsions, as well as various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington's Pharm.Sci., 15th Ed. (Mack Publ.Co., Easton).

[0100] The "subject" of diagnosis or treatment is a cell or an animal, such as a mammal, or a human. The subject is not limited to a particular species, and includes non-human animals that are the subject of diagnosis or treatment, those that are subject to infection, or animal models, such as monkeys, rats, mice, murines such as chinchillas, canines such as dogs, lagomorphs such as rabbits, farm animals, sport animals, and pets. Human patients are also included in this term.

[0101] The term "tissue" is used herein to refer to tissue of a living or dead organism, or any tissue derived from or designed to mimic a living or dead organism. Tissues may be healthy, diseased, and / or have genetic mutations. Biological tissues may include any single tissue (e.g., a collection of cells that may be interconnected), or a group of tissues that make up an organ or part or region of an organism's body. Tissues may include homogenous cellular material, or tissues may be composite structures such as those found in regions of the body including the chest, which may include, for example, lung tissue, skeletal tissue, and / or muscle tissue. Exemplary tissues include, but are not limited to, tissues derived from the liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidney, brain, bile duct, duodenum, abdominal aorta, iliac vein, heart, and intestine (including any combination thereof).

[0102] As used herein, "treating" a disease in a subject or "treatment" of a disease in a subject refers to (1) preventing symptoms or disease from occurring in a subject who is predisposed to or has not yet shown symptoms of the disease, (2) arresting or halting the development of the disease, or (3) alleviating a disease or symptoms of the disease or causing a regression of the disease or symptoms of the disease. As understood in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results. In the present technology, beneficial or desired results can include, but are not limited to, one or more of the following: alleviation or relief of one or more symptoms, reduction in the severity of a symptom (including a disease), a stabilized (i.e., non-worsening) state of a symptom (including a disease), a delay or slowdown of a symptom (including a disease), progression, alleviation or remission, state and remission (whether partial or complete) of a symptom (including a disease), whether detectable or undetectable.

[0103] As used herein, the term "effective amount" is intended to mean an amount sufficient to achieve a desired effect. In the context of therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the symptoms in question, as well as the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to pharmaceutical compositions. In the context of gene therapy, in some embodiments, the effective amount is an amount sufficient to cause partial or complete restoration of function of a gene that is missing in a subject. In other embodiments, the effective amount of AAV viral particles is an amount sufficient to cause expression of a gene in a subject. In some embodiments, the effective amount is the amount required to increase galactose metabolism in a subject in need of increased galactose metabolism. Those skilled in the art will be able to determine the appropriate amount depending on these and other factors.

[0104] In some embodiments, the effective amount will depend on the size and nature of the application in question.The effective amount also depends on the nature and sensitivity of the intended subject and the method used.Those skilled in the art will be able to determine the effective amount based on these and other considerations.The effective amount may include one or more administrations of the composition depending on the embodiment.

[0105] As used herein, the term "administer" or "administration" is intended to mean the delivery of a substance to a subject, such as an animal or human. Administration can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods for determining the most effective means and dosage of administration are known to those skilled in the art and vary with the composition used for treatment, the purpose of the treatment, and the age, health, or sex of the subject being treated. Single or multiple administrations can be performed, with the dose level and pattern being selected by the treating physician or, in the case of pets and animals, by the treating veterinarian. Appropriate dosage formulations and methods for administering drugs are known in the art. Routes of administration can also be determined, and methods for determining the most effective route of administration are known to those skilled in the art and vary with the composition used for treatment, the purpose of the treatment, the health or disease stage of the subject being treated, and the target cell or tissue. Non-limiting examples of routes of administration include intravenous, intraarterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intraventricular, subretinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal and inhalation. MODES FOR CARRYING OUT THE DISCLOSURE AAV vectors, capsids and preparation methods

[0106] Provided herein is a recombinant polynucleotide or adeno-associated virus ("AAV") vector that comprises, consists essentially of, or consists of a polynucleotide sequence encoding galactose-1-phosphate uridyl transferase ("GALT"). In one embodiment, the polynucleotide sequence encoding GALT comprises, consists essentially of, or consists of a nucleotide sequence that is at least 85%, or 90%, or 95%, or 97%, or 99% identical to SEQ ID NO:1. In one embodiment, the sequence is at least 85%, or 90%, or 95%, or 97%, or 99% identical to SEQ ID NO:1, provided that at least one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or more, or all of the nucleotides modified from the wild-type sequence are not modified from SEQ ID NO:1. In another aspect, the polynucleotide sequence encoding GALT comprises, consists essentially of, or consists of the nucleotide sequence set forth in SEQ ID NO: 1. In another embodiment, the polynucleotide sequence encodes the amino acid sequence of SEQ ID NO: 4, or an equivalent thereof.

[0107] In one embodiment, the vector is a self-complementary vector or a single-stranded DNA (ssDNA) vector.

[0108] In one embodiment, AAV comprises, consists essentially of, or consists of AAV, such as scAAV or ssAAV, flanked by two inverted terminal repeats (ITRs).These ITRs form hairpins at the end of the sequence to act as a primer to initiate second strand synthesis before the subsequent steps of infection can begin.The synthesis of second strand is thought to be one of several obstacles to efficient infection.Additional advantages of scAAV include increased and extended transgene expression in vitro and in vivo.Thus, in one embodiment, AAV further comprises two ITRs.

[0109] Non-limiting examples of recombinant AAV backbones for making vectors include AAV vector serotypes from the group of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B or AAV rh74. In further embodiments, the vector backbone is an AAV9 serotype, rh74 serotype or a modified AAVrh74 serotype.

[0110] The polynucleotide sequence encoding GALT is optionally functionally linked to promoter, tissue-specific regulatory element or constitutive promoter.Non-limiting examples of constitutive promoter include, for example, Rous sarcoma virus (RSV) LTR promoter (optionally with RSV enhancer), cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter or EF1 promoter.In a specific embodiment, β-actin promoter is chicken β-actin ("CBA") promoter. In another embodiment, the promoter is selected from a CMV promoter, an EF1a promoter, an SV40 promoter, a PGK1 (human or mouse) promoter, a P5 promoter, a Ubc promoter, a human beta-actin promoter, a CAG promoter, a TRE promoter, a UAS promoter, an Ac5 promoter, a polyhedrin promoter, a CaMKIIa promoter, a Gal1 promoter, a TEF1, a GDS promoter, an ADH1 promoter, a CaMV35S promoter, a Ubi promoter, an H1 promoter, a U6 promoter, or an alpha-1-antitrypsin promoter.

[0111] In further embodiments, the recombinant polynucleotide or AAV further comprises a polynucleotide encoding an enhancer element. Non-limiting examples include the CMV enhancer, the WPRE, and the RSV enhancer.

[0112] In certain embodiments, the recombinant polynucleotide or AAV vector comprises, consists essentially of, or consists of a nucleotide sequence at least 85%, or 90%, or 95%, or 97%, or 99% identical to SEQ ID NO:2 or SEQ ID NO:3. In one embodiment, the sequence is at least 85%, or 90%, or 95%, or 97%, or 99% identical to SEQ ID NO:2 or 3, provided that at least one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or more, or all of the nucleotides modified from the wild-type sequence are unmodified from SEQ ID NO:2 or 3. In further embodiments, the recombinant AAV vector comprises, consists essentially of, or consists of the nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3.

[0113] The recombinant polynucleotides or AAV vectors described herein can further comprise a detectable or purification marker. The polynucleotides and / or vectors can be contained as a composition comprising the vector and a carrier, such as a preservative or a pharma- ceutically acceptable carrier.

[0114] AAV vector delivery currently relies on the use of serotype selection for tissue targeting based on the natural tropism of the virus or by direct injection into target tissue.When systemic delivery is required to achieve maximum therapeutic effect, serotype selection is the only option available for tissue targeting combined with tissue-specific promoter.Therefore, AAV vector can be packaged into capsid with tissue tropism.

[0115] In some embodiments, the vector is packaged in a viral capsid that is a wild-type or modified capsid particle. In one embodiment, the disclosure provides capsid proteins, isolated polynucleotides, methods for preparing capsid proteins, recombinant viral particles, and recombinant expression systems for generating viral particles. In one embodiment, the viral capsid protein comprises, consists essentially of, or consists of a wild-type capsid protein, or a viral capsid protein modified by an amino acid substitution or insertion of 1-7 amino acids. In some embodiments, the viral capsid protein is VP1 of AAV9, AAV PHP.B, or modified AAVrh74, as appropriate. For example, AAV PHP.B has amino acid 498 of AAV9 VP1 modified from asparagine to lysine to reduce liver tropism. In a further embodiment, the modification comprises an asparagine to isoleucine substitution or equivalent modification at amino acid position 502 of VP1 of AAVrh74. In some embodiments, the modification comprises a tryptophan to arginine substitution at amino acid 505 of VP1 of AAVrh74. In some embodiments, the modification is an insertion of peptide YIG or YIGSR at amino acid position 591 of VP1 of AAVrh74. In some embodiments, the peptide has high affinity for α7β1 integrin and / or is located in a region likely to alter normal rh74 receptor binding. Equivalents of these polypeptides and polynucleotides encoding equivalents of these peptides are also provided, where the equivalents have at least 85%, or 90%, or 95%, or 97%, or 99% sequence identity, provided that one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or all of the amino acids and polynucleotides are unaltered from the mutated sequence.

[0116] Viruses, such as AAV, can be packaged using viral packaging systems such as retrovirus, adenovirus, herpesvirus or baculovirus packaging systems.In some embodiments, packaging is achieved by using helper virus or helper plasmid and cell line.Helper virus or helper plasmid contains elements and sequences that facilitate the delivery of genetic material into cells.In another aspect, helper plasmid or the polynucleotide that comprises helper plasmid is stably integrated into the genome of packaging cell line, so that packaging cell line does not need additional transfection with helper plasmid.

[0117] The helper plasmid may, for example, contain at least one viral helper DNA sequence derived from a replication-deficient viral genome that encodes in trans all virion proteins required for packaging replication-deficient AAV, for producing virion proteins capable of packaging replication-deficient AAV at high titers without producing replication-competent AAV. The viral DNA sequence lacks the region encoding the original enhancer and / or promoter of the viral 5'LTR of the virus, lacks both the psi functional sequence and the 3'LTR involved in packaging the helper genome, but encodes an exogenous polyadenylation site, e.g., the SV40 polyadenylation site, as well as an exogenous enhancer and / or promoter directing efficient transcription in the cell type in which virus production is desired. The virus is a leukemia virus, such as Moloney murine leukemia virus (MMLV), human immunodeficiency virus (HIV) or gibbon ape leukemia virus (GALV).

[0118] The exogenous enhancer and promoter can be the human cytomegalovirus (HCMV) immediate early (IE) enhancer and promoter, the Moloney murine sarcoma virus (MMSV) enhancer and promoter (U3 region), the Rous sarcoma virus (RSV) U3 region, the spleen focus forming virus (SFFV) U3 region, or the HCMV IE enhancer linked to the native Moloney murine leukemia virus (MMLV) promoter. The helper plasmid can consist of two retroviral helper DNA sequences encoded by a plasmid-based expression vector, for example, the first helper sequence contains cDNAs encoding the gag and pol proteins of ecotropic MMLV or GALV, and the second helper sequence contains cDNAs encoding the env protein. The Env gene determining the host range may be derived from xenotropic, amphotropic, ecotropic, polytropic (mink focus forming) or 10A1 murine leukemia virus env proteins, or from genes encoding gibbon ape leukemia virus (GALV env proteins, human immunodeficiency virus env (gp160) protein, vesicular stomatitis (Stomatitus) virus (VSV) G protein, human T-cell leukemia (HTLV) type I and II env gene products, chimeric envelope genes derived from combinations of one or more of the above env genes, or chimeric envelope genes encoding the cytoplasmic and transmembrane env gene products and monoclonal antibodies directed against specific surface molecules on the desired target cells.

[0119] In the packaging process, to produce high titer recombinant retrovirus-containing supernatant, the helper plasmid and the plasmids encoding the AAV viral proteins are transiently co-transfected into a first population of mammalian cells capable of producing virus, such as human embryonic kidney cells, e.g., 293 cells (ATCC No. CRL1573, ATCC, Rockville, Md.). In another method of the invention, this first population of transiently transfected cells is then co-cultured with mammalian target cells to transduce the foreign gene into the target cells with high efficiency. Packaging

[0120] The present invention also provides a viral packaging system comprising the vector as described above, whose backbone is derived from a plasmid or virus, a packaging plasmid, and an envelope plasmid. The packaging plasmid contains nucleosides, capsid and matrix proteins. Examples of packaging plasmids are also described in patent literature, such as U.S. Pat. Nos. 7,262,049; 6,995,258; 7,252,991 and 5,710,037, which are incorporated herein by reference. The system also contains a plasmid encoding a pseudotyped envelope protein provided by the envelope plasmid. A pseudotyped viral vector consists of a vector particle that carries or contains functional parts of glycoproteins derived from other enveloped viruses. See, for example, U.S. Pat. No. 7,262,049, which is incorporated herein by reference. In a preferred embodiment, the envelope plasmid encodes an envelope protein that does not allow the viral particle to bind nonspecifically to cells or cell populations. The specificity of the virus particle is provided by the antibody binding domain inserted into the particle.Examples of suitable envelope proteins include, but are not limited to, those that contain the Staph. aureus ZZ domain.The selection of the glycoprotein to be used in the envelope is determined in part by the antibody to which the particle can be conjugated.

[0121] The present disclosure also provides suitable packaging cell line.In one embodiment, packaging cell line is HEK-293 cell line.Other suitable cell lines are known in the art and described in, for example, U.S. Patent Nos. 7,070,994; 6,995,919; 6,475,786; 6,372,502; 6,365,150 and 5,591,624, each of which is incorporated herein by reference.

[0122] The present invention further provides a method for making pseudotyped AAV particles, comprising, or consisting essentially of, or consisting of transducing a packaging cell line with a viral system as described above under suitable conditions for packaging viral vectors.Such conditions are known in the art and are briefly described herein.Pseudotyped viral particles can be isolated from cell supernatant using methods known to those skilled in the art, such as centrifugation.Such isolated particles are further provided by the present invention.

[0123] The present invention further provides an isolated polynucleotide and / or AAV viral particle produced by this method. The pseudotyped viral particle is described herein and comprises, consists essentially of, or consists of a polynucleotide encoding a GALT protein or an equivalent thereof (e.g., SEQ ID NO:4 or an equivalent of SEQ ID NO:4 as described above).

[0124] The isolated pseudotyped particles can be conjugated to one or more antibodies or antibody fragments (e.g., fragments containing at least the Fc domain) that retain the ability to bind a preselected cellular receptor.

[0125] The antibody is not species specific. In other words, the antibody may be polyclonal or monoclonal, and may be murine, ovine, human or other species. Furthermore, the antibody may be a chimeric or humanized antibody. host cell

[0126] Further provided is an isolated cell or population of cells comprising, consisting essentially of, or consisting of the isolated polynucleotides, viral particles, vectors and packaging systems described above and incorporated herein by reference. In one aspect, the isolated cell is a packaging cell line.

[0127] Also provided is an isolated cell or population of cells comprising, consisting essentially of, or consisting of a polynucleotide sequence encoding a GALT protein or equivalent as described herein and a constitutive or inducible promoter controlling expression of the nucleic acid encoding GALT. In one embodiment, the promoter is an inducible promoter as described herein. In another aspect, the promoter is a constitutive promoter as described herein. In a further aspect, the nucleic acid encoding GALT comprises, consists essentially of, or consists of SEQ ID NO:1 or its equivalent. In one aspect, the sequence is at least 85%, or 90%, or 95%, or 97%, or 99% identical to SEQ ID NO:1, provided that at least one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or more, or all of the nucleotides modified from the wild-type sequence are not modified from SEQ ID NO:1. In a further embodiment, the isolated cell further comprises, consists essentially of, or consists of a nucleic acid encoding a tetracycline activating protein and a promoter that regulates the expression of the tetracycline activating protein. In one embodiment, the promoter that regulates the expression of the tetracycline activating protein is a constitutive promoter. In a related embodiment, the promoter is a phosphoglycerate kinase promoter (PGK) or a CMV promoter.

[0128] In certain embodiments, the isolated cell comprises, or consists essentially of, or consists of a nucleic acid or biological equivalent comprising a polynucleotide of SEQ ID NO:1 encoding a GALT protein. In a related embodiment, the biological equivalent of GALT comprises a nucleic acid that hybridizes to the complement of SEQ ID NO:1 under high stringency conditions and encodes a GALT protein (e.g., SEQ ID NO:4). In another embodiment, the biological equivalent comprises a nucleic acid having at least 80% sequence identity, or at least 85% sequence identity, or at least 90% sequence identity, or at least 92% sequence identity, or at least 95% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity with SEQ ID NO:1, and in one aspect, at least one or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten or more, or all of the nucleotides modified from the wild-type sequence are not modified from SEQ ID NO:1 (see FIG. 4). In a further embodiment, the GALT protein is a wild-type human GALT protein.

[0129] The isolated cells described herein may be any of the cells of species from the following groups: mouse, rat, rabbit, monkey, cow, sheep, pig, dog, cat, farm animals, sports animals, pets, horses and primates, and may in particular be human cells.

[0130] In certain embodiments, the isolated cells described herein contain a specific level of GALT protein. The level of GALT protein can be achieved by selecting an appropriate constitutive promoter that produces a desired level of protein, or by using an inducible system that regulates the amount of protein produced. These promoters and inducible systems have been previously described. In one embodiment, the isolated cells are at least about 1×10-7 ng, about 3×10-7 ng, about 5×10-7 ng, about 7×10-7 ng, about 9×10-7 ng, about 1×10-6 ng, about 2×10-6 ng, about 3×10-6 ng, about 4×10-6 ng, about 6×10-6 ng, about 7×10-6 ng, about 8×10-6 ng, about 9×10-6 ng, about 10×10-6 ng, about 12×10-6 ng, about 14×10-6 ng, about 16×10-6 ng, about 18×10-6 ng, about 20×10-6 ng, about 25×10-6 ng, about 30×10-6 ng, about 35×10-6 ng, about 40×10-6 ng, about 45×10-6 ng, about 50×10-6 ng, about 55×10-6 ng, about 60×10-6 ng, about 70×10-6 ng, about 80×10-6 ng, about 90×10-6 ng, about 100×10-6 ng, about 120×10-6 ng, about 140×10-6 ng, about 160×10-6 ng, about 180×10-6 ng, about 200×10-6 ng, about 25×10-6 ng, about 30×10-6 ng, about 35×10-6 ng, about 40×10-6 ng, about 45×10-6 ng, about 50×10-6 ng, about 55 10-6 ng, about 50×10-6 ng, about 55×10-6 ng, about 60×10-6 ng, about 65×10-6 ng, about 70×10-6 ng, about 75×10-6 ng, about 80×10-6 ng, about 85×10-6 ng, about 90×10-6 ng, about 95×10-6 ng, about 10×10-5 ng, about 20×10-5 ng, about 30×10-5 ng, about 40×10-5 ng, about 50×10-5 ng, about 60×10-5 ng, about 70×10-5 ng, about 80×10-5 ng or about 90×10-5 ng of GALT protein, or alternatively may comprise, or consist essentially of, or consist of GALT protein. Therapeutic Compositions and Methods

[0131] The present disclosure provides therapeutic gene vector compositions and methods of treatment for treating galactosemia. Galactosemia is a rare disease caused by a recessive single gene defect in the GALT gene (galactose-1-phosphate uridyltransferase). See ghr.nlm.nih.gov / gene / GALT. Galactosemia affects approximately 1 in 45,000 newborns in the United States. Fortunately, galactosemia is detected early in national newborn screening programs. The only treatment is to remove all sources of lactose and galactose from the child's diet. Early treatment with galactose restriction is beneficial and life-saving in severe forms of the disease, but the low levels of galactose contained in other foods and from endogenously produced galactose are not sufficient to treat galactosemia. Long-term complications, such as speech and cognitive impairment and disability due to neuromuscular and ovarian toxicity, are common.

[0132] Various embodiments of vectors are described herein.In one embodiment, the vectors and methods of using the vectors provided herein comprise, consist essentially of, or consist of an effective amount of recombinant adeno-associated virus ("AAV") vectors comprising, consisting essentially of, or consisting of the polynucleotide sequence encoding galactose-1-phosphate uridyl transferase ("GALT") provided by the present disclosure.In one aspect, the polynucleotide sequence encoding GALT comprises at least 85%, or 90%, or 95%, or 97%, or 99% identical nucleotide sequence to SEQ ID NO:1. In one aspect, the sequence is at least 85%, or 90%, or 95%, or 97%, or 99% identical to SEQ ID NO:1, provided that at least one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or more, or all of the nucleotides modified from the wild type sequence are unmodified from SEQ ID NO:1 (see FIG. 4). In another aspect, the polynucleotide sequence encoding GALT comprises the nucleotide sequence shown in SEQ ID NO:1. In another embodiment, the polynucleotide sequence encodes the amino acid sequence of SEQ ID NO:4. In one aspect, the polynucleotide and / or AAV vector contains a reporter gene, either luciferase or GFP, for determining the biodistribution of the vector. In another embodiment, the polynucleotide and / or AAV vector comprises, consists essentially of, or consists of a polynucleotide sequence comprising a GALT gene sequence from human (e.g., NCBI Reference Sequence: NM_000155.3 or NM_001258332.1), mouse (e.g., NCBI Reference Sequence: NM_001302511.1 or NM_016658.3), rat (e.g., NCBI Reference Sequence: NM_001013089.2), chimpanzee (e.g., NCBI Reference Sequence: XM_003951414.4 or XM_001163419.6) or other species.In another embodiment, the polynucleotide and / or AAV vector comprises, consists essentially of, or consists of a polynucleotide sequence comprising a codon-optimized version of the human GALT gene (SEQ ID NO:1 and FIG. 4). One of the AAV vectors is identified as pscAAV-CB-hGALT (SEQ ID NO:2). In another embodiment, the AAV vector is identified as pAAV-CB-hGALT-WPREv2 (SEQ ID NO:3). The pscAAV-CB-hGALT vector comprises a constitutive CBA promoter with a small SV40 intron driving a codon-optimized version of the human GALT gene (galactose-1-phosphate uridyl transferase) (SEQ ID NO:1 and FIG. 4).

[0133] In one aspect, the AAV vector comprises a polynucleotide sequence encoding a human GALT protein (e.g., SEQ ID NO: 4), a mouse GALT protein (e.g., NCBI Reference Sequence NP_001289440.1), a rat GALT protein (e.g., NCBI Reference Sequence: NP_001013107.1), a chimpanzee GALT protein (e.g., NCBI Reference Sequence: XP_003951463.1), or a GALT protein from another species. In another embodiment, the AAV vector comprises a polynucleotide sequence encoding a human GALT protein (e.g., SEQ ID NO: 4).

[0134] In one embodiment, the AAV is the AAV9 serotype.Another serotype or modified capsid virus can be used to optimize neurotropism.Another vector includes modified AAV9 serotype vectors for higher neurotropism than standard AAV9, such as PHP.B, which uses the Cre-lox recombination system to identify vectors that target neuronal cells.Alternatively, AAV9 PHP.B has amino acid 498 of VP1 modified from asparagine to lysine to reduce liver tropism.Further variants of AAVrh74 with several amino acids mutated can be used for very broad tissue tropism, including the brain.

[0135] In one embodiment, the AAV vector is contained within a modified viral capsid protein that comprises, consists essentially of, or consists of a viral capsid protein modified by an amino acid substitution or insertion of 1-7 amino acids. Applicant has generated three mutants. One of the mutants (AAVmut4, asparagine to isoleucine at amino acid 502 of the VP1 capsid) generally increases gene delivery to all tissues tested, with up to 56-fold (3-56-fold increase depending on the tissue) higher transduction efficiency. Another mutant (AAVmut5, tryptophan to arginine at amino acid 505 of the VP1 capsid) increases gene delivery to the heart almost 50-fold over AAVrh74. The third mutant (AAVYIG591) targets a receptor found primarily on satellite cells, which are considered muscle stem cells, regardless of satellite cell tropism. In particular, based on the Applicant's knowledge of the AAV crystal structure and alpha7beta1 integrin from which AAVYIG591 is designed, it is believed to have a higher affinity for skeletal muscle and a lower affinity for the liver.

[0136] Without being bound by theory, the applicant expects that a therapeutic effect in a patient will be achieved by increasing the effective dose reaching the muscle without increasing the overall dose to the patient. By reducing the total dose required to achieve a therapeutic effect, less viral antigen will be delivered to the patient, ideally reducing the immune response to the vector and improving safety. Producing enough gene therapy medicines to conduct late-stage clinical trials is a major hurdle in further development. Reducing the dose required to achieve a therapeutic effect would result in reduced manufacturing needs, reduced manufacturing costs, faster clinical trial development, and improved ability to treat more patients.

[0137] Thus, the present disclosure relates to AAV vectors contained within modified capsid proteins, isolated polynucleotides, methods for preparing modified capsid proteins, recombinant viral particles, and recombinant expression systems for the generation of modified viral particles. One aspect of the present disclosure relates to modified viral capsid proteins comprising, consisting essentially of, or consisting of a viral capsid protein modified by amino acid substitution or insertion of 1-7 amino acids. In some embodiments, the viral capsid protein is optionally VP1 of AAV9, AAV PHP.B, or AAVrh74. In further embodiments, the modification comprises an asparagine to isoleucine substitution or equivalent modification at amino acid position 502 of VP1 of AAVrh74. In some embodiments, the modification comprises a tryptophan to arginine substation at amino acid 505 of VP1 of AAVrh74. In some embodiments, the modification targets a receptor found primarily on satellite cells, optionally muscle stem cells. In some embodiments, the modification is an insertion of the peptide YIG at amino acid position 591 of VP1 of AAVrh74. In some embodiments, this peptide is located in a region that has high affinity for α7β1 integrin and / or is likely to alter normal rh74 receptor binding.

[0138] Two exemplary vector plasmid backbones are disclosed herein. The first vector contains CBA promoter / enhancer driven expression of a reporter fusion protein composed of luciferase and enhanced yellow fluorescent protein. In one embodiment, the luciferase and enhanced yellow fluorescent protein sequences are removed. This vector can be packaged as a single-stranded virus inside a standard AAV9 serotype capsid or mutant capsid. In another embodiment, the AAV contains a self-complementary vector with a CBA promoter / enhancer driving expression of a codon-optimized human GALT gene (SEQ ID NO:1) or its equivalent. This vector can be packaged in a standard AAV9 serotype capsid. This vector can provide high levels of human GALT protein. The luc-EYFP reporter virus can be assessed for biodistribution and gene expression by in vivo luciferase staining at various time points using Xenogen IVIS (or similar) or by harvesting tissues at various time points and assaying gene expression for either EYFP or luciferase activity. Samples may be further obtained and vector genome quantification by qPCR evaluated. Administration can begin in neonatal animals (day 1 or 2 after birth) that develop disease. In animal models, mouse pups can be injected via the facial vein (see jove.com / video / 52037 / intravenous-injections-in-neonatal-mice) and vector biodistribution can be looked for by GFP expression after 4 weeks. Homozygous GALT-deficient mice born to mothers fed a normal chow diet can be a good indicator of vector tropism and biodistribution in knockout (KO) mouse models.

[0139] The present disclosure also provides compositions comprising a carrier and one or more of modified proteins, polynucleotides, vectors, plasmids, host cells or expression systems.In addition, kits are provided that include one or more of modified proteins, polynucleotides, vectors, plasmids, host cells or expression systems and instructions for use.Compositions can be formulated for specific modes of administration. Administration

[0140] Administration of the recombinant polynucleotide, vector (e.g., AAV), viral particle or composition of the present disclosure can be performed in one dose, continuously or intermittently throughout the duration of treatment. Administration can be through any suitable mode of administration, including but not limited to localized, intravenous, intraarterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intraventricular, subretinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal and inhalation. In one embodiment, the recombinant vector or composition is administered by intramuscular or intravenous injection. In another embodiment, the recombinant AAV vector or composition is administered systemically. In another embodiment, the recombinant AAV vector or composition is administered parentally by injection, infusion or implantation. In one aspect, the AAV vector or GALT polynucleotide is delivered locally to the liver.

[0141] The method of determining the most effective means and dosage of administration is known to those skilled in the art and varies according to the composition used for treatment, the purpose of treatment and the subject being treated. Single or multiple administrations can be carried out with the dosage level and pattern selected by the treating physician. It should be noted that the dosage can be influenced by the route of administration. Appropriate dosage formulations and methods of administering drugs are known in the art. Non-limiting examples of such suitable dosages can be a minimum of 1E+9 vector genomes to a maximum of 1E+17 vector genomes per administration.

[0142] In some embodiments of the methods described herein, the number of viral particles (e.g., AAV) administered to a subject is about 10 9 ~about 10 17 In certain embodiments, the range is about 10 10 ~about 10 12 , about 10 11 ~about 10 13 , about 10 11 ~ approx. 1012, approx. 10 11 ~about 10 14 , about 5×10 11 ~Approx. 5×10 12 or about 10 12 ~about 10 13 of viral particles are administered to the subject.

[0143] In further embodiments, the polynucleotides, viral particles and compositions of the present disclosure can be administered in combination with other treatments, such as approved treatments appropriate for galactosemia and its associated disorders or symptoms. Non-limiting examples include treatment of galactosemia with a viral vector or composition of the present disclosure while reducing or eliminating lactose and / or galactose from the subject's diet.

[0144] Successful treatment and / or repair is determined when one or more of the following is detected: alleviation or improvement of one or more symptoms of the disease, disorder or condition of the treated subject, reduction in the severity of the disease, disorder or condition of the subject, stable (i.e., not worsening) state of the disease, disorder or condition, delay or slowing of the progression of the disease, disorder or condition, and improvement or alleviation of the disease, disorder or condition. In some embodiments, successful treatment is determined by detecting the presence of the repaired target polynucleotide in one or more cells, tissues or organs isolated from the subject. In some embodiments, successful treatment is determined by detecting the presence of the polypeptide encoded by the repaired target polynucleotide in one or more cells, tissues or organs isolated from the subject.

[0145] In one embodiment, recombinant polynucleotide and / or viral vector can repair GALT gene in subject.In some embodiments, the ratio of repaired target polynucleotide or polypeptide to unrepaired target polynucleotide or polypeptide in successfully treated cell, tissue, organ or subject is about 1.5:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 20:1, about 50:1, about 100:1, about 1000:1, about 10,000:1, about 100,000:1 or about 1,000,000:1. The amount or ratio of repaired target polynucleotides or polypeptides can be determined by any method known in the art, including, but not limited to, Western blot, Northern blot, Southern blot, PCR, sequencing, mass spectrometry, flow cytometry, immunohistochemistry, immunofluorescence, fluorescent in situ hybridization, next generation sequencing, immunoblot, and ELISA. kit

[0146] In some embodiments, the polynucleotides, agents, vectors or compositions described herein can be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic or research applications. In some embodiments, the kits of the present disclosure include one or more of the modified viral capsid proteins, isolated polynucleotides, vectors, host cells, recombinant viral particles, recombinant expression systems, modified AAVs, modified cells, isolated tissues, compositions or pharmaceutical compositions described herein.

[0147] In some embodiments, the kit further includes instructions for use. Specifically, such kits may include one or more agents described herein, together with instructions that describe the intended use and proper use of these agents. As an example, in one embodiment, the kit may include instructions for mixing one or more components of the kit, and / or for isolating and mixing samples and applying to a subject. In certain embodiments, the agents in the kit are pharmaceutical formulations and dosages that are suitable for a particular use and method of administering the agents. Kits for research purposes may contain components in appropriate concentrations or amounts to carry out various experiments.

[0148] The kits can be designed to facilitate the use of the methods described herein and can take many forms. Each composition of the kit can be provided in liquid form (e.g., in solution) or solid form (e.g., dry powder), as applicable. In certain cases, some of the compositions can be configurable or otherwise processable (e.g., into an active form), for example, by the addition of a suitable solvent or other species (e.g., water or cell culture medium), which may or may not be provided with the kit. In some embodiments, the compositions can be provided in a preservation solution (e.g., a cryopreservation solution). Non-limiting examples of preservation solutions include DMSO, paraformaldehyde, and CryoStor® (Stem Cell Technologies, Vancouver, Canada). In some embodiments, the preservation solution contains an amount of a metalloprotease inhibitor.

[0149] As used herein, "instructions" can define instruction and / or promotional elements, and typically include written instructions on or accompanying packaging of the claimed methods, recombinant vectors, or compositions. Instructions can also include any oral or electronic instructions provided in any format that clearly identifies to a user that the instructions are to be associated with the kit, such as audiovisual (e.g., videotape, DVD, etc.), internet and / or web-based communication means, etc. In some embodiments, the written instructions are in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, and the instructions can also reflect approval by the agency of manufacture, use, or sale for animal administration.

[0150] In some embodiments, the kit contains any one or more of the components described herein in one or more containers. Thus, in some embodiments, the kit may include a container housing the agent described herein. The agent may be in liquid, gel, or solid (powder) form. The agent may be aseptically prepared, packaged in a syringe, and shipped refrigerated. Alternatively, it may be contained in a vial or other container for storage. A second container may have another agent prepared aseptically. Alternatively, the kit may contain an active agent premixed and shipped in a syringe, vial, tube, or other container. The kit may have one or more or all of the components required to administer the agent to a subject, such as a syringe, topical application device, or IV needle tube and bag.

[0151] The therapeutic method described herein can be combined with suitable diagnostic technology to identify and select patients for the therapeutic method.For example, genetic testing can be provided to identify mutations in GALT gene.Therefore, patients with mutations can be identified as suitable for the treatment. EXAMPLES

[0152] In Vitro Administration Viral particles are produced by standard triple transfection methods using a plasmid for the vector genome, plasmids for the AAV rep and cap genes, and an Ad helper plasmid that provides the adenoviral helper functions necessary to generate AAV. These three plasmids are transfected into HEK293 cells grown in serum-free suspension using PEI. Four days after transfection, viruses are harvested and purified by standard methods. Effective doses range from 1012 to 1014 Vg / kg when administered intravenously based on similar disease models and serotypes. AAVmut4 contains modifications to the region responsible for liver-specific binding, conferring broader tropism to the virus for many other target tissues, including the central nervous system and muscle.

[0153] The plasmid pAAV-CB-GALT-WPRE-Kan (Figure 1) was transfected into HEK293 cells by transient transfection, and two days later the cells were lysed and the harvested proteins were run on an acrylamide gel, transferred to a nylon membrane, and probed with an anti-human GALT antibody followed by an IR dye-labeled secondary antibody for detection. As shown in Figure 3, lane 1 is a molecular weight marker with sizes indicated on the Y axis. Lane 2 is 1 μl of GALT transfected cell lysate, lane 3 is 0.1 μl of GALT transfected cell lysate, lane 4 is 5 μl of lysate of HEK293 cells transfected with lucEYFP reporter plasmid used as transfection and cell control, also showing small amounts of endogenously expressed native human GALT protein, lane 5 is empty, lane 6 is HepG2 cell lysate showing native human GALT protein, lane 7 is empty, lanes 8-10 show increasing concentrations of bacterially expressed GALT protein standard with slightly higher mobility due to the tag used for purification. In vivo administration

[0154] First, the GALT gene therapy approach disclosed herein is highly innovative, since there is no AAV-mediated or any gene-based therapy approved to treat the long-term complications associated with classical galactosemia. Second, in addition to this technology, the disclosed therapy can use local gene delivery to the liver to normalize abnormal galactose metabolism in distant organs.

[0155] A novel GALT gene-trapped (GalT-deficient) mouse model was used as described in Tang, M. et al., Eur. J. Hum. Genet, (2014):1172-1179. Complete absence of GALT activity was confirmed in homozygous GalT gene-trapped mice using an LC-MS / MS procedure (Li, Y. et al., Mol. Genet. Metab. (2011) 102(1):33-40. Further characterization of homozygous GalT gene-trapped mice revealed galactose sensitivity in newborn GalT-deficient pups, reduced fertility in adult females, impaired motor function and growth retardation in GalT-deficient mice of both sexes (Tang et al., (2014), supra; Balakrishnan, B. et al., (2016) 470(1):205-212; Chen et al., (2017) J. Inherit. Metab. Dis. 40(1):131-137).

[0156] As shown in Figure 6, transduction of the AAV9-GALT vector (see Figures 1 and 2) into the HEK293 cell line resulted in abundant production of GALT protein. Expression of GALT protein can be altered as needed by replacing the strong CBA promoter / enhancer with a promoter that produces near physiological levels of GALT protein.

[0157] Biodistribution: It has been established that AAV9 vectors have a broad biodistribution, including the central nervous system (CNS), whereas AAV8 primarily targets the liver in mice. Thus, administration of GALT and codon-optimized GALT can be performed using AAV8 vectors. Delivery of vectors via the vasculature of 4-week-old wild-type and GalT-deficient mice allows for the assessment of differences in biodistribution between the two groups. First, 1E+12Vg of each AAV vector expressing a lucEYFP reporter construct (N=5 per sex per group, N based on FDA guidelines for toxicity / biodistribution studies as described in the document entitled "Guidance for Industry: Gene Therapy Clinical Trials-Observing Subjects for Delayed Adverse Events") is performed via the tail vein into male and female mice. Mice are imaged weekly for 4 weeks before sacrifice. Controls are age- and sex-matched animals injected with vectors without the GALT gene insert. After 4 weeks, animals are sacrificed, perfused with fixative, and the brain, heart, kidneys, liver, eyes, and gonads are isolated. Tissues are sectioned for microscopic staining for EYFP, and a portion of each tissue is used for qPCR measurement of vector genomes per µg of genomic DNA. In another controlled administration, mice are challenged with 10% and 20% galactose in the diet.

[0158] Dosage Study: Separately, with 12 mice per group (N=6 per group per sex) plus a control group injected with empty vector of the selected serotype, male and female 4-week-old GalT-deficient mice are intravenously injected with one of three different doses of AAV9-GALT (or AAV8-GALT) vector at 1E+10, 1E+11 or 1E+12 vg per mouse, respectively. Animals are monitored for up to six months and examined for potential improvements in motor coordination and reproductive fitness. Animals are also monitored and assayed for overt adverse physiological effects of the treatment, such as weight loss or inactivity.

[0159] Behavioral analysis: Mice (n=12 per group) are tested for behavioral capabilities at 6 months of age to quantify the functional impact of treatment on neurological disorders. Behavioral tests include cognitive and swimming abilities in the Morris water maze, and motor function on the rotarod. To determine motor deficits associated with ataxia in mice, a modified rotarod is used that forces animals to walk along the rod with alternating limbs, instead of passively using their body for support. For training, the speed is gradually increased to 6 RPM in 2 min. After a 2 min rest period, mice are tested at 6 RPM for up to 2 min (or less if the animal fails the test). Testing can be repeated for each mouse at 12 RPM to increase the load compared to wild-type (WT) normal mice. Each mouse is tested three times at each speed after a 5 min rest period. At the end of 6 months, all mice will be euthanized and assessed for GALT gene expression by Western and vector genome content by qPCR in various regions of the brain and other tissues along with H&E histology for pathology by a certified pathologist.

[0160] Fertility assessment of female mice: The reproductive fitness of female mice is assessed by regularly monitoring the estrous cycle and counting the number of follicles at the end of 6 months. Signs of normalization of the estrous cycle and follicle count alone are good enough indicators of improved fertility in these animals. Equivalent

[0161] While the invention has been described in conjunction with the above embodiments, it should be understood that the foregoing description and examples are intended to illustrate, but not to limit, the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art.

[0162] Furthermore, where features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also hereby described in terms of any individual member or subgroup of members of the Markush group.

[0163] All technical and patent publications referenced herein are incorporated by reference. [ka] [ka] SEQ ID NO:1 Codon-optimized GALT sequence [ka] SEQ ID NO:2 - pAAV-CB-hGALT Mutated ITR: Nucleotides (NT): 1-106 CMV enhancer: NT:153-432 Chicken β-actin promoter: NT 439-708 Modified SV40 intron: 774-833 Human GALT: 1004-2143 Bovine growth hormone polyadenylation signal: NT 2186-2332 ITR:NT:2412-2552 Ampicillin resistance gene: NT 3455-4315 Plasmid origin of replication (ori): NT:4470-5089 [ka] [ka] [ka] SEQ ID NO:3: pAAV-CB-hGALT-WPREv2-KAN Mutated ITR: nucleotides (NT): 1-145 CMV enhancer: NT:190-551 Chicken β-actin promoter: NT 552-834 Chimera Intron: 929-1103 Human GALT: 1160-2299 Woodchuck hepatitis virus position response element (WPRE): NT 2334-2927 PolyA signal (BGHpA): NT 2942-3176 ITR:NT:3449-3592 KAN gene: NT 5145-5960 [ka] [ka] [ka] [ka] SEQ ID NO:4: [ka] SEQ ID NO:5: Rh74 VP1 amino acid sequence [ka] SEQ ID NO:6 Rh74 VP1 DNA [ka] [ka] SEQ ID NO:7-KAN gene translation product [ka] AAV rh74 consensus sequence alignment AAVrh74 AAVrh74-N502I-capsid Rh74 YIG591 cap protein [ka] [ka]

Claims

[Claim 1] The invention described in the specification.

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