Adeno-associated vectors and virions for the treatment of galactosemia and methods of use and manufacture - Patents.com

Abstract

Provided are recombinant adeno-associated virus (rAAV) vectors containing a transgene that expresses galactose-1-phosphate uridylyltransferase (GALT); virions containing the vectors (rAAV virions); methods for their production, methods of their use, including methods for treating galactosemia, GALT deficiency, and symptoms resulting therefrom; and kits.

Description

[Technical field]

[0001] Viral constructs, particles, and compositions for use in treating galactose-1-phosphate uridylyltransferase (GALT) deficiency are provided. [Background technology]

[0002] Galactosemia is a type of disease caused by an inability to metabolize galactose; specifically, type 1 galactosemia is a disease caused by pathogenic variants in the GALT gene, which encodes the enzyme galactose-1-phosphate uridylyltransferase (GALT). Thus, type 1 galactosemia may also be known as galactose-1-phosphate uridylyltransferase deficiency. GALT performs the second step of the Leloir pathway of galactose metabolism. In that step, GALT reversibly interconverts uridine diphosphate glucose and galactose-1-phosphate to glucose-1-phosphate and uridine diphosphate galactose.

[0003] Type 1 galactosemia can be classified into three disorders. The first, classic galactosemia, occurs when an individual has pathogenic variants in both GALT alleles resulting in loss of expression of GALT or complete or near complete loss of GALT catalytic activity (approximately 1% residual activity). Classic galactosemia is an autosomal recessive disorder. The second category of type 1 galactosemia is described as "clinical variant" galactosemia, in which patients maintain 1%-10% of normal enzyme activity levels in red blood cells or other tissues. The third type, biochemical variant galactosemia, called Duarte galactosemia, occurs when an individual is compound heterozygous for the GALT alleles, a combination that results in the expression of approximately 25% of the GALT activity levels seen in individuals with two normal GALT alleles. An example of a mutation causing classic galactosemia is an A to G nucleotide substitution in exon 6 of the GALT gene, resulting in the substitution of arginine for glutamine at position 188 of the protein.

[0004] The type 1 galactosemia disease process begins in utero (Holton 1995). Patients with type 1 galactosemia, who have GALT enzyme activity <10% of normal levels, are at risk for neonatal death if galactose (mainly from lactose) is not removed from the diet. Signs of type 1 galactosemia in newborns include feeding difficulties, failure to thrive, jaundice, and liver damage, which can lead to liver failure in some cases (Berry 2021; Rubio-Gozalbo et al. 2019). The GalNet registry collected data from 509 patients in 15 countries between December 2014 and July 2018, and showed that 26% of patients experienced cataracts during the neonatal period (Rubio-Gozalbo et al. 2019). The timing, amount, and duration of exposure to galactose influence the extent of damage in galactosemic infants, with early exposure to large amounts of galactose for a long period causing the greatest damage. Morbidity during the first 10 days of life has been reported to reach up to 75% in neonates with classical galactosemia, with most deaths due to sepsis caused by Escherichia coli, before the initiation of newborn screening practices and dietary restrictions. Damage from the galactosemia-sepsis complex extends beyond hepatocyte necrosis, hemosiderosis, fatty regeneration, and hepatocyte acinar formation to pancreatic islet hyperplasia, renal cortical necrosis, periventricular leukomalacia, and meningitis (Kotb et al. 2019). Infants who are rapidly switched from breast milk or standard formula to soy-based or other low-galactose formulas generally recover quickly, without the most severe acute complications, such as sepsis, liver failure, and neonatal death (Berry 2021, Welling et al. 2017).

[0005] Evidence indicates that long-term complications in patients with classical galactosemia begin during pregnancy and are present in very early childhood. Despite dietary interventions, patients still experience significant long-term complications of the disease resulting from endogenous galactose production of 1-2 grams per day (Bosch 2006 and Berry 2021). Clinical evaluation of patients with type 1 galactosemia shows early onset of symptoms. These patients are more likely to have neurological deficits, motor, cognitive, and speech and language pathology compared to age-matched controls, evident in children as young as 48 months of age (Ozgun et al. 2019). Data from the GalNet registry also demonstrate that motor symptoms (tremor) are present in 23.8% of patients from one year of age to preschool age. These data support the need for early intervention strategies to mitigate or significantly reduce long-term disease outcomes (Rubio-Gozalbo et al. 2019), made feasible by newborn screening for classical galactosemia present in many developed countries.

[0006] The GalNet registry also provides information on the overall impact of these long-term complications based on 509 patients evaluated (Rubio-Gozalbo et al. 2019 and Berry 2021). Neurological, cognitive, and behavioral complications were common in patients enrolled in the GalNet registry. Among the 52.2% of patients reported to have a global developmental delay, 85.0% of patients reported brain disorders, including multiple cognitive and psychiatric complications, and in the case of language and speech disorders, 66.4% of patients reported them. Language and speech disorders were more commonly reported in younger male patients (p=0.034) (Rubio-Gozalbo et al. 2019). In total, 52.0% of patients had neurological complications, such as tremor (31.0%), general dyskinesia (27.0%), ataxia (12.2%), seizures (8.1%), and dystonia (7.5%). General dyskinesis was most frequently reported in the preschool age, but ataxia, seizures, and dystonia were reported at any age. According to the GalNet registry, 44.4% of patients exhibited mental (psychiatric) and behavioral problems, including anxiety disorders (22.3%), depression (12.5%), attention-deficit / hyperactivity disorder (7.3%), and autism spectrum disorder (6.0%) (Rubio-Gozalbo et al., 2019). Growth in childhood and early adolescence is also significantly delayed, with a significant number of patients having height below the 10th percentile and weight below the 50th percentile (Waggoner et al., 1990).

[0007] One of the most common and severe complications in patients with classical galactosemia is speech and voice disorders (Hughes et al., 2009; Rubio- Gozalbo et al., 2019; and Waggoner et al., 1990), with more severe childhood apraxia of speech (CAS) reported in 24–63% of children with type 1 galactosemia (Shriberg et al., 2011a; Waggoner et al., 1990; Waisbren et al., 2012; and Webb et al., 2003). Children with speech disorders secondary to galactosemia are conservatively estimated to be at 6–8 times higher risk and at 180 times higher risk of CAS compared with children with speech disorders of unknown etiology (Potter et al., 2008; Shriberg et al., 2011b). CAS is a motor speech disorder caused by disruptions in higher-level motor commands, neuromuscular disorders, or both, and is characterized by inconsistent consonant and vowel errors, difficulty transitioning between articulatory actions, and inappropriate prosody during speech (Duffy 2005 and American Speech-Language-Hearing Association 2007). CAS is caused by disruptions in higher-level motor commands, primarily in the left hemisphere's Broca's area, supplementary motor area, or insula, and can involve the cerebellum and basal ganglia, leading to difficulties planning and regulating sequences of speech actions (Potter 2011). CAS is not a disease that will be cured as the child grows. Rather, children with CAS will not improve without treatment. Approximately 50-78% of children with classic galactosemia have been reported to have developmental language disorder (DLD) (Potter et al. 2008, Rubio- Gozalbo et al. 2019, and Waggoner et al. 1990). Children with DLD have difficulty using and / or understanding language and have poorer language skills than their peers, even within a similar IQ range. Children with DLD may have difficulty interacting with their peers, talking about their feelings, and learning in school. DLD is usually first identified and treated in childhood, but it does not usually go away as a child grows older (Norbury et al. 2016).Many of the neurological complications of classic galactosemia are multifactorial and patients often present with multiple comorbidities simultaneously (i.e., cognitive impairment and language delay). Improved treatments are needed for these patients. Summary of the Invention

[0008] Provided is a nucleic acid vector construct for the production of recombinant adeno-associated virus (AAV) virions encoding GALT, which is used to generate recombinant AAV virions or particles that are administered to a subject suffering from galactosemia for rAAV gene therapy to deliver nucleic acid encoding GALT to cells of the subject, thereby increasing GALT activity in the subject and treating and ameliorating the disease.

[0009] The present invention provides an AAV vector comprising an expression cassette having a nucleotide sequence encoding human galactose-1-phosphate uridylyltransferase (hGALT) operably linked to one or more regulatory elements that facilitate expression of the hGALT coding sequence and a polyadenylation (poly(A)) tail signal, wherein the promoter comprises a cytomegalovirus (CMV) early enhancer / chicken β-actin / rabbit β-globin splice acceptor (CAG) promoter or an elongation factor-1 (EF-1) promoter, and the poly(A) tail signal comprises a bovine growth hormone (bGH) poly(A) tail signal or a simian virus 40 (SV40) poly(A) tail signal flanked by inverted terminal repeat (ITR) nucleotide sequences. The hGALT may have the amino acid sequence of SEQ ID NO:1, may be encoded by a nucleic acid having a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO:2 encoding hGALT or its reverse complement, and may have the nucleotide sequence of SEQ ID NO:2. The vector may also contain a WPRE element located between the hGALT coding sequence and the polyA signal sequence. The ITR sequences may be wild-type sequences, such as the AAV2 ITRs, or may have modified ITR sequences to generate a double-stranded "self-complementary" AAV vector.

[0010] Provided is a particular expression cassette (i.e., a recombinant AAV genome having a transgene, regulatory sequences, and ITR sequences) that has SEQ ID NO:3 (pscAAV-CAG-hGALT), SEQ ID NO:4 (pAAV2 ITR-CAG-hGALT), or SEQ ID NO:5 (pAAV2 ITR-EF1a-hGALT) (or a reverse complement thereof), or may have at least 85% identity to the nucleotide sequence of SEQ ID NO:3 (pscAAV-CAG-hGALT), SEQ ID NO:4 (pAAV2 ITR-CAG-hGALT), or SEQ ID NO:5 (pAAV2 ITR-EF1a-hGALT), which encodes and expresses human GALT.

[0011] Particular expression cassettes (without flanking ITR sequences) are provided that have SEQ ID NO: 19 (pscAAV CAG-hGALT), SEQ ID NO: 20 (pAAV2 ITR-CAG-hGALT), or SEQ ID NO: 21 (pAAV2 ITR EF1a-hGALT) (or a reverse complement thereof), or may have at least 85% identity to the nucleotide sequence of SEQ ID NO: 19 (pscAAV-CAG-hGALT), SEQ ID NO: 20 (pAAV2 ITR-CAG-hGALT), or SEQ ID NO: 21 (pAAV2 ITR-EF1a-hGALT), which encode and express human GALT.

[0012] Also provided are plasmids containing these expression cassettes, including plasmid pAAV2 ITR-CAG-hGALT-KanR shown in Figure 1, the nucleotide sequence of which (also the nucleotide sequence of SEQ ID NO: 7 and its reverse complement) is shown in Figure 2; plasmid pAAV2 ITR-EF1a-hGALT-KanR shown in Figure 3, the nucleotide sequence of which (also the nucleotide sequence of SEQ ID NO: 8) is shown in Figure 4; and plasmid pscAAV-CAG-hGALT-KanR shown in Figure 5, the nucleotide sequence of which (also the nucleotide sequence of SEQ ID NO: 6) is shown in Figure 6.

[0013] Also provided are recombinant AAV virions and AAV capsids comprising the recombinant AAV genome (expression cassette) described herein that encodes hGALT (e.g., an expression cassette of SEQ ID NO: 3, 4, or 5). The AAV capsid may be an AAV9 capsid (amino acid sequence of SEQ ID NO: 18).

[0014] Also provided are methods of treating galactosemia or increasing galactose metabolism in a subject, particularly a human subject, in need thereof. Also included are methods of alleviating disease states in a subject suffering from galactosemia by administering the rAAV virions described herein, including jaundice, hepatosplenomegaly, hepatocellular failure, hypoglycemia, renal tubular dysfunction, hypotonia, sepsis, cataracts, ataxia, tremor, decreased bone density, or primary ovarian failure. Provided are pharmaceutical compositions and methods of administration, including but not limited to intravenous or intrathecal administration, that are used to treat galactosemia or increase galactose metabolism in a subject in need thereof.

[0015] Host cells and methods for producing the recombinant AAV virions described herein are also provided.

[0016] Also provided are methods for reducing galactose levels in a subject in need of reduced galactose levels, methods for reducing galactitol levels in a subject in need of reduced galactitol levels, methods for reducing galactose-1-phosphate (Gal-1P) levels in a subject in need of reduced galactitol levels, methods for inhibiting cataract formation or promoting cataract resorption in a subject in need of inhibiting cataract formation or promoting cataract resorption, methods for limiting the severity of cataract formation in a subject in need of limiting the severity of cataract formation, and methods for preventing growth inhibition in a subject in need of preventing growth inhibition.

[0017] Also provided are methods for increasing GALT enzyme activity in the liver, brain, and skeletal muscle in a subject in need thereof.

[0018] Embodiment 1. A recombinant adeno-associated virus (AAV) vector comprising an expression cassette, the expression cassette having a nucleotide sequence encoding human galactose-1-phosphate uridylyltransferase (hGALT) operably linked to one or more regulatory elements, the regulatory elements driving expression of the hGALT coding sequence and a polyadenylation (poly(A)) tail signal, the promoter comprising a cytomegalovirus (CMV) early enhancer / chicken β-actin / rabbit β-globin splice acceptor (CAG) promoter or an elongation factor-1 (EF-1) promoter, the poly(A) tail signal comprising the bovine growth hormone (bGH) poly(A) tail signal flanked by inverted terminal repeat (ITR) nucleotide sequences or the simian virus 40 (SV40) poly(A) tail signal.

[0019] 2. The recombinant AAV vector of embodiment 1, wherein the hGALT has the amino acid sequence of SEQ ID NO:1.

[0020] 3. The recombinant AAV vector of embodiment 1 or 2, wherein the nucleic acid encoding GALT comprises a nucleic acid having at least 85% identity to the nucleotide sequence of SEQ ID NO: 2, or its reverse complement.

[0021] 4. The recombinant AAV vector of any one of embodiments 1 to 3, wherein said nucleic acid encoding GALT has or consists of the nucleotide sequence of SEQ ID NO: 2 or its reverse complement.

[0022] 5. The recombinant AAV vector of any one of embodiments 1-4, wherein the promoter comprises the nucleotide sequence of SEQ ID NO:9 or SEQ ID NO:10, or a reverse complement thereof, and the poly A signal sequence comprises the nucleotide sequence of SEQ ID NO:15 or SEQ ID NO:16, or a reverse complement thereof.

[0023] 6. The recombinant AAV vector of any one of embodiments 1 to 5, comprising a WPRE element.

[0024] 7. The recombinant AAV vector of any one of embodiments 1-6, wherein the ITRs comprise a 5' AAV2 ITR having the nucleotide sequence of SEQ ID NO:11 and a 3' AAV2 ITR having the nucleotide sequence of SEQ ID NO:12, or a reverse complement thereof.

[0025] 8. The recombinant AAV vector of any one of embodiments 1 to 6, wherein the ITRs comprise a 5' ITR having the nucleotide sequence of SEQ ID NO: 11 and a modified self-complementary 3' ITR having the nucleotide sequence of SEQ ID NO: 13, or a reverse complement thereof.

[0026] 9. The recombinant AAV vector of any one of embodiments 1-8, comprising an expression cassette comprising a nucleic acid having at least 85% identity to the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or the reverse complement thereof, encoding human GALT.

[0027] 10. The recombinant AAV vector of any one of embodiments 1 to 8, comprising an expression cassette comprising a nucleic acid encoding human GALT having at least 85% identity to the nucleotide sequence of SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or its reverse complement.

[0028] 11. The recombinant AAV vector of any one of embodiments 1-9, comprising an expression cassette comprising a nucleic acid having the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or a reverse complement thereof.

[0029] 12. The recombinant AAV vector of any one of embodiments 1-8 or embodiment 10, comprising an expression cassette comprising a nucleic acid having the nucleotide sequence of SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or a reverse complement thereof.

[0030] 13. A recombinant self-complementary AAV (scAAV) vector comprising the recombinant AAV vector of any one of embodiments 1-12 comprising scAAV ITRs.

[0031] 14. A recombinant AAV virion comprising: 1) an AAV capsid; and 2) a recombinant AAV vector of any one of embodiments 1-13, wherein the AAV capsid protein encapsulates the recombinant AAV vector.

[0032] 15. The AAV virion of embodiment 14, wherein the AAV capsid has an amino acid sequence having at least 85% identity to SEQ ID NO: 18 (AAV9).

[0033] 16. The AAV virion of embodiment 15, wherein the AAV capsid has the amino acid sequence of SEQ ID NO:18.

[0034] 17. A method for treating galactosemia in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an AAV virion of any one of embodiments 14-16.

[0035] 18. A method for increasing galactose metabolism in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an AAV virion of any one of embodiments 14 to 16.

[0036] 19. A method for alleviating a disease state in a subject suffering from galactosemia, comprising administering to the subject a therapeutically effective amount of an AAV virion of any one of embodiments 14-16, wherein the disease state comprises jaundice, hepatosplenomegaly, hepatocellular failure, hypoglycemia, renal tubular dysfunction, hypotonia, sepsis, cataracts, ataxia, tremor, decreased bone density, or primary ovarian failure.

[0037] 20. The method of any one of embodiments 17-19, wherein said administering comprises intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intraventricular, subretinal, intravitreal, intra-articular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, or by inhalation.

[0038] 21. The method of any one of embodiments 17-19, wherein said administering comprises intravenous or intrathecal administration.

[0039] 22. An AAV vector plasmid comprising: 1) an origin of replication; and 2) a recombinant AAV vector of any one of embodiments 1-13.

[0040] 23. The AAV vector plasmid of embodiment 22, comprising a nucleic acid having at least 85% identity to the nucleotide sequence of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, and encoding human GALT.

[0041] 24. The AAV vector plasmid of embodiment 22, comprising a nucleic acid having at least 85% identity to the nucleotide sequence of SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, and encoding human GALT.

[0042] 25. The AAV vector plasmid of embodiment 22 or 23, having the nucleotide sequence of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0043] 26. The AAV vector plasmid of embodiment 22 or 24, having the nucleotide sequence of SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

[0044] 27. A cell comprising the AAV vector plasmid of any one of embodiments 22 to 26 and a second plasmid having a nucleotide sequence encoding rep and cap, wherein the cap encodes VP1, VP2, and VP3, and the rep encodes rep78, rep68, rep52, and rep40.

[0045] 28. The cell of embodiment 27, wherein the cap is an AAV9 cap.

[0046] 29. A method for producing AAV virions, comprising culturing a host cell comprising the AAV vector plasmid of any one of embodiments 22-26, a second plasmid encoding cap encoding VP1, VP2, and VP3, and rep encoding rep78, rep68, rep52, and rep40, and additional adenovirus helper functions under conditions sufficient to produce AAV virions, and isolating the AAV virions produced by the host cell.

[0047] 30. A method for reducing galactose levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of AAV virions of any one of embodiments 14 to 16.

[0048] 31. A method for reducing galactitol levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of AAV virions of any one of embodiments 14 to 16.

[0049] 32. A method for reducing galactose-1-phosphate (Gal-1P) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of AAV virions of any one of embodiments 14 to 16.

[0050] 33. The method of any one of embodiments 30-32, wherein galactose levels, galactitol levels, and / or Gal-1P levels are reduced in the liver, muscle, brain, eye, ovary, red blood cells, and / or plasma.

[0051] 34. The method of embodiment 33, wherein the levels of galactose and galactitol in plasma are reduced.

[0052] 35. The method of embodiment 33, wherein the levels of galactose, galactitol, and Gal-1P in red blood cells are reduced.

[0053] 36. A method for increasing GALT protein expression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of AAV virions of any one of embodiments 14 to 16.

[0054] 37. A method for increasing GALT enzyme activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of AAV virions of any one of embodiments 14 to 16.

[0055] 38. The method of any one of embodiments 36-37, wherein GALT protein expression and / or GALT enzyme activity is increased in the liver, muscle, brain, eye, red blood cells, and / or plasma.

[0056] 39. The method of any one of embodiments 36-37, wherein GALT protein expression and / or GALT enzyme activity in the brain is increased.

[0057] 40. The method of embodiment 39, wherein GALT protein expression and / or GALT enzyme activity is increased in cortical and / or cerebral tissue.

[0058] 41. The method of embodiment 40, wherein GALT protein expression and / or GALT enzyme activity in neuronal and / or glial cells is increased.

[0059] 42. The method of embodiment 41, wherein GALT protein expression and / or GALT enzyme activity in Purkinje neurons is increased.

[0060] 43. A method for improving motor coordination and / or neuromuscular coordination in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an AAV virion of any one of embodiments 14-16.

[0061] 44. A method for improving motor activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an AAV virion of any one of embodiments 14-16.

[0062] 45. A method for improving spatial learning in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an AAV virion of any one of embodiments 14 to 16.

[0063] 46. ​​A method for reducing and / or reversing ovarian failure in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an AAV virion of any one of embodiments 14-16.

[0064] 47. A method for regulating follicle-stimulating hormone, luteinizing hormone, and / or anti-Mullerian hormone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an AAV virion of any one of embodiments 14-16.

[0065] 48. A method for inhibiting cataract formation or promoting cataract resorption in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an AAV virion of any one of embodiments 14-16.

[0066] 49. A method for limiting the severity of cataract formation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an AAV virion of any one of embodiments 14-16.

[0067] 50. The method of embodiment 48 or 49, wherein the therapeutically effective amount of AAV virions is administered prior to the onset of cataracts.

[0068] 51. A method for reducing prepubertal growth retardation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an AAV virion of any one of embodiments 14-16.

[0069] 52. A method for improving weight gain in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an AAV virion of any one of embodiments 14 to 16.

[0070] 53. A method for reducing the severity of a visceral neuromuscular disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an AAV virion of any one of embodiments 14-16.

[0071] 54. A method for reducing the severity of a neurological disorder and / or a socio-emotional disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an AAV virion of any one of embodiments 14-16.

[0072] 55. The method of embodiment 54, wherein said neurological disorder and / or said socio-emotional disorder is locomotor function.

[0073] 56. The method of embodiment 55, wherein the locomotor function is tremor and / or ataxia.

[0074] 57. The method of embodiment 54, wherein said neurological disorder and / or said socio-emotional disorder is anxiety.

[0075] 58. The method of embodiment 54, wherein said neurological disorder and / or said socio-emotional disorder is depression.

[0076] 59. The method of any one of embodiments 17-21 or 30-58, wherein the AAV virion is administered before the onset of puberty.

[0077] 60. The method of embodiment 59, wherein the AAV virion is administered to a young pediatric subject or a pediatric subject.

[0078] 61. The method of any one of embodiments 17-21 or 30-60, further comprising monitoring levels of galactose, GAL-1P, and / or galactitol. [Brief description of the drawings]

[0079] [Figure 1]1 shows a map of plasmid pAAV2 ITR-CAG-hGALT-KanR, which contains two AAV2 inverted terminal repeats (ITRs) (SEQ ID NO: 11 and 12) flanking an expression region containing a cytomegalovirus (CMV) early enhancer / chicken β-actin / rabbit β-globin splice acceptor (CAG) promoter (SEQ ID NO: 9), a nucleic acid encoding human GALT (hGALT) (SEQ ID NO: 2), a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) (SEQ ID NO: 14), and a bovine growth hormone polyadenylation (bGH poly(A)) signal sequence (SEQ ID NO: 15). The plasmid further contains an ampicillin resistance gene (AmpR) promoter controlling expression of a kanamycin resistance gene sequence (KanR), a CMV origin of replication (ori), and a catabolite activator protein (CAP) binding site, a lactose (lac) promoter controlling the lac operator, and an M13 reverse primer region. [Figure 2-1] The double-stranded nucleotide sequence (SEQ ID NO:7) of the pAAV2 ITR-CAG-hGALT-KanR plasmid, which contains the nucleotide sequence encoding hGALT (SEQ ID NO:2) and the amino acid sequence of hGALT (SEQ ID NO:1), is provided. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 2-3] This is a continuation of Figure 2-1. [Figure 2-4] This is a continuation of Figure 2-1. [Figure 2-5] This is a continuation of Figure 2-1. [Figure 2-6] This is a continuation of Figure 2-1. [Diagram 2-7] This is a continuation of Figure 2-1. [Figure 2-8] This is a continuation of Figure 2-1. [Diagram 3]1 shows a map of plasmid pAAV2 ITR-EF1-hGALT-KanR, which contains two AAV2 ITRs flanking an expression region with the elongation factor 1 alpha (EF-1 alpha) promoter (SEQ ID NO: 10), a nucleic acid encoding hGALT, a WPRE, and a bGH poly(A) signal sequence. The plasmid further contains the AmpR promoter controlling expression of the KanR gene sequence, a CMV origin of replication (ori), and a CAP binding site, the lac promoter controlling the lac operator, and an M13 reverse primer region. [Figure 4-1] The double-stranded nucleotide sequence (SEQ ID NO:8) of the pAAV2 ITR-EF1-hGALT-KanR plasmid, which contains the nucleotide sequence encoding hGALT (SEQ ID NO:2) and the amino acid sequence of hGALT (SEQ ID NO:1), is provided. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 4-3] This is a continuation of Figure 4-1. [Figure 4-4] This is a continuation of Figure 4-1. [Figure 4-5] This is a continuation of Figure 4-1. [Figure 4-6] This is a continuation of Figure 4-1. [Figure 4-7] This is a continuation of Figure 4-1. [Figure 4-8] This is a continuation of Figure 4-1. [Diagram 5] 1 shows a map of the plasmid pscAAV-CAG-hGALT-KanR, which contains one AAV2 ITR upstream of an expression region containing a CAG promoter, a nucleic acid sequence encoding hGALT, a WPRE, a Simian Virus 40 (SV40) poly(A) signal sequence (SEQ ID NO: 16), and a self-complementary AAV (scAAV) ITR (SEQ ID NO: 13). The plasmid further contains the AmpR promoter controlling expression of the KanR gene sequence, a CMV origin of replication (ori), and a CAP binding site, a lac promoter controlling the lac operator, and an M13 reverse primer region. [Figure 6-1]The double-stranded nucleotide sequence of the pscAAV-CAG-hGALT-KanR plasmid (SEQ ID NO:6) is provided, which contains the nucleotide sequence encoding hGALT (SEQ ID NO:2) and the amino acid sequence of hGALT (SEQ ID NO:1). [Figure 6-2] This is a continuation of Figure 6-1. [Figure 6-3] This is a continuation of Figure 6-1. [Figure 6-4] This is a continuation of Figure 6-1. [Figure 6-5] This is a continuation of Figure 6-1. [Figure 6-6] This is a continuation of Figure 6-1. [Figure 7] Concentrations of Gal-1P, a biomarker of galactosemia, in brain tissue (A and B), skeletal muscle tissue (C and D), RBCs (E and F), and liver tissue (G) at 12 weeks after treatment from wild-type (WT / WT) and GALT null (GG / GG) mice at 4 and 12 weeks after treatment with high dose AAV9-CAG-hGalt (1.15×10 vg / kg), low dose AAV9-CAG-hGalt (3.74×10 vg / kg), or AAV9-CAG-hGalt formulation buffer vehicle control. AAV9-CAG-hGalt significantly reduces Gal-1P levels in brain, skeletal muscle, RBCs, and liver of GALT null mice. [Figure 8] Figure 1 shows concentrations of galactose, a biomarker of galactosemia, in brain tissue (A and B), skeletal muscle tissue (C and D), RBCs (E and F), and plasma (H and I) from wild-type (WT / WT) and GALT null (GG / GG) mice at 4 and 12 weeks after treatment with high dose AAV9-CAG-hGalt (1.15x1014 vg / kg), low dose AAV9-CAG-hGalt (3.74x1013 vg / kg), or AAV9-CAG-hGalt formulation buffer vehicle control, and in liver tissue (G) at 12 weeks after treatment. AAV9-CAG-hGalt significantly reduces galactose levels in brain, skeletal muscle, RBCs, plasma, and liver of GALT null mice. [Figure 9]Figure 1 shows concentrations of galactitol, a biomarker of galactosemia, in brain tissue (A and B), skeletal muscle tissue (C and D), RBCs (E and F), and plasma (H and I) from wild-type (WT / WT) and GALT null (GG / GG) mice at 4 and 12 weeks after treatment with high dose AAV9-CAG-hGalt (1.15x1014 vg / kg), low dose AAV9-CAG-hGalt (3.74x1013 vg / kg), or AAV9-CAG-hGalt formulation buffer vehicle control, and in liver tissue (G) at 12 weeks after treatment. AAV9-CAG-hGalt significantly reduces galactitol levels in brain, skeletal muscle, RBCs, plasma, and liver of GALT null mice. [Figure 10] Immunohistochemical assessment of GALT protein expression in liver (A), muscle (B), and brain (C) from wild-type (WT / WT) and GALT null (GG / GG) mice at 4 and 12 weeks after treatment with high dose AAV9-CAG-hGalt (1.15×10 vg / kg), low dose AAV9-CAG-hGalt (3.74×10 vg / kg), or AAV9-CAG-hGalt formulation buffer vehicle control. AAV9-CAG-hGalt induced GALT enzyme expression in GALT null mouse pups. [Figure 11A] 1 shows the experimental design for the behavioral study of Logical Example 2. [Figure 11B] 1 shows the experimental design for the safety study of logical example 2. [Figure 11C] 1 shows the experimental design for the fertility study of Logical Example 2. [Figure 12-1]FIG. 12A shows GALT protein expression by immunohistochemistry in wild-type (WT / WT) and GALT null (M3 / M3) rats at 14 and 35 days after treatment with high dose AAV9-CAG-hGalt (1.16×10 vg / kg), low dose AAV9-CAG-hGalt (3.82×10 vg / kg) or AAV9-CAG-hGalt formulation buffer vehicle control. Panel A shows GALT protein staining of liver tissue sections at 14 and 35 days after treatment and quantification of GALT positive staining. Panel B shows GALT protein staining in skeletal muscle tissue at 35 days after treatment. Panel C shows co-staining of GALT protein (brown) and astrocyte marker GFAP (purple) in brain cortex tissue at 14 and 35 days after treatment. Panel D shows co-staining of GALT protein (brown) and either astrocyte marker GFAP (purple) in cerebellar tissue at 14 and 35 days after treatment. Administration of AAV9-CAG-hGalt induced GALT enzyme expression in GALT null rat pups. [Figure 12-2]FIG. 12B shows GALT protein expression by immunohistochemistry in wild-type (WT / WT) and GALT null (M3 / M3) rats at 14 and 35 days after treatment with high dose AAV9-CAG-hGalt (1.16×10 vg / kg), low dose AAV9-CAG-hGalt (3.82×10 vg / kg) or AAV9-CAG-hGalt formulation buffer vehicle control. Panel A shows GALT protein staining of liver tissue sections at 14 and 35 days after treatment and quantification of GALT positive staining. Panel B shows GALT protein staining in skeletal muscle tissue at 35 days after treatment. Panel C shows co-staining of GALT protein (brown) and astrocyte marker GFAP (purple) in brain cortex tissue at 14 and 35 days after treatment. Panel D shows co-staining of GALT protein (brown) and either astrocyte marker GFAP (purple) in cerebellar tissues at 14 and 35 days after treatment. Administration of AAV9-CAG-hGalt induced GALT enzyme expression in GALT null rat pups. Figure 12C shows GALT protein expression by immunohistochemistry in wild-type (WT / WT) and GALT null (M3 / M3) rats at 14 and 35 days after treatment with high dose AAV9-CAG-hGalt (1.16x1014vg / kg), low dose AAV9-CAG-hGalt (3.82x1013vg / kg) or AAV9-CAG-hGalt formulation buffer vehicle control. Panel A shows GALT protein staining of liver tissue sections at 14 and 35 days after treatment and quantification of GALT positive staining. Panel B shows GALT protein staining in skeletal muscle tissue at day 35 after treatment. Panel C shows co-staining of GALT protein (brown) and astrocyte marker GFAP (purple) in brain cortex tissue at days 14 and 35 after treatment. Panel D shows co-staining of GALT protein (brown) and either astrocyte marker GFAP (purple) in cerebellum tissue at days 14 and 35 after treatment. Administration of AAV9-CAG-hGalt induced GALT enzyme expression in GALT null rat pups.FIG. 12D shows GALT protein expression by immunohistochemistry in wild-type (WT / WT) and GALT null (M3 / M3) rats at 14 and 35 days after treatment with high dose AAV9-CAG-hGalt (1.16×10 vg / kg), low dose AAV9-CAG-hGalt (3.82×10 vg / kg) or AAV9-CAG-hGalt formulation buffer vehicle control. Panel A shows GALT protein staining of liver tissue sections at 14 and 35 days after treatment and quantification of GALT positive staining. Panel B shows GALT protein staining in skeletal muscle tissue at 35 days after treatment. Panel C shows co-staining of GALT protein (brown) and astrocyte marker GFAP (purple) in brain cortex tissue at 14 and 35 days after treatment. Panel D shows co-staining of GALT protein (brown) and either astrocyte marker GFAP (purple) in cerebellar tissue at 14 and 35 days after treatment. Administration of AAV9-CAG-hGalt induced GALT enzyme expression in GALT null rat pups. [Figure 13] GALT enzyme activity assessment in liver (A and B), brain (C and D), skeletal muscle (E and F), and eye tissue (G and H) of wild-type (WT / WT) and GALT null (M3 / M3) rats at days 14 and 35 after treatment with high dose AAV9-CAG-hGalt (1.16×10 vg / kg), low dose AAV9-CAG-hGalt (3.82×10 vg / kg), or AAV9-CAG-hGalt formulation buffer vehicle control. AAV9-CAG-hGalt induced GALT enzyme activity in GALT null rat pups. [Figure 14]Concentrations of Gal-1P, a biomarker of galactosemia, in the liver (A and B), brain (C and D), skeletal muscle (E and F), ovaries (G and H), and RBCs (I and J) of wild-type (WT / WT) and GALT null (M3 / M3) rats at days 14 and 35 after treatment with high dose AAV9-CAG-hGalt (1.16×10 vg / kg), low dose AAV9-CAG-hGalt (3.82×10 vg / kg), or AAV9-CAG-hGalt formulation buffer vehicle control. Concentrations of Gal-1P in the eye (K) at day 14 after treatment. AAV9-CAG-hGalt significantly reduces Gal-1P levels in the liver, brain, muscle, ovaries, RBCs, and eyes of GALT null rat pups. [Figure 15] Concentrations of galactose, a biomarker of galactosemia, in liver (A and B), brain (C and D), plasma (E and F), muscle (G and H), ovaries (I and J), eyes (K and L), and RBCs (M and N) of wild-type (WT / WT) and GALT null (M3 / M3) rats at days 14 and 35 after treatment with high dose AAV9-CAG-hGalt (1.16×10 vg / kg), low dose AAV9-CAG-hGalt (3.82×10 vg / kg), or AAV9-CAG-hGalt formulation buffer vehicle control. AAV9-CAG-hGalt significantly reduces galactose levels in liver, brain, plasma, muscle, ovaries, and eyes of GALT null rat pups. [Figure 16] Concentrations of galactitol, a biomarker of galactosemia, in liver (A and B), brain (C and D), plasma (E and F), muscle (G and H), ovary (I and J), eye (K and L), and RBC (M and N) of wild-type (WT / WT) and GALT null (M3 / M3) rats at days 14 and 35 after treatment with high dose AAV9-CAG-hGalt (1.16×10 vg / kg), low dose AAV9-CAG-hGalt (3.82×10 vg / kg), or AAV9-CAG-hGalt formulation buffer vehicle control. AAV9-CAG-hGalt significantly reduces galactitol levels in liver, brain, plasma, muscle, ovary, and eye of GALT null rat pups. [Figure 17] Cataract scoring (A and B) and cataract incidence (C) of wild-type (WT / WT) and GALT null (M3 / M3) rat eyes at 14 and 35 days after treatment with high dose AAV9-CAG-hGalt (1.16×10 vg / kg), low dose AAV9-CAG-hGalt (3.82×10 vg / kg) or AAV9-CAG-hGalt formulation buffer vehicle control. Eyes without cataracts are scored as 0, and higher cataract scores indicate increased cataract severity. N represents individual eyes. Data represent unique animals as cataracts are scored at sacrifice. AAV9-CAG-hGalt reduces cataract incidence and severity in GALT null rat pups. [Figure 18] Figure 1 shows daily body weights of wild-type (WT / WT) and GALT-null (M3 / M3) rats from P2 to P37 treated with high dose AAV9-CAG-hGalt (1.16 x 10 vg / kg), low dose AAV9-CAG-hGalt (3.82 x 10 vg / kg) or AAV9-CAG-hGalt formulation buffer vehicle control. AAV9-CAG-hGalt improves weight gain in GALT-null rat pups. [Figure 19] FIG. 1. Experimental design for in-life and post-mortem evaluations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0080] definition When interpreting the detailed description and claims, any of "comprising," "consisting of," "consisting essentially of," "selected from the group consisting of," "at least selected from the group consisting of," "at least one selected from the group consisting of," as well as "is," "being," and "are," or equivalents thereof, should be understood to support any other alternative to what is stated, in light of any other alternative. For example, when "comprising A, B, or C" is stated, it supports embodiments including any alternative, in light of any alternative, such as "A, B, or C," "consisting of A, B, and C," "essentially consisting of A, B, or C," "at least selected from the group consisting of A, B, and C," "at least one selected from the group consisting of A, B, and C," or equivalents thereof.

[0081] Additionally, statements of "or" contemplate and support "one or more," "one or a combination thereof," or "and," as in "and / or." For example, "A, B, or C" contemplates and supports embodiments that include A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, and A, B, and C in combination. Additionally, in statements of "closed" language (e.g., consisting of) and statements of "open" language (e.g., comprising) enumerations such as "A, B, or C" also contemplate one or a combination of the enumeration, unless otherwise indicated. For example, "consisting of A, B, or C" contemplates and supports embodiments that include A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, and A, B, and C in combination. The statement "and / or" contemplates and supports all combinations of the enumeration (i.e., "A, B, and / or C" contemplates "A, B, and C") as well as "one or more" or "one or a combination thereof." For example, "A, B, and C" contemplates A alone, B alone, C alone, A in combination with B and C, A in combination with B, A in combination with C, and B in combination with C.

[0082] For example, a statement listing alternatives with "and," such as "selected from the group consisting of," contemplates and supports the listed combination unless expressly stated otherwise. For example, "selected from the group consisting of A, B, and C" contemplates and supports "selected from the group consisting of A, B, C, and combinations thereof," and is in the same scope as "at least one selected from the group consisting of A, B, and C," or the "group" is understood to include A alone, B alone, C alone, the combination of A and B, the combination of A and C, the combination of B and C, and the combination of A, B, and C.

[0083] The phrase "at least selected from the group consisting of A, B, and C" supports the embodiment, considering that the statement "at least" covers the same scope as "including members of the group consisting of A, B, and C" or includes embodiments in which "consisting of" modifies the meaning of only the "group" rather than "selected from the group."

[0084] Furthermore, the recitation of an element in an embodiment also contemplates and supports the explicit exclusion of that element from the embodiment. For example, "comprising A, B, or C" supports embodiments that include A or B, but specifically exclude C.

[0085] As used herein, the articles "a," "an," and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. As an example, "an element" means one element or more than one element. For example, "comprising an A, a B, or a C" contemplates and supports embodiments including two or more A, two or more B, and two or more C.

[0086] Unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood by those skilled in the art to which the embodiments belong. Although preferred materials and methods are described, it is understood that any methods and materials similar or equivalent to those described can be used in the practice of the embodiments. The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. In describing and claiming the present invention, the following terms are used.

[0087] The term "about" as used herein when referring to a measurable value, such as an amount, temporal duration, and the like, is intended to encompass variations of +-20% or +-10% from the specified value, more preferably +-5%, even more preferably +-1%, and even more preferably +-0.1%, as such variations are appropriate to practice the embodiments.

[0088] The term "cell line" as used herein refers to a population of cells that have the ability to grow and divide continuously or over a long period of time in vitro. In many cases, a cell line is a clonal population derived from a single progenitor cell. Furthermore, karyotypic changes may occur naturally or be induced during storage or transfer of such a clonal population. Thus, cells derived from the cell line referred to may not be strictly identical to their ancestor cells or cultures, and the cell line referred to includes such variants.

[0089] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0090] The term "host cell" as used herein is any cell that carries or can carry a substance of interest. In many cases, the host cell can be a mammalian cell (e.g., a non-human primate, rodent, or human cell). In some embodiments, the host cell can be a mammalian cell, a yeast cell, a bacterial cell, an insect cell, a plant cell, or a fungal cell. The host cell can be used as a recipient for AAV helper constructs, AAV plasmids encoding recombinant AAV genomes containing transgenes, accessory function vectors, or other transfer DNA involved in the production of recombinant AAV (rAAV) virions. This term includes the progeny of the original transfected cell. Thus, "host cell" as used herein can refer to a cell transfected with an exogenous DNA sequence. It is understood that the progeny of a single parent cell may not necessarily be completely identical to the original parent in morphology or in genome or total DNA complement due to natural, accidental, or deliberate mutations.

[0091] "Identity" as used herein refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, such as between two polypeptide molecules or between two nucleic acid molecules (e.g., polynucleotides). If two amino acid sequences have the same residue at the same position, e.g., if a position is occupied by arginine in each of the two polypeptide molecules, then they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions. For example, if half of the positions in the two sequences (e.g., 5 positions in a polymer that is 10 amino acids in length) are identical, then the two sequences are 50% identical. If 90% of the positions (e.g., 9 out of 10 positions) are matched or identical, then the two amino acid sequences are 90% identical. In the case of an insertion or deletion, it is understood that identity is readjusted after the insertion or deletion to what was considered to be non-identical at the time of the insertion or deletion.

[0092] By "substantially identical" it is meant that a polypeptide or nucleic acid molecule exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, or even 99% identical at the amino acid level or at the nucleic acid level to the sequence used for comparison.

[0093] The term "modified" as used herein means that the state or structure of a molecule or cell of the invention has been altered. Molecules can be modified in many ways, including chemical, structural, and functional ways. Cells can be modified by the introduction of nucleic acids.

[0094] The term "modulate" as used herein means to mediate a detectable increase or decrease in the level of a response in a subject compared to the level of the response in the subject in the absence of a treatment or compound and / or compared to the level of the response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a natural signal or response in a subject, preferably a human, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0095] "Nucleic acid" as used herein is interchangeable with "polynucleotide" or "specific sequence of nucleotides". These terms refer to a discrete sequence that performs a specific function in a cell, directly or indirectly. The function includes encoding a sequence of a gene that is transcribed into mRNA and translated into a protein, and controlling said transcription (i.e., by a promoter) and / or translation (i.e., by a microRNA). Nucleic acids essentially have a sequence. Thus, "a nucleic acid comprising SEQ ID NO:X" can be used to consider and support "a nucleic acid having the sequence of SEQ ID NO:X". In recombinant molecular biology, it is possible to combine discrete nucleic acids. In some embodiments, a nucleic acid encoding a protein can be linked to a promoter (which is a nucleic acid) and a cis-acting element of a viral vector (i.e., an inverted terminal repeat (ITR) that is also a nucleic acid). For convenience, "nucleic acid" can be used to refer to a separate element within a larger nucleic acid, which can also be referred to as a "polynucleotide", "expression region" (i.e., a polynucleotide comprising a promoter and a nucleic acid encoding a protein), or a "vector" (see definition below).

[0096] "Encoding" refers to the inherent property of a nucleic acid to serve as a template for the direct (i.e., sense strand) or indirect (i.e., antisense strand) synthesis of a peptide, polypeptide, protein, or another nucleic acid (i.e., rRNA, tRNA, microRNA). A nucleic acid "encodes" whether it is a sense strand, an antisense strand, or a double-stranded segment. The sense strand directly encodes the rRNA, tRNA, microRNA, or mRNA. The mRNA then serves as a template for translation of the peptide, polypeptide, or protein. The antisense strand is generally considered to be the reverse complement and is sometimes referred to in the art as the "non-coding" strand (although for present purposes "non-coding" is a misnomer since the non-coding strand still "encodes" genetic information by serving as a template for the polymerization of new sense strands, thereby perpetuating that genetic information during semi-conservative replication). For semi-conservative replication, the two single strands in a double-stranded nucleic acid are separated, and a new strand is polymerized from the information from each single-stranded nucleic acid (i.e., the single-stranded template), whether one single-stranded template is the sense strand (e.g., used to transcribe an mRNA, thereby translating or encoding a protein, or directly) or the antisense strand. By perpetuating the genetic information, the antisense strand still encodes the genetic information, for example, for a protein. Thus, a "nucleic acid encoding X" includes the sense and antisense sequences or strands, whether X is a peptide, polypeptide, or protein, or whether X is a sequence encoding rRNA, tRNA, microRNA, antisense RNA, etc.

[0097] Furthermore, since nucleic acids can be synthesized by natural or artificial processes (e.g., recombinant biology, molecular biology), such as transcription, reverse transcription, and replication, a "nucleic acid encoding X" includes RNA, DNA, and combinations thereof.

[0098] Thus, a described nucleic acid sequence contemplates and supports its complementary, reverse complement, and double-stranded forms. That is, a "nucleic acid comprising SEQ ID NO:X" should be understood to contemplate and support a "nucleic acid having the reverse complement of SEQ ID NO:X" or a "nucleic acid comprising SEQ ID NO:X'" unless otherwise indicated, using nomenclature with a prime symbol such as "'". For example, a "nucleic acid comprising SEQ ID NO:X", where SEQ ID NO:X is 5'-ATGCC-3', contemplates and supports the reverse complement of SEQ ID NO:X, specifically 5'-GGCAT-3.

[0099] As noted above, the nucleic acid sequences described contemplate and support conversion between its RNA and DNA forms. For example, SEQ ID NO:X is "5'-ATGCC-3'", and 5'-AUGCC-3', as well as its reverse complement, 5'-GGCAU-3, are contemplated and supported.

[0100] With respect to AAV vectors or AAV virions, the incorporation of reverse complement sequences and double-stranded segments into the definition of "nucleic acid" above, and the use of the term "encoding" to include sense and antisense strands as above, is intended to incorporate the means by which an AAV vector can introduce an exogenous nucleic acid sequence encoding a nucleic acid or protein into a cell. In some embodiments, it is further intended to incorporate the process by which such incorporation results in expression of such a nucleic acid (i.e., miRNA or antisense RNA) or protein (i.e., galactose-1-phosphate uridylyltransferase (GALT)).

[0101] For example, the nucleic acid coding for a protein and the AAV vector containing the nucleic acid coding for the protein can be included. When a typical (i.e. naturally occurring) AAV vector coding for one sense strand or one antisense strand of the nucleic acid coding for the protein enters a cell, the inverted terminal repeat (ITR) primes the synthesis of the sequence that is reverse-complementary to the sense strand or antisense strand of the nucleic acid coding for the protein. Thus, regardless of whether the sense or antisense type is first introduced into the cell, polymerization forms a double-stranded DNA segment that includes the sense strand and the antisense strand. In this regard, the entire nucleic acid, including the ITR coding for a protein and the sense and antisense nucleic acids, can be a single-stranded DNA that loops around itself to form a double-stranded segment, and the base pairs of the sense and antisense nucleic acids coding for the protein can be aligned.

[0102] This segment of double-stranded DNA achieves transcription of mRNA and translation of protein from the sense strand of said DNA, regardless of whether the AAV vector contained only the sense strand or only the antisense strand when it first entered the cell. In this regard, an "AAV vector comprising a nucleic acid encoding protein X" includes, contemplates and supports embodiments in which the nucleic acid is a sense strand encoding protein X, an antisense strand encoding protein X, a double-stranded nucleic acid encoding protein X, and a single-stranded nucleic acid comprising a sense strand and an antisense strand, where the sense strand and the antisense strand form a segment of double-stranded nucleic acid.

[0103] The phrase "operably linked" refers to the binding between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter.For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.For example, a promoter is operably linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence.

[0104] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery necessary to initiate specific transcription of a polynucleotide sequence. In some instances, this sequence may be a core promoter, while in other instances, this sequence may also include, or may be, only an enhancer and / or other regulatory elements required for expression of a gene product.

[0105] In certain instances, a promoter may contain enhancer elements, exons, and introns from one or a variety of viruses and animals. Thus, the term "promoter" should be understood to be not limited to non-expressed sequences, does not exclude non-expressed sequences that are between expressed sequences (i.e., introns), and does not exclude only enhancers, so long as the combination of sequences used to construct the promoter is capable of initiating specific transcription of a polynucleotide sequence.

[0106] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specific for a gene product, allows the gene product to be produced in a cell under most or all physiological conditions of the cell without the need to add exogenous factors or introduce a different phenotype into the cell. The constitutive promoter can be cell-specific as long as it is produced in a specific or target cell under most or all physiological conditions of the cell. For example, a telencephalic neuron-specific promoter is calcium / calmodulin-dependent protein kinase II (CaMKII). The CAG promoter and the elongation factor 1 (EF1) promoter are examples of constitutive promoters in a wide range of target cell types.

[0107] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only if an inducer corresponding to the promoter is present in the cell. For example, the promoter in the cyclooxygenase-2 gene is considered to be a peripherally inducible promoter.

[0108] As used herein, the term "recombinant cell" refers to a cell into which an exogenous DNA segment has been introduced, such as a DNA segment that results in the transcription of a biologically active polypeptide or the production of a biologically active nucleic acid, such as RNA.

[0109] "Target gene" refers to a nucleic acid that encodes a target protein that is expressed in a target cell when a vector carrying the target gene enters the cell. Target genes include naturally occurring polymorphisms (i.e., variants) and artificial modifications to wild-type genes, so long as the target protein is still expressed. Examples of such artificial modifications include codon optimization.

[0110] "Target protein" refers to an artificial or naturally occurring protein of interest that is introduced into a host cell by a vector. In some embodiments, the target protein encoded in the genome of a host cell is non-functional due to a polymorphism in the gene sequence that causes mistranscription, missense, or mistranslation of a portion of the gene, resulting in reduced or no target protein being produced, or a non-functional target protein being produced (i.e., the polymorphism causes a premature stop codon), or attenuated activity of the target protein encoded by and expressed from the genome of interest.

[0111] In some embodiments, the target protein comprises galactose-1-phosphate uridylyltransferase (GALT). GALT is an enzyme that interconverts (i.e., a reversible enzymatic reaction) uridine diphosphate glucose and galactose-1-phosphate to glucose-1-phosphate and uridine diphosphate galactose (i.e., the second step of the Leloir pathway of galactose metabolism). It is understood and contemplated that "GALT" encompasses naturally occurring forms (i.e., human GALT) and non-naturally occurring GALT (i.e., amino acid additions, deletions, or substitutions of GALT that increase or decrease activity compared to naturally occurring GALT), so long as the enzyme referred to as GALT has at least the enzymatic activity described above. In one embodiment, the non-natural GALT has at least or less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200% of the activity of the corresponding natural GALT, where "corresponding" contemplates and supports the addition, deletion, or substitution applied. In an embodiment, the GALT is human GALT, and in some embodiments, the GALT has the amino acid sequence of SEQ ID NO:1. In alternative embodiments, GALT has an amino acid sequence having at least 99%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 1 and has GALT activity. In another embodiment, hGALT is encoded by a nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence encoding hGALT having at least 99%, 95%, 90%, 85%, or 80% identity to SEQ ID NO: 2 and having the amino acid sequence of SEQ ID NO: 2, or an hGALT having an amino acid sequence having at least 99%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 1 and having GALT activity.

[0112] In some embodiments, the subject is deficient in GALT encoded and expressed by the subject's genome, for example due to autosomal recessive inheritance of two GALT deficient genes. In some embodiments, the subject has classical galactosemia. In some embodiments, the subject has attenuated GALT activity. In some embodiments, the subject has Duarte galactosemia. In some embodiments, the subject has an unstable form of the GALT enzyme compared to individuals without the polymorphism, regardless of whether the polymorphism causes reduced enzyme activity. In some embodiments, the subject has a mutation in a promoter regulating GALT transcription, which causes reduced transcription of the mRNA encoding GALT, causing reduced expression of GALT (i.e., reduced GALT protein). In some embodiments, the subject has a combination of one gene encoding a deficient GALT activity and another gene encoding attenuated GALT activity. That is, in some embodiments, the subject's genotype is heterozygous for the classical variant and the Duarte variant. In some embodiments, the subject has one or both of an A to G mutation in exon 6 of the GALT gene that changes Glu188 to arginine and an A to G mutation in exon 10 that changes Asn314 to aspartic acid.

[0113] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a gene-encoding or gene-specific polynucleotide, causes a gene product to be produced in a cell only if the cell is substantially a cell of the tissue type corresponding to the promoter.

[0114] A "vector" is a nucleic acid capable of delivering a target gene into a cell, and includes not only an expression region (i.e., a promoter and a nucleic acid encoding a protein or even a nucleic acid), but also several cis-acting genetic elements that provide for expression within the cell, replication within the cell, or a combination thereof, for packaging into virions.

[0115] As an example, the inverted terminal repeats (ITRs) from adeno-associated virus (AAV) provide the nucleic acid encoding the target protein to be packaged in the AAV virion, and thus constitute a vector when bound to the nucleic acid encoding the target protein. The ITRs also provide other cis-acting functions for the expression of the nucleic acid encoding the target protein in the host cell once the vector has entered the host cell. Such cis-acting functions of ITRs include assisting in the formation of concatemers for genome insertion; initiating second strand formation in the case of single-stranded (ss)AAV (ssAAV) vectors; or initiating replication and transcription in the case of ssAAV and self-complementary (sc)AAV (scAAV) vectors. In this regard, AAV ITRs can be characterized based on the nucleic acid sequences that provide such cis-acting functions from the serotype of AAV. That is, an ITR isolated from the AAV2 serotype can be known as an AAV2 ITR, even though the ITRs do not generally contribute to the serotype of AAV.

[0116] Furthermore, the scAAV ITR (e.g., SEQ ID NO: 13) is formed by mutating or altering the wild-type AAV2 ITR (e.g., SEQ ID NO: 11, the 5' adjacent ITR), in particular the terminal resolution site (trs) in the D sequence responsible for signaling packaging (packaging sequence), but it is understood that the scAAV ITR provides the function of generating an AAV vector containing the expression region and its reverse complement before packaging into an AAV virion, which is not provided by the wild-type AAV ITR. Without wishing to be bound by a particular theory, it is believed that when a producer cell expresses an AAV vector plasmid, an AAV vector containing the expression region and two ITRs (one of which is the scAAV ITR) is produced. Due to the negating mutation to trs in the D sequence, DNA polymerase can polymerize from the expression region and the reverse complement of the remaining ITR. This results in a scAAV vector. In some embodiments, the scAAV vector is then packaged into a recombinant scAAV virion.

[0117] The scAAV vectors may exhibit at least 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, or 140-fold or less efficient transduction than a corresponding vector that contains wild-type AAV2 ITRs and does not contain the complement of the expressed region. Without wishing to be bound by theory, it is believed that synthesis of the double-stranded segment in the ssAAV vector by the host cell is the rate-limiting step in ssAAV vector transduction, and that by providing the expressed region and its reverse complement, the scAAV ITRs (and vectors containing the scAAV ITRs) increase the efficiency of transduction as described above.

[0118] As an example, a plasmid may contain an origin of replication (from cytomegalovirus) that allows replication of a target gene within a cell, and thus such a plasmid is a vector. The viral genetic code provides a nucleic acid sequence or a protein encoded therein that allows insertion of a gene of interest into the host genome, thereby allowing replication of the target gene within the host cell's genome at the same time that the host cell replicates its genome.

[0119] "Expression vector" refers to a vector that includes an expression region. The expression region includes a recombinant polynucleotide that includes a nucleic acid that controls expression (i.e., a promoter) and a coding nucleic acid. The coding nucleic acid includes a nucleic acid that encodes a protein. Generally, the promoter is operably linked to the nucleic acid encoding the target protein in a manner that can promote expression of the protein once the vector has entered a host cell. In some embodiments, the promoter can be operably linked by ensuring that there are no codon misalignments.

[0120] Ranges: Throughout this disclosure, various aspects of the invention can be presented in a range format. The description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, any description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, a description of a range such as 1-6 should be considered to include the specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6.

[0121] The nucleotide and amino acid sequences provided herein are shown in Table 1.

[0122] AAV vectors In some embodiments, a recombinant adeno-associated virus (AAV) vector comprises an expression region and at least two inverted terminal repeats (ITRs), where the expression region comprises a promoter and a nucleic acid encoding galactose-1-phosphate uridylyltransferase (GALT), the promoter is operably linked to expression of GALT, and the at least two ITRs flank the expression region, and the expression region may further comprise other regulatory sequences, such as enhancers, polyA signals, intron sequences, and WPRE sequences.

[0123] In some embodiments, GALT has an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identical to the amino acid sequence of SEQ ID NO: 1; or has 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof therein. In some embodiments, GALT has the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encoding GALT comprises a nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to SEQ ID NO:2 or its reverse complement; or having 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof therein. In some embodiments, the nucleic acid encoding GALT comprises or consists of SEQ ID NO:2 or its reverse complement.

[0124] In some embodiments, the ITR, or at least two ITRs, comprise AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, or AAV9 ITR, and in some embodiments may be scAAV ITR. In some embodiments, the ITR, or at least two ITRs, comprise AAV2 ITR or scAAV ITR. In some embodiments, the ITR, or at least two ITRs, comprise AAV2 ITR and scAAV ITR. In some embodiments, the scAAV ITR is an AAV2 ITR that lacks at least one functional terminal resolution site (trs) in the D sequence. In some embodiments, the scAAV ITR lacks at least one functional trs. In some embodiments, the scAAV ITR has at least one substitution, addition, or deletion in at least one trs, and at least one substitution, addition, or deletion confers dysfunction to at least one trs. In some embodiments, the AAV ITRs have at least one D sequence deleted. In some embodiments, the deleted D-sequence is at the 3' end of the ITR (ssD[-]). In some embodiments, the deleted D-sequence is at the 5' end of the ITR (ssD[+]). In some embodiments, the ssD[-] sequence has at least one substitution, deletion, or addition that prevents binding of the 52-kDa-FK506 binding protein (FKBP52). In some embodiments, the AAV ITRs have a D-sequence substituted for a transcription factor binding site. In some embodiments, the transcription factor binding site has an S sequence. In some embodiments, the S sequence comprises a Foxd3 binding site or a NF-μE1 binding site. In some embodiments, the transcription factor binding site or S sequence comprises a GATA-1 and GATA-2 binding site.In some embodiments, the ITR, or at least two ITRs, comprise a nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to a reverse complement thereof, including SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 12; or having 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof therein. In some embodiments, the ITR, or at least two ITRs, comprise SEQ ID NO: 11, SEQ ID NO: 12, or a reverse complement thereof. Indeed, the adjacent ITRs may be reverse complementary to each other (or their respective reverse complements), such that the ITR at the 5' end of the expression cassette has the nucleotide sequence of SEQ ID NO: 11 and the ITR at the 3' end of the expression cassette has the nucleotide sequence of SEQ ID NO: 12. In the case of a self-complementary vector, the ITR at the 5' end of the expression cassette has the mutated sequence of SEQ ID NO: 13 and the ITR at the 3' end of the expression cassette is, for example, an unmodified ITR and an AAV2 ITR having the nucleotide sequence of SEQ ID NO: 11 at the 5' end of the expression cassette.

[0125] In some embodiments, the sequence encoding GALT is operably linked to a constitutive promoter. In some embodiments, the promoter is a CAG promoter. The CAG promoter is a composite synthetic promoter that includes a CMV early enhancer element, a chicken β-actin promoter, a chicken β-actin promoter, and the first exon and first intron of the chicken β-actin gene and a splice acceptor of the rabbit β-globin gene. See, for example, Miyazaki et al, Gene 79:269-277 (1989) and Niwa et al, Gene 108:193-199 (1991). In certain embodiments, the CAG "promoter" can have the nucleotide sequence of SEQ ID NO:9 or at least a 200, 300, 400, 500, or 600 nucleotide fragment (or its reverse complement, as appropriate) that exhibits promoter activity to promote expression of a target gene in an appropriate tissue. In another embodiment, the promoter is an EF1a or EF1α promoter, which may or may not contain the EF-1α intron A sequence. In a particular embodiment, the EF1a or EF1α promoter has the nucleotide sequence of SEQ ID NO: 10 (or the nucleotide sequence of the first 230 nucleotides of SEQ ID NO: 10, not including the EF1α intron A). Alternatively, the EF1α promoter is at least a 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotide fragment of SEQ ID NO: 10, and has promoter activity to promote expression of a target gene (or its reverse complement, as appropriate) in an appropriate tissue.In some embodiments, the promoter comprises a nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to SEQ ID NO:9, SEQ ID NO:10, or its reverse complement; or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof therein to promote GALT gene expression in appropriate tissues. In some alternative embodiments, the promoter comprises or consists of a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, a simian virus (SV40) promoter, a dihydrofolate reductase promoter, a β-actin promoter, a phosphoglycerol kinase (PGK) promoter, a P5 promoter, a Ubc promoter, a tetracycline response element promoter, a UAS promoter, an Ac5 promoter, a polyhedrin promoter, a calmodulin-dependent protein kinase II-α (CaMKIIα) promoter, a galactose promoter, a GALT promoter, a GDS promoter, an alcohol dehydrogenase promoter, an H1 promoter, a U6 promoter, or an alpha-1-antitrypsin promoter. In some embodiments, the β-actin promoter is a chicken β-actin ("CBA") promoter or a human β-actin promoter.

[0126] In some embodiments, the expression region is in an antisense (e.g., reverse complement) orientation, or a sense orientation. In some embodiments, the vector comprises two or more expression regions. In some embodiments, the two or more expression regions comprise one in an antisense orientation and another in a sense orientation (i.e., as in scAAV).

[0127] In some embodiments, the expression region further comprises a nucleic acid encoding a polyadenylation (poly(A)) signal 3' of the target gene coding sequence, such that the expressed mRNA has a poly A tail. In some embodiments, the nucleic acid encoding the poly(A) signal comprises the bovine growth hormone (bGH) poly(A) tail signal or the simian virus 40 (SV40) poly(A) tail signal. In some embodiments, the poly A signal has the nucleotide sequence of SEQ ID NO:15 (bGH poly A signal) or SEQ ID NO:16 (SV40 poly A signal) (or its reverse complement), and the poly A signal has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to SEQ ID NO:15, SEQ ID NO:16, or its reverse complement; or has 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof.

[0128] In some embodiments, the expression cassette further comprises a regulatory element that can enhance expression of the target gene. In one embodiment, the expression region or expression cassette further comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE). In some embodiments, the WPRE is downstream (3') of the GALT coding sequence and upstream (5') of the poly(A) tail signal. In some embodiments, the WPRE comprises a nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to SEQ ID NO: 14 (or its reverse complement), which enhances target gene expression; or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof therein. In some embodiments, the WPRE comprises SEQ ID NO: 14 (or its reverse complement). In another embodiment, the expression cassette has an intron or chimeric intron sequence that enhances target gene expression. The intron sequence can be inserted between the promoter and the hGALT coding sequence. In certain embodiments, the intron has a nucleotide sequence of SEQ ID NO:17 (or its reverse complement), or a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to SEQ ID NO:17 (or its reverse complement) that enhances target gene expression; or having 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof therein.

[0129] In some embodiments, the recombinant AAV vector can be packaged into recombinant AAV virions.

[0130] In some embodiments, a recombinant self-complementary AAV (scAAV) vector is provided. In some embodiments, the scAAV comprises an expression region, at least three ITRs, and a nucleic acid that is reverse complementary to the expression region. In some embodiments, the at least three ITRs comprise at least one scAAV ITR. In some embodiments, the scAAV vector is in the following order: at least one of the three ITRs, the expression region, the scAAV ITR, a nucleic acid that is reverse complementary to the expression region, and at least one of the three ITRs. In some embodiments, the at least three ITRs comprise at least two scAAV ITRs. In some embodiments, the scAAV vector is in the following order: at least one of the two scAAV ITRs, the expression region, at least one ITR, a nucleic acid that is reverse complementary to the expression region, and at least another one of the two scAAV ITRs. In some embodiments, a scAAV vector plasmid is provided, the scAAV plasmid encoding the scAAV. In some embodiments, the scAAV vector plasmid encoding the scAAV comprises at least two ITRs and an expression region, where at least one of the at least two ITRs is an scAAV ITR. In an embodiment, as described above, the 5' ITR can have the nucleotide sequence of SEQ ID NO: 11, and the modified ITR is located at the 3' end of the expression cassette and can have the nucleotide sequence of SEQ ID NO: 13. Figures 5 and 6 show plasmids and their nucleotide sequences, respectively, that can produce scAAV.

[0131] Thus, an expression cassette is provided that can be incorporated into an AAV vector for gene replacement expression of a target gene. In a particular embodiment, the expression cassette can have elements arranged as follows: 5'AAV2ITR-CAG promoter sequence-hGALT coding sequence-SV40 polyA signal sequence-3'scAAV2 ITR. In an embodiment, the expression cassette has the nucleotide sequence of SEQ ID NO: 3. In another embodiment, the gene expression cassette can have elements arranged as follows: 5'AAV2ITR-CAG promoter (CMV enhancer-CBA promoter-intron sequence)-hGALT coding sequence-WPRE sequence-bGH polyA signal sequence-3'AAV2ITR sequence. In an embodiment, the expression cassette has the nucleotide sequence of SEQ ID NO: 4 (or its reverse complement). In another embodiment, the expression cassette can have elements arranged as follows: 5'ITR-EF1α promoter sequence-hGALT coding sequence-WPRE sequence-bGH polyA signal sequence-3'AAV2 ITR. In an embodiment, the expression cassette has the nucleotide sequence of SEQ ID NO: 5 (or its reverse complement).In an embodiment, the expression cassette has the nucleotide sequence of SEQ ID NO: 5 (or its reverse complement).

[0132] In some embodiments, the scAAV vector is then packaged into recombinant AAV virions.

[0133] In some embodiments, the expression region comprises a nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or a reverse complement thereof; or 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof therein, and is an expression cassette that expresses hGALT in a suitable human tissue. In some embodiments, the expression region or expression cassette has SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or a reverse complement thereof.

[0134] AAV Vector Plasmids In some embodiments, AAV vector plasmids are provided that can be used to prepare recombinant AAV viral particles having a recombinant genome with a nucleotide sequence encoding a target gene operably linked to a regulatory element that facilitates expression in an appropriate tissue. The plasmids provided herein generally have an origin of replication and a selectable marker, allowing the plasmid to replicate and be used in a host cell to generate the recombinant AAV viral particles described herein. Exemplary plasmids, and their sequences, are shown in Figures 1-6. Plasmids provided herein include plasmids that contain the expression cassettes described herein. In particular, the AAV vector plasmids can contain an expression cassette having a nucleotide sequence of SEQ ID NO: 3, 4, or 5. In certain embodiments, the AAV vector plasmid comprises a nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or less identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21; or having 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions, additions, deletions, or combinations thereof therein. The AAV vector plasmid may have the nucleotide sequence of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0135] In some embodiments, the AAV vector plasmid further comprises a bacterial expression region. In some embodiments, the bacterial expression region comprises a bacterial promoter and a nucleic acid encoding a bacterial selection region. In some embodiments, the nucleic acid encoding the bacterial selection region is operably linked to the bacterial promoter. In some embodiments, the nucleic acid encoding the bacterial selection region comprises a nucleic acid encoding an antibiotic resistance gene or protein. In some embodiments, the antibiotic resistance gene or protein comprises an ampicillin resistance gene (AmpR) or a kanamycin resistance gene sequence (kanR). In some embodiments, the bacterial promoter comprises an AmpR promoter or a KanR promoter. In some embodiments, the AAV vector plasmid further comprises an origin of replication. In some embodiments, the origin of replication comprises a CMV origin of replication (ori). In some embodiments, the AAV vector plasmid further comprises a eukaryotic expression region. In some embodiments, the eukaryotic expression region comprises a eukaryotic promoter and a nucleic acid encoding a eukaryotic selection region. In some embodiments, the nucleic acid encoding the eukaryotic selection region is operably linked to the eukaryotic promoter. In some embodiments, the eukaryotic promoter comprises a nucleic acid catabolite activator protein (CAP) binding site or a lactose (lac) promoter. In some embodiments, the eukaryotic selection region comprises a lac operator. In some embodiments, the plasmid comprises an M13 reverse primer region.

[0136] AAV virion In some embodiments, a recombinant AAV virion is provided.In some embodiments, the AAV virion comprises AAV capsid protein and a recombinant AAV vector having a nucleotide sequence encoding hGALT operably linked to a regulatory element.In some embodiments, the recombinant AAV vector is a recombinant scAAV vector.In some embodiments, the recombinant AAV or scAAV vector is single-stranded DNA.In some embodiments, the AAV capsid protein encapsulates the recombinant AAV vector.

[0137] In some embodiments, the AAV capsid proteins include VP1, VP2, and VP3. The capsid preferably has tropism for appropriate cells and tissues, such as, for example, neural tissue, CNS, liver, etc. In some embodiments, the capsid proteins include AAV1 capsid protein, AAV2 capsid protein, AAV3 capsid protein, AAV4 capsid protein, AAV5 capsid protein, AAV6 capsid protein, AAV7 capsid protein, AAV8 capsid protein, AAV9 capsid protein, AAV-DJ capsid protein, AAV-DJ / 8 capsid protein, AAV-Rh10 capsid protein, AAV-retrocapsid protein, AAV-PHP.B capsid protein, AAV8-PHP.eB capsid protein, or AAV-PHP.S capsid protein. In certain embodiments, the rAAV particles have an AAV9 capsid protein, e.g., having the amino acid sequence of SEQ ID NO: 18. Alternatively, the capsid protein has an amino acid sequence that is 99%, 98%, 95%, 90%, or 85% identical to the AAV9 capsid and has the tropism and transduction activity of the AAV9 capsid protein.

[0138] In some embodiments, the isolated nucleic acids and / or rAAVs described herein may be modified and / or selected to enhance targeting of the isolated rAAV to a target tissue (e.g., CNS). Non-limiting methods of modification and / or selection include AAV capsid serotypes (e.g., AAV9), tissue-specific promoters, and / or targeting peptides. In some embodiments, the isolated nucleic acids and rAAVs disclosed herein may comprise an AAV capsid serotype with enhanced targeting to CNS tissues (e.g., AAV9). In some embodiments, the isolated nucleic acids and rAAVs described herein may comprise a tissue-specific promoter. In some embodiments, the isolated nucleic acids and rAAVs described herein may comprise an AAV capsid serotype and tissue-specific promoter with enhanced targeting to CNS tissues. Although AAV9 targets CNS tissues, the rAAV9 vector may transduce other non-CNS tissues, and thus the transgene may be expressed in both the CNS and other tissues outside the CNS under the control of a promoter such as a CAG promoter.

[0139] Methods for obtaining recombinant AAV with desired capsid proteins can be obtained, for example, from US Patent Application Publication No. 2003 / 0138772. Typically, the methods involve culturing a host cell that contains a nucleic acid sequence encoding an AAV capsid protein or a fragment thereof, a functional rep gene, sufficient helper functions to allow packaging of the recombinant AAV vector into the AAV capsid protein, and a recombinant AAV vector plasmid containing the AAV vector. Typically, the capsid protein is a structural protein encoded by the cap gene of AAV. In some embodiments, when the capsid protein comprises VP1, VP2, and VP3, the VP1, VP2, and VP3 are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2, and VP3 are about 87 kDa, about 72 kDa, and about 62 kDa, respectively. In some embodiments, upon translation, the capsid protein forms a spherical 60-mer protein shell around the viral genome. In some embodiments, the capsid protein protects the viral genome, delivers the genome, and / or interacts with the host cell, hi some embodiments, the capsid protein delivers the viral genome to the host in a tissue-specific manner.

[0140] In some embodiments, the components to be cultured in a host cell to package a recombinant AAV vector into an AAV capsid may be provided in trans to the host cell. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell engineered to contain one or more of the required components. In certain embodiments, a host cell is provided that contains a recombinant AAV construct or plasmid with a target gene sequence, a plasmid providing the AAV rep and cap gene sequences, and optionally a construct providing adenoviral helper proteins to produce recombinant viral particles.

[0141] In some embodiments, such stable host cells may contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein in the discussion of regulatory elements suitable for use with transgenes. In some embodiments, the selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be made that is derived from 293 cells (containing El helper functions under the control of a constitutive promoter) but contains the rep and / or cap proteins under the control of an inducible promoter. Still other stable host cells may be made by one of skill in the art.

[0142] The recombinant AAV vectors, rep sequences, cap sequences, and helper functions useful for producing the rAAV described herein can be delivered to the packaging host cell using any suitable genetic element (vector, e.g., plasmid). The selected genetic element can be delivered by any suitable method, including those described herein. The methods used to construct any of the components disclosed herein are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for producing rAAV virions are known, and the present disclosure does not limit the selection of a suitable method. See, e.g., K. Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745.

[0143] In some embodiments, recombinant AAV can be produced using a triple transfection method (described in U.S. Pat. No. 6,001,650). Typically, recombinant AAV can be produced by transfecting a host cell with a recombinant AAV vector (including a transgene) packaged in an AAV particle, an AAV helper function vector, and an auxiliary function vector. The AAV helper function vector encodes "AAV helper function" sequences (i.e., rep and cap) that function in trans for efficient AAV replication and encapsidation with the cap gene encoding the capsid protein of the desired serotype, e.g., AAV9 capsid. In some embodiments, the AAV helper function vector can support efficient AAV vector production without producing any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use in the present disclosure include pHLP19, described in U.S. Patent No. 6,001,650, and pRep6cap6 vector, described in U.S. Patent No. 6,156,303, both of which are incorporated herein by reference in their entirety. Accessory function vectors encode nucleotide sequences for non-AAV derived viral and / or cellular functions (i.e., "accessory functions") on which AAV depends for replication. Accessory functions encompass functions required for AAV replication, including, but not limited to, portions involved in transcription of AAV genes, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and activation of AAV capsid assembly. Viral-based accessory functions can be derived from any of the well-known helper viruses, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0144] Cells Transfected host cells are disclosed herein. The term transfection refers to the uptake of foreign DNA by a cell, and a cell is transfected when foreign DNA is introduced through the cell membrane. Examples of transfection methods include Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more foreign nucleic acids, such as nucleotide integration vectors and other nucleic acid molecules, into a suitable host cell.

[0145] In one aspect, a cell is provided. In some embodiments, the cell comprises an AAV second plasmid and an AAV vector plasmid. In some embodiments, the AAV second plasmid comprises rep and cap. In some embodiments, cap encodes VP1, VP2, and VP3. In some embodiments, rep encodes rep78, rep68, rep52, and rep40. In some embodiments, the AAV vector plasmid comprises a recombinant AAV vector or a recombinant scAAV vector. In some embodiments, cap is an AAV9 cap. In some embodiments, the AAV vector plasmid comprises an expression cassette of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0146] In another aspect, a method for producing AAV virions is provided. In some embodiments, the method includes transfecting at least one of a second plasmid and a vector plasmid or an AAV vector construct into a cell. In some embodiments, the vector plasmid or the AAV vector construct includes a recombinant AAV vector or a recombinant scAAV vector. In some embodiments, the second plasmid includes cap and rep. In some embodiments, cap encodes VP1, VP2, and VP3. In some embodiments, rep encodes rep78, rep68, rep52, and rep40. In some embodiments, the vector plasmid or the AAV vector construct includes an expression cassette having a nucleotide sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or a nucleotide sequence of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0147] Treatment method In some embodiments, the AAV virion transduces a cell, or a cell of a tissue or organ. In some embodiments, upon transduction, the AAV vector is released from encapsulation by the capsid protein. In some embodiments, the AAV vector is released into the cytosol or nucleus of the cell. In some embodiments, the transcription machinery of the cell binds to the promoter of the AAV vector. In some embodiments, the cell expresses a nucleic acid encoding a protein. In some embodiments, the cell expresses a protein. In some embodiments, the cell expresses a nucleic acid encoding GALT. In some embodiments, the cell expresses GALT. In some embodiments, GALT undergoes its enzymatic activity (i.e., at least the forward or reverse reaction of interconversion of uridine diphosphate glucose and galactose-1-phosphate to glucose-1-phosphate and uridine diphosphate galactose).

[0148] In some embodiments, a method is provided for treating at least one of the symptoms of galactosemia, insufficient galactose metabolism, GALT deficiency, GALT deficiency, or insufficient galactose metabolism in a subject in need of treatment of at least one of the symptoms of galactosemia, insufficient galactose metabolism, GALT deficiency, GALT deficiency, or insufficient galactose metabolism. In some embodiments, a method is provided for increasing galactose metabolism in a subject. In some embodiments, a method is provided for alleviating a disease state in a subject suffering from galactosemia. In some embodiments, the disease state comprises jaundice, hepatosplenomegaly, hepatocellular failure, hypoglycemia, renal tubular dysfunction, hypotonia, sepsis, cataracts, ataxia, tremor, decreased bone density, or primary ovarian insufficiency. In some embodiments, the method comprises administering a therapeutically effective amount of AAV virions to the subject.

[0149] In some embodiments, the subject has type 1 galactosemia. In some embodiments, the subject does not express GALT protein or expresses undetectable levels of GALT protein. In some embodiments, the subject lacks GALT enzyme activity or has undetectable levels of GALT enzyme activity. In some embodiments, the subject has an increased level of galactose compared to subjects without galactosemia. In some embodiments, the subject has an increased level of GAL-1P compared to subjects without galactosemia. In some embodiments, the subject has an increased level of galactitol compared to subjects without galactosemia. In some embodiments, the subject has a reduced level of expression of GALT protein compared to subjects without galactosemia. In some embodiments, the subject has a reduced level of GALT enzyme activity compared to subjects without galactosemia.

[0150] In some embodiments, a method is provided for reducing galactose levels in a subject in need of such reduction. "Reduced levels" includes a reduction in levels compared to subjects not treated with gene therapy encoding hGALT (including subjects receiving a low galactose diet) or compared to levels characteristic of subjects of similar age and weight as determined in natural history studies of galactosemia patients, which provides a therapeutic difference to the patient by improving one or more symptoms of the disease. The reduced levels can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% reduction compared to baseline. The decrease in level can be about 10% to about 90%, about 20% to about 90%, about 40% to about 90%, about 50% to about 60%, about 70% to about 90%, about 80% to about 90%, or about 90% to about 99% compared to the baseline.

[0151] In some embodiments, the levels of galactose in the liver are reduced by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% compared to subjects prior to administration or compared to similarly situated subjects identified in natural history studies, which reduction provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, the levels of galactose in muscle are reduced by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% compared to subjects prior to administration or compared to similarly situated subjects identified in natural history studies, which reduction provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, the level of galactose in the brain is reduced by about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% compared to subjects prior to administration or compared to similarly situated subjects identified in natural history studies, which reduction provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, the level of galactose in RBCs is reduced by about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% compared to subjects prior to administration or compared to similarly situated subjects identified in natural history studies, which reduction provides a therapeutic difference to the patient by improving one or more symptoms of the disease.In some embodiments, the level of galactose in the ovary is reduced by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% compared to the subject prior to administration or compared to similarly situated subjects identified in natural history studies, which provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, the level of galactose in the eye is reduced by about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% compared to the subject prior to administration or compared to similarly situated subjects identified in natural history studies, which provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, the level of galactose in plasma is reduced by about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% compared to subjects prior to administration or compared to similarly situated subjects identified in natural history studies, which reduction provides a therapeutic difference to the patient by improving one or more symptoms of the disease.

[0152] In some embodiments, there is provided a method for reducing galactitol levels by administering to a subject in need thereof a pharmaceutical composition comprising a recombinant AAV as described herein.In some embodiments, there is provided a method for reducing GAL-1P levels by administering to a subject in need thereof a pharmaceutical composition comprising a recombinant AAV as described herein.

[0153] In some embodiments, the level of galactose is reduced in the liver, muscle, brain, and / or plasma, which provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, the level of galactose is reduced in the liver, muscle, brain, eyes, ovaries, red blood cells, and / or plasma. In some embodiments, the level of galactitol is reduced in the liver, muscle, brain, and / or plasma. In some embodiments, the level of galactitol is reduced in the liver, muscle, brain, eyes, ovaries, red blood cells, and / or plasma. In some embodiments, the level of GAL-1P is reduced in the liver, muscle, brain, and / or plasma, which provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, the level of GAL-1P is reduced in the liver, muscle, brain, eyes, ovaries, red blood cells, and / or plasma, which provides a therapeutic difference to the patient by improving one or more symptoms of the disease. The reduction in levels can be achieved within 1 month, 2 months, 3 months, 6 months, 1 year, or 2 years of gene therapy administration.

[0154] In some embodiments, the levels of galactose are reduced in the liver, muscle, brain, and / or plasma compared to similarly situated subjects, which reduction provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, the levels of galactitol are reduced in the liver, muscle, brain, and / or plasma compared to similarly situated subjects not administered a gene therapy therapy described herein. In some embodiments, the levels of GAL-1P are reduced in the liver, muscle, brain, and / or plasma compared to similarly situated subjects not administered a gene therapy therapy described herein. In some embodiments, the levels of galactose are reduced in the liver, muscle, brain, eye, ovary, and / or plasma compared to similarly situated subjects, which reduction provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, the levels of galactitol are reduced in the liver, muscle, brain, eye, ovary, red blood cells, and / or plasma compared to similarly situated subjects not administered a gene therapy therapy described herein. In some embodiments, the levels of GAL-1P are reduced in liver, muscle, brain, eye, ovary, and / or plasma compared to similarly situated subjects not administered the gene therapy regimen described herein. By "similarly situated subjects" is meant subjects that have similar galactose levels (within about 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than about 1%) in all tissues or in one or more of liver, muscle, brain, eye, ovary, and / or plasma as the subject undergoing the treatment prior to administration of gene therapy. In some embodiments, similarly situated subjects have similar mutations that result in reduced or absent expression of GALT protein and / or GALT enzyme activity as the subject undergoing the treatment.

[0155] In some embodiments, the levels of galactose are reduced in the liver, muscle, brain, and / or plasma compared to similarly situated subjects not treated with a gene therapy regimen described herein but on a low galactose diet. In some embodiments, the levels of galactitol are reduced in the liver, muscle, brain, and / or plasma compared to similarly situated subjects not treated with a gene therapy regimen described herein but on a low galactose diet. In some embodiments, the levels of GAL-1P are reduced in the liver, muscle, brain, and / or plasma compared to similarly situated subjects not treated with a gene therapy regimen described herein but on a low galactose diet.

[0156] In some embodiments, the levels of galactose are reduced in the liver, muscle, brain, eyes, ovaries, and / or plasma compared to similarly situated subjects not treated with a gene therapy regimen described herein but receiving a low galactose diet. In some embodiments, the levels of galactitol are reduced in the liver, muscle, brain, eyes, ovaries, and / or plasma compared to similarly situated subjects not treated with a gene therapy regimen described herein but receiving a low galactose diet. In some embodiments, the levels of GAL-1P are reduced in the liver, muscle, brain, eyes, ovaries, and / or plasma compared to similarly situated subjects not treated with a gene therapy regimen described herein but receiving a low galactose diet.

[0157] In some embodiments, the levels of galactose are reduced in the liver, muscle, brain, and / or plasma, compared to levels in similarly situated subjects identified in natural history studies. In some embodiments, the levels of galactitol are reduced in the liver, muscle, brain, and / or plasma, compared to levels in similarly situated subjects identified in natural history studies. In some embodiments, the levels of GAL-1P are reduced in the liver, muscle, brain, and / or plasma, compared to levels in similarly situated subjects identified in natural history studies.

[0158] In some embodiments, the levels of galactose are reduced in the liver, muscle, brain, eyes, ovaries, red blood cells, and / or plasma, compared to levels in similarly situated subjects identified in natural history studies. In some embodiments, the levels of galactitol are reduced in the liver, muscle, brain, eyes, ovaries, red blood cells, and / or plasma, compared to levels in similarly situated subjects identified in natural history studies. In some embodiments, the levels of GAL-1P are reduced in the liver, muscle, brain, eyes, ovaries, red blood cells, and / or plasma, compared to levels in similarly situated subjects identified in natural history studies.

[0159] In some embodiments, the level of Gal-1P in liver is reduced by about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% compared to the subject prior to administration or compared to similarly situated subjects identified in natural history studies, which reduction provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, the level of Gal-1P in muscle is reduced by about 40%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% compared to the subject prior to administration or compared to similarly situated subjects identified in natural history studies. In some embodiments, the level of Gal-1P in the brain is reduced by about 30%, about 40%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% compared to subjects prior to administration or similarly situated subjects identified in natural history studies, which provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, the level of Gal-1P in RBCs is reduced by about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% compared to subjects prior to administration or similarly situated subjects identified in natural history studies, which provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, the level of Gal-1P in the ovary is reduced by about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% compared to subjects prior to administration or similarly situated subjects identified in natural history studies, which provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, the level of Gal-1P in the eye is reduced by about 35%, about 40%, about 45%, or about 50% compared to subjects prior to administration or similarly situated subjects identified in natural history studies, which provides a therapeutic difference to the patient by improving one or more symptoms of the disease.

[0160] In some embodiments, liver galactitol levels are reduced by about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% compared to subjects prior to administration or compared to similarly situated subjects identified in natural history studies, which reduction provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, galactitol levels in muscle are reduced by about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% compared to subjects prior to administration or compared to similarly situated subjects identified in natural history studies, which reduction provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, the levels of galactitol in the brain are reduced by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% compared to subjects prior to administration or compared to similarly situated subjects identified in natural history studies, which reduction provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, plasma galactitol levels are reduced by about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% compared to subjects prior to administration or compared to similarly situated subjects identified in natural history studies, which reduction provides a therapeutic difference to the patient by improving one or more symptoms of the disease.In some embodiments, the level of galactitol in RBCs is reduced by about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% compared to subjects prior to administration or similarly situated subjects identified in natural history studies, which provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, the level of galactitol in ovaries is reduced by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% compared to subjects prior to administration or similarly situated subjects identified in natural history studies, which provides a therapeutic difference to the patient by improving one or more symptoms of the disease. In some embodiments, galactitol levels in the eye are reduced by about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% compared to subjects prior to administration or compared to similarly situated subjects identified in natural history studies, which reduction provides a therapeutic difference to the patient by improving one or more symptoms of the disease.

[0161] In some embodiments, the method for increasing GALT protein expression in a subject who needs to increase GALT protein expression is provided.The increase in level can be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200% increase compared to baseline. The increase in levels can be about 10% to about 200%, about 20% to about 200%, about 40% to about 200%, about 50% to about 200%, about 70% to about 200%, about 80% to about 200%, about 90% to about 200%, about 100% to about 200%, about 110% to about 200%, about 120% to about 200%, about 130% to about 200%, about 150% to about 200%, about 160% to about 200%, about 170% to about 200%, or about 180% to about 200% increase compared to baseline. In some embodiments, GALT protein expression is increased in liver, muscle, brain, eye, and / or plasma compared to GALT protein expression levels prior to administration of gene therapy or compared to levels in similarly situated subjects as determined in natural history studies. In some embodiments, GALT protein expression is increased in the brain. In some embodiments, GALT protein expression is increased in cortical and / or cerebral tissue. In some embodiments, GALT protein expression is increased in neuronal and / or glial cells. In some embodiments, GALT protein expression is increased in Purkinje neurons. In some embodiments, GALT protein expression is increased in plasma. In some embodiments, GALT protein expression is increased in red blood cells. In all cases, the increase is relative to GALT protein expression levels prior to gene therapy administration or to levels in similarly situated subjects as determined in natural history studies, and the decrease provides a therapeutic difference to the patient by improving one or more symptoms of the disease.

[0162] In some embodiments, methods are provided for increasing GALT enzyme activity in a subject in need thereof. In some embodiments, GALT enzyme activity is increased in liver, muscle, eye, plasma, and / or brain. In some embodiments, GALT enzyme activity is increased in brain. In some embodiments, GALT enzyme activity is increased in cortical and / or cerebral tissue. In some embodiments, GALT enzyme activity is increased in neuronal and / or glial cells. In some embodiments, GALT enzyme activity is increased in Purkinje neurons. In some embodiments, GALT enzyme activity is increased in liver. In some embodiments, GALT enzyme activity is increased in muscle. In some embodiments, GALT enzyme activity is increased in plasma. In some embodiments, GALT enzyme activity is increased in red blood cells. In all cases, the increase is relative to GALT protein expression levels prior to gene therapy administration or to levels in similarly situated subjects as determined in natural history studies, and the decrease provides a therapeutic difference to the patient by improving one or more symptoms of the disease.

[0163] In some embodiments, GALT enzyme activity in liver is increased by about 1000%, about 1500%, about 2000%, about 2500%, about 3000%, about 3500%, about 4000%, about 4500%, about 5000%, about 5500%, about 6000%, about 6500%, about 7000%, or about 7500% compared to WT / WT subjects without GALT enzyme deficiency. In some embodiments, GALT enzyme activity in brain is increased by about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, or about 550% compared to WT / WT subjects without GALT enzyme deficiency. In some embodiments, GALT enzyme activity in skeletal muscle is increased by about 2000%, about 2500%, about 3000%, about 3500%, about 4000%, about 4500%, about 5000%, about 5500%, about 6000%, about 6500%, about 7000%, about 7500%, about 8000%, about 8500%, about 9000%, about 9500, or about 10,000% compared to WT / WT subjects without GALT enzyme deficiency. In some embodiments, GALT enzyme activity in eye is increased by about 90%, about 100%, about 130%, about 150%, about 200%, about 250%, or about 300% compared to WT / WT subjects without GALT enzyme deficiency.

[0164] In some embodiments, GALT enzyme activity in the liver is increased by about 35%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200% compared to a WT / WT subject without GALT enzyme deficiency. In some embodiments, GALT enzyme activity in the brain is increased by about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% compared to a WT / WT subject without a GALT enzyme deficiency. In some embodiments, GALT enzyme activity in muscle is increased by about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, or about 250% compared to a WT / WT subject without a GALT enzyme deficiency.

[0165] In some embodiments, a method of improving motor coordination and / or neuromuscular coordination in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of an AAV virion of the present disclosure, wherein the improvement in the motor and / or neuromuscular disorder is reduced compared to a similarly situated subject not administered the gene therapy regimen. Motor coordination and / or neuromuscular coordination can be assessed according to methods known in the art.

[0166] In some embodiments, a method of improving motor performance in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of an AAV virion of the present disclosure, which improves motor performance compared to a similarly situated subject not administered a gene therapy regimen. Motor performance can be assessed according to methods known in the art.

[0167] In some embodiments, a method of improving spatial learning is provided, comprising administering to a subject a therapeutically effective amount of an AAV virion of the present disclosure, wherein the improvement in spatial learning is improved compared to a similarly situated subject not administered a gene therapy regimen. Spatial learning can be assessed according to methods known in the art.

[0168] In some embodiments, a method of reducing and / or reversing ovarian failure is provided comprising administering to a subject a therapeutically effective amount of an AAV virion of the present disclosure, wherein the reduction and / or reversal of ovarian failure is relative to a similarly situated subject not administered a gene therapy regimen. Ovarian failure can be assessed according to methods known in the art.

[0169] In some embodiments, a method of modulating follicle stimulating hormone, luteinizing hormone, and / or anti-Mullerian hormone is provided comprising administering to a subject a therapeutically effective amount of an AAV virion of the present disclosure, wherein the modulation of follicle stimulating hormone, luteinizing hormone, and / or anti-Mullerian hormone is relative to a similarly situated subject not administered a gene therapy regimen. Modulation of follicle stimulating hormone, luteinizing hormone, and / or anti-Mullerian hormone can be assessed according to methods known in the art.

[0170] In some embodiments, a method is provided for inhibiting cataract formation or promoting cataract resorption in a subject in need of inhibiting cataract formation or promoting cataract resorption. In some embodiments, a method is provided for limiting the severity or risk of cataract formation or promoting cataract resorption in a subject in need of inhibiting cataract formation or promoting cataract resorption, where the severity and / or risk of cataract formation is reduced compared to before gene therapy administration or compared to similarly situated subjects identified in natural history studies. In some embodiments, a method is provided for limiting the severity or risk of cataract formation or promoting cataract resorption in a subject in need of inhibiting cataract formation or promoting cataract resorption, where the severity and / or risk of cataract formation is reduced compared to similarly situated subjects not administered gene therapy. In some embodiments, the AAV virion is administered before cataract develops.

[0171] In some embodiments, methods are provided that improve (i.e., increase the rate or amount of) weight gain in a subject in need thereof compared to the subject's rate of weight gain prior to administration of gene therapy or compared to the rate of weight gain in a similarly situated subject not administered gene therapy. In some embodiments, methods are provided that reduce prepubertal growth retardation in a subject in need thereof.

[0172] In some embodiments, methods are provided for reducing the severity of visceral neuromuscular disorders, where the severity of the visceral neuromuscular disorder is reduced compared to before administration of gene therapy or compared to similarly situated subjects identified in natural history studies. In some embodiments, methods are provided for reducing the severity of visceral neuromuscular disorders in a subject in need thereof, where the severity and / or risk of visceral neuromuscular disorder is reduced compared to similarly situated subjects not administered gene therapy. Visceral neuromuscular disorders can be assessed according to methods known in the art.

[0173] In some embodiments, methods are provided for reducing the severity of neurological and / or socio-emotional disorders, where the severity of the neurological and / or socio-emotional disorders is reduced compared to before gene therapy administration or compared to the level of similarly situated subjects identified in natural history studies. In some embodiments, methods are provided for reducing the severity of neurological and / or socio-emotional disorders, where the severity is reduced compared to similarly situated subjects not administered gene therapy. Neurological and / or socio-emotional disorders include locomotor function / activity, e.g., tremor and ataxia, anxiety and / or depression. Neurological and / or socio-emotional disorders can be assessed according to methods known in the art.

[0174] In some embodiments, the AAV virion is administered before the onset of puberty. In some embodiments, the AAV virion is administered to a young pediatric subject. In some embodiments, the "young pediatric subject" is about 4 years old, about 3.5 years old, about 3 years old, about 2.5 years old, about 2 years old, about 1.5 years old, about 1 year old, or about 6 months old.

[0175] In some embodiments, the AAV virions are administered to a pediatric subject who is about 5 years old, about 6 years old, about 7 years old, about 8 years old, about 9 years old, about 10 years old, about 11 years old, about 12 years old, about 13 years old, about 14 years old, about 15 years old, about 16 years old, or about 17 years old.

[0176] In some embodiments, the methods described herein further comprise monitoring levels of galactose, GAL-1P, and / or galactitol. Methods for measuring levels of galactose, GAL-1P, and / or galactitol are known in the art and include immunohistochemistry, enzyme assays, mass spectrometry (GC / MS and LC / MS), and the like.

[0177] In some embodiments, the subject has a GALT mutation that includes at least one of Q188R, N314D, L218L, S135L, K285N, L195P, T138M, Y209C, and IVS2-2A>G. In some embodiments, the subject has a deletion of about 5 kb in one or both genes encoding GALT, which deletion was first identified in individuals of Ashkenazi Jewish ancestry. In some embodiments, the subject has classic galactosemia, clinical variant galactosemia, or Duarte galactosemia.

[0178] In some embodiments, administering or treating comprises intravenous, intraarterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intraventricular, subretinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, or by inhalation. In some embodiments, the method comprises contacting an AAV virion, or an effective amount thereof, with at least one of the liver, spleen, muscle, kidney, blood, lens, eye, cerebellum, brain stem, basal ganglia, hypothalamus, preoptic area, hippocampus, striatum, cortex, motor cortex, prefrontal cortex, somatosensory cortex, temporal cortex, visual cortex, occipital lobe, temporal lobe, parietal lobe, frontal lobe, bone, reproductive organs, ovary, testis, skin surface, prostate, uterus, or pancreas of a subject.

[0179] In some embodiments, the administered, treated, contacted, or effective amount is at least 10 4 , 10 5 , 10 6 , 10 7 , 10 8, 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 or less than plaque forming units (PFU) (PFU / mL) or virions of AAV.

[0180] In some embodiments, the number of administrations or treatments may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or less. In some embodiments, successful treatment and / or repair is determined when one or more of the following are detected: alleviation or amelioration of one or more symptoms of the disease, disorder, or condition being treated; reduction in the severity of the disease, disorder, or condition being treated; stabilization (i.e., not worsening) of the disease, disorder, or condition; delay or slowing of the progression of the disease, disorder, or condition; improvement or alleviation of the disease, disorder, or condition. In some embodiments, successful treatment is determined by detecting the presence of a 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 a polypeptide encoded by the repaired target polynucleotide in one or more cells, tissues, or organs isolated from the subject.

[0181] In some embodiments, recombinant AAV (rAAV) virions may be administered in a composition at a concentration of at least or less than 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0% weight, volume, or molar ratio.

[0182] In some embodiments, the composition comprises a suitable carrier. A suitable carrier can be selected for the indication for which the rAAV is targeted. For example, one suitable carrier includes saline, which can be formulated with various buffer solutions (e.g., phosphate buffered saline). Other suitable examples of carriers include, but are not limited to, sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The compositions disclosed herein can optionally include other pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to the rAAV virions and carrier(s). Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0183] In certain embodiments, rAAV virions are administered in a pharmaceutical composition comprising phosphate buffered saline (PBS), pH 7.3, and 0.001% pharma- ceutically acceptable non-ionic surfactant, such as Pluronic F-68 (PF68), or other suitable pharma- ceutically acceptable buffer or excipient. The formulation may be frozen until ready for use, then thawed and administered. In some embodiments, the pharmaceutical composition is 10 mM Tris, 150 mM NaCl, 0.02% poloxamer 188, 1 mM MgCl 2(adjusted to pH 8.0). In some embodiments, the composition may further comprise a sugar, sorbitol, or trehalose. In some embodiments, the sugar is at least 0.0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3.0% by weight, volume, or molar ratio, or less. In some embodiments, the sugar is sucrose, glucose, or lactose. In some embodiments, sorbitol is at least 0.0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3.0% weight, volume, or molar ratio, or less. In some embodiments, trehalose is at least 0.0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3.0% weight, volume, or molar ratio, or less.

[0184] In some embodiments, the compositions disclosed herein may include rAAV virions alone or in combination with one or more other viruses (e.g., a second rAAV virion encoding one or more different transgenes). In some embodiments, the compositions may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAV virions, each of which has one or more different transgenes.

[0185] Recombinant AAV virions can be administered in sufficient amounts to transfect cells of the desired tissue and provide sufficient levels of gene transfer and expression without undue adverse effects. In some embodiments, acceptable routes of administration include, but are not limited to, direct delivery to a selected organ (e.g., injection into the liver, skeletal muscle), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parenteral routes of administration. In some embodiments, the route of administration can be by intraventricular injection. Routes of administration can be combined as needed. The dose of rAAV virion required to achieve a particular "therapeutic effect", e.g., units of dose in genome copies per kilogram of body weight (GC / kg), units of dose in genome copies per brain volume, and units of dose in genome copies per CSF volume, will vary based on a variety of factors, including, but not limited to, the route of administration of the rAAV virion, the level of gene or RNA expression required to achieve the therapeutic effect, the particular disease or disorder being treated, and the stability of the gene or RNA product.

[0186] In some embodiments, an effective amount of rAAV virions is an amount sufficient to infect an animal and target the desired tissue. The effective amount depends primarily on factors such as the subject's species, age, weight, health, and tissue to be targeted, and therefore may vary between animals and tissues. For example, an effective amount of rAAV virions is about 10 6 ~10 16 genome copy number (e.g., 1 × 10 6 ~1×10 16 In the methods disclosed herein, a therapeutically effective amount can range from about 1 ml to about 100 ml of a solution containing 6×10 13 vg / kg~6×10 14 vg / kg, 7 × 10 13 vg / kg, 8×10 13 vg / kg, 9×10 13 vg / kg, 1×10 14 vg / kg, 2×10 14 vg / kg, 3 × 1014 vg / kg, 4 × 10 14 vg / kg, or 5 × 10 14 vg / kg (or genome copies per brain volume, CSF volume, or other measure appropriate for ICV or ICM delivery). In some embodiments, 11 ~10 12 Dosages of about 10 / kg or an appropriate measured rAAV genome copy number may be appropriate. 11 ~10 13 Dosages of about 10 / kg or an appropriate measured rAAV genome copy number may be appropriate. 11 ~10 14 Dosages of about 10 / kg or an appropriate measured rAAV genome copy number may be appropriate. 11 ~10 15 Dosages of about 1×10 / kg or an appropriate measured rAAV genome copy number may be appropriate. 14 Dosage of vector genome (vg) copies / kg or an appropriate measure may be appropriate. In some embodiments, the dose may be varied or reduced when specifically targeting one or more brain region(s). In some embodiments, the dose may be about 10 7 ~10 8 Dosage of rAAV genome copies / kg or an appropriate measure may be appropriate. In some embodiments, about 10 8 ~10 9 Dosage of rAAV genome copies / kg or an appropriate measure may be appropriate. In some embodiments, about 10 9 ~10 10 Dosage of rAAV genome copies / kg or an appropriate measure may be appropriate. In some embodiments, about 10 10 ~10 11 Dosing of rAAV genome copies / kg or other suitable measure may be appropriate.

[0187] In some embodiments, a potential side effect of administering an AAV virion to a subject may be an immune response in the subject to the AAV virion, including inflammation, which may depend on the route of administration, particularly if the administration of the AAV virion is systemic. In some embodiments, the subject may be immunosuppressed prior to administration of one or more rAAVs described herein.

[0188] As used herein, "immunosuppressed" or "immunosuppression" refers to a reduction in the activation or effectiveness of the immune response in a subject. Immunosuppression can be induced in a subject using one or more (e.g., a plurality, such as 2, 3, 4, 5, or more) agents, including, but not limited to, rituximab, methylprednisolone, prednisolone, sirolimus, immunoglobulin injections, prednisone, methotrexate, interleukin-6 inhibitors, anti-interleukin-6 antibodies, interleukin-6 receptor inhibitors, anti-interleukin-6 receptor antibodies, and any combination thereof.

[0189] In some embodiments, the methods disclosed herein further include inducing immunosuppression in the subject (e.g., administering one or more immunosuppressants) prior to administering an rAAV virion (e.g., an rAAV virion or pharmaceutical composition disclosed herein) to the subject. In some embodiments, the subject is immunosuppressed (e.g., immunosuppression is induced in the subject) for about 30 days to about 0 days (e.g., any time between 30 days prior to administration of the rAAV virion, inclusive) prior to administering the rAAV virion to the subject. In some embodiments, the subject is pretreated with an immunosuppressant (e.g., rituximab, sirolimus, and / or prednisone) for at least 7 days.

[0190] In some embodiments, the subject's immunosuppression is maintained during and / or after administration of the rAAV virion or pharmaceutical composition, hi some embodiments, the subject can be immunosuppressed (e.g., administered one or more immunosuppressive agents) for between one day and one year after administration of the rAAV virion or pharmaceutical composition.

[0191] In some embodiments, the rAAV virion composition comprises a virion that is specifically present in the composition at a high concentration (e.g., −10 13 The rAAV is formulated to reduce aggregation of AAV virions in compositions present at concentrations of 1000 ng / ml or more. Methods for reducing rAAV aggregation include, for example, the addition of detergents, adjusting pH, adjusting salt concentration, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178).

[0192] In some embodiments, these formulations may contain at least about 0.1% or more of the active compound, although the percentage of the active ingredient(s) may of course vary and may conveniently be from about 1 or 2% to about 70% or 80% or more by weight or volume of the total formulation. Naturally, the amount of active compound in each therapeutically useful composition may be prepared so that a suitable dosage is obtained in any unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be considered by those skilled in the art of preparing such pharmaceutical formulations. In light of this, various dosages and treatment regimens may be desired.

[0193] In some embodiments, it may be desirable to deliver the rAAV virions in a suitably formulated pharmaceutical composition disclosed herein subcutaneously, intrapancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, or orally, intraperitoneally, intracerebroventricularly, or by inhalation. In some embodiments, the administration techniques described in U.S. Patent Nos. 5,543,158, 5,641,515, and 5,399,363 may be used to deliver the rAAV. In some embodiments, the preferred mode of administration may be by intracerebroventricular or intrathecal injection.

[0194] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, or mixtures thereof in oil. These preparations contain a preservative to prevent the growth of microorganisms under ordinary storage and use conditions. In many cases, the form can be sterile and fluid to the extent that it can be easily squirted. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars or sodium chloride, may be included. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0195] In the case of administration of injectable aqueous solutions, for example, the solution can be suitably buffered if necessary, and the liquid diluent is first made isotonic with sufficient saline or glucose. These particular aqueous solutions can be suitable for intravenous, intramuscular, subcutaneous, intracerebroventricular, or intraperitoneal administration. In this connection, a sterile aqueous medium can be used. For example, a dosage can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection fluid or injected at the proposed site of injection (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

[0196] Sterile injectable solutions are prepared by incorporating the required amount of active rAAV virions in a suitable solvent with various other ingredients listed herein, followed by filter sterilization as necessary. In general, dispersions can be prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other required ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preparation method can be vacuum drying and freeze-drying techniques, which can produce a powder consisting of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating agent, diluent, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that do not cause allergic or similar adverse reactions when administered to a host.

[0197] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc., can be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, transgenes delivered by rAAV vectors can be formulated for delivery either encapsulated in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, etc.

[0198] Such formulations can be used for the introduction of pharma- ceutically acceptable formulations of the nucleic acids or rAAV constructs disclosed herein. The formation and use of liposomes is generally known to those skilled in the art. Recently, liposomes with improved serum stability and circulation half-time have been developed (U.S. Pat. No. 5,741,516). In addition, various methods have been described for liposomes and liposome-like preparations as potential drug carriers (U.S. Pat. Nos. 5,567,434, 5,552,157, 5,565,213, 5,738,868, and 5,795,587).

[0199] Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are not subject to the DNA length constraints typical of virus-based delivery systems. Liposomes have been effectively used to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors, and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials have been completed that validate the efficacy of liposome-mediated drug delivery.

[0200] Liposomes are formed from phospholipids that disperse in aqueous media and spontaneously form multilamellar centripetal bilayer vesicles, also called multilamellar vesicles (MLVs). MLVs generally have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs), which have diameters in the range of 200-500 angstroms and contain aqueous solution in their cores.

[0201] Alternatively, nanocapsule formulations of rAAV virions can be used. Nanocapsules can generally encapsulate substances stably and reproducibly. To avoid side effects caused by polymer overloading in cells, such ultrafine particles (approximately 0.1 pm in size) should be designed using polymers that can be degraded in vivo. Biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements are considered for use.

[0202] In addition to the delivery methods described above, the following techniques are also contemplated as alternative methods for delivering the rAAV composition to a host.

[0203] Sonophoresis (e.g., ultrasound) has been used as a device to enhance the rate and efficacy of drug penetration into and through the circulatory system, as described in U.S. Patent No. 5,656,016. Other drug delivery alternatives under consideration include intraosseous injection (U.S. Patent No. 5,779,708), microchip devices (U.S. Patent No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Patent Nos. 5,770,219 and 5,783,208), and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0204] In some embodiments, the methods may include administering one or more additional therapeutic agents to a subject to whom the rAAV or pharmaceutical composition described herein has been administered.

[0205] In some embodiments, administration of a rAAV virion described herein to a subject enhances expression of GALT by 10-fold compared to a control. In some embodiments, administration of a rAAV virion described herein to a subject enhances expression of GALT by 5-fold to 100-fold compared to a control (e.g., 5-fold to 10-fold, 10-fold to 15-fold, 10-fold to 20-fold, 15-fold to 25-fold, 20-fold to 30-fold, 25-fold to 35-fold, 30-fold to 40-fold, 35-fold to 45-fold, 40-fold to 60-fold, 50-fold to 75-fold, 60-fold to 80-fold, 75-fold to 100-fold compared to a control).

[0206] In some embodiments, administration of a rAAV virion described herein to a subject promotes a 5%-200% (e.g., 5-50%, 25-75%, 50-100%, 75-125%, 100-200%, or 100-150%, etc.) increase in expression of GALT in the subject compared to a control subject (e.g., promotes expression of GALT in the CNS of the subject).

[0207] As used herein, "treating" refers to the application or administration of a composition (e.g., an isolated nucleic acid or rAAV described herein) to a subject having a disease or disorder associated with low levels of GALT expression (e.g., GALT deficiency) for the purpose of curing, curing, ameliorating, alleviating, altering, relieving, promoting, ameliorating, or affecting the disorder, symptoms of the disease, or predisposition to the disease.

[0208] Alleviating a disease associated with low levels of GALT expression (e.g., GALT deficiency) includes delaying the onset or progression of the disease or reducing the severity of the disease. Alleviating a disease does not necessarily require a curative outcome. As used herein, "delaying" the onset of a disease means delaying, preventing, slowing, retarding, stabilizing, and / or postponing the progression of the disease. This delay may vary in duration depending on the disease history and / or the individual being treated. A method of "delaying" or alleviating the onset of cancer, or delaying the onset of a disease, is a method that reduces the probability of developing one or more symptoms of the disease within a given time frame and / or reduces the extent of symptoms within a given time frame compared to not using the method. Such comparisons are typically based on clinical studies with a sufficient number of subjects to obtain statistically significant results.

[0209] In particular, administration of the rAAV virions described herein to a human subject suffering from a GALT deficiency will result in a reduction in one or more biomarkers or characteristics of the disease within 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, 50 weeks, or within one year of administration.

[0210] "Onset" or "progression" of a disease refers to the initial symptoms and / or subsequent progression of a disease. Onset of a disease can be detectable and evaluated using standard clinical techniques well known in the art. However, onset also refers to undetectable progression. As used herein, the term onset or progression refers to the biological course of a condition. Onset includes occurrence, recurrence, and onset. As used herein, "onset" or "onset" of a disease can be associated with low levels of GALT expression (e.g., GALT deficiency).

[0211] In some embodiments, the subject has or is suspected to have a disease or disorder associated with low levels of GALT expression (e.g., GALT deficiency). In some embodiments, the subject with a disease or disorder associated with low levels of GALT expression (e.g., GALT deficiency) has at least one GALT allele with a loss-of-function mutation (e.g., associated with GALT deficiency). In some embodiments, the GALT allele with a loss-of-function mutation (e.g., associated with GALT deficiency) comprises a frameshift mutation, a splice site mutation, a missense mutation, a truncation mutation, or a nonsense mutation. The subject may have two GALT alleles with the same loss-of-function mutation (homozygous state) or two GALT alleles with different loss-of-function mutations (compound heterozygous state). In certain embodiments, the subject is a carrier of GALT deficiency, and in certain embodiments, is heterozygous for the loss-of-function alleles described herein.

[0212] In some embodiments, the rAAV virions disclosed herein can be administered in sufficient amounts to transduce cells of the desired tissue and provide sufficient levels of gene transfer and expression without undue adverse effects. Pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to a selected organ (e.g., the central nervous system) by ICV or administration into the cisterna magna, oral, inhalation (including intranasal and intratracheal delivery), intraocular, intraventricular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes. Routes of administration can be combined as needed.

[0213] kit Disclosed herein are kits that include any of the agents described herein. In some embodiments, any of the agents disclosed herein can be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic, or research applications. The kits can include one or more containers housing the components of the present disclosure and instructions for use. In particular, such kits can include one or more agents described herein, along with instructions describing the intended use and proper use of these agents. In some embodiments, the agents in the kits can be in pharmaceutical formulations and dosages suitable for the particular use and method of administration of the agent. For research purposes, the kits can contain components in concentrations or amounts suitable for conducting various experiments.

[0214] Also disclosed herein is a kit for producing rAAV virions.In some embodiments, the kit can include a container that contains the isolated nucleic acid encoding GALT1 protein or a part thereof.In some embodiments, the kit can further include instructions for producing rAAV virions.In some embodiments, the kit further includes at least one container that contains the recombinant AAV vector that contains transgene (i.e., GALT).

[0215] In some embodiments, the kit can include a container that contains the above-mentioned recombinant AAV virion.In some embodiments, the kit can further include a container that contains a pharmaceutically acceptable carrier.For example, the kit can include one container that contains rAAV virion and a second container that contains a suitable buffer for injecting rAAV virion into a subject.In some embodiments, the container can be a syringe.

[0216] In some embodiments, the kits can be designed to facilitate the use of the methods described herein by researchers and can take many forms. Each of the compositions of the kit may be provided in liquid form (e.g., solution) or solid form (e.g., dry powder), as applicable. In some embodiments, some compositions may be configurable or processable (e.g., into an active form), for example, by the addition of appropriate solvents or other species (such as water or cell culture medium) that may or may not be provided with the kit. As used herein, "instructions for use" defines an instructional and / or promotional component, and can typically be accompanied by written instructions on or associated with the packaging of the present disclosure. Instructions for use can also include oral or electronic instructions provided in any manner, such as audiovisual (e.g., videotape, DVD, etc.), internet, and / or web-based communications, etc., that clearly identify to the user that the instructions are to be associated with the kit. The written instructions may be in a form prescribed by a government agency regulating the manufacture, use, or sale of drugs or biological products, and these instructions may also reflect approval by the agency of the manufacture, use, or sale for animal administration.

[0217] The kits disclosed herein may contain any one or more of the components described herein in one or more containers. In some embodiments, the kits may include instructions for mixing one or more components of the kit and / or for isolating and mixing a sample and applying it to a subject. The kits may include a container that contains an agent described herein. The agent may be in liquid, gel, or solid (powder) form. The agent may be sterilely prepared and 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 that is sterilely prepared. Alternatively, the kit may contain an active agent that is premixed and shipped in a syringe, vial, tube, or other container. The kits may have one or more or all of the components required to administer the agent to an animal, such as a syringe, topical application device, or intravenous (iv) needle tube and bag, especially in the case of kits for producing certain somatic cell animal models.

[0218] In some embodiments, the methods disclosed herein include transfecting cells with total cellular DNA isolated from tissues potentially carrying proviral AAV genomes in very small amounts and supplementing with helper virus functions (e.g., adenovirus) to initiate and / or boost transcription of AAV rep and cap genes in the transfected cells. In some embodiments, RNA from the transfected cells provides a template for RT-PCR amplification of cDNA and detection of novel AAV. When transfecting cells with total cellular DNA isolated from tissues potentially carrying proviral AAV genomes, it is often desirable to supplement the cells with factors that promote AAV gene transcription. For example, the cells may also be infected with a helper virus, such as an adenovirus or a herpes virus. In some embodiments, the helper functions may be provided by an adenovirus. The adenovirus may be a wild-type adenovirus and may be of human or non-human origin, such as non-human primate (NHP) origin. Similarly, adenoviruses known to infect non-human animals (e.g., chimpanzees, mice) can also be used in the methods of the present disclosure (see, e.g., U.S. Pat. No. 6,083,716). In addition to wild-type adenoviruses, recombinant viruses or non-viral vectors (e.g., plasmids, episomes, etc.) carrying the necessary helper functions can be utilized. Such recombinant viruses are known in the art and can be prepared according to published techniques. See, e.g., U.S. Pat. Nos. 5,871,982 and 6,251,677, which describe hybrid Ad / AAV viruses. Various adenovirus strains are available from the American Type Culture Collection, Manassas, Va., or available upon request from various commercial and industrial sources. Additionally, sequences of many such strains are available from various databases, including, e.g., PubMed and GenBank.

[0219] The cell can also be transfected with a vector (e.g., a helper vector) that provides helper functions for AAV. The vector that provides helper functions can provide adenoviral functions, including, for example, Ela, Elb, E2a, E4ORF6. The sequences of the adenoviral genes that provide these functions can be obtained from any known adenovirus serotype, such as serotypes 2, 3, 4, 7, 12, and 40, and further include any of the currently identified human types known in the art. Thus, in some embodiments, the method includes transfecting the cell with a vector that expresses one or more genes required for AAV replication, AAV gene transcription, and / or AAV packaging.

[0220] In some embodiments, the isolated capsid gene can be used to construct and package a recombinant AAV vector, and the functional characteristics associated with the novel capsid protein encoded by the gene can be determined using methods well known in the art. For example, the isolated capsid gene can be used to construct and package a recombinant AAV (rAAV) vector containing a reporter gene (e.g., B-galactosidase, GFP, luciferase, etc.). The rAAV vector can then be delivered to an animal (e.g., a mouse), and the tissue targeting properties of the isolated capsid gene can be determined by examining the expression of the reporter gene in various tissues of the animal (e.g., heart, liver, kidney). Other methods for characterizing the isolated capsid gene are disclosed herein, and still others are well known in the art.

[0221] The kits may have various forms, such as blister pouches, shrink-wrap pouches, vacuum sealable pouches, sealable thermoformed trays, or similar pouch or tray forms, with loosely packed accessories within the pouch, one or more tubes, containers, boxes, or bags. The kits may be sterilized after the accessories are added, allowing the individual accessories within the container to be unpacked in other ways. The kits may be sterilized using any suitable sterilization technique, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kits may also include other components, such as containers, cell culture media, salts, buffers, reagents, syringes, needles, cloths such as gauze for applying or removing disinfectants, disposable gloves, support for medication prior to administration, etc., depending on the particular application.

[0222] The instructions included in the kit may include methods for detecting potential AAV in cells. Additionally, kits of the present disclosure may include instructions, negative and / or positive controls, containers for samples, diluents and buffers, sample preparation tubes, and printed or electronic tables of reference AAV sequences for sequence comparison. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15] EXAMPLES

[0223] Example 1 GALT null mice are a genetic model of galactosemia. C57Bl / 6J female mice were mated with infertile male mice and hormonally prepared for implantation of genetically modified blastocysts. The GALT gene in these blastocysts was trapped by replacing a critical exon with an inactive but easily traceable sequence. The resulting mice were bred for homozygous trapping of the GALT gene. All GALT null mice have type 1 galactosemia prenatally, as they are bred from other GALT null mice. The timing of neonatal intervention at P9 targets the period of disease onset prior to extensive organ damage and is intended to represent a comparable developmental stage for the younger human pediatric patient population.

[0224] This animal model reproduces many of the features of galactosemia seen in humans, including lack of GALT enzyme activity and elevated Gal-1P levels in RBCs, reduced fertility in females, motor impairment demonstrated via rotarod performance, and high mortality of pups (>70%) when dams were fed a galactose-rich diet (similar to the increased neonatal mortality seen in galactosemia patients when dietary restriction was not implemented; see Table 2). Furthermore, as seen in galactosemia patients, GALT null mice grew slower than wild-type controls, especially when fed galactose (Tang et al., 2014). [Table 2] The primary objectives were to evaluate the efficacy of AAV9-CAG-hGalt in lowering blood and tissue metabolites and to assess the biodistribution of AAV9-CAG-hGalt in a mouse model of galactosemia. Two doses of AAV9-CAG-hGalt were examined. A total of 39 animals (32 GALT-null, 7 GALT-null) were administered two doses (3.74 × 10 13 vg / kg and 1.15×10 14Mice were administered AAV9-CAG-hGalt at 100 mg / kg (vg / kg). AAV9-CAG-hGalt formulation buffer (vehicle)-treated and wild-type mice served as control arms. Endpoints for this study included improvement of GALT function (enzyme activity) (Table 3 below), attenuation of type 1 galactosemia biomarkers (i.e., GALT, Gal-1P, galactitol, and galactose, assessed using non-qualified test methods), and biodistribution in various organs measured via GALT immunohistochemistry and pending PCR data after 4 and 12 weeks of treatment administration.

[0225] Assessment of Gal-1P levels in GALT null mouse pups in response to treatment with AAV9-CAG-hGalt showed a significant reduction in brain tissue at both 4 weeks (low dose: 48% and high dose: 59%) and 12 weeks (low dose: 28% and high dose: 40%) compared to GALT null vehicle-treated mice (Figures 7A and B). No changes in Gal-1P levels in skeletal muscle were observed at either dose at 4 weeks post-treatment (Figure 7C), but a significant 38% reduction in Gal-1P levels in skeletal muscle of animals receiving the high dose of AAV9-CAG-hGalt was observed at 12 weeks post-treatment (Figure 7D). Similarly, no changes in Gal-1P levels in RBCs were observed at either dose at 4 weeks of treatment (Figure 7E), but a significant decrease was observed in Gal-1P levels in RBCs from AAV9-CAG-hGalt-treated animals (low dose: 45.8% and high dose: 70.3%) at 12 weeks of treatment (Figure 7F). Liver showed a highly significant decrease in Gal-1P at 12 weeks (72% and 77% decrease at low and high doses, respectively, compared to vehicle), with data at 4 weeks still pending.

[0226] In line with the observed reduction in Gal-1P, a significant decrease in galactose levels was observed in brain tissue at both 4 weeks (low dose: 55% and high dose: 76%) and 12 weeks (low dose: 37% and high dose: 56%) compared to GALT null vehicle-treated mice (Figure 8A and B). A significant decrease in galactose levels in skeletal muscle was also observed at both 4 weeks (low dose: 34% and high dose: 51%) and 12 weeks (low dose: 45% and high dose: 52%) after treatment (Figure 8C and D). Galactose levels in RBCs were significantly decreased at both 4 weeks (low dose: 70% and high dose: 54%) and 12 weeks (low dose: 46.4% and high dose: 48.8%) after treatment (Figure 8E and F). Plasma galactose levels were significantly reduced at both 4 weeks (low dose: 86% and high dose: 89%) and 12 weeks (low dose: 80% and high dose: 78%) after treatment (Figure 8H and I). Liver showed a highly significant reduction in galactose at 12 weeks (72% and 73% reduction for low and high doses, respectively, over vehicle) (Figure 8G), with data from 4 weeks still pending.

[0227] Galactitol levels in brain tissue were significantly decreased at both 4 weeks (low dose: 27% and high dose: 48%) and 12 weeks (high dose: 33%) compared to GALT null vehicle-treated mice (Figures 9A and B). Galactitol levels in skeletal muscle were also significantly decreased at both 4 weeks (low dose: 58% and high dose: 67%) and 12 weeks (low dose: 74% and high dose: 78%) after treatment (Figures 9C and D). Galactitol levels in plasma were also significantly decreased at both 4 weeks (low dose: 25% and high dose: 39%) and 12 weeks (low dose: 27% and high dose: 37%) after treatment (Figures 9H and I). However, no significant changes were observed in galactitol in RBCs at either 4 or 12 weeks after treatment (Figures 9E and F). In the liver, there was a highly significant reduction in galactitol at 12 weeks (28% and 57% reduction at the low and high doses, respectively, compared to vehicle) (FIG. 9G), with data from 4 weeks still pending.

[0228] Evaluation of GALT enzyme expression in multiple tissues by immunohistochemistry showed a strong induction of GALT protein expression after treatment with AAV9-CAG-hGalt in GALT null mice at P37 and P93 (4 and 12 weeks after treatment with AAV9-CAG-hGalt, respectively) compared to no GALT protein staining in vehicle-treated GALT null animals (Figure 10). In the case of liver, GALT protein staining in wild-type and GALT null animals treated with AAV9-CAG-hGalt demonstrates increased GALT protein expression compared to the respective vehicle-treated controls (Figure 10A). Both low- and high-dose treatment with AAV9-CAG-hGalt in GALT null animals results in strong GALT protein expression at P37 and a notably sustained GALT staining at P93. For skeletal muscle, GALT protein staining shows a strong induction of GALT protein expression following treatment with AAV9-CAG-hGalt in GALT null animals at P37, with GALT staining strongly persisting at P93 (Figure 10B). For the brain, GALT protein staining in GALT null mice demonstrates AAV9-CAG-hGalt-induced GALT protein expression at P37, which persists until P93 (Figure 10C). High levels of GALT protein staining are observed in animals treated with high doses compared to low levels in low dose treatments, demonstrating a dose-dependent increase in AAV9-CAG-hGalt brain transduction. Neuronal and glial cell transduction is observed throughout the brain with different efficiencies depending on the structure, as shown by representative data from the cortex (Figure 10C).

[0229] Evaluation of GALT enzyme activity in liver, brain, and muscle (e.g., by quantification of Gal-1P to UDP-gal conversion by HPLC) showed a strong induction of GALT enzyme activity after treatment with AAV9-CAG-hGalt in GALT null mice at P93 (12 weeks after treatment with AAV9-CAG-hGalt, respectively) compared to the absence of GALT enzyme activity in vehicle-treated GALT null animals (Table 3). In the case of liver, GALT enzyme activity in GALT null animals demonstrates increased GALT enzyme activity in both low-dose (53.4%) and high-dose (159%) treated animals compared to vehicle-treated controls (1.3%). In the case of brain, GALT enzyme activity in GALT null animals demonstrates increased GALT enzyme activity in both low-dose (50%) and high-dose (28%) treated animals compared to vehicle-treated controls (2.6%). In muscle, GALT enzyme activity in GALT null animals demonstrates increased GALT enzyme activity in both low dose (147%) and high dose (177%) treated animals compared to vehicle treated controls (8%). [Table 3] A single intravenous delivery of AAV9-CAG-hGalt into P9 mice results in robust transgene expression in key tissues throughout the body until the last time point examined (12 weeks post-dosing). Increased GALT enzymes in tissues are accompanied by metabolite reductions in these tissues, as reductions in Gal-1P, galactose, and galactitol over 12 weeks were also observed extensively in AAV9-CAG-hGalt-treated animals. The approximately three-fold difference between the low and high doses did not result in similar fold changes in metabolite levels, suggesting that similar potential effects can be expected at any dose. Finally, transduction of RBCs has been demonstrated elsewhere (Pasi et al., 2020), but sustained transgene expression in non-nucleated cells is unlikely to be expected. For this experiment, the experimental results suggest that changes in RBC metabolites are likely a product of their environment and lifespan. These data suggest that although RBCs can be used to measure metabolites, they are more likely to represent changes in galactose metabolism, particularly Gal-1P, in tissues instead of changes to galactose metabolic functions within the RBCs.

[0230] Example 2 Previous studies (Tang et al., 2014 and Balakrishnan et al., 2020) have demonstrated that the GalT gene trap mice used in this example demonstrate both the biochemical / metabolic markers associated with classical galactosemia and many of the long-term complications experienced by patients, including growth retardation, motor impairment, and reduced fertility. Previous studies (Balakrishnan et al., 2020) have also demonstrated that intravenously administered GALT mRNA has the ability to restore GALT activity in the liver, resulting in a reduction in galactose-1-phosphate (Gal-1P) and galactose in plasma in a mouse model.

[0231] The goal of this study in GALT-deficient mice is to determine the efficacy of AAV9-CAG-hGalt, an AAV9-based GALT gene therapy, to correct disease-associated phenotypes in a mouse model of galactosemia. This study will be performed with material manufactured in a clinical process (non-GMP) designed to enhance potency while reducing impurities at 200L scale.

[0232] The work is divided into three arms. i. Behavioral (neuromuscular and cognitive) ii. Safety (clinically relevant blood and tissue pathology) iii. Fertility (reproductive hormones and pregnancy) Each arm will have a follow-up period of up to 6 months. In-life assessments with GALT activity measured in tissues at the time of euthanasia, as well as longitudinal blood and endpoint tissue levels of CG-related metabolites (galactitol, Gal-1P, and galactose) will be evaluated. Histological tissues and GALT expression in tissues defined by immunohistochemistry will also be followed in endpoint samples. CT scans and clinical chemistry for bone density may also be performed in the same mouse models.

[0233] AAV9-CAG-hGalt is an adeno-associated virus vector-based gene therapy suspension for parenteral administration. It is a recombinant non-replicating AAV9 vector containing a self-complementary transgene encoding the human galactose-1-phosphate uridylyltransferase (GALT) protein under the control of the cytomegalovirus enhancer / chicken-β-actin hybrid promoter. Three different concentrations of AAV9-CAG-hGalt are provided in multi-use sterile cryovials without preservatives. For the purposes of this study, a control substance of AAV9-CAG-hGalt is provided in multi-use sterile vials without preservatives. The solution filled in the vials consists of the same excipient mixture utilized to formulate and fill the test substances. Each test substance vial contains enough material to dose up to two mice. This proof-of-concept study will be conducted as a blinded trial where relevant personnel will be blinded to treatment group and dose. Table 4 below will be labeled with a random blinding identifier such as FRM-X, which stands for "Formulation X," omitting the dose group. [Table 4] Material is stored at ≤ -60°C. Each vial is thawed immediately prior to use at room temperature, and material is administered within a maximum of 2 hours after thawing. Thawed vials will not be refrozen, and any unused material in the open vial will be discarded with biohazard waste.

[0234] Both behavioral and reproductive cohorts are designed as dose ranging and efficacy studies. High, medium and low doses will be tested in all three study arms along with a vehicle control. All animals at weaning will be fed a 2.5% galactose diet.

[0235] The study will be conducted as a blinded study, and study site personnel involved in the conduct of the study (including treatment administration procedures and in-life and post-mortem evaluations and analyses) will be blinded to treatment and dosage. Blinding will be lifted following completion of all in-life activities and post-mortem evaluations. In addition to blinding, a vehicle control cohort will be included throughout the study to minimize the influence of confounding variables and to compare with the AAV9-CAG-hGalt-treated cohort.

[0236] Behavioral Research Arm A complete list of mouse animal numbers by behavioral arm, genotype, treatment group, and sacrifice time point is outlined in Table 5 below. [Table 5]

[0237] The main focus of this study is the rescue of GALT expression and behavioral phenotype. The study will test whether AAV9-CAG-hGalt reduces the incidence and prevents neurological complications and cognitive impairment. To examine the effect of AAV9-CAG-hGalt on behavioral improvement, each animal will receive one intravenous treatment of AAV9-CAG-hGalt at P9 (9 days after estimated time of birth). AAV9-CAG-hGalt formulation buffer (vehicle / control substance)-treated mice will serve as the control arm. Three endpoints are planned for the behavioral study. Evaluations may be performed at + / - 1 week.

[0238] Rotarod test Rotarod testing is performed to assess changes in motor / neuromuscular coordination and is performed monthly for a total of six assessments until age 6 months. Rotarod evaluation begins one month after intravenous (IV) administration of test or control substances in the following order:

[0239] Acclimatization: Subjects are removed from their home cage to the testing room at least 30 min prior to training or testing to minimize the effects of stress on behavior during testing.

[0240] Training: To minimize the effects of stress on behavior during testing, subjects are transferred to the testing room in their home cages and allowed to acclimate for 15 min. To ensure that all subjects are able to walk forward on the rotarod, subjects are trained the day before the first test. Subjects are placed on the rotarod, which rotates at a constant speed of 4 RPM, for a minimum of 180 seconds. If the subject falls off the rotarod during the training trial, the subject is placed back on the rotarod. Once the training trial is over, subjects are returned to their home cage for testing the next day.

[0241] Testing: The rotarod is set to accelerate from 4 to 40 RPM over a total of 300 seconds (increasing approximately 1 RPM every 8 seconds), reaching a maximum speed of 40 RPM at 300 seconds. The subject is placed on the rotarod at a starting speed of 4 RPM. If the subject falls off the rotarod before the start of the test, the subject is placed back on the rotarod. Once all subjects are on the rotarod, the assay begins and the latency to fall and the velocity (RPM) at which the subject falls are recorded. If they return, the subject undergoes one passive rotation (grasping the rod and performing a full rotation without walking) and continues the test. A second passive rotation is recorded as a failure, and the latency and velocity at which the subject falls are recorded. The subject is then returned to the home cage and apparatus between each trial. A total of three trials are performed, separated by a 5 minute rest interval. The average and peak latencies to fall across the three trials are calculated and plotted.

[0242] Reverse Screen Test Reversal screen testing is performed to assess changes in motor strength / coordination and is performed monthly for a total of six assessments until age 6 months. Reversal screen testing begins one month after intravenous (IV) administration of test or control substances. Assessments may be performed + / - 1 week.

[0243] Acclimatization: Subjects are removed from their home cage to the testing room at least 30 min prior to training or testing to minimize the effects of stress on behavior during testing.

[0244] Testing: To minimize the effects of stress on behavior during testing, subjects in their home cages are transferred to the testing room and allowed to acclimate for 15 or 30 min. The subject is placed in the center of the wire mesh screen and, with the mouse's head initially lowered, the screen is rotated to the inverted position in one movement. Once the screen is fully inverted, the test and timer begin. The screen is stably positioned at a height of at least 40 cm-50 cm above the surface, ensuring that the subject is as secure as possible in grasping the inverted screen to avoid jumping off it. The time is recorded when the mouse falls off the mesh onto the surface of the pad below (latency to fall).

[0245] Morris Water Maze Test Morris water maze test is performed to evaluate the improvement of spatial learning in GALT gene trap mice. Animals are evaluated at both 3 and 6 months after intravenous (IV) administration of test or control substances. Evaluations may be performed + / - 1 week.

[0246] Acclimatization: Subjects are removed from their home cage to the testing room at least 30 min prior to training or testing to minimize the effects of stress on behavior during testing.

[0247] Training: Phase 1: Cued swimming (2–3 trials, day 1, platform 5 mm above water surface). Subjects are lowered into the desired pre-set quadrant in the pool for each 60-s trial. Training is repeated twice. Video capture begins once the subject is lowered into the pool. If the platform is found and attached, the subject is allowed to remain motionless for 20 seconds, then rescued, towel-dried, and transferred to a warmed cage. If the platform is not found or attached, the subject is gently guided to the platform with gloved hands, allowed to remain motionless for 20 seconds, then rescued, towel-dried, and transferred to a warmed cage. After transferring the mouse, use an aquarium net to remove any debris between each trial.

[0248] Training: Phase 2: Swimming without cues (2 trials, days 2-4, platform 5 mm below water surface). Subjects are lowered into their desired quadrant in the pool for 60 s trials each. Repeat trials by randomizing mouse quadrant placement as shown in Table 6 below. [Table 6] Once the subject is lowered into the pool, video capture begins. If the platform is found and attached, the subject is allowed to remain for 20 seconds, then rescued, towel-dried, and transferred to a warmed cage. If the platform is not found or attached, the subject is gently guided to the platform with gloved hands, allowed to remain for 20 seconds, then rescued, towel-dried, and transferred to a warmed cage. After the mouse is transferred, a water bath net is used to remove any debris between each trial.

[0249] Testing: Phase 3: Probe Trial (1 trial, day 5, platform removal). Subjects are lowered into a random quadrant (1, 2, 4) in the pool for each 180 second trial. Video capture begins once the subject is lowered into the pool. After the 180 seconds have elapsed, the subject is rescued, towel dried, and transferred to a heated cage. After the mouse is transferred, a fish tank net is used to remove any debris between each trial.

[0250] Figure 11 is a chart showing the experimental design and data collection points. In-life evaluation of animals metabolites for RBC GALT, RBC galactitol, RBC galactose, plasma galactose, plasma galactitol, and plasma Gal-1P will be measured at 1, 2, 3, and 6 months after treatment. 50% of the mice in all groups will be euthanized at 3 months after treatment, and the remaining 50% will be euthanized at 6 months after treatment. Tissue samples collected at termination (brain, liver, ovaries, muscle) (collected at 3 and 6 months after treatment) will be evaluated histologically to confirm positive GALT expression (n>3 at each time point). RBCs and collected tissues (brain, liver, ovaries, muscle) will also be evaluated for both ddPCR and metabolite biochemistry (n>6 at each time point, 3M / 3F, when possible).

[0251] Growth of all animals assessed by weight is measured postnatally. Mice weights are collected every 3 days starting at P9 until 1 month (P12, P15, P18, P21, P24, P27, P30, P33, P36, P39) and then weighed weekly (i.e., P46, P53, P60, etc.). Weights will then be grouped under the circumstances of obtaining weights + / - 1 day from the designated date (i.e., P21 + / - 1 day). Computed tomography scans of bone density are performed on euthanized animals (n=6 / group, 3 and 6 months).

[0252] Reproductive Research Arm A complete list of mouse animal numbers by reproductive arm, genotype, treatment group, and sacrifice time point is outlined in Table 7 below. [Table 7] The primary focus of this study will be to reduce and restore ovarian failure / fertility in females. To examine efficacy, each animal will receive one IV treatment of test or control substance on P9 (9 days after estimated time of birth). Endpoints for this study include changes in reproductive hormones (follicle stimulating hormone, luteinizing hormone, and anti-mullerian hormone) assessed at the beginning and end of the study.

[0253] The breeding set-up is performed using the procedure described in Balakrishnan et al., 2019 (section 1.7). Determination of estrous cycle using cytology is evaluated during the last 2 weeks before breeding and at the end of the study to ensure that blood and tissue samples collected for analysis are synchronized. Females are transferred to a breeding cage and paired with one WT male (no treatment). Females are then mated consecutively from 11 weeks to 6 months of age or until a minimum of three litters are obtained. Females in the breeding cage are followed for visual or palpable signs of pregnancy. Planned studies will allow to determine pregnancy gain, time to pregnancy, litter size after birth, and survival rate of pups. Ovarian tissue samples collected after termination are evaluated histologically (n ≥ 3). Collected ovaries are also evaluated for attenuation in metabolite biochemistry (n ≥ 5).

[0254] Safety dose study arm A complete list of mouse animal numbers for safety arms, genotypes, treatment groups, and sacrifice time points is outlined in Table 8 below. [Table 8]

[0255] The primary focus of this study arm is to determine the preclinical safety profile in a preclinical pharmacology model of galactosemia. To examine efficacy, each animal will receive one IV treatment of test or control substance on day P9. Animals will be euthanized approximately 30 days and approximately 180 days after treatment. Euthanasia may occur up to + / - 6 days to ensure overnight shipment and delivery of samples collected for clinical chemistry. Endpoints for this study include full necropsy and histology, as well as biodistribution on a comprehensive list of organs (listed under the tissue collection table). Safety of AAV9-CAG-hGalt will also be evaluated by hematology and clinical chemistry panels.

[0256] Registration Breeding pairs are housed in a single cage and are from homozygous parents, so all litters are enrolled. There is no planned cross-breeding. Not all pups from the same litter will receive the same treatment. Using the procedures described in Balakrishnan et al., 2019 (section 1.7), individual pups from the same litter are identified and differentiated. Study personnel will assign random dose levels to pups within litters. The total number of pups enrolled across the various arms is shown in Table 9. [Table 9] To ensure that the correct volume of AAV vector is administered, all pups are weighed and injected with the required 5 μL / g dose. Pups that appear sick will not be injected as part of the study. This study will be performed as a blinded study.

[0257] Evaluation items For unscheduled SACS, follow section 5.5 and collect the organs listed under the tissue collection table.

[0258] Mice are randomly assigned to outcome measurements for scheduled necropsy in the behavioral and reproductive arms. Animals designated for metabolite analysis are deeply anesthetized and blood is collected via transcardiac puncture. Collected whole blood is separated into plasma and RBCs by centrifugation. Plasma is aliquoted for metabolite analysis (described below) and reproductive hormone analysis (females only). At each time point, n>6 (3M / 3F, in behavioral arms when possible) are designated for tissue, plasma, and RBC metabolite / biochemical analysis and GALT activity.

[0259] The remaining animals in the group (n>3, if possible) are assigned to histology, GALT IHC, and ddPCR. For scheduled necropsies in the reproductive cohort, animals will further undergo transcardial perfusion with PBS after completion of transcardial blood collection. For the reproductive arm, ovarian tissue samples will be evaluated histologically (n>3). Harvested ovaries will also be evaluated for attenuation in metabolite biochemistry (n>5). For scheduled necropsies in the safety cohort, animals will be deeply anesthetized and blood will be collected via transcardial puncture for complete blood count and clinical chemistry.

[0260] Additionally, at scheduled necropsies, blood samples will be collected from the GG and WT "safety" treatment groups for hematology and clinical chemistry evaluation at IDEXX Bioanalytics (Sacramento, Calif.) as described below.

[0261] A comprehensive CBC including reticulocyte HGB will be analyzed. The following variables will be evaluated: white blood cells (WBC), neutrophils (% and absolute), band cells (% and absolute), lymphocytes (% and absolute), monocytes (% and absolute), eosinophils (% and absolute), basophils (% and absolute), red blood cells (RBC), hematocrit (HCT), total hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), reticulocyte count (% and absolute), reticulocyte hemoglobin content, platelet count, and platelet estimate. Clinical chemistry endpoints were as follows: alanine aminotransferase (ALT), albumin (ALB), albumin:globulin ratio, alkaline phosphatase (ALP), aspartate aminotransferase (AST), bicarbonate, bilirubin (conjugated), bilirubin (unconjugated), total bilirubin (TBIL), blood urea nitrogen (BUN), BUN:creatinine ratio, calcium (Ca), chloride (Cl), cholesterol (CHOL), creatine kinase (CK), creatinine (CREA), globulin (GLB), glucose (GLU), phosphorus (PHOS), potassium (K), sodium (Na), sodium:potassium ratio, and total protein (TP). For all study arms, tissue samples are collected and either flash frozen or immersed in 10% neutral buffered formalin for a minimum of 48 hours and a maximum of 72 hours. Tissues collected for fixation are collected in histology cassettes and placed in 125 ml screw-cap containers containing enough 10% neutral buffered formalin to completely submerge the cassettes. Fixed tissues are sent for histological processing, GALT IHC, H&E staining, and anatomic pathology. Frozen tissues are cut into three aliquots of approximately 25 mg each and then placed into three round-bottom 2 ml tubes. Tubes are labeled with animal ID and tissue. Tubes are flash frozen in liquid nitrogen and then maintained at ≦-60°C until analysis.

[0262] Mouse body weights are collected every 3rd day starting at P9 until 1 month (P12, P15, P18, P21, P24, P27, P30, P33, P36, P39), and then weighed weekly thereafter (i.e., P46, P53, P60, etc.).

[0263] GALT activity analysis in selected tissues (RBC / brain / liver / ovary / muscle / plasma) will be performed using LCMS-MS.

[0264] GALT metabolite (galactose, galactitol, Gal-1P) analysis in selected tissues (RBC / brain / liver / ovary / muscle / plasma) will be performed using LC-MS / MS. Each sample will be analyzed once and the results will be treated as a single data point. Samples will not be pooled for analysis.

[0265] Samples are immersed and fixed in 10% neutral buffered formalin for a minimum of 48 hours and a maximum of 72 hours at room temperature, then transferred to 70% ethanol and stored at room temperature until shipping. After fixation, tissues are shipped to Invicro for histological processing, H&E, and GALT immunohistochemistry.

[0266] Biodistribution of transgene DNA and RNA will be assessed by ddPCR on frozen tissues. All frozen tissue samples for vector DNA and RNA ddPCR analysis will be shipped to BioAggregix in batches on dry ice. Archived samples will be stored at ≤-60°C until the end of the project after the study is completed.

[0267] Plasma samples obtained from the reproductive arm will be analyzed for reproductive hormones (AMH, LH, FSH) using ELISA kits.

[0268] CT scans of selected animals (n=6 per group) will be performed at the Preclinical Imaging Core Facility at the University of Utah.

[0269] ≥0.200 mL of whole blood is collected for hematological evaluation (LTT-MINI Top tube 0.5 mL), vortex mixed, and stored at approximately 4° C. For clinical chemistry, ≥0.5 mL of whole blood is added to a serum collection tube (0.8 mL SST-MINI Gold Top tube), then gently inverted 5-10 times after collection, the sample allowed to clot for 15-20 minutes at room temperature, centrifuged at 2,500 rpm for 10 minutes to remove off-clot serum, combined with a new unspiked sample transfer tube (yielding >200 μL of serum) and stored at approximately 4° C.

[0270] A complete list of organs harvested for the safety arm and unscheduled deaths is outlined in Table 10 below. [Table 10]

[0271] Example 3 We performed a study to evaluate the efficacy of AAV2 / 9 expressing the human GALT gene (AAV9-CAG-hGalt) for reducing cataracts in a rat model of classical galactosemia and to correlate cataracts with both the levels of GALT activity in tissues and the levels of CG-related metabolites (galactitol, galactose-1-phosphate (Gal-1P), and galactose) in tissues and blood.

[0272] GALT null rats are a genetic model of galactosemia type 1. Using clustered regularly interspaced short palindromic repeats (CRISPR) gene editing, a two-base pair insertion mutation (GaltM3) was introduced into exon 6 of the Galt locus in Sprague Dawley rats. Rats carrying the homozygous GaltM3 mutation were then crossed to generate GALT null rats.

[0273] If untreated, GaltM3 homozygotes lack GALT enzyme activity at all ages. Pups are also born with low birth weight and remain smaller than wild-type or heterozygous controls until puberty, at which point they "catch up" similarly to humans with type 1 galactosemia. This rat genetic model has 100% penetrance for the lack of GALT activity and abnormal accumulation of galactose metabolites, and therefore all GALT null rats have a prenatal basis for disease similar to humans. The timing of neonatal intervention at P2 for this study targets the period of disease onset prior to extensive organ damage, with the intention that the young human pediatric patient population represents a comparable developmental stage. GALT null rats also mimic the characteristics of galactosemia seen in humans, such as the mild growth retardation, cataracts, and motor and cognitive impairments described above, which are detected in mature animals. As in humans, these phenotypic outcomes show incomplete penetrance and expressivity differences between individual rats, but are clearly present when comparing cohorts of animals. Within 24 hours of birth, GALT null rat pups exhibit increased birth weight and levels of toxic metabolites associated with type 1 galactosemia (Rasmussen et al., 2020).

[0274] A summary of the rat models and their relevance to the human condition is shown in Table 11. [Table 11] The primary objectives of this study were to evaluate the efficacy of AAV9-CAG-hGalt in reducing metabolites in blood and tissues, determine the biodistribution and GALT enzyme activity levels in tissues, and evaluate end-organ damage in the lens of a rat model with galactosemia. GALT null mice have low GALT activity and high levels of galactose and its metabolites (galactose, galactitol, and Gal-1P) in blood, liver, and brain. Mirroring human patients with galactosemia, GALT null rats further develop cataracts. Notably, metabolic derangements at birth and cataract development established by age P17 in the rat model support early intervention targeting the critical period of disease development. Thus, P2 rats were dosed targeting the disease period after galactose metabolic abnormalities are recognized but before extensive organ damage has begun, representing a developmental stage comparable to the young human pediatric patient population. Additionally, rats at the P2 time point provide an early time window for IV dosing after disease onset.

[0275] Wild-type and GALT-null rats were administered AAV9-CAG-hGalt at two doses (3.82 × 10) via IV dosing in the tail vein on day P2. 13 vg / kg and 1.16×10 14AAV9-CAG-hGalt formulation buffer (vehicle) treated rats served as the control arm. Endpoints for this study included biodistribution in selected organs measured via GALT IHC (described below) and ddPCR (data pending) at 14 and 35 days post-treatment, increase in GALT enzyme activity in tissues, attenuation of biomarkers of type 1 galactosemia (i.e., GALT, Gal-1P, galactitol, galactose; assessed using a non-certified test method), and assessment of cataract incidence and severity. Automated ddPCR methods are known in the art and include, for example, the BioRad Droplet Digital PCR System.

[0276] Assessment of GALT enzyme expression in multiple tissues by immunohistochemistry showed a strong induction of GALT protein expression at ages P16 and P37 (days 14 and 35 after treatment with AAV9-CAG-hGalt, respectively) in GALT null rats compared to no GALT protein staining in vehicle-treated GALT null animals (Figure 12). In the case of liver, anti-GALT immunohistochemistry (IHC) of wild-type and GALT null animals treated with AAV9-CAG-hGalt demonstrates increased GALT protein expression compared to their respective vehicle-treated controls (Figure 12A). Both low- and high-dose treatment of GALT null animals with AAV9-CAG-hGalt resulted in strong GALT protein expression at P16 and persistent, albeit reduced, but significant (as measured by H-score) GALT staining at P37. Interestingly, livers from vehicle-treated WTs also showed a decrease in GALT immunoreactivity over the course of the study. In skeletal muscle, GALT IHC shows a strong induction of GALT protein expression after treatment with AAV9-CAG-hGalt in GALT null animals at P37 (Figure 12B). Skeletal muscle at P16 was not examined. GALT IHC demonstrated that in the brain, GALT protein induced by AAV9-CAG-hGalt was expressed throughout the brain at P16 and persisted until P37, as demonstrated by representative data in the cortex and cerebellum (Figures 12C and D). High levels of GALT protein staining were observed in animals treated with the high dose compared to animals treated with the low dose, demonstrating a dose-dependent increase in AAV9-CAG-hGalt brain transduction. Co-staining of brown GALT protein with purple astrocyte glial marker GFAP (glial fibrillary acidic protein) demonstrated robust transduction of both neurons and glial cells in the cortex (Figure 12D). A dose-dependent increase in Purkinje neuron transduction throughout the cerebellum was also observed after AAV9-CAG-hGalt administration (FIG. 12D). Ovaries from vehicle- and AAV9-CAG-hGalt-treated null rats were negative for GALT immunoreactivity (not shown), whereas signal was observed in wild-type tissue.

[0277] The increase in GALT immunoreactivity in tissues also corresponded to an increase in enzyme activity. GALT enzyme activity data (FIG. 13), measured by quantifying the conversion of Gal-1P to UDP-gal by HPLC in both P16 and P37 examined tissues, show a highly significant increase compared to GALT null rats. In liver (FIGS. 13A and B), GALT enzyme activity was significantly increased at 14 days after treatment (low dose: 47-fold increase over wild type, high dose: 70-fold increase over wild type) and remained at high levels at 35 days after treatment (low dose: 6.4-fold increase over wild type, high dose: 13.7-fold increase over wild type). GALT enzyme activity was also significantly increased in brain tissue at 14 days after treatment (FIGS. 13C and D) (low dose: 1.9-fold increase and high dose: 5.1-fold increase over wild type) and remained at 35 days after treatment (low dose: 1.4-fold increase over wild type, high dose: 2.1-fold increase over wild type). Similarly, skeletal muscle tissue demonstrated a significant increase in GALT enzyme activity at 14 days post-treatment (FIGS. 13E and F) (low dose: 23-fold increase over wild type and high dose: 108-fold increase over wild type) that was maintained at 35 days post-treatment (low dose: 48.5-fold increase over wild type, high dose: 84-fold increase over wild type). Finally, GALT enzyme activity reached near wild-type levels in the eyes of AAV9-CAG-hGalt-treated animals both 14 and 35 days post-injection (FIGS. 13G and H).

[0278] Positive expression of GALT was confirmed biochemically by GALT enzyme activity assays (liver, brain, skeletal muscle, and eye) and histologically (liver, muscle, and brain) that could subsequently lead to a reduction in the deleterious metabolic triggers of galactosemia (galactose, galactitol, and Gal-1P). To test this hypothesis, metabolites were examined in tissues of M3 and WT rats treated with AAV9-CAG-hGalt and vehicle. Levels of GALT null rat pups (M3) upon treatment with AAV9-CAG-hGalt show a significant reduction in Gal-1P in liver tissue at P16 (low dose: 95% and high dose: 93%). The reduction in Gal-1P in liver was maintained at least through P37 (low dose: 95% and high dose: 97%) compared to GALT null vehicle-treated rats (Figures 14A and B). No change in Gal-1P levels in the brain at P16 was observed after treatment with low dose AAV9-CAG-hGalt, whereas the high dose resulted in a significant decrease in Gal-1P in the brain at P16 (Fig. 14C). Furthermore, a significant decrease in Gal-1P levels in the brain of rats treated with AAV9-CAG-hGalt was observed at both low and high doses at P37 (low dose: 72% and high dose: 89%) (Fig. 14D). Interestingly, despite the apparent lack of GALT expression in AAV9-CAG-hGalt-treated ovaries by IHC, there was an 82% and 67% decrease in Gal-1P in ovaries from AAV9-CAG-hGalt low and high dose animals, respectively, from vehicle levels at P16 (Fig. 14G). At P37, Gal-1P levels in the ovaries of vehicle-treated null animals were decreased without a corresponding decrease in Gal-1P in vector-treated null animals compared to vehicle at P16 (Figure 14H). A significant decrease in Gal-1P levels in RBCs (low dose: 52% and high dose: 34%) was observed in vector-treated null rats at P16 (Figure 14I). However, no significant decrease in Gal-1P levels in RBCs was observed in null rats at P37 (Figure 14J). Notably, Gal-1P levels in the eyes of vector-treated null rats at P16 were significantly decreased with both doses of AAV9-CAG-hGalt (low dose: 37% and high dose: 42%).Gal-1P data from the eye are pending for the P37 time point.

[0279] Galactose levels were also evaluated to determine whether the positive observations of GALT expression corresponded to a decrease in galactose. The data (Figure 15) show that vehicle-treated GALT null pups had high galactose levels in liver (Figures 15A and B), brain (Figures 15C and D), plasma (Figures 15E and F), muscle (Figures 15G and H), ovaries (Figures 15I and J), eyes (Figures 15K and L), and RBCs, whereas nearly all tissues from animals treated with AAV9-CAG-hGalt showed a significant decrease in galactose levels at both P16 and P37, whereas ovaries from null animals administered high dose AAV9-CAG-hGalt showed a trend toward significance (p=0.0602).

[0280] The effect of AAV9-CAG-hGalt on galactitol, a key metabolite involved in the formation of cataracts in galactosemia patients, was also evaluated. Similar to the observed decrease in Gal-1P and galactose levels, galactitol levels also decreased in response to treatment with AAV9-CAG-hGalt. Galactitol levels (Figure 16) showed a statistically significant decrease in all examined tissues (liver, brain, plasma, muscle, ovary, and eye) from AAV9-CAG-hGalt-treated null animals compared to vehicle at both examined time points (days 14 and 35) after injection. A significant decrease in galactitol levels in RBCs was observed at day 14 after treatment with high dose AAV9-CAG-hGalt, and at days 14 and 35 after treatment with both low and high doses of AAV9-CAG-hGalt (Figure 16).

[0281] Evaluation of AAV9-CAG-hGalt-treated GALT null rat pups at P16 and P37 showed significantly reduced cataract severity (FIGS. 17A and B) and incidence (FIG. 17C).

[0282] Furthermore, an overall increase in body weight was also observed in GALT null rat pups treated with AAV9-CAG-hGalt (Figure 18).

[0283] Systemic delivery of AAV9-CAG-hGalt to GALT null rats resulted in widespread transgene expression in tissues known to be transduced by AAV9. Importantly, expression levels were robustly maintained at or above wild-type levels of staining intensity and enzyme activity during the time points examined during the experiment. Increases in GALT enzymes corresponded to decreases in galactosemia-related metabolites in AAV9-CAG-hGalt-treated null rats. Notably, decreases in metabolites in the eye were associated with reduced incidence and severity of cataracts in null rats administered AAV9-CAG-hGalt. Vehicle metabolite levels spontaneously decreased with increasing age. This decrease was likely due to multiple factors, including a transition from breast milk to a low-galactose diet, and possibly a decrease in endogenous galactose production with age and upregulation of alternative galactose metabolic pathways. Overall, these data indicate that AAV9-CAG-hGalt treatment is highly effective in reducing galactosemia-associated metabolites in well- and poorly-transduced tissues, leading to improved cataract incidence and severity. [Table 12]

[0284] Example 4 The aim of the proposed study is to perform a proof-of-concept study in GALT null rats using material produced at scale by a clinical grade process to evaluate the efficacy of AAV9-CAG-hGalt at three dose levels in preventing the development of long-term cataracts and attenuating other phenotypes including prepubertal growth retardation, visceral neuromuscular impairment by grip strength measurement, and neurological or socio-emotional impairment via open field and forced swim assessments. We aim to correlate these in-life assessments with GALT activity measured in tissues at the time of euthanasia with longitudinal blood and endpoint tissue levels of CG-related metabolites (galactitol, Gal-1P, and galactose). GALT expression in histology and tissues defined by immunohistochemistry will also be followed in endpoint samples.

[0285] AAV9-CAG-hGalt is an adeno-associated virus vector-based gene therapy suspension for parenteral administration. It is a recombinant non-replicating AAV9 vector containing a self-complementary transgene encoding the human galactose-1-phosphate uridylyltransferase (GALT) protein under the control of the cytomegalovirus enhancer / chicken-β-actin hybrid promoter. For the purposes of this study, AAV9-CAG-hGalt is produced and provided in multi-use sterile frozen vials without preservative at three different concentrations. A vehicle control is also provided in a multi-use sterile vial without preservative. The solution filled into the vehicle control vial consists of the same excipient mixture used to formulate and fill AAV9-CAG-hGalt. Each vial contains enough material to be administered to up to four newborn rat pups.

[0286] The study will be conducted as a blinded trial where relevant personnel will be blinded to the treatment group and dose. The blinded study and control substances are outlined in Table 12 below, omitting the dose groups and labeled with a random blinding identifier such as FRM-X, which stands for "Formulation X." [Table 13] The vials are stored at ≦−60° C. Each vial is thawed immediately prior to use at room temperature, with the active drug being administered within a maximum of 2 hours after thawing.

[0287] Research Plan Experimental Design A complete list of rat animal numbers, genotypes, treatment groups, and sacrifice time points is outlined in Table 13 below. [Table 14] Rats are dosed at P2 (24-48 hours from estimated birth time) and subsequently weighed to calculate the exact dose (7 µL of material / gram pup mass). Rats undergo in-life evaluations at monthly intervals and are collected for post-mortem evaluations either at 14, 35, or approximately 180 days post-dosing. The experimental design for both in-life and post-mortem evaluations is outlined in Figure 19.

[0288] Breeding: All rats used in this study are Sprague-Dawley (outbred). GALT null pups (M3) are obtained by mating confirmed GALT null parents; wild-type (WT) pups are obtained by mating confirmed WT parents. The galt genotype of all experimental animals is confirmed by sending tissue samples (tail snip, ear punch, or other tissue samples) to Transnetyx (https: / / www.transnetyx.com), which performs all rat genotyping. The relevant mutant Galt allele is M3 (Rasmussen et al. 2020).

[0289] Registration: To be enrolled in the study, pups must weigh at least 5 grams, appear healthy (pink and "wiggly"), warm, and have visible milk spots (evidence of feeding). Both sexes of pups are included in each comparison group in a ratio as close to 1:1 as possible. Pups from each litter are randomly assigned to the relevant treatment group so that each group contains pups of the correct genotype from two or more litters. Comparison group numbers are shown in Table 13 above. Between 24 and more than 48 hours after birth, pups are weighed and marked (ink number on pup's bottom + ink on paws for two-factor ID), visually scored for the presence of milk spots, and visually scored as male or female. If enrollment criteria are met, each pup is assigned to a treatment group and is not blinded to sex and genotype, but treatment groups remain blinded, except for non-treatment.

[0290] Pups assigned to treatment groups (other than "untreated") are given the appropriate treatment. After injection and recovery, each pup is returned to its nest with its littermates and mother. Each following day, each pup is weighed and recorded in ink. Sex determination is confirmed approximately at postnatal day 10, when signs become more evident (e.g., females develop ventral nipple buds, but males do not). Just prior to weaning, pups are ear punched for identification.

[0291] All pups are weaned in same-sex pairs or triplicates on postnatal day 24 and caged with Lab Diet 5053 chow and water ad libitum.

[0292] Evaluation items: Cataract Data Collection: While rats are alive, slit lamp examinations are performed and both eyes are photographed dilated at monthly post-dose time points (approximately 30 day intervals). Immediately after euthanasia, eyes are manually scored for the presence or severity of cataract using a 4-point scale defined in Rasmussen et al., JIMD 2020.

[0293] Weight Data Collection: Rats are weighed daily until 1 month of age, then weekly (± 1 day) until euthanasia.

[0294] Grip Strength Data Collection: Rats are assessed for grip strength at monthly time points (approximately 30 day intervals) after dosing using a separately constructed grip strength testing apparatus. Specifically, each rat is placed face-on on a clean metal wire grid, supported by low friction roller bearings (Skelang 1 inch; zs1818), and seated on a smooth "runway." One end of the grid is attached to a dynamometer spring scale (QWORK WD3854) so ​​that as the grid is displaced, the spring stretches and records the force on the scale. For each rat, a specific spring scale is used, e.g., a 20 Newton scale for older rats and a 5 or 10 N scale for younger (weaker) rats, so that the expected reading is near the middle of the scale's range. With the rat gripping the grid with all four paws and the two front paws at the proximal edge of the grid, the examiner gently tugs on the base of the rat's tail and continues pulling until the rat begins to slip. Grip strength is measured with a Newton spring as the maximum force registered just before the rat begins to lose grip. At each monthly evaluation, each rat is tested once per day for four consecutive days.

[0295] Six months after dosing, a single additional grip strength assessment is performed using a digital dynamometer (Columbus Instruments) attached to a rat pull bar. Once the rat grasps the pull bar, the examiner gently pulls on the base of the rat's tail and continues pulling until the rat begins to slip. Peak grip strength is measured automatically by the digital dynamometer and recorded for data analysis.

[0296] Open field data collection: Rats undergo open field testing 6 months after dosing (approximately day 180) on two consecutive days using a separate rectangular open field arena approximately 3 feet by 4 feet. Rats are placed in the open field within the peripheral area and allowed to freely explore the arena for 10 minutes, which is video recorded from above for data analysis. Once the evaluation is completed, rats are returned to their home cage. After each run, any feces are removed and the arena is thoroughly wiped and washed to prevent soiling that may affect the next open field evaluation.

[0297] Forced Swim Test: Rats undergo forced swim test assessment over two days at 6 months post-dosing (approximately day 180). The forced swim test is conducted in a cylindrical tank half-filled with room temperature water, and the rat is unable to reach the bottom or top of the tank while swimming at the surface. On day 1 of the forced swim test, individual rats are placed in the water-filled tank and undergo a 15-minute acclimation run, followed by a second 5-minute test run on day 2 (24 hours later). Both day 1 and day 2 runs are video recorded and analyzed for activity and immobility times. After each run, the wet rats are gently dried and then returned to their home cage.

[0298] Tail vein blood collection: Up to approximately 500 μL of blood is collected monthly from the tail vein of each rat over one month of age. Blood is collected into a sterile syringe and immediately transferred to a small sample tube prefilled with sodium heparin to prevent clotting. Tail vein blood samples are processed as described following euthanasia.

[0299] Rats are anesthetized with isoflurane and euthanized with PBS whole body perfusion for tissue collection at 14, 35, or approximately 180 days (± 1 day) after treatment. Table 14 [Table 15] Vector DNA / RNA / archive sample collection method: All frozen tissue samples for vector DNA and RNA ddPCR analysis are shipped to BioAggregix in batches on dry ice. Sample storage is ≦-60°C.

[0300] Brain (left hemisphere) - A 2 mm thick slice was collected starting 2 / 10 of the total distance from the anterior end of the left brain hemisphere and divided into three approximately equal pieces of at least 10-25 mg each (the most dorsal piece was for vector DNA, the next piece was for RNA, and the most ventral piece was for storage). Each piece was placed into a pre-labeled round-bottom 2 ml microcentrifuge tube, then flash frozen and kept at ≦-60°C.

[0301] A 2 mm thick slice was taken from the largest liver lobe immediately proximal to the slice taken for histology / IHC (see below) and divided into three pieces of at least 25 mg each (for vector DNA, RNA, and storage, respectively). Each piece was placed into a pre-labeled round-bottom 2 mL microcentrifuge tube and then flash frozen and kept at ≦-60°C.

[0302] Skeletal muscle - A 2 mm thick slice was collected from the gastrocnemius muscle of the hind limb just proximal to the slice collected for histology / IHC (see below) and divided into three fragments of approximately 10-25 mg each (for vector DNA, RNA, and storage, respectively). Each fragment was placed into a pre-labeled round-bottom 2 mL microcentrifuge tube, then flash frozen and kept at ≤-60°C.

[0303] The blood-washed red cell pellet is reserved for ddPCR analysis and should be kept at ≦-60°C.

[0304] Collection Method for Histology / IHC: Samples are immersion fixed in 10% neutral buffered formalin at room temperature for up to 72 hours, then transferred to 70% ethanol and stored at room temperature until shipping. After fixation, tissues are shipped for histological processing, H&E, and GALT immunohistochemistry. Tissues collected include:

[0305] Intact brain (right hemisphere) 2-5mm thick slices of liver from the largest lobe Slices of gastrocnemius muscle 2-5 mm thick Right ovary or testis (only available in animals at T35 and T180) Collection methods for biochemical analysis (quantification of GALT activity and galactose, galactitol, and Gal-1p): Tissues for biochemical analysis are homogenized, aliquoted, flash frozen and stored at ≦−60° C. Biochemical analysis is performed. Samples include:

[0306] Blood (plasma and washed RBCs) Brain (left hemisphere) Liver (sections not taken for vector DNA / RNA / retention or histology / IHC) Ovaries or testes (both from young animals combined with the left ovary or testis from an aged animal) ·eye If rats previously enrolled in a study need to be prematurely sacrificed or are found dead, blood samples (plasma and washed RBCs) should be collected and, if possible, a necropsy procedure should be performed. If rigor mortis has been shown to set in in animals found dead, a necropsy procedure should preferably be performed solely for the purpose of fixed tissue collection.

[0307] Metabolite data from the mouse and rat studies described above are shown in Table 15. [Table 16-1] [Table 16-2] References All publications, patent applications, patents, and other references (e.g., sequence database reference numbers) mentioned herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences mentioned herein are incorporated by reference, for example, in any table herein. Unless otherwise indicated, sequence accession numbers identified herein, including in any table herein, refer to the current database entries as of the filing date of this application. When a gene or protein refers to multiple sequence accession numbers, all sequence variants are encompassed.

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Claims

1. 1. A pharmaceutical composition for treating galactosemia in a subject in need thereof, the pharmaceutical composition comprising a therapeutically effective amount of a recombinant AAV virion, the recombinant AAV virion comprising: 1) an AAV capsid; and 2) a recombinant AAV vector, the recombinant AAV vector comprising an expression cassette, the expression cassette having a nucleotide sequence encoding human galactose-1-phosphate uridylyltransferase (hGALT) operably linked to one or more regulatory elements, the regulatory elements encoding the hGALT nucleotide sequence and polyadenylation (poly(A)). the pharmaceutical composition, which promotes expression of a tail signal, wherein the promoter comprises a cytomegalovirus (CMV) early enhancer / chicken β-actin / rabbit β-globin splice acceptor (CAG) promoter or an elongation factor-1 (EF-1) promoter, the poly(A) tail signal comprises a bovine growth hormone (bGH) poly(A) tail signal or a simian virus 40 (SV40) poly(A) tail signal, the expression cassette is flanked by inverted terminal repeat (ITR) nucleotide sequences, and the AAV capsid protein encapsidates the recombinant AAV vector.

2. 1. A pharmaceutical composition for increasing galactose metabolism, GALT protein expression, and / or GALT enzyme activity in a subject in need thereof, the pharmaceutical composition comprising a therapeutically effective amount of a recombinant AAV virion, the recombinant AAV virion comprising: 1) an AAV capsid; and 2) a recombinant AAV vector, the recombinant AAV vector comprising an expression cassette having a nucleotide sequence encoding human galactose-1-phosphate uridylyltransferase (hGALT) operably linked to one or more regulatory elements, the regulatory elements comprising:

1. The pharmaceutical composition of claim 1, wherein the expression cassette is flanked by inverted terminal repeat (ITR) nucleotide sequences, and the AAV capsid protein encapsidates the recombinant AAV vector.

2. The pharmaceutical composition of claim 1, wherein the expression cassette is flanked by inverted terminal repeat (ITR) nucleotide sequences, and the AAV capsid protein encapsidates the recombinant AAV vector.

3. A pharmaceutical composition for alleviating a disease state in a subject suffering from galactosemia, the pharmaceutical composition comprising a therapeutically effective amount of a recombinant AAV virion, the recombinant AAV virion comprising: 1) an AAV capsid; and 2) a recombinant AAV vector, the recombinant AAV vector comprising an expression cassette, the expression cassette having a nucleotide sequence encoding human galactose-1-phosphate uridylyltransferase (hGALT) operably linked to one or more regulatory elements, the regulatory elements comprising the hGALT nucleotide sequence. a promoter comprising a cytomegalovirus (CMV) early enhancer / chicken β-actin / rabbit β-globin splice acceptor (CAG) promoter or an elongation factor-1 (EF-1) promoter, the poly(A) tail signal comprising a bovine growth hormone (bGH) poly(A) tail signal or a simian virus 40 (SV40) poly(A) tail signal, and the expression cassette is flanked by inverted terminal repeat (ITR) nucleotide sequences; The pharmaceutical composition, wherein the disease state comprises jaundice, hepatosplenomegaly, hepatocellular failure, hypoglycemia, renal tubular dysfunction, hypotonia, sepsis, cataracts, ataxia, tremor, decreased bone density, or primary ovarian insufficiency.

4. 1. A pharmaceutical composition for reducing galactose levels, galactitol levels, and / or galactose-1-phosphate (Gal-1P) in a subject in need thereof, the pharmaceutical composition comprising a therapeutically effective amount of a recombinant AAV virion, the recombinant AAV virion comprising: 1) an AAV capsid; and 2) a recombinant AAV vector, the recombinant AAV vector comprising an expression cassette having a nucleotide sequence encoding human galactose-1-phosphate uridylyltransferase (hGALT) operably linked to one or more regulatory elements. the regulatory element drives expression of the hGALT nucleotide sequence and a polyadenylation (poly(A)) tail signal, the promoter comprising a cytomegalovirus (CMV) early enhancer / chicken β-actin / rabbit β-globin splice acceptor (CAG) promoter or an elongation factor-1 (EF-1) promoter, the poly(A) tail signal comprising a bovine growth hormone (bGH) poly(A) tail signal or a simian virus 40 (SV40) poly(A) tail signal, the expression cassette is flanked by inverted terminal repeat (ITR) nucleotide sequences, and the AAV capsid protein encapsidates the recombinant AAV vector.

5. A pharmaceutical composition for improving motor strength, motor coordination and / or neuromuscular coordination in a subject with galactosemia, said pharmaceutical composition comprising a therapeutically effective amount of a recombinant AAV virion, said recombinant AAV virion comprising 1) an AAV capsid, and 2) a recombinant AAV vector, said recombinant AAV vector comprising an expression cassette, said expression cassette having a nucleotide sequence encoding human galactose-1-phosphate uridylyltransferase (hGALT) operably linked to one or more regulatory elements, said regulatory elements encoding said hGALT nucleotide sequence and polyadenylation (poly( A)) The pharmaceutical composition, wherein the promoter promotes expression of a tail signal, wherein the promoter comprises a cytomegalovirus (CMV) early enhancer / chicken β-actin / rabbit β-globin splice acceptor (CAG) promoter or an elongation factor-1 (EF-1) promoter, the poly(A) tail signal comprises a bovine growth hormone (bGH) poly(A) tail signal or a simian virus 40 (SV40) poly(A) tail signal, the expression cassette is flanked by inverted terminal repeat (ITR) nucleotide sequences, and the AAV capsid protein encapsidates the recombinant AAV vector.

6. A pharmaceutical composition for improving spatial learning in a subject with galactosemia, said pharmaceutical composition comprising a therapeutically effective amount of a recombinant AAV virion, said recombinant AAV virion comprising: 1) an AAV capsid; and 2) a recombinant AAV vector, said recombinant AAV vector comprising an expression cassette, said expression cassette having a nucleotide sequence encoding human galactose-1-phosphate uridylyltransferase (hGALT) operably linked to one or more regulatory elements, said regulatory elements encoding said hGALT nucleotide sequence and a polyadenylation (poly(A)) tail signal. the promoter comprises a cytomegalovirus (CMV) early enhancer / chicken β-actin / rabbit β-globin splice acceptor (CAG) promoter or an elongation factor-1 (EF-1) promoter; the poly(A) tail signal comprises a bovine growth hormone (bGH) poly(A) tail signal or a simian virus 40 (SV40) poly(A) tail signal; the expression cassette is flanked by inverted terminal repeat (ITR) nucleotide sequences; and the AAV capsid proteins encapsidate the recombinant AAV vector.

7. A pharmaceutical composition for reducing and / or reversing ovarian failure or regulating follicle-stimulating hormone, luteinizing hormone, and / or anti-Mullerian hormone in a subject with galactosemia, said pharmaceutical composition comprising a therapeutically effective amount of a recombinant AAV virion, said recombinant AAV virion comprising: 1) an AAV capsid; and 2) a recombinant AAV vector, said recombinant AAV vector comprising an expression cassette, said expression cassette having a nucleotide sequence encoding human galactose-1-phosphate uridylyltransferase (hGALT) operably linked to one or more regulatory elements, said regulatory elements comprising said hGALT nucleoside.

10. The pharmaceutical composition of claim 1, wherein the promoter comprises a cytomegalovirus (CMV) early enhancer / chicken β-actin / rabbit β-globin splice acceptor (CAG) promoter or an elongation factor-1 (EF-1) promoter, the poly(A) tail signal comprises a bovine growth hormone (bGH) poly(A) tail signal or a simian virus 40 (SV40) poly(A) tail signal, the expression cassette is flanked by inverted terminal repeat (ITR) nucleotide sequences, and the AAV capsid proteins encapsidate the recombinant AAV vector.

8. A pharmaceutical composition for inhibiting cataract formation, limiting the severity of cataract formation, or promoting cataract resorption in a subject with galactosemia, said pharmaceutical composition comprising a therapeutically effective amount of a recombinant AAV virion, said recombinant AAV virion comprising: 1) an AAV capsid; and 2) a recombinant AAV vector, said recombinant AAV vector comprising an expression cassette, said expression cassette having a nucleotide sequence encoding human galactose-1-phosphate uridylyltransferase (hGALT) operably linked to one or more regulatory elements, said regulatory elements encoding said hGALT nucleotide sequence and polyadenylation. the promoter comprises a cytomegalovirus (CMV) early enhancer / chicken β-actin / rabbit β-globin splice acceptor (CAG) promoter or an elongation factor-1 (EF-1) promoter, the poly(A) tail signal comprises a bovine growth hormone (bGH) poly(A) tail signal or a simian virus 40 (SV40) poly(A) tail signal, the expression cassette is flanked by inverted terminal repeat (ITR) nucleotide sequences, and the AAV capsid proteins encapsidate the recombinant AAV vector.

9. A pharmaceutical composition for reducing prepubertal growth retardation or improving weight gain in a subject with galactosemia, the pharmaceutical composition comprising a therapeutically effective amount of a recombinant AAV virion, the recombinant AAV virion comprising: 1) an AAV capsid; and 2) a recombinant AAV vector, the recombinant AAV vector comprising an expression cassette having a nucleotide sequence encoding human galactose-1-phosphate uridylyltransferase (hGALT) operably linked to one or more regulatory elements, the regulatory elements controlling the hGALT nucleotide sequence and polyadenylation (polyadenylation). (A)) The pharmaceutical composition, wherein the promoter promotes expression of a tail signal, wherein the promoter comprises a cytomegalovirus (CMV) early enhancer / chicken β-actin / rabbit β-globin splice acceptor (CAG) promoter or an elongation factor-1 (EF-1) promoter, the poly(A) tail signal comprises a bovine growth hormone (bGH) poly(A) tail signal or a simian virus 40 (SV40) poly(A) tail signal, the expression cassette is flanked by inverted terminal repeat (ITR) nucleotide sequences, and the AAV capsid protein encapsidates the recombinant AAV vector.

10. A pharmaceutical composition for reducing the severity of visceral neuromuscular disorders, or the severity of neurological disorders and / or socio-emotional disorders in a subject with galactosemia, said pharmaceutical composition comprising a therapeutically effective amount of a recombinant AAV virion, said recombinant AAV virion comprising: 1) an AAV capsid; and 2) a recombinant AAV vector, said recombinant AAV vector comprising an expression cassette, said expression cassette having a nucleotide sequence encoding human galactose-1-phosphate uridylyltransferase (hGALT) operably linked to one or more regulatory elements, said regulatory elements encoding said hGALT nucleotide sequence and a polyadenylation factor. the promoter comprises a cytomegalovirus (CMV) early enhancer / chicken β-actin / rabbit β-globin splice acceptor (CAG) promoter or an elongation factor-1 (EF-1) promoter, the poly(A) tail signal comprises a bovine growth hormone (bGH) poly(A) tail signal or a simian virus 40 (SV40) poly(A) tail signal, the expression cassette is flanked by inverted terminal repeat (ITR) nucleotide sequences, and the AAV capsid proteins encapsidate the recombinant AAV vector.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the hGALT has the amino acid sequence of SEQ ID NO:

1.

12. The pharmaceutical composition of any one of claims 1 to 10, wherein the nucleotide sequence encoding GALT has at least 85% identity to the nucleotide sequence of SEQ ID NO: 2 or its reverse complement.

13. The pharmaceutical composition according to any one of claims 1 to 10, wherein the nucleotide sequence encoding GALT comprises or consists of the nucleotide sequence of SEQ ID NO: 2 or its reverse complement.

14. 11. The pharmaceutical composition of any one of claims 1 to 10, wherein the promoter comprises the nucleotide sequence of SEQ ID NO:9 or SEQ ID NO:10, or its reverse complement, and the poly A signal sequence comprises the nucleotide sequence of SEQ ID NO:15 or SEQ ID NO:16, or its reverse complement.

15. A pharmaceutical composition described in any one of claims 1 to 10, wherein the expression cassette further comprises a WPRE element.

16. the ITRs are i) a 5' AAV2 ITR having the nucleotide sequence of SEQ ID NO:11, or its reverse complement, and a 3' AAV2 ITR having the nucleotide sequence of SEQ ID NO:12, or its reverse complement; or ii) a 5' ITR having the nucleotide sequence of SEQ ID NO: 11, or its reverse complement, and a modified self-complementary 3' ITR having the nucleotide sequence of SEQ ID NO: 13, or its reverse complement. The pharmaceutical composition according to any one of claims 1 to 10, comprising:

17. A pharmaceutical composition described in any one of claims 1 to 10, wherein the expression cassette comprises a nucleotide sequence encoding human GALT, having at least 85% identity to the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or its reverse complement.

18. A pharmaceutical composition described in any one of claims 1 to 10, wherein the expression cassette comprises a nucleotide sequence encoding human GALT that has at least 85% identity to the nucleotide sequence of SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or its reverse complement.

19. A pharmaceutical composition described in any one of claims 1 to 10, wherein the expression cassette comprises a nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or a reverse complement thereof.

20. A pharmaceutical composition described in any one of claims 1 to 10, wherein the expression cassette comprises the nucleotide sequence of SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or a reverse complement thereof.

21. The pharmaceutical composition according to any one of claims 1 to 10, wherein the recombinant AAV vector is a self-complementary AAV vector (scAAV).

22. 11. The pharmaceutical composition of any one of claims 1 to 10, wherein the AAV capsid has an amino acid sequence having at least 85% identity to SEQ ID NO: 18 (AAV9).

23. The pharmaceutical composition of any one of claims 1 to 10, wherein the AAV capsid has the amino acid sequence of SEQ ID NO:

18.

24. The pharmaceutical composition of any one of claims 1 to 10, wherein the pharmaceutical composition is administered intravenously, intra-arterially, intramuscularly, intracardially, intrathecally, subventricularly, epidurally, intracerebrally, intraventricularly, subretinally, intravitreally, intra-articularly, intraocularly, intraperitoneally, intrauterinely, intradermally, subcutaneously, transdermally, transmucosally, or by inhalation.

25. The pharmaceutical composition of any one of claims 1 to 10, wherein administration of the pharmaceutical composition reduces galactose levels, galactitol levels, and / or Gal-1P levels in the liver, muscle, brain, eyes, ovaries, red blood cells, and / or plasma of a subject.

26. The pharmaceutical composition of any one of claims 1 to 10, wherein administration of the pharmaceutical composition increases GALT protein expression and / or GALT enzyme activity in the liver, muscle, brain, cortex, cerebral tissue, Purkinje neurons, nerve cells, glial cells, eyes, red blood cells, and / or plasma of a subject.

27. ​​A pharmaceutical composition described in any one of claims 1 to 10, wherein the pharmaceutical composition is administered before the onset of cataracts in a subject.

28. 11. The pharmaceutical composition of claim 10, wherein the neurological and / or socio-emotional disorder is anxiety and / or depression, the neurological and / or socio-emotional disorder is locomotor function, and the locomotor function is tremor and / or ataxia.

29. The pharmaceutical composition of any one of claims 1 to 10, wherein the pharmaceutical composition is administered before the onset of puberty or the subject is a young pediatric subject or a child subject.

30. The pharmaceutical composition of any one of claims 1 to 10, wherein the levels of galactose, GAL-1P, and / or galactitol are monitored in the subject after administration of the pharmaceutical composition.

31. The pharmaceutical composition of any one of claims 1 to 10, wherein the AAV virion comprises a recombinant AAV vector comprising 1) an AAV9 capsid having the amino acid sequence of SEQ ID NO:18, and 2) an expression cassette having the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, and wherein the AAV capsid protein encapsidates the recombinant AAV vector.

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