Compositions useful in treatment of krabbe disease

Abstract

To provide improved treatment for Krabbe disease patients.SOLUTION: There is provided herein a composition comprising a recombinant adeno-associated virus (rAAV), where the rAAV comprises: (a) an AAV capsid targeting cells of a central nervous system and (b) a vector genome comprising (i) a signal peptide, and a sequence encoding a mature human galactosylceramidase protein of a specific amino acid sequence under control of a regulatory sequence directing expression of the protein, and (ii) an AAV inverted terminal repeat sequence necessary for packaging the vector genome into the AAV capsid, where the vector genome is packaged in the AAV capsid.SELECTED DRAWING: None

Description

[Background technology]

[0001] Adeno-associated virus (AAV), a member of the parvovirus family, is a small, non-enveloped, icosahedral virus with a single-stranded linear DNA (ssDNA) genome approximately 4.7 kilobases (kb) long. The wild-type genome contains inverted terminal repeats (ITRs) at both ends of the DNA strand, as well as two open reading frames (ORFs), rep and cap. Rep is composed of four overlapping genes that encode the rep proteins required for the AAV life cycle, and cap contains overlapping nucleotide sequences of the capsid proteins VP1, VP2, and VP3, which self-assemble to form a capsid with icosahedral symmetry.

[0002] Recombinant adeno-associated virus (rAAV) vectors derived from replication-defective human parvoviruses have been described as suitable vehicles for gene delivery. Typically, functional rep and cap genes are removed from the vector, resulting in a replication-defective vector. These functions are provided in the vector production system but are not present in the final vector.

[0003] To date, there are several different well-characterized AAVs isolated from humans or non-human primates (NHPs). Different serotypes of AAV have been found to exhibit different transfection efficiencies and tropisms for different cells or tissues. Many different AAV clades have been described in WO2005 / 033321, including clade F, which has been identified with only three members: AAV9, AAVhu31, and AAVhu32. A structural analysis of AAV9 has been reported by MADiMattia et al, J. Virol. (June 2009) and others. 2012) vol.86 no.12 6947-6958. In this paper, it is reported that AAV9 has 60 copies (total) of three variable proteins (vps), which are encoded by the cap gene and have overlapping sequences. These include VP1 (87 kDa), VP2 (73 kDa), and VP3 (62 kDa), which are present in a predicted ratio of 1:1:10, respectively. The entire sequence of VP3 is within VP2, and all of VP2 is within VP1. VP1 has a unique N-terminal domain. Refined coordinates and structure factors are available from the RCSB PDB database under the accession number 3UX1.

[0004] Several different AAV9 variants have been engineered to detarget or target different tissues. See, for example, N. Pulicheria, "Engineering Liver-detargeted AAV9 Vectors for Cardiac and Musculoskeletal Gene Transfer", Molecular Therapy, Vol, 19, no. 6, p. 1070-1078 (June 2011). The development of AAV9 variants to deliver genes across the blood-brain barrier has also been reported. See, e.g., BE Everman et al, Nature Biotech, Vol. 34, No. 2, p204-211 (published online 1 Feb 2016) and Caltech press release, A. Wetherston, www.neurology-central.com / 2016 / 02 / 10 / successful-delivery-of-genes-through-the-blood-brain-barrier / , accessed 10 / 05 / 2016. See also WO2016 / 0492301 and US8,734,809.

[0005] AAVhu68 was identified following amplification of the capsid gene from a natural source and has recently been identified as a new AAV capsid. See also WO2018 / 160582. This AAV is within phylogenetic group F, as is AAV9.

[0006] Krabbe disease (globoid cell leukodystrophy, GLD) is an autosomal recessive lysosomal storage disorder (LSD) caused by mutations in the gene encoding the hydrolase galactosylceramidase (GALC) (Wenger DA, et al. (2000) Mol Genet Metab. 70(1):1-9). This enzyme is involved in the degradation of certain galactolipids, including galactosylceramide (ceramide) and galactosylsphingosine (psychosine), which are found almost exclusively in the myelin sheath. In Krabbe disease, GALC deficiency causes toxic accumulation of psychosine (but not galactosylceramide) in lysosomes (Svennerholm et al., 1980). Psychosine accumulation is particularly toxic to myelin-producing oligodendrocytes in the CNS and Schwann cells in the PNS, resulting in rapid and widespread death of these cell types. Myelin degradation in both the CNS and PNS is accompanied by gliosis of reactive astrocytes and infiltration of multinucleated giant macrophages ("globoid cells") (Suzuki K. (2003) J Child Neurol. 18(9): 595-603). Galactosylceramide does not accumulate in the absence of GALC activity, mainly due to hydrolysis by another enzyme, GM1 ganglioside β-galactosidase (Kobayashi T., et al. (1985) J Biol Chem. 260(28): 14982-7) and death of oligodendrocytes, which contribute to the cessation of galactosylceramide synthesis (Svennerholm L., et al. (1980) J Lipid Res. 21(1): 53-64).

[0007] Currently, the only disease-modifying treatment available for Krabbe disease is hematopoietic stem cell transplantation (HSCT), which is often provided by umbilical cord blood transplantation (UCBT), allogeneic peripheral blood stem cells, or allogeneic bone marrow. Treatment of patients with infantile Krabbe disease using HSCT has been only modestly successful, and they typically become symptomatic before their first birthday. When performed after the onset of overt symptoms in infantile Krabbe disease, HSCT provides only minimal neurological improvement and does not substantially improve survival (Escolar ML, et al. (2005) N Engl J Med. 352(20):2069-81). HSCT can be effective when performed in presymptomatic patients, but still results in poor motor outcomes (Escolar ML, et al. (2005) N Engl J Med. 352(20):2069-81). Med.352(20):2069-81, Wright MD,et al.(2017)Neurology.89(13):1365-1372, van den Broek BTA,et al.(2018)Blood Adv.2(1):49-60). Infants who received transplants before 30 days of age had better survival and functional outcomes compared to infants who received transplants later (Allewelt H.,et al.(2018)Biol Blood Marrow Transplant.24(11):2233-2238). Presymptomatic transplantation has been reported to result in significantly better outcomes compared with untreated or treated infantile Krabbe disease patients after symptom onset, with progressive central myelination, normal receptive language, attenuation of symptom severity, and longer survival (Escolar ML,et al.(2005)N Engl J Med.352(20):2069-81; Duffner PK,et al.(2009)Genet Med.11(6):450-4; Wright MD,et al.(2017)Neurology.89(13):1365-1372). Nevertheless, most children who are treated before the onset of symptoms remain well below average for height and weight and have progressive gross motor delays ranging from mild spasticity to inability to walk independently (Escolar ML, et al. (2005) N Engl J Med. 352(20):2069-81; Duffner PK, et al. (2009 )Genet Med.11(6):450-4). Some children also have residual disabilities, including acquired microcephaly, the need for gastrostomy, and dysarthria (Duffner PK,et al.(2009)Genet Med.11(6):450-4). Furthermore, HSCT appears to only affect CNS-specific disease pathology. Clinical features associated with PNS pathology, such as peripheral neuropathy, remain unaffected by HSCT. These results highlight the limitations of HSCT, particularly in the early-onset form, where the rapid progression of the disease exceeds the time required for hematopoietic stem cells to engraft, migrate to the CNS, differentiate, and provide a therapeutic effect through GALC secretion and cross-correction (i.e., the process in which enzymes secreted by corrective cells are taken up by GALC-deficient cells).

[0008] There remains a need in the art for improved treatments for patients with Krabbe disease. Summary of the Invention

[0009] Compositions are provided that include a recombinant adeno-associated virus (rAAV), comprising an AAV capsid that targets cells of the central nervous system, and (i) a coding sequence for galactosylceramidase that encodes a mature galactosylceramidase protein having the amino acid sequence of SEQ ID NO: 10 under the control of sequences directing expression of the protein, and (ii) a vector genome that includes AAV inverted terminal repeats necessary for packaging the vector genome into the AAV capsid, wherein the vector genome is packaged into the AAV capsid. In certain embodiments, the AAV capsid is an AAVhu68 capsid. In certain embodiments, the coding sequence has a nucleic acid sequence of SEQ ID NO: 9, or a sequence that is 95%-99.9% identical thereto. In certain embodiments, the coding sequence encodes a mature protein of SEQ ID NO: 10, and an exogenous signal peptide suitable for human cells of the central nervous system. In certain embodiments, the regulatory sequence includes a beta-actin promoter, an intron, and / or a rabbit globin polyA. In a specific embodiment, the composition comprises a rAAV having the vector genome CB7.CI.hGALC.rBG.

[0010] In certain embodiments, a recombinant adeno-associated virus is provided, comprising an AAV capsid for targeting cells of the central nervous system, and a vector genome comprising (i) a coding sequence for galactosylceramidase encoding a mature galactosylceramidase protein having the amino acid sequence of SEQ ID NO: 10 under the control of regulatory sequences directing expression of the mature galactosylceramidase protein, and (ii) an AAV inverted terminal end sequence required for packaging the vector genome into the AAV capsid. In certain embodiments, the AAV capsid is an AAVhu68 capsid. In certain embodiments, the coding sequence has a nucleic acid sequence of SEQ ID NO: 9, or a sequence 95%-99.9% identical thereto. In certain embodiments, the coding sequence encodes a mature protein of SEQ ID NO: 10, and an exogenous signal peptide suitable for human cells of the central nervous system. In certain embodiments, the regulatory sequences comprise a beta-actin promoter, an intron, and / or a rabbit globin polyA. In certain embodiments, the vector genome is CB7.CI.hGALC.RBG.

[0011] In certain embodiments, a composition is provided, comprising a stock of rAAV useful for treating Krabbe disease. In certain embodiments, a use of a composition is provided, comprising a stock of rAAV in the preparation of a medicament. In certain embodiments, a composition is provided, comprising a stock of rAAV useful for treating peripheral nerve dysfunction and / or treating Krabbe disease. In certain embodiments, the rAAV can be administered as a combination therapy with hematopoietic stem cell therapy, bone marrow transplantation, and / or substrate reduction therapy.

[0012] In a specific embodiment, a coding sequence for galactosylceramidase encoding a signal peptide and a mature human galactosylceramidase having the amino acid sequence of SEQ ID NO: 10 (aa 43 to 685) are provided. A plasmid comprising the ctosylceramidase protein is provided. In a specific embodiment, the plasmid comprises the nucleic acid sequence of SEQ ID NO: 9, or a sequence that is 95% to 99.9% identical thereto.

[0013] In certain embodiments, the use of a composition for treating Krabbe disease is provided, which corrects peripheral nerve dysfunction that causes respiratory insufficiency and loss of motor function caused by Krabbe disease, or delays the onset or frequency of attacks caused by Krabbe disease, comprising administering a composition comprising a stock of recombinant adeno-associated virus (rAAV) to a patient, wherein the rAAV comprises (a) an AAV capsid that targets cells in the central nervous system, and (b) a vector genome that comprises a galactosylceramidase coding sequence that encodes a mature galactosylceramidase protein having the amino acid sequence of SEQ ID NO: 10 under the control of a regulatory sequence that directs protein expression, wherein the vector genome further comprises an AAV inverted terminal sequence that is necessary for packaging the vector genome into the AAV capsid, and the vector genome is packaged into the AAV capsid. In certain embodiments, the patient has late infantile Krabbe disease (LIKD). In certain embodiments, the patient has juvenile Krabbe disease (JKD). In certain embodiments, the patient has adolescent / adult onset Krabbe disease. In certain embodiments, rAAV is administered as a combination therapy with hematopoietic stem cell transplantation (HSCT), bone marrow transplantation, and / or matrix synthesis suppression therapy. In certain embodiments, rAAV is delivered via intrathecal, intraventricular, or intraparenchymal administration.

[0014] In certain embodiments, the provided compositions are formulated for intrathecal, intraventricular, or intraparenchymal administration.In certain embodiments, the compositions are administered as a single dose via computed tomography (CT)-guided suboccipital injection into the cisterna magna (intracisternal).

[0015] These and other aspects of the present invention will become apparent from the following detailed description of the invention. [Brief description of the drawings]

[0016] [Figure 1]An alignment of the capsid sequences of AAV9 (SEQ ID NO:4) and AAVhu68 (SEQ ID NO:2) is provided. The two amino acids that differ between AAV9 and AAVhu68 capsids are located in the VP1 (67, 157) and VP2 (157) regions of the capsid. Abbreviations: AAV9: adeno-associated virus serotype 9, AAVhu68: adeno-associated virus serotype hu68, VP1: viral protein 1, VP2: viral protein. [Diagram 2] A schematic diagram of the CB7.CI.hGALC.rBG vector genome is shown. A linear map shows the vector genome, designed to express human GALC under the control of the ubiquitous CB7 promoter. CB7 consists of a hybrid between the CMV IE enhancer and the chicken β-actin (CB) promoter. Abbreviations: CMV IE sa: Itomegalovirus immediate early, GALC: Galactosylceramidase, ITR: Inverted terminal repeats, PolyA: Polyadenylation, rBG: Rabbit β-globin. [Diagram 3] 1 shows the vector map of pENN AAV.CB7.CI.RBG(p1044), with an engineered cGALC gene (cGALCco) inserted. [Figure 4A] Neuropathological and behavioral phenotypic progression in Twitcher mice (twi / twi). Mice show accumulation of cytotoxic psychosine followed by infiltration of PNS and CNS white matter by phagocytic globoid cells. After an initial period of myelination, demyelination is observed in the PNS and to a lesser extent in the CNS, resulting from death of myelinating Schwann cells and oligodendrocytes, respectively. The behavioral phenotype appears around PND20 consisting of tremors, seizures, and hindlimb weakness followed by paralysis and weight loss, necessitating euthanasia around PND40. (Adapted from Nicaise AM,et al.(2016)J Neurosci Res.94(11):1049-61). Abbreviations: CNS: central nervous system; PND: postnatal days; PNS: peripheral nervous system; twi, seizure loss-of-function allele. [Figure 4B] 1 shows the study design for evaluation of AAV.CB7.cGALCco.rBG gene therapy using the Twitcher mouse model. [Diagram 5] Survival curves following intravenous or intracerebroventricular administration of rAAVhu68.hGALC to presymptomatic Twitcher mice are shown. IV administration of a high dose of rAAVhu68.hGALC (1.00×10 GC [equivalent to 1.00×10 GC / kg]) to neonatal Twitcher mice on PND0 resulted in a median survival of 49 days (N=6). ICV administration of a 5-fold lower dose of rAAVhu68.hGALC (2.00×10 GC) to neonatal Twitcher mice on PND0 resulted in a median survival of 61.5 days (N=10). Survival of Twitcher mice administered rAAVhu68.hGALC was compared to that of age-matched control Twitcher mice administered vehicle (PBS) ICV (N=8). Abbreviations: GC: genome copies, ICV: intracerebroventricular, IV: intravenous, N: number of animals, PND: postnatal days. [Figure 6] Survival curves following intraventricular delivery of GALC to presymptomatic Twitcher mice using different AAV capsids. AAVhu68 vector (rAAVhu68.hGALC) administered ICV at a dose of 2.00x1010 GC on PND0 resulted in a median survival of 61.5 days (N=10). AAV1, AAV3b, or AAV5 vectors administered ICV at a dose of 2.00x1010 GC on PND0 resulted in a median survival of less than 60 days (AAV1: 57 days [N=6], AAV3b: 51 days [N=9], AAV5: 51 days [N=6]), whereas control Twitcher mice administered vehicle only (PBS) ICV on PND0 showed a median survival of 43 days (N=8). Abbreviations: AAV3b: AAV serotype 3b, AAV5: AAV serotype 5, AAV1: AAV serotype 1, and AAVhu68: AAV serotype hu68 (rAAVhu68.hGALC); GC: genome copies; ICV: intracerebroventricular; N: number of animals; PBS: phosphate buffered saline; PND: postnatal days. [Figure 7]Neuromotor function after intracerebroventricular administration of different doses of rAAVhu68.hGALC in presymptomatic Twitcher mice. Presymptomatic Twitcher mice (twi / twi) were administered rAAVhu68.hGALC ICV at doses of 2.00×1010 GC (N=10), 5.00×1010 GC (N=12), or 1.00×1011 GC (N=12) on PND0. The doses per gram of brain mass (0.15 g for neonatal mice) corresponded to 1.30×1011 GC / g, 3.30×1011 GC / g, and 6.70×1011 GC / g, respectively. As controls, PBS was administered ICV to age-matched pre-symptomatic Twitcher mice (twi / twi) (N=8) and age-matched non-affected mice (twi / + or + / +, N=14) on PND0. On PND35, neuromotor function was assessed by the latency (seconds) to fall of mice running on an accelerating rod that initially rotated at 5 RPM and increased to 40 RPM over 120 seconds. **p<0.01 determined by one-way ANOVA followed by Dunn's multiple comparison test. Abbreviations: ANOVA: analysis of variance; GC: genome copies; ICV: intracerebroventricular; N: number of animals; PBS: phosphate buffered saline; PND: postnatal days; RPM: revolutions per minute. [Figure 8] Survival curves after intracerebroventricular administration of different doses of rAAVhu68.hGALC in presymptomatic Twitcher mice. Control Twitcher mice (twi / twi) administered vehicle (PBS) ICV had a median survival of 43 days (N=8). Twitcher mice (twi / twi) administered rAAVhu68.hGALC ICV at PND0 had a median survival of 61.5 days at a dose of 2.00x1010GC (N=10), 99 days at a dose of 5.00x1010GC (N=12), and 130 days at a dose of 1.00x1011GC (N=12). The dose of rAAVhu68.hGALC per gram of brain mass (0.15 g for newborn mice) corresponded to 1.30 × 10 GC / g, 3.30 × 10 GC / g, and 6.70 × 10 GC / g, respectively. Abbreviations: GC: genome copies, ICV: intracerebroventricular, N: number of animals, PBS: phosphate buffered saline, PND: postnatal days. [Figure 9] Neuromotor function following intraventricular administration of rAAVhu68.hGALC to symptomatic Twitcher mice. Presymptomatic neonatal Twitcher mice (twi / twi) received rAAVhu68.hGALC ICV at a dose of 1.00x1011 GC (N=12) on PND0. Early symptomatic Twitcher mice (twi / twi) received rAAVhu68.hGALC ICV at a dose of either 1.00x1011 GC (N=12) or 2.00x1011 GC (N=11) on PND12. Late symptomatic Twitcher mice (twi / twi) received rAAVhu68.hGALC ICV at a higher dose of 2.00x1011 GC (N=16) on PND21. Age-matched non-diseased mice (twi / + and + / +, N=27) and diseased Twitcher mice (twi / twi, N=15) received vehicle (PBS) ICV as a control on PND12. On PND35, neuromotor function was assessed by the latency (seconds) to fall of mice running on an accelerating rod that initially rotated at 5 RPM and increased to 40 RPM over 120 seconds. **p<0.01 and ***p<0.001 determined by one-way ANOVA followed by Dunn's multiple comparison test. Abbreviations: ANOVA: analysis of variance; GC: genome copies; ICV: intracerebroventricular; N: number of animals; PBS: phosphate buffered saline; PND: postnatal days; RPM: revolutions per minute. [Figure 10]Survival curves following intracerebroventricular administration of rAAVhu68.hGALC in symptomatic Twitcher mice. Early symptomatic Twitcher mice (twi / twi) administered rAAVhu68.hGALC ICV at either 1.00x1011 GC (N=12) or 2.00x1011 GC (N=11) on PND12 had a median survival of 71 or 81 days, respectively. Late symptomatic Twitcher mice (twi / twi) administered rAAVhu68.hGALC ICV at 2.00x1011 GC (N=16) on PND21 had a median survival of 51.5 days. In comparison, control Twitcher mice (twi / twi, historical controls, N=8 from Study 1 [PND0] and N=4 from Study 2 [PND12]) administered vehicle (PBS) ICV on either PND0 or PND12 showed a median survival of less than 50 days. Abbreviations: GC: genome copies, ICV: intracerebroventricular, N: number of animals, PBS: phosphate-buffered saline, PND: postnatal days. [Figure 11A-11B]Clinical scoring and neuromotor function after intracerebroventricular administration of rAAVhu68.hGALC in symptomatic Twitcher mice. Early symptomatic Twitcher mice (twi / twi, N=9) were administered rAAVhu68.hGALC at a dose of 2.00×10 GC ICV on PND12. Age-matched early symptomatic Twitcher mice (twi / twi, N=9), unaffected Twitcher heterozygous mice (twi / +, N=10), and wild-type mice (N=8) were administered PBS ICV on PND12 as a control. (FIG. 11A) Each mouse was evaluated daily for clasping ability, gait, tremor, kyphosis, and fur quality using clinical scoring assessments starting on PND22 until necropsy on PND40. Each animal was assigned a cumulative score, which was then normalized by AAV treatment during the study. Higher scores indicate worse clinical status. (FIG. 11B) At PND35, neuromotor function was assessed by the time (seconds) to fall of mice running on an accelerating rod that initially rotated at 5 RPM and increased to 40 RPM over 120 seconds. *p<0.05 determined by one-way ANOVA followed by Dunn's multiple comparison test. Abbreviations: GC: genome copies; ICV: intracerebroventricular; N: number of animals; PBS: phosphate buffered saline; PND: postnatal days; RPM: revolutions per minute. [Figure 12] Histology of the sciatic nerve after intraventricular administration of rAAVhu68.hGALC in symptomatic Twitcher mice. Early symptomatic Twitcher mice (twi / twi, N=9) received rAAVhu68.hGALC at a dose of 2.00×1011 GC ICV on PND12. Age-matched early symptomatic Twitcher mice (twi / twi, N=9) and unaffected wild-type mice (N=8) received PBS ICV on PND12 as a control. Twenty-eight days later, on PND40, mice were necropsied and samples of the sciatic nerve were obtained. Luxol blue and PAS reactive staining were performed on tissue sections to visualize myelin (dark staining) and globoid cells (light staining), respectively. Abbreviations: GC: genome copies, ICV: intracerebroventricular, N: number of animals, PBS: phosphate buffered saline, PAS: periodic acid Schiff, PND: postnatal days. [Figures 13A-13C] GALC activity 28 days after intracerebroventricular administration of rAAVhu68.hGALC in symptomatic Twitcher mice on postnatal day 12 is shown. Early symptomatic Twitcher mice (twi / twi, N=9) received rAAVhu68.hGALC at a dose of 2.00×1011 GC ICV on PND12. Age-matched early symptomatic Twitcher mice (twi / twi, N=9) and unaffected wild-type mice (N=8) received PBS ICV on PND12 as a control. 28 days later, on PND40, mice were necropsied and brain, liver, and serum samples were obtained. Levels of GALC enzyme activity (relative FU) were quantified using a fluorophore-based assay. Abbreviations: FU: fluorescent units, GALC: galactosylceramidase, GC: genome copies, ICV: intracerebroventricular, N: number of animals, PBS: phosphate buffered saline, PND: postnatal days. [Figure 14A-14B] Preliminary survival curves after combined therapy of rAAVhu68.hGALC and bone marrow transplantation are shown. Twitcher mice (twi / twi) were treated with BMT only (N=13, PND10), rAAVhu68.hGALC only (N=12, PND0, or N=13, PND12, ICV, 1.00×1011 GC), rAAVhu68.hGALC followed by BMT (N=7, PND0 and PND10, respectively), or BMT followed by rAAVhu68.hGALC (N=7, PND10 and PND12, respectively). Twitcher mice (twi / twi) receiving PBS served as preexisting controls only (N=8, study 1, PND0; N=4, study 2, PND12). Interim survival results are shown and experiments are still ongoing. Abbreviations: BMT: bone marrow transplant, GC: genome copies, ICV: intracerebroventricular, N: number of animals, PBS: phosphate buffered saline, PND: postnatal days [Figure 15]The progression of the neuropathology and behavioral phenotype of Crabbyne is shown (Wenger DA,et al.(1999)J Hered.90(1):138-42; Bradbury A.,et al.(2016)Neuroradiol J.29(6):417-424; Bradbury AM,et al.(2016b)94(11):1007-17; Bradbury AM,et al.(2018)Hum Gene Ther.29(7):785-801). Dashed lines indicate that data for the previous time point for the specified phenotype is not described. *Asterisks refer to demyelination observed in histology. Abbreviations: BAER: brainstem auditory evoked response, CNS: central nervous system, MRI: magnetic resonance imaging, NCV: nerve conduction velocity, PNS: peripheral nervous system. [Figure 16] The design of the first first-in-human phase 1 / 2 clinical trial is shown. In cohort 1 (low dose) and cohort 2 (high dose), 3 subjects will be dosed each, followed by mandatory safety committee review after subjects #3 and #6. After enrollment of the first subject in cohort 1 and cohort 2, the next 2 subjects in each cohort will be enrolled simultaneously, with an interval of 30 days. Six subjects in cohort 3 (MTD) will be enrolled simultaneously, without staggered dosing. Abbreviations: FIH: first-in-human, LTFU: long-term follow-up, MTD: maximum tolerated dose. [Figure 17] A decision tree for safety assessment of the proposed Phase 1 / 2 study is shown. *Study discontinuation criteria include occurrence of more than one subject with grade 3 or higher adverse events (AEs) related to the investigational drug or ICM injection procedure as assessed by the investigator. **Medical review will be performed by the medical monitor with the principal investigator. ***Subjects in cohort 3 will be enrolled simultaneously. Dosing at the MTD will not be staggered with a 4-week safety observation period between each subject. No safety committee review will be required after the first 3 subjects in cohort 3 are enrolled. Abbreviations: AE: adverse event; ICM: intracisternal; MTD: maximum tolerated dose; SRT: safety review trigger. [Figures 18A-18C] A table showing the schedule of events for the Phase 1 / 2 study is shown. [Figure 19] Shown is brain engraftment of GFP+ donor cells in the cerebellum of wild-type and Twitcher (Krabbe) mice 8 weeks after HSCT. [Figure 20] Comparison of serum GALC activity in Twitcher mice administered rAAVhu68 with either engineered GALC (cGALCco) or native canine GALC (cGALnat) sequences. Improved survival was observed in Twitcher mice administered rAAVhu.cGALCco compared to rAAVhu68 with the native sequence. [Figure 21] A linear vector map of the trans-plasmid pAAV2 / hu68.KanR (p0068) is shown. Abbreviations: AAV2: adeno-associated virus serotype 2, AAVhu68: adeno-associated virus serotype hu68, bp: base pairs, Cap: capsid, KanR: kanamycin resistance, Ori: origin of replication, Rep: replicase. [Fig. 22A-22B] The adenovirus helper plasmid pAdDeltaF6(KanR) is shown. (FIG. 22A) Derivation of the helper plasmid pAdΔF6 from the parent plasmid pBHG10 via the intermediates pAdΔF1 and pAdΔF5. (FIG. 22B) The ampicillin resistance gene of pAdΔF6 was replaced with the kanamycin resistance gene to obtain pAdΔF6(Kan). [Figure 23] 1 shows the study design for evaluation of AAV.CB7.cGALCco.rBG gene therapy in Crabbean cats. [Figures 24A-24C] Survival and enzyme secretion into CSF ​​after ICM administration of AAVhu68.cGALC to Crabbyne mice. (Figure 24A) Survival curve (ongoing). (Figure 24B and Figure 24C) Measurement of GALC activity in CSF using fluorescent substrate (Marker Gene Technologies, Inc., Cat. No. M2774). [Fig. 25A-25D]FIG. 25A shows nerve conduction velocity (NCV) in the tibial motor nerve (FIG. 25A), radial sensory nerve (FIG. 25B), sciatic motor nerve (FIG. 25C), and ulnar motor nerve (FIG. 25D) in the Crabbesian dog after administration of AAVhu68.cGALC. Periodic NCV recordings show delayed (FIG. 25A) or undetected (FIG. 25B) signals in the Crabbesian sham-treated dogs, while all four rAAVhu68.cGALC-treated animals have normalized velocities similar to age-matched WT control dogs. [Figure 25E] 13 shows the results of neurological examination of sham and rAAVhu68.cGALC-treated Crabbinone mice. [Figures 26A-26C] Results from histology of brain sections from sham- and AAVhu68.cGALC-treated Crabbei mice are shown: (FIG. 26A) Luxol blue staining of myelin; (FIG. 26B) IBA1 immunostaining (microglial marker) and (FIG. 26C) its quantification. [Figure 27] Body weight curves of wild-type (vehicle-treated) and Krabbein mice receiving either vehicle or AAVhu68.cGALC are shown. [Fig. 28A-28B] Safety monitoring of CSF and sensory neurons in sham-operated Krabbejn and wild-type dogs, and Krabbejn administered AAVhu68.cGALC. (FIG. 28A) CSF pleocytosis. (FIG. 28B) Histology of dorsal root ganglion from AAVhu68.cGALC-treated Krabbejn. [Figure 29A] MRI measurements are shown in sham-operated Crabbei dogs and wild-type dogs, and Crabbei dogs administered AAVhu68.cGALC. [Figure 29B] The results of cumulative scoring of the MRI measurements in FIG. 29A are shown. [Diagram 30]1 shows a flow diagram of the manufacturing process for vector production. Abbreviations: AEX: Anion Exchange, CRL: Charles River Laboratories, ddPCR: Digital Droplet Polymerase Chain Reaction, DMEM: Dulbecco's Modified Eagle's Medium, DNA: Deoxyribonucleic Acid, FFB: Final Formulation Buffer, GC: Genome Copy, HEK293: Human Embryonic Kidney 293 Cells, ITFFB: Intrathecal Final Formulation Buffer, PEI: Polyethylenimine, SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, TFF: Tangential Flow Filtration, USP: United States Pharmacopeia, WCB: Working Cell Bank. [Diagram 31] Figure 1 shows a manufacturing process flow diagram for vector formulation. Abbreviations: Ad5: adenovirus serotype 5, AUC: analytical ultracentrifugation, BDS: bulk drug substance, BSA: bovine serum albumin, CZ: Crystal Zenith, ddPCR: droplet digital polymerase chain reaction, E1A: early region 1A (gene), ELISA: enzyme-linked immunosorbent assay, FDP: final formulation, GC: genome copies, HEK293: human embryonic kidney 293 cells, ITFFB: intrathecal final formulation buffer, KanR: kanamycin resistance (gene), MS: mass spectrometry, NGS: next generation sequencing, qPCR: quantitative polymerase chain reaction, SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis, TCID50: tissue culture infectious dose 50%, UPLC: ultraperformance liquid chromatography, USP: United States Pharmacopeia. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Recombinant adeno-associated viruses (rAAV) expressing human galactosylceramidase (GALC) protein, as well as compositions comprising rAAV and uses thereof, are provided. In certain embodiments, rAAV.hGALC provides for the first time a disease-modifying treatment for symptomatic infantile Krabbe patients (Infantile Krabbe Disease, EIKD). In certain embodiments, rAAV.hGALC provides a treatment for presymptomatic infantile patients. In certain embodiments, rAAV.hGALC provides a therapy that can correct peripheral nerves that cause respiratory failure and loss of motor function. In certain embodiments, rAAV.hGALC provides an additional option for the treatment of late-onset patients whose benefit-risk ratio is not favorable for hematopoietic stem cell transplantation (HSCT), which is currently the only disease-modifying treatment.

[0018] As used herein, "rAAV.GALC" refers to a rAAV having an AAV capsid and a vector genome that includes at least the coding sequence of the galactosylceramidase protein (enzyme) packaged therein. rAAVhu68.GALC refers to a rAAV whose AAV capsid is an AAVhu68 capsid, as defined herein. The following examples also show other AAV capsids.

[0019] The term "cGALC" refers to the coding sequence expressing canine GALC and is used in the following examples for studies in dogs. Canine GALC has a 26 bp signal peptide and a full length protein of 669 amino acids.

[0020] The term "hGALC" refers to the coding sequence for human GALC.

[0021] Isoform 1 of hGALC is the canonical sequence and is 685 amino acids long. Its amino acid sequence is reproduced in SEQ ID NO:6. The mature protein is located at about amino acid 43 to about 685, with the signal peptide located at positions 1 to 42, although some suggest that the initiating Met is at position 17 rather than position 1. Although multiple isoforms of GALC (isoforms 1 to 5) are known and over 30 natural variants have been described, the inventors have found that a variation having a threonine (T) to alanine (A) mutation at position 641 is particularly desirable. This sequence is provided in SEQ ID NO:10. This variant is the protein sequence encoded by the coding sequence of human galactosylceramidase (hGALC) and is exemplified in the rAAV and vector genome examples provided herein. Galactosylceramidase (GALC) is also known as galactocerebrosidase, and these names are used interchangeably. In certain embodiments, this variant may be used in enzyme replacement therapy or combination therapy.

[0022] As used herein, "CB7.CI.hGALC.rBG" refers to a vector genome. CB7.CI.hGALC.rBG refers to a vector genome (e.g., as shown in FIG. 2) that comprises a coding sequence for human GALC under the control of the ubiquitous CB7 promoter and includes at least a CMV IE (cytomegalovirus immediate early) enhancer, a chimeric intron, and a rabbit β-globin (rBG) polyA sequence, all of which are flanked by 5′ and 3′ ITRs. In certain embodiments, CB7.CI.hGALC.rBG comprises a coding sequence for GALC that encodes a mature GALC protein having the amino acid sequence of SEQ ID NO: 10. In certain embodiments, CB7.CI.hGALC.rBG comprises a coding sequence for GALC that comprises a nucleic acid sequence of SEQ ID NO: 9, or a sequence 95%-99.9% identical thereto. In yet another embodiment, the CB7.CI.hGALC.rBG vector genome comprises SEQ ID NO: 19. In certain embodiments, CB7.CI.hGALC.rBG comprises a coding sequence for the mature protein of SEQ ID NO: 10 and an exogenous signal peptide.

[0023] In certain embodiments, fusion proteins are contemplated that include at least mature GALC (aa1-17, or aa1-42) with all or a portion of the native signal peptide removed and replaced with an exogenous signal peptide. Such fusion proteins may include an exogenous signal peptide and at least the mature human GALC protein (e.g., amino acids 43-695 of SEQ ID NO:6 or SEQ ID NO:10). In certain embodiments, the fusion protein includes an exogenous signal peptide that is suitable for human cells of the CNS (i.e., a signal peptide in which the native signal peptide has been replaced to improve production, intracellular trafficking, and / or secretion of the protein (i.e., hGALC) in cells present in the human CNS). Suitable exogenous signal peptides for human cells of the CNS include, but are not limited to, those found naturally in the secretory signal peptides of immunoglobulins (e.g., IgG), cytokines (e.g., IL-2, IL12, IL18, etc.), insulin, albumin, β-glucuronidase insulin, alkaline protease, von Willebrand factor (VWF), or fibronectin (see also, e.g., www.signalpeptide.de / index.php?m=listspdb_mammalia).

[0024] The present invention also encompasses nucleic acid sequences encoding the GALC proteins provided herein (e.g., SEQ ID NO:6, SEQ ID NO:10, or fusion proteins comprising mature GALC). In certain embodiments, the coding sequence is a cDNA sequence encoding the protein. However, the corresponding RNA sequence is also encompassed.

[0025] In certain embodiments, the nucleic acid coding sequence has a cDNA sequence of SEQ ID NO: 5, or a sequence 95% to 99.9% identical thereto, or a fragment thereof. Suitable fragments include the coding sequence of the mature protein (from about nt 127 to about nt 2058), or the coding sequence of the mature protein with a fragment of the signal peptide (e.g., from about nt 54 to about nt 2058). In certain embodiments, the coding sequence has a nucleic acid sequence encoding the mature hGALC of SEQ ID NO: 5 (nt 127 to 2058) or a fusion protein including its leader or an exogenous leader, or a sequence 95% to 99.9% identical thereto. In certain embodiments, the coding sequence has a nucleic acid sequence encoding the mature hGALC of SEQ ID NO: 5 (nt 127 to 2058), or a sequence 95% to 99.9% identical thereto, or a fragment thereof (including the leader sequence and a fragment of mature hGALC). In certain embodiments, the coding sequence encodes a full-length human GALC protein having the amino acid sequence of SEQ ID NO: 10. In a specific embodiment, the coding sequence encodes the hGALC leader (nucleic acids 1-126) and mature protein (encoded by nucleic acids 127-2058) of SEQ ID NO:5.

[0026] In certain embodiments, the expression cassette comprises one or more miRNA target sequences that repress expression of hGALC in the dorsal root ganglia (drg) (see, e.g., International Patent Application No. PCT / US19 / 67872, filed February 12, 2020, which is incorporated herein by reference). (Incorporated herein by reference).

[0027] As used herein, Krabbe disease (also known as globoid cell leukodystrophy (GLD)) is a lysosomal storage disorder caused by mutations affecting the activity of galactosylceramidase (GALC), the enzyme responsible for the breakdown of galactolipids in myelin. Several types of Krabbe disease have been described, depending on the severity of the enzyme deficiency. The most to least severe forms of the enzyme deficiency are early infantile Krabbe disease (EIKD), defined by onset at or below 6 months of age, late infantile Krabbe disease (LIKD), defined by onset between 7 and 12 months of age, juvenile Krabbe disease (JKD), defined by onset between 13 months and 10 years of age, and adolescent / adult onset Krabbe disease.

[0028] In certain embodiments, an effective amount of the rAAV.GALC vector increases the level of the GALC enzyme in the CSF to within about 30% to about 100% of normal levels. In other embodiments, an effective amount of the rAAV.GALC vector increases the level of the GALC enzyme in the plasma to within about 30% to about 100% of normal levels. In certain embodiments, lower amounts of increased CSF or plasma levels of GALC are observed, but improvement is observed in one or more of the symptoms associated with Krabbe disease, as described herein.

[0029] "Recombinant AAV" or "rAAV" is a DNAse-resistant viral particle that contains two elements, an AAV capsid, and a vector genome that contains at least a non-AAV coding sequence packaged within the AAV capsid. Unless otherwise specified, this term can be used interchangeably with the phrase "rAAV vector". rAAV is a "replication-defective virus" or "viral vector" because it lacks any functional AAV rep gene or functional AAV cap gene and cannot generate progeny. In certain embodiments, the only AAV sequence is the AAV inverted terminal repeat (ITR), which is typically located at the 5' and 3' extreme ends of the vector genome to allow the genes and regulatory sequences located between the ITRs to be packaged within the AAV capsid.

[0030] As used herein, "vector genome" refers to a nucleic acid sequence that is packaged inside the rAAV capsid that forms the viral particle. Such a nucleic acid sequence includes AAV inverted terminal repeats (ITRs). In the examples herein, the vector genome includes, at least 5' to 3', the AAV 5'ITR, a coding sequence, and the AAV 3'ITR. ITRs from AAV2, a source AAV different from the capsid, or other than the full-length ITRs can be selected. In certain embodiments, the ITRs are from the same AAV source as the AAV that provides the rep function or trans-complementing AAV during production. Additionally, other ITRs can be used. Additionally, the vector genome includes a control sequence that directs the expression of a gene product. Suitable components of the vector genome are discussed in more detail herein.

[0031] AAVhu68 As described in the following examples, the rAAV provided herein comprises an AAVhu68 capsid. See, for example, WO2018 / 160582, which is incorporated herein by reference. AAVhu68 is within phylogenetic group F. AAVhu68 (SEQ ID NO:2) differs from another phylogenetic group F virus, AAV9 (SEQ ID NO:4), by two encoded amino acids at positions 67 and 157 of vp1. In contrast, other phylogenetic group F AAVs (AAV9, hu31, hu31) have Ala at position 67 and Ala at position 157.

[0032] rAAVhu68 consists of the capsid and vector genome of AAVhu68. In one embodiment, a composition comprising rAAVhu68 comprises a heterogeneous population of vp1 proteins, a heterogeneous population of vp2 proteins, and a heterogeneous population of vp3 proteins. As used herein, the term "heterologous" or any grammatical variation thereof, when used to refer to the vp capsid protein, refers to a population of non-identical members, e.g., having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences. SEQ ID NO:2 provides the encoded amino acid sequence of the AAVhu68 vp1 protein. The AAVhu68 capsid includes subpopulations within the vp1, vp2, and vp3 proteins, which have modifications from the predicted amino acid residues of SEQ ID NO:2. These subpopulations include at least certain deamidated asparagine (N or Asn) residues. For example, certain subpopulations include at least one, two, three, or four highly deamidated asparagine (N) positions in the asparagine-glycine pairs of SEQ ID NO:2, and optionally further include other deamidated amino acids, where the deamidation results in amino acid changes and other optional modifications. Various combinations of these and other modifications are described herein.

[0033] As used herein, a "subpopulation" of vp proteins, unless otherwise specified, refers to a group of vp proteins that have at least one defined common characteristic and that consists of at least one group member and fewer than all members of a reference group.

[0034] For example, a "subpopulation" of vp1 proteins is at least one (1) vp1 protein in an assembled AAV capsid, but less than all vp1 proteins, unless otherwise specified. A "subpopulation" of vp3 proteins can be one (1) vp3 protein less than all vp3 proteins in an assembled AAV capsid, unless otherwise specified. For example, vp1 proteins can be a subpopulation of vp proteins, vp2 proteins can be a separate subpopulation of vp proteins, and vp3 can be a further subpopulation of vp proteins in an assembled AAV capsid. In another example, vp1, vp2, and vp3 proteins can include subpopulations with, for example, at least one, two, three, or four highly deamidated asparagines, e.g., different modifications at asparagine-glycine pairs.

[0035] Unless otherwise specified, extensive deamidation refers to at least 45% deamidation, at least 50% deamidation, at least 60% deamidation, at least 65% deamidation, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, up to about 100% deamidation at a reference amino acid position compared to the predicted amino acid sequence at the reference amino acid position (e.g., at amino acid 57 of SEQ ID NO:2, at least 80% of the asparagine may be deamidated based on the total vp1 protein, or at amino acid 409 of SEQ ID NO:2, 20% of the asparagine may be deamidated based on the total vp1, vp2, and vp3 proteins). Such percentages may be determined using 2D gels, mass spectrometry techniques, or other suitable techniques.

[0036] As provided herein, each deamidated N of SEQ ID NO:2 can independently be aspartic acid (Asp), isoaspartic acid (isoAsp), aspartate, and / or an interconverted blend of Asp and isoAsp, or a combination thereof. Any suitable ratio of α- and isoaspartic acid can be present. For example, in certain embodiments, the ratio can be 10:1 to 1:10 aspartic acid to isoaspartic acid, about 50:50 aspartic acid:isoaspartic acid, or about 1:3 aspartic acid:isoaspartic acid, or another selected ratio. In certain embodiments, one or more glutamines (Q) of SEQ ID NO:2 can be deamidated to glutamic acid (Glu), i.e., α-glutamic acid, γ-glutamic acid (Glu), or a blend of α- and γ-glutamic acid (which can be interconverted via a common glutarimide intermediate). Any suitable ratio of α- and γ-glutamic acid can be present. For example, in certain embodiments, In some embodiments, the ratio can be between 10:1 and 1:10 alpha to gamma, about 50:50 alpha:gamma, or about 1:3 alpha:gamma, or another selected ratio.

[0037] Thus, rAAVhu68 includes subpopulations within the rAAVhu68 capsid of vp1, vp2, and / or vp3 proteins that have deamidated amino acids, including, at a minimum, at least one subpopulation that includes at least one highly deamidated asparagine. Additionally, other modifications may include isomerization at specifically selected aspartic acid (D or Asp) residue positions. In yet other embodiments, modifications may include amidation at Asp positions.

[0038] In certain embodiments, the AAVhu68 capsid comprises a subpopulation of vp1, vp2, and vp3 having at least 4 to at least about 25 deamidated amino acid residue positions, of which at least 1-10% are deamidated compared to the encoded amino acid sequence of SEQ ID NO: 2. The majority of these may be N residues. However, Q residues may be deamidated.

[0039] In certain embodiments, the AAVhu68 capsid is further characterized by one or more of the following: The AAVhu68 capsid protein is an AAVhu68 vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1-736 of SEQ ID NO:2; a vp1 protein produced from SEQ ID NO:1, or a vp1 protein produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO:1 encoding the predicted amino acid sequence of 1-736 of SEQ ID NO:2; an AAVhu68 vp2 protein produced by expression from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 138-736 of SEQ ID NO:2; a vp2 protein produced from a sequence that includes at least nucleotides 412-2211 of SEQ ID NO:1, or a vp2 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 412-2211 of SEQ ID NO:1 encoding the predicted amino acid sequence of at least about amino acids 138-736 of SEQ ID NO:2; and / or an AAVhu68 vp2 protein produced by expression from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:2. vp3 protein, a vp3 protein produced from a sequence including at least nucleotides 607-2211 of SEQ ID NO:1, or a vp3 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607-2211 of SEQ ID NO:1, which encodes the predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:2.

[0040] Additionally or alternatively, AAV capsids are provided that optionally include a heterogeneous population of vp1 proteins that include a valine at position 157, optionally a heterogeneous population of vp2 proteins that include a valine at position 157, and a heterogeneous population of vp3 proteins, where at least a subpopulation of vp1 and vp2 proteins include a valine at position 157, and optionally further include a glutamic acid at position 67, based on the numbering of the vp1 capsid of SEQ ID NO: 2. Additionally or alternatively, AAVhu68 capsids are provided that include a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 2, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about amino acids 138-736 of SEQ ID NO: 2, and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 203-736 of SEQ ID NO: 2, where the vp1, vp2, and vp3 proteins include subpopulations with amino acid modifications.

[0041] The vp1, vp2, and vp3 proteins of AAVhu68 are typically expressed as alternative splice variants encoded by the same nucleic acid sequence that encodes the full-length vp1 amino acid sequence (amino acids 1-736) of SEQ ID NO: 2. Optionally, the vp1 coding sequence is used alone to express the vp1, vp2, and vp3 proteins. Alternatively, this sequence may be co-expressed with one or more of a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence of SEQ ID NO:2 (about aa203-736) without the vp1 unique region (about aa1 to about aa137) and / or the vp2 unique region (about aa1 to about aa202), or a complementary strand thereto, a corresponding mRNA or tRNA (about nt607 to about nt2211 of SEQ ID NO:1), or a sequence that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:1 encoding aa203-736 of SEQ ID NO:2. Additionally or alternatively, the vp1 and / or vp2 coding sequences can be co-expressed with a nucleic acid sequence or a complementary strand thereof encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO:2 (about aa 1 to about 137) without the vp1 unique region (about aa 1 to about 137), the corresponding mRNA or tRNA (e.g., nt 412 to 22121 of SEQ ID NO:1), or a sequence that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:1 encoding about aa 138 to 736 of SEQ ID NO:2.

[0042] As described herein, rAAVhu68 has rAAVhu68 capsids produced in a production system that expresses capsids from an AAVhu68 nucleic acid sequence encoding the vp1 amino acid sequence of SEQ ID NO:2, and, optionally, additional nucleic acid sequences (e.g., sequences encoding vp3 proteins without the vp1 and / or vp2 unique regions). rAAVhu68 resulting from production using a single nucleic acid sequence vp1 produces a heterogeneous population of vp1, vp2, and vp3 proteins. rAAVhu68 capsids contain subpopulations within the vp1, vp2, and vp3 proteins that have modifications from the predicted amino acid residues of SEQ ID NO:2. These subpopulations include at least deamidated asparagine (N or Asn) residues. For example, the asparagine in an asparagine-glycine pair is highly deamidated.

[0043] In one embodiment, the AAVhu68 vp1 nucleic acid sequence has the sequence of SEQ ID NO: 1, or a complementary strand thereto, e.g., a corresponding mRNA or tRNA. In certain embodiments, vp2 and / or vp3 proteins may additionally or alternatively be expressed from a nucleic acid sequence different from vp1, e.g., to alter the ratio of vp proteins in a selected expression system. In certain embodiments, a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence of SEQ ID NO: 2 (about aa203-736) or a complementary strand thereto, a corresponding mRNA or tRNA (about nt607 to about nt2211 of SEQ ID NO: 1), without the vp1 unique region (about aa1 to about aa137) and / or the vp2 unique region (about aa1 to about aa202), is also provided. In certain embodiments, a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO:2 (about aa 138-736) without the vp1 unique region (about aa 1 to about 137), or a complementary strand thereto, the corresponding mRNA or tRNA (nt 412-2211 of SEQ ID NO:1) is also provided.

[0044] However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO:2 may be selected for use in producing rAAVhu68 capsids. In certain embodiments, the nucleic acid sequence has a nucleic acid sequence of SEQ ID NO:1, or a sequence at least 70%-99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO:1, and encodes SEQ ID NO:2. In certain embodiments, the nucleic acid sequence has a nucleic acid sequence of SEQ ID NO:1, or a sequence at least 70%-99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO:1 from about nt 412 to about nt 2211 of SEQ ID NO:1, and encodes the vp2 capsid protein (about aa 138-736) of SEQ ID NO:2. In certain embodiments, the nucleic acid sequence has a nucleic acid sequence of SEQ ID NO:1 from about nt 607 to about nt 2211 of SEQ ID NO:1, and encodes the vp2 capsid protein (about aa 138-736) of SEQ ID NO:2. nt 2211, or a sequence at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the nucleic acid sequence of from about nt 607 to about nt 2211 of SEQ ID NO:1, and encodes the vp3 capsid protein (about aa 203 to 736) of SEQ ID NO:2.

[0045] It is within the skill of the art to design a nucleic acid sequence encoding this rAAVhu68 capsid, including DNA (genomic or cDNA), or RNA (e.g., mRNA). In certain embodiments, a nucleic acid sequence encoding the AAVhu68 vp1 capsid protein is provided in SEQ ID NO:1. In other embodiments, a nucleic acid sequence that is 70%-99.9% identical to SEQ ID NO:1 may be selected to express the AAVhu68 capsid protein. In certain other embodiments, the nucleic acid sequence is at least about 75% identical, at least 80% identical, at least 85%, at least 90%, at least 95%, at least 97% identical, or at least 99%-99.9% identical to SEQ ID NO:1. Such nucleic acid sequences may be codon optimized for expression in a selected system (i.e., cell type) and can be designed by a variety of methods. This optimization can be performed using methods available online (e.g., GeneArt), published methods, or companies that provide codon optimization services, such as DNA2.0 (Menlo Park, CA). One codon optimization method is described, for example, in U.S. International Patent Publication No. WO 2015 / 012924, which is incorporated herein by reference in its entirety. See also, for example, U.S. Patent Publication No. 2014 / 0032186 and U.S. Patent Publication No. 2006 / 0136184. Preferably, the entire length of the open reading frame (ORF) of the product is modified. However, in some embodiments, only a fragment of the ORF may be modified. By using one of these methods, a frequency can be applied to any given polypeptide sequence to produce a nucleic acid fragment of a codon-optimized coding region that encodes a polypeptide. Several options are available for performing the actual changes to the codons or for synthesizing the codon-optimized coding region designed as described herein. Such modification or synthesis can be performed using standard and routine molecular biology operations well known to those skilled in the art. In one approach, a series of complementary oligonucleotide pairs, each 80-90 nucleotides in length and spanning the length of the desired sequence, are synthesized by standard methods.These oligonucleotide pairs are synthesized such that upon annealing, 80-90 base pair double stranded fragments containing sticky ends are formed, e.g., each oligonucleotide of the pair is synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10 or more bases beyond the region complementary to the other oligonucleotide of the pair. The single stranded end of each pair of oligonucleotides is designed to anneal with the single stranded end of another pair of oligonucleotides. The oligonucleotide pairs are annealed, then approximately 5-6 of these double stranded fragments are annealed together via the cohesive single stranded ends, which are then ligated together and cloned into a standard bacterial cloning vector, e.g., a TOPO® vector available from Invitrogen Corporation, Carlsbad, Calif. The constructs are then sequenced by standard methods. Several of these constructs are prepared consisting of 5-6 fragments of the 80-90 base pair fragments ligated together, i.e., approximately 500 base pair fragments, so that the entire desired sequence is represented in a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and ligated together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector and sequenced. Additional methods will be readily apparent to those of skill in the art. Additionally, gene synthesis is readily available commercially.

[0046] In certain embodiments, the asparagine (N) of the N-G pairs of the vp1, vp2, and vp3 proteins of rAAVhu68 are highly deamidated. In the case of the rAAVhu68 capsid protein, four residues (N57, N329, N452, N512) are highly deamidated. The deamidation levels of N94, N253, N270, N304, N409, N477, and Q599 routinely show deamidation levels of more than 70% across lots, and in most cases, more than 90%. Additional asparagine residues (N94, N253, N270, N304, N409, N477, and Q599) also show deamidation levels of up to about 20% across various lots. Deamidation levels were initially identified using trypsin digestion and verified with chymotrypsin digestion.

[0047] In certain embodiments, the rAAVhu68 capsid comprises a subpopulation of AAV vp1, vp2, and / or vp3 capsid proteins that have at least four asparagine (N) positions in the rAAVhu68 capsid protein that are highly deamidated. In certain embodiments, about 20-50% of N-N pairs (excluding N-N-triplets) exhibit deamidation. In certain embodiments, the first N is deamidated. In certain embodiments, the second N is deamidated. In certain embodiments, the deamidation is about 15% to about 25% deamidation. Deamidation at Q at position 259 of SEQ ID NO:2 is about 8% to about 42% of the AAVhu68 vp1, vp2, and vp3 capsid proteins of the AAVhu68 protein.

[0048] In certain embodiments, the rAAVhu68 capsid is further characterized by amidation at D297 of the vp1, vp2, and vp3 proteins. In certain embodiments, about 70% to about 75% of the D at position 297 of the vp1, vp2, and / or vp3 proteins of the AAVhu68 capsid are amidated, based on the numbering of SEQ ID NO:2. In certain embodiments, at least one Asp of vp1, vp2, and / or vp3 of the capsid is isomerized to D-Asp. Such isomers are generally present in an amount of less than about 1% of the Asps of one or more of residue positions 97, 107, 384, based on the numbering of SEQ ID NO:2.

[0049] In certain embodiments, rAAVhu68 has an AAVVhu68 capsid with vp1, vp2, and vp3 proteins, with subpopulations containing combinations of one, two, three, four, or more deamidated residues at the positions shown in the table below. rAAV deamidation can be determined using 2D gel electrophoresis, and / or mass spectrometry, and / or protein modeling techniques. Online chromatography can be performed on an Acclaim PepMap column and Thermo UltiMate 3000 RSLC system (Thermo Fisher Scientific) coupled to a Q Exactive HF with a NanoFlex source (Thermo Fisher Scientific). MS data are acquired using the data-dependent Top-20 method of the Q Exactive HF, which dynamically selects the most abundant and not yet sequenced precursor ions from the survey scan (200-2000 m / z). Sequencing was performed via higher energy collisional dissociation fragmentation with a target value of 1e5 ions determined with predictive autoincrement control, followed by precursor isolation with a window of 4 m / z. Survey scans were acquired with a resolution of 120,000 at m / z 200. The resolution of the HCD spectrum can be set to 30,000 with a maximum ion injection time of 50 ms, normalized collision energy of 30, and m / z of 200. The S-lens RF level can be set to 50 to optimize the transmission of the m / z region occupied by peptides from the digest. Precursor ions can be single, unassigned, or excluded with 6 or more charge states from the fragmentation selection. BioPharma Finder 1.0 software (Thermo Fischer Scientific) can be used to analyze the acquired data. For peptide mapping, searches are performed using a single-entry protein FASTA database with carbamidomethylation set as the fixed modification, oxidation, deamidation, and phosphorylation set as variable modifications, 10 ppm mass accuracy, high protease specificity, and a confidence level of 0.8 for MS / MS spectra. Examples of suitable proteases may include, for example, trypsin or chymotrypsin. Deamidation reduces the mass of the intact molecule by 10 ppm, while chymotrypsin reduces the mass of the intact molecule by 10 ppm. Mass spectrometric identification of deamidated peptides is relatively straightforward, since the mass area of ​​the deamidated peptide is 0.984 Da (mass difference of -OH and -NH2 groups). The percentage of deamidation of a particular peptide is determined by dividing the mass area of ​​the deamidated peptide by the sum of the areas of the deamidated and native peptides. Given the number of possible deamidation sites, isobars that are deamidated at different sites may co-migrate in a single peak. Thus, fragment ions derived from peptides with multiple potential deamidation sites can be used to identify or distinguish between multiple deamidation sites. In these cases, the relative intensities within the observed isotopic patterns can be used to specifically determine the relative abundance of different deamidated peptide isomers. This method assumes that the fragmentation efficiency for all isomeric species is the same and is independent at the deamidation site. It will be understood by those skilled in the art that several variations of these exemplary methods can be used. For example, suitable mass analyzers may include quadruple flight mass analyzers (QTOF), such as Waters Xevo or Agilent 6530, or Orbitrap instruments, such as Orbitrap Fusion or Orbitrap Velos (Thermo Fisher). Suitable liquid chromatography systems include, for example, Acquity UPLC systems from Waters or Agilent systems (1100 or 1200 series). Suitable data analysis software may include, for example, MassLynx (Waters), Pinpoint and Pepfinder (Thermo Fischer Scientific), Mascot (Matrix Science), Peaks DB (Bioinformatics Solutions). Still other techniques may be described, for example, in X. Jin et al, Hu Gene Therapy Methods, Vol. 28, No. 5, pp. 255-267, published online on June 16, 2017. [Table 1-1] [Table 1-2]

[0050] In certain embodiments, the AAVhu68 capsid is characterized as having a capsid protein in which at least 45% of the N residues are deamidated at at least one of positions N57, N329, N452, and / or N512, based on the numbering of the amino acid sequence of SEQ ID NO: 2. In certain embodiments, at least about 60%, at least about 70%, at least about 80%, or at least 90% of the N residues are deamidated at one or more of these NG positions (i.e., N57, N329, N452, and / or N512, based on the numbering of the amino acid sequence of SEQ ID NO: 2). In these and other embodiments, the AAVhu68 capsid is characterized as having a population of proteins in which about 1% to about 20% of the N residues have deamidation at one or more of positions N94, N253, N270, N304, N409, N477, and / or Q599, based on the numbering of the amino acid sequence of SEQ ID NO: 2. In certain embodiments, AAVhu68 comprises at least a subpopulation of vp1, vp2, and / or vp3 proteins that are deamidated at one or more of positions N35, N57, N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N329, N336, N409, N410, N452, N477, N515, N598, Q599, N628, N651, N663, N709, N735, or combinations thereof, based on the numbering of the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the capsid protein may have one or more amidated amino acids.

[0051] Still other modifications are observed, most of which do not involve conversion of an amino acid to a different amino acid residue. Optionally, at least one Lys in vp1, vp2, and vp3 of the capsid is acetylated. Optionally, at least one Asp in vp1, vp2, and / or vp3 of the capsid is isomerized to D-Asp. Optionally, at least one S (Ser, serine) in vp1, vp2, and / or vp3 of the capsid is phosphorylated. Optionally, at least one T (Thr, threonine) in vp1, vp2, and / or vp3 of the capsid is phosphorylated. Optionally, at least one W (trp, tryptophan) in vp1, vp2, and / or vp3 of the capsid is oxidized. Optionally, at least one M (Met, methionine) in vp1, vp2, and / or vp3 of the capsid is oxidized. In certain embodiments, the capsid protein has one or more phosphorylations. For example, a particular vp1 capsid protein may be phosphorylated at position 149.

[0052] In certain embodiments, the rAAVhu68 capsid is a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:2, wherein the vp1 proteins comprise a glutamic acid (Glu) at position 67 and / or a valine (Val) at position 157, optionally comprising a vp1 protein comprising a valine (Val) at position 157. AAVhu68 capsids include a heterogeneous population of p2 proteins, and a heterogeneous population of vp3 proteins. The AAVhu68 capsids include at least one subpopulation in which at least 65% of the asparagine (N) of the asparagine-glycine pairs located at position 57 of the vp1 protein, and at least 70% of the asparagine (N) of the asparagine-glycine pairs at positions 329, 452, and / or 512 of the vp1, v2, and vp3 proteins are deamidated, based on the residue numbering of the amino acid sequence of SEQ ID NO:2, where the deamidation results in an amino acid change. As discussed in more detail herein, the deamidated asparagine may be deamidated to aspartic acid, isoaspartic acid, an interconverting aspartic acid / isoaspartic acid pair, or a combination thereof. In certain embodiments, rAAVhu68 is further characterized by one or more of the following: (a) each of the vp2 proteins is independently a product of a nucleic acid sequence encoding at least the vp2 protein of SEQ ID NO:2, (b) each of the vp3 proteins is independently a product of a nucleic acid sequence encoding at least the vp3 protein of SEQ ID NO:2, (c) the nucleic acid sequence encoding the vp1 protein is SEQ ID NO:1, or a sequence at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO:1 and encodes the amino acid sequence of SEQ ID NO:2. Optionally, the sequence is used alone to express the vp1, vp2, and vp3 proteins. Alternatively, the sequence may be AAVhu68 of SEQ ID NO:2, which does not have the unique region of vp1 (from about aa1 to about aa137) and / or the unique region of vp2 (from about aa1 to about aa202). It may be co-expressed with one or more of a nucleic acid sequence encoding the amino acid sequence of vp3 (approximately aa 203 to 736) or a complementary strand thereto, a corresponding mRNA or tRNA (e.g., from about nt 607 to about nt 2211 of SEQ ID NO: 1), or a sequence that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1 encoding aa 203 to 736 of SEQ ID NO: 2. Additionally or alternatively, the vp1 and / or vp2 coding sequences may be co-expressed with a nucleic acid sequence or a complementary strand thereof encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO:2 (about aa 1 to about 137) without the vp1 unique region (about aa 1 to about 137), the corresponding mRNA or tRNA (e.g., nt 412 to 2211 of SEQ ID NO:1), or a sequence that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:1 encoding about aa 138 to 736 of SEQ ID NO:2. Additionally or alternatively, the rAAVhu68 capsid may comprise, based on the numbering of SEQ ID NO:2, positions N57, N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N329, N336, N409, N410, N452, N477, N512, N515, N598, Q599, N628, N651, N652, N653, N654, N655, N656, N657, N658, N659, N660, N661, N662, N663, N664, N665, N666, N667, N668, N669, N670, N671, N672, N673, N674, N675, N676, N677, N678, N679, N680, N681, N682, N683, N684, N685, N686, N687, N688, N689, N690, N691, N692, N693, N694, N695, N696, N697, N698, N699, N700, N701, N702, N703, N704, N705, N706, N707, N708, N710, N711, N712, N715, N720, N721, N722, N723, N724, N725, N730, N731, N732, N733, N (e) the rAAVhu68 capsid comprises at least a subpopulation of vp1, vp2, and / or vp3 proteins that are deamidated at one or more of positions N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N330, N331, N332, N333, N334, N335, N336, N337, N338, N339, N340, N341, N342, N343, N344, N345, N346, N347, N348, N349, N350, N351, N352, N353, N353, N354, N355, N356, N357, N358, N359, N360, N361, N362, N363, N364, N365, N366, N367, N368, N369, N370, N371, N372, N373, N374, N375, N376, N377, N378, N379, N380, N381, N382, N383, N384, N385, N386, N387, N388, N389, N390, N391, N392, N393, N394, N395, N396, N397, N398, N399, N400, N401, N402, N403, N404, N405, N406, N407, N408, N409 (f) the rAAVhu68 capsid comprises a subpopulation of vp1 proteins in which between 65% and 100% of the Ns at position 57 of the vp1 protein are deamidated based on the numbering of SEQ ID NO:2; (g) the rAAVhu68 capsid comprises a subpopulation of vp1 proteins in which between 75% and 100% of the Ns at position 57 of the vp1 protein are deamidated based on the numbering of SEQ ID NO:2; (h) the rAAVhu68 capsid comprises a subpopulation of vp1 proteins in which between 80% and 100% of the Ns at position 329 are deamidated based on the numbering of SEQ ID NO:2; (i) the rAAVhu68 capsid comprises a subpopulation of vp1, vp2, and / or vp3 proteins in which between 80% and 100% of the N at position 452 are deaminated based on the numbering of SEQ ID NO:2; and (j) the rAAVhu68 capsid comprises a subpopulation of vp1, vp2, and / or vp3 proteins in which between 80% and 100% of the N at position 512 are deaminated based on the numbering of SEQ ID NO:2. (k) the rAAV comprises about 60 total capsid proteins in a ratio of about 1 vp1 protein:about 1-1.5 vp2 protein:3-10 vp3 protein; and (l) the rAAV comprises about 60 total capsid proteins in a ratio of about 1 vp1 protein:about 1 vp2 protein:3-9 vp3 protein.

[0053] In certain embodiments, AAVhu68 is modified to change the glycine of an asparagine-glycine pair to reduce deamidation. In other embodiments, the asparagine is changed to a different amino acid, for example, glutamine, which is deamidated at a slower rate, or an amino acid lacking an amide group (e.g., glutamine and asparagine contain an amide group), and / or an amino acid lacking an amine group (e.g., lysine, arginine, and histidine contain an amide group). As used herein, an amino acid lacking an amide or amine side group refers to, for example, glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, phenylalanine, tyrosine, or tryptophan, and / or proline. The modification as described may be in one, two, or three of the asparagine-glycine pairs found in the amino acid sequence of the encoded AAVhu68. In certain embodiments, such modification is not made in all four of the asparagine-glycine pairs. Thus, methods are provided for reducing deamidation of rAAVhu68 and / or engineered rAAVhu68 variants having a lower deamidation rate. Additionally, or alternatively, one or more other amide amino acids may be changed to a non-amide amino acid to reduce deamidation of rAAVhu68.

[0054] These amino acid modifications may be made by conventional genetic engineering techniques. For example, a nucleic acid sequence may be generated that includes a modified AAVhu68 vp codon, where one to three of the codons encoding glycine (asparagine-glycine pairs) at positions 58, 330, 453, and / or 513 of SEQ ID NO:2 are modified to encode an amino acid other than glycine. In certain embodiments, a nucleic acid sequence that includes a modified asparagine codon may have one to three of the asparagine-glycine pairs located at positions 57, 329, 452, and / or 512 of SEQ ID NO:2 engineered to encode an amino acid other than asparagine. Each modified codon may encode a different amino acid. Alternatively, one or more of the altered codons may encode the same amino acid. In certain embodiments, these modified AAVhu68 nucleic acid sequences may be used to generate mutant rAAVhu68 with capsids that include less deamidation than native hu68 capsids. Such mutated rAAVhu68 may reduce immunogenicity and / or increase stability upon storage, particularly storage in suspension form. As used herein, "codon" refers to three nucleotides in a sequence that codes for an amino acid.

[0055] As used herein, "encoded amino acid sequence" refers to predicted amino acids based on translation of known DNA codons of a reference nucleic acid sequence that are translated into amino acids. The table below illustrates DNA codons and the 20 common amino acids, showing both the one-letter code (SLC) and the three-letter code (3LC). [Table 2]

[0056] rAAVhu68 capsids may be useful in certain embodiments. For example, such capsids may be used to generate monoclonal antibodies and / or reagents useful in assays for monitoring AAVhu68 concentration levels in gene therapy patients. Techniques for producing useful anti-AAVhu68 antibodies, labeling such antibodies or empty capsids, and suitable assay formats are known to those skilled in the art.

[0057] In certain embodiments, the nucleic acid sequence of SEQ ID NO: 1 or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% of the sequence encoding the amino acid sequence of vp1 of SEQ ID NO: 2 including modifications described herein (e.g., deamidated amino acids) is produced using the sequence depicted in SEQ ID NO: 2. In certain embodiments, the vp1 amino acid sequence is reproduced in SEQ ID NO: 2.

[0058] As used herein, the term "clade" in relation to a group of AAVs refers to a group of AAVs that are phylogenetically related to each other and have at least a 75% bootstrap value (out of at least 1000 repeats) and a similarity to each other based on an alignment of AAV vp1 amino acid sequences. and a Neighbor-Joining algorithm with a Poisson-corrected distance measure of 0.05 or less. The Neighbor-Joining algorithm has been described in the literature. See, e.g., Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York (2000). Computer programs are available that can be used to implement this algorithm. For example, the MEGA v2.1 program implements a modified Nei-Gojobori method. Using these techniques and computer programs, and the sequence of the AAV vp1 capsid protein, one of skill in the art can readily determine whether a selected AAV falls within one clade identified herein, another clade, or is outside these clades. See, e.g., G Gao, et al, J Virol, 2004 Jun;78(10:6381-6388 (identifying clades A, B, C, D, E, and F), and GenBank accession numbers AY530553 to AY530629. See also WO2005 / 033321.

[0059] As used herein, an "AAV9 capsid" is a self-assembled AAV capsid consisting of multiple AAV9 vp proteins. AAV9 vp proteins are typically expressed as alternative splice variants encoded by the nucleic acid sequence of SEQ ID NO: 3, which encodes the amino acid sequence of vp1 of SEQ ID NO: 4 (GenBank Accession No. AAS99264). These splice variants result in proteins of different lengths of SEQ ID NO: 4. In certain embodiments, an "AAV9 capsid" includes an AAV having an amino acid sequence 99% identical to AAS99264 or 99% identical to SEQ ID NO: 4. See also US7906111 and WO2005 / 033321. As used herein, an "AAV9 variant" includes, for example, variants described in WO2016 / 049230, US8,927,514, US2015 / 0344911, and US8,734,809.

[0060] Methods for producing capsids and therefore coding sequences, as well as methods for producing rAAV viral vectors, have been described. See, e.g., Gao, et al., Proc. Natl. Acad. Sci. USA 100(10), 6081-6086 (2003) and US2013 / 0045186A1.

[0061] The terms "substantial homology" or "substantial similarity," when referring to a nucleic acid or a fragment thereof, when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity in at least about 95-99% of the aligned sequence. Preferably, the homology is over the full length sequence, or its open reading frame, or another suitable fragment that is at least 15 nucleotides in length. Examples of suitable fragments are described herein.

[0062] The terms "sequence identity," "percent sequence identity," or "percent identical" in the context of nucleic acid sequences refer to residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison can be over the entire length of a genome, the entire length of a gene coding sequence, or a fragment of at least about 500-5000 nucleotides, desirably. However, identity between smaller fragments, e.g., of at least about 9 nucleotides, usually at least about 20-24 nucleotides, at least about 28-32 nucleotides, at least about 36 nucleotides or more, may also be desired. Similarly, "percent sequence identity" can be readily determined for amino acid sequences over the entire length of a protein or fragments thereof. Preferably, the fragments are at least about 8 amino acids in length and can be up to about 700 amino acids in length. Examples of suitable fragments are described herein.

[0063] The terms "substantial homology" or "substantial similarity," when referring to an amino acid or a fragment thereof, when optimally aligned with another amino acid (or its complementary strand) with appropriate amino acid insertions or deletions, there is amino acid sequence identity in at least about 95-99% of the aligned sequence. Preferably, the homology is over the full length sequence, or a protein thereof, e.g., cap protein, rep protein, or a fragment thereof that is at least 8 amino acids in length, or more preferably, at least 15 amino acids in length. Examples of suitable fragments are described herein.

[0064] The term "highly conserved" means at least 80% identity, preferably at least 90% identity, more preferably greater than 97% identity. Identity is readily determined by those of skill in the art using algorithms and computer programs known to those of skill in the art.

[0065] Generally, when referring to "identity", "homology", or "similarity" between two different adeno-associated viruses, the "identity", "homology", or "similarity" is determined with reference to "aligned" sequences. An "aligned" sequence or "alignment" refers to multiple nucleic acid or protein (amino acid) sequences, often including corrections for missing or additional bases or amino acids, as compared to a reference sequence. In the examples, the AAV alignment is performed using the published AAV9 sequence as a reference point. The alignment is performed using a variety of multiple sequence alignment programs, either public or commercially available. Examples of such programs include "Clustal Omega", "Clustal W", "CAP Sequence Assembly", "MAP", and "MEME", which are accessible through web servers on the Internet. Other sources of such programs are known to those of skill in the art. Alternatively, the Vector NTI utility is also used. There are also several algorithms known in the art that can be used to measure nucleotide sequence identity, including those included in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG version 6.1. Fasta™ provides alignment and percent sequence identity of the best overlap region between query sequence and search sequence. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta™ with its default parameters (word size 6 and NOPAM factor for scoring matrix) as provided in GCG version 6.1 (hereby incorporated by reference). Multiple sequence alignment programs such as "Clustal Omega", "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match-Box" programs are also available for amino acid sequences. Generally, any of these programs are used with default settings, but if necessary, those skilled in the art can change these settings.Alternatively, one of skill in the art can utilize another algorithm or computer program that provides at least the level of identity or alignment as provided by the referenced algorithms and programs. See, e.g., JD Thomson et al, Nucl. Acids. Res., "A. See, “Comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

[0066] rAAV vectors As described above, the AAVhu68 sequence and protein are useful for the production of rAAV and are also useful as recombinant AAV vectors, which may be antisense delivery vectors, gene therapy vectors, or vaccine vectors. V capsids (e.g., AAV capsids having variant amino acids at positions 67, 157, or both, relative to the numbering of the vp1 capsid protein in SEQ ID NO:2) can be used to engineer rAAV vectors for delivery of several suitable nucleic acid molecules to target cells and tissues.

[0067] The genomic sequence that is packaged in AAV capsid and delivered to host cell typically consists of at least a transgene, its regulatory sequence, and AAV inverted terminal repeat (ITR). Both single-stranded AAV and self-complementary (sc) AAV are included in rAAV. A transgene is a nucleic acid coding sequence that is heterologous to the vector sequence and codes for a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor), or other gene product of interest.

[0068] In particular, the present disclosure provides an rAAV comprising the coding sequence of human galactosylceramidase (GALC). In some embodiments, the coding sequence is an engineered GALC coding sequence. In some embodiments, the coding sequence is the sequence of cGALC gene (cGALCco) of SEQ ID NO:9.

[0069] The nucleic acid coding sequence is operably linked to the regulatory element in a manner that allows transcription, translation, and / or expression of the transgene in cells of the target tissue. In some embodiments, the regulatory sequence comprises a beta-actin promoter, an intron, and rabbit globin polyA. In certain embodiments, the regulatory sequence has SEQ ID NO: 13. In certain embodiments, the regulatory sequence has SEQ ID NO: 15. In certain embodiments, the regulatory sequence has SEQ ID NO: 16.

[0070] The AAV sequence of the vector typically contains cis-acting 5' and 3' inverted terminal repeat sequences (see, e.g., BJ Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are approximately 145 bp in length. Preferably, substantially the entire sequence encoding the ITRs is used in the molecule, although some minor modification of these sequences is tolerated. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., Sambrook et al, "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J. Virol., 70:520 532 (1996)). One example of such a molecule utilized in the present invention is a "cis-acting" plasmid containing a transgene with a selected transgene sequence and associated regulatory elements flanked by 5' and 3' AAV ITR sequences. In one embodiment, the ITRs are from a different AAV than the one that supplies the capsid. In one embodiment, the ITR sequences are from AAV2. A shortened version of the 5' ITR, called ΔITR, has been described, in which the D sequence and terminal separation site (trs) are deleted. In other embodiments, full-length AAV 5' and 3' ITRs are used. However, ITRs from other AAV origins may be selected. If the origin of the ITRs is from AAV2 and the AAV capsid is from another AAV origin, the resulting vector may be referred to as pseudotyped. However, other configurations of these elements may also be suitable.

[0071] In addition to the major elements described above for recombinant AAV vectors, the vectors also contain the necessary conventional control elements operably linked to the transgene in a manner that allows for its transcription, translation, and / or expression in cells transfected with the plasmid vector or infected with the virus produced by the invention. As used herein, "operably linked" sequences include both expression control sequences that are adjacent to the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0072] Regulatory control elements typically include promoter sequences located as part of expression control sequences, for example between selected 5'ITR sequences and coding sequences. Constitutive promoters, regulatable promoters [see, for example, WO2011 / 126808 and WO2013 / 04943], tissue-specific promoters, or promoters that respond to physiological cues can be used in the vectors described herein. The promoter may be selected from different sources, for example, the human cytomegalovirus (CMV) immediate early enhancer / promoter, the SV40 immediate early enhancer / promoter, the JC polymomavirus promoter, the myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoter, the herpes simplex virus (HSV-1) latency-associated promoter (LAP), the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, the neuron-specific promoter (NSE), the platelet-derived growth factor (PDGF) promoter, hSYN, the melanin-concentrating hormone (MCH) promoter, CBA, the matrix metalloprotein promoter (MPP), and the chicken beta-actin promoter. In addition to the promoter, the vector may include one or more other suitable transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals, sequences that stabilize cytoplasmic mRNA, such as WPRE, sequences that increase translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and, if necessary, sequences that enhance secretion of the encoded product. An example of a suitable enhancer is the CMV enhancer. Other suitable enhancers include those appropriate for the desired target tissue indication. In one embodiment, the expression cassette includes one or more expression enhancers. In one embodiment, the expression cassette includes two or more expression enhancers. These enhancers may be the same or different from each other. For example, the enhancer may include a CMV immediate early enhancer. This enhancer may be present in two copies located adjacent to each other. Alternatively, the double copies of the enhancer are separated by one or more sequences.In yet another embodiment, the expression cassette further comprises an intron, for example, a chicken beta actin intron. Other suitable introns include those known in the art, for example, described in WO2011 / 126808. Examples of suitable polyA sequences include, for example, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyA. Optionally, one or more sequences may be selected to stabilize the mRNA. One example of such a sequence is a modified WPRE sequence, which may be engineered upstream of the polyA sequence and downstream of the coding sequence [see, for example, MA Zanta-Boussif, et al, Gene Therapy (2009) 16:605-619.

[0073] These rAAVs are particularly suitable for gene delivery for therapeutic purposes and immunization, including inducing protective immunity. In addition, the compositions of the present invention can be used to produce desired gene products in vitro. For in vitro production, the desired product (e.g., protein) can be obtained from the desired culture after transfecting host cells with rAAVs containing molecules encoding the desired product and culturing the cell culture under conditions that allow expression. The expressed product can then be purified and isolated, if desired. Suitable techniques for transfection, cell culture, purification, and isolation are known to those skilled in the art.

[0074] rAAV vector production For use in producing AAV viral vectors (e.g., recombinant (r)AAV), the expression cassette can be carried on any suitable vector, e.g., a plasmid, that is delivered to a packaging host cell. Plasmids useful in the present invention can be engineered to be suitable for in vitro replication and packaging in prokaryotic, insect, mammalian cells, among others. Suitable transfection techniques and packaging host cells are known and / or can be readily designed by one of skill in the art.

[0075] Methods for generating and isolating AAV suitable for use as a vector are known in the art.See generally, for example, Grieger & Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications," Adv. Biochem. Engin / Biotechnol. 99:119-145; Buning et al., 2008, "Recent developments in adeno-associated virus vector technology," J. Gene Med. 10:717-733, and the references cited below (each of which is incorporated herein by reference in its entirety).The ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the expression cassette in order to package genes into virions.The cap and rep genes can be supplied in trans.

[0076] In one embodiment, the expression cassette described herein is engineered into a genetic element (e.g., shuttle plasmid) that introduces the immunoglobulin construct sequence carried thereon into a packaging host cell to produce a viral vector. In one embodiment, the selected genetic element can be delivered to the AAV packaging cell by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Stable AAV packaging cells can also be created. Alternatively, the expression cassette can be used to generate viral vectors other than AAV, or for the production of a mixture of antibodies in vitro. The methods used to create such constructs are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Molecular Cloning: A Laboratory Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0077] The term "AAV intermediate" or "AAV vector intermediate" refers to assembled rAAV capsids that lack the desired genomic sequence packaged therein. These may be referred to as "empty" capsids. Such capsids may contain no detectable genomic sequence of an expression cassette or may contain only partially packaged genomic sequence that is insufficient to achieve expression of a gene product. These empty capsids are non-functional for introducing a gene of interest into a host cell.

[0078] The recombinant adeno-associated virus (AAV) described herein may be produced using known techniques. For example, see WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, US7588772B2. Such methods include culturing a host cell that contains an expression cassette consisting of a nucleic acid sequence encoding an AAV capsid protein, a functional rep gene, at a minimum an AAV inverted terminal repeat (ITR) and a transgene, and sufficient helper functions to allow the expression cassette to be packaged into an AAV capsid protein. Methods for producing capsids, coding sequences therefor, and methods for producing rAAV viral vectors have been described. For example, see Gao, et al, Proc. Natl. Acad. Sci. USA100(10), 6081-6086(2003) and US2013 / 0045186A1.

[0079] In one embodiment, a producer cell culture useful for producing recombinant rAAVhu68 is provided. Such cell cultures contain nucleic acids that express rAAVhu68 capsid proteins in host cells, nucleic acid molecules suitable for packaging into rAAVhu68 capsids (e.g., AAVhu68 capsids, which contain a vector genome including AAV ITRs, and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences that direct expression of the product in a host cell, as well as sufficient AAV rep and adenovirus helper functions to permit packaging of the nucleic acid molecule into a recombinant AAVhu68 capsid. In one embodiment, the cell culture is comprised of mammalian cells (e.g., human embryonic kidney 293 cells, among others) or insect cells (e.g., baculovirus).

[0080] Suitably, the rep function is provided by AAV from the same source as the ITRs present in the vector genome, or from another source (e.g., AAVhu68) that packages the vector genome into the AAV capsid. In a particular embodiment, the rep protein is from AAV2. In another embodiment, the rep protein is a heterologous rep protein other than AAVhu68rep, such as, but not limited to, AAV1 rep protein, AAV2 rep protein, AAV3 rep protein, AAV4 rep protein, AAV5 rep protein, AAV6 rep protein, AAV7 rep protein, AAV8 rep protein, or rep78, rep68, rep52, rep40, rep68 / 78, and rep40 / 52, or fragments thereof, or from another source. Any of these AAVhu68 or mutant AAV capsid sequences may be under the control of exogenous regulatory control sequences that direct their expression in the producer cell.

[0081] In one embodiment, the cells are produced in suitable cell culture (e.g., HEK293) cells. The methods for producing gene therapy vectors described herein include methods well known in the art, such as the production of plasmid DNA used for the production of gene therapy vectors, the production of vectors, and the purification of vectors. In some embodiments, the gene therapy vector is an AAV vector, and the produced plasmids are AAV cis-plasmids encoding the AAV genome and gene of interest, AAV trans-plasmids containing the AAV rep and cap genes, and adenovirus helper plasmids. The vector production process may include method steps such as initiation of cell culture, passaging of cells, seeding of cells, transfection of cells with plasmid DNA, medium exchange with serum-free medium after transfection, and harvesting of cells and culture medium containing vector. The harvested vector-containing cells and culture medium are referred to herein as crude cell harvest. In yet another system, the gene therapy vector is introduced into insect cells by infection with a baculovirus-based vector. For a review of these production systems, see generally, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entireties. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of which are incorporated by reference in their entireties: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065.

[0082] In a specific embodiment, the manufacturing process for rAAV.hGALC involves transient transfection of HEK293 cells with plasmid DNA. Single or multiple batches are produced by PEI-mediated triple transfection of HEK293 cells in a PALL iCELLis bioreactor. Harvested AAV material is further purified by clarification, TFF, affinity chromatography, and purification in a single-use closed bioprocessing system, when possible. The product was purified by sequential chromatography, followed by anion exchange chromatography.

[0083] The crude cell harvest is then subjected to process steps such as concentration of the vector harvest, diafiltration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, filtration of the microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration to prepare bulk vector.

[0084] A two-step high salt affinity chromatography purification, followed by anion exchange resin chromatography, is used to purify the vector drug product and remove empty capsids. These methods are described in detail in International Patent Application No. PCT / US2016 / 065970, filed December 9, 2016, and its priority documents, U.S. Patent Application No. 62 / 322,071, filed April 13, 2016, and U.S. Patent Application No. 62 / 226,357, filed December 11, 2015, entitled "Scalable Purification Method for AAV9," which are incorporated herein by reference. For AAV8, the purification method is described in International Patent Application No. PCT / US2016 / 065976, filed December 9, 2016, and its priority documents, U.S. Patent Application No. 62 / 322,098, filed April 13, 2016, and U.S. Patent Application No. 62 / 266,341, filed December 11, 2015, and for rh10, the purification method is described in International Patent Application No. PCT / US16 / 66013, filed December 9, 2016, and its priority documents, U.S. Patent Application No. 62 / 322,055, filed April 13, 2016, and U.S. Patent Application No. 62 / 322,055, filed December 11, 2015, and "Scalable Purification Method for AAV8," filed December 9, 2016, and its priority documents, U.S. Patent Application No. 62 / 266,341, filed December 11, 2015, and "Scalable Purification Method for AAV8," filed December 9, 2016, and its priority documents, U.S. Patent Application No. No. 62 / 266,347, entitled "Scalable Purification Method for AAVrh10," and for AAV1, International Patent Application No. PCT / US2016 / 065974, filed December 9, 2016, and its priority documents, U.S. Patent Application No. 62 / 322,083, filed April 13, 2016, and U.S. Patent Application No. 62 / 26,351, filed December 11, 2015, entitled "Scalable Purification Method for AAV1," all of which are incorporated herein by reference.

[0085] To calculate the empty and filled particle content, the VP3 band volume for a selected sample (e.g., in the examples herein, a preparation purified on an iodixanol gradient, where number of GC = number of particles) is plotted against the GC particles loaded. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volume of the test article peak. The number of particles (pt) per 20 μL loaded is then multiplied by 50 to obtain particles (pt) / mL. Pt / mL is divided by GC / mL to obtain the ratio of particles to genome copies (pt / GC). Pt / mL to GC / mL gives empty pt / mL. Empty pt / mL is divided by pt / mL and then multiplied by 100 to obtain the percentage of empty particles.

[0086] In general, methods for assaying AAV vector particles, including empty capsids and packaged genomes, are known in the art. See, e.g., Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., J. Immunol. 1999, 11:1322-1330; See, e.g., et al., Molec. Ther. (2003) 7:122-128. To test for denatured capsids, the method involves subjecting the processed AAV stock to SDS-polyacrylamide gel electrophoresis in any gel capable of separating the three capsid proteins (e.g., a gradient gel with 3-8% Tris-acetate in buffer), then running the gel until the sample material is separated, and blotting the gel onto a nylon or nitrocellulose membrane (preferably nylon). An anti-AAV capsid antibody is then coupled to a primary antibody that binds to the denatured capsid proteins, preferably an anti- An AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody, is used (Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody is then used that binds to the primary antibody and includes a means for detecting binding with the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound to the antibody, most preferably a sheep anti-mouse IgG antibody covalently bound to horseradish peroxidase. To semi-quantitatively determine the binding between the primary antibody and the secondary antibody, a method for detecting binding is used, preferably a detection method that can detect radioisotope radiation, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit. For example, in SDS-PAGE, samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and capsid proteins were resolved in a precast gradient polyacrylamide gel (e.g., Novex). Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions, or other suitable staining methods, i.e., SYPRO Ruby or Coomassie staining. In one embodiment, the concentration of AAV vector genome (vg) in the column fractions can be measured by quantitative real-time PCR (Q-PCR). Samples are diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the samples are further diluted and amplified using a TaqMan™ fluorogenic probe specific for the primers and the DNA sequence between the primers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 sequence detection system. Plasmid DNA containing the same sequence as contained in the AAV vector is used to create a standard curve in the Q-PCR reaction. The cycle threshold (Ct) value obtained from the sample was used to determine the vector genome titer by normalizing it to the Ct value of the plasmid standard curve.Digital PCR-based end-point assays can also be used.

[0087] In one embodiment, an optimized q-PCR method is used that utilizes a broad-spectrum serine protease, such as proteinase K (e.g., commercially available from Qiagen). More specifically, the optimized qPCR genomic titer assay is similar to the standard assay, except that after DNase I digestion, the sample is diluted with protease K buffer and treated with protease K, followed by heat inactivation. Suitably, the sample is diluted with an amount of protease K buffer equal to the sample size. The protease K buffer can be concentrated two-fold or more. Typically, the protease K treatment is about 0.2 mg / mL, but can vary from 0.1 mg / mL to about 1 mg / mL. The treatment step is generally carried out at about 55° C. for about 15 minutes, but may be carried out at lower temperatures (e.g., about 37° C. to about 50° C.) for longer times (e.g., about 20 minutes to about 30 minutes), or at higher temperatures (e.g., up to about 60° C.) for shorter times (e.g., about 5 to 10 minutes). Similarly, heat inactivation is generally at about 95° C. for about 15 minutes, but may be performed at lower temperatures (e.g., about 70 to about 90° C.) and for longer times (e.g., about 20 minutes to about 30 minutes). The sample is then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described for standard assays.

[0088] Additionally or alternatively, droplet digital PCR (ddPCR) can be used.For example, the method for measuring single-stranded and self-complementary AAV vector genome titer by ddPCR is described.See, for example, M. Lock et al, Hu Gene Therapy Methods.2014 Apr;25(2):115-25.doi:10.1089 / hgtb.2013.131.Epub 2014 Feb 14.

[0089] Briefly, a method for separating rAAVhu68 particles with packaged genome sequences from genome-deleted AAVhu68 intermediates involves subjecting a suspension containing recombinant AAVhu68 viral particles and AAVhu689 capsid intermediates to high performance liquid chromatography, in which the AAVhu68 viral particles and AAVhu68 intermediates are bound to a strong anion exchange resin equilibrated at a pH of 10.2 and subjected to a salt gradient while monitoring the eluate for UV absorbance at about 260 and about 280. The pH can range from about 10.0 to 10.4, although less optimal for rAAV9hu68. In this method, AAVhu68 complete capsids are recovered from fractions that elute when the ratio of A260 / A280 reaches an inflection point. In one example, for the affinity chromatography step, the diafiltered product may be applied to Capture Select™ Poros-AAV2 / 9 affinity resin (Life Technologies), which efficiently captures the AAV2 / hu68 serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flows through the column, while the AAV particles are efficiently captured.

[0090] Compositions and Uses Provided herein are compositions comprising at least one rAAV.hGALC stock (e.g., rAAVhu68 stock or mutant rAAV stock) and optional carriers, excipients and / or preservatives. A rAAV stock refers to multiple rAAV vectors that are the same, e.g., in the amounts described below in the discussion of concentrations and dosage units.

[0091] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharma- ceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like can be used to introduce the compositions of the present invention into suitable host cells. In particular, the rAAV vector delivery vector genome can be formulated for delivery either encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, and the like.

[0092] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, for example, an aqueous liquid suspension that is buffered to a physiologically compatible pH and salt concentration.Optionally, one or more surfactants are present in the formulation.In another embodiment, the composition can be delivered as a concentrate that is diluted for administration to a subject.In other embodiments, the composition can be lyophilized and reconstituted when administered.

[0093] A suitable surfactant or combination of surfactants may be selected from among non-ionic surfactants that are non-toxic. In one embodiment, a primary hydroxyl terminated bifunctional block copolymer surfactant is selected, such as Pluronic® F68 [BASF], also known as Poloxamer 188, with a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers may be selected, i.e., non-ionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycaprylic acid glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are generally designated by the letter "P" (for poloxamer) followed by three digits. They are named using a polyoxyethylene copolymer, where the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 gives the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

[0094] The vector is administered in an amount sufficient to transfect cells and provide sufficient levels of gene transfer and expression to provide therapeutic effects without undue adverse effects or with medically acceptable physiological effects, which can be determined by those skilled in the art. Conventional and pharmacologic acceptable routes of administration include, but are not limited to, direct delivery to the desired organ (e.g., liver (optionally via hepatic artery), lung, heart, eye, kidney), oral, inhalation, intranasal, intrathecal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parental routes of administration. Routes of administration may be combined if desired.

[0095] The dosage of the viral vector depends primarily on factors such as the condition being treated, the age, weight, and health of the patient, and may therefore vary between patients. For example, a therapeutically effective human dosage of a viral vector is generally about 1×10 9 ~1×10 16 The range of solutions containing the concentration of genome viral vector.The dosage may be adjusted to balance the therapeutic effect against any side effects, and the dosage may vary depending on the therapeutic application that the recombinant vector is used for.The expression level of the transgene can be monitored to determine the dosage frequency that results in the viral vector (preferably the AAV vector containing a minigene).Optionally, the dosage regimen similar to that described for therapeutic purposes can be used for immunization using the composition of the present invention.

[0096] The replication-defective virus composition is formulated in a dosage unit, which for a human patient is approximately 1.0×10 9 GC~approx. 1.0×10 16 GC, including amounts of replication defective virus (to treat an average subject weighing 70 kg), all integer or fractional amounts within that range, preferably 1.0×10 12 GC~1.0×10 14 In one embodiment, the composition comprises at least 1x10 per dose, including all integer or fractional amounts within the range. 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , or 9x10 9 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10, 7×10 10 , 8×10 10 , or 9×10 10 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , or 9×10 11 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , or 9×10 12 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , or 9×10 13 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 14 , 2×10 14 , 3×10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , or 9×10 14In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 15 , 2×10 15 , 3×10 15 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 , or 9×10 15 In one embodiment, for human applications, the dose is formulated to include all integers in the range or decimals, 1 x 10 per dose 10 ~Approx. 1×10 12 In one embodiment, for human applications, the dose is in the range of 1.4×10 per dose, including all integers or decimals within the range. 13 ~Approx. 4×10 14 It may be in the GC range.

[0097] These above doses may be administered in various volumes of the carrier, excipient, or buffer formulation ranging from about 25 to about 1000 microliters, or higher volumes including all numbers within that range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume of the carrier, excipient, or buffer is at least about 25 μL. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 75 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 225 μL. In yet another embodiment, the volume is about 250 μL. In yet another embodiment, the volume is about 275 μL. In yet another embodiment, the volume is about 300 μL. In yet another embodiment, the volume is about 325 μL. In another embodiment, the capacity is about 350 μL. In another embodiment, the capacity is about 375 μL. In another embodiment, the capacity is about 400 μL. In another embodiment, the capacity is about 450 μL. In another embodiment, the capacity is about 500 μL. In another embodiment, the capacity is about 550 μL. In another embodiment, the capacity is about 600 μL. In another embodiment, the capacity is about 650 μL. In another embodiment, the capacity is about 700 μL. In another embodiment, the capacity is about 700-1000 μL.

[0098] The therapeutically effective intrathecal / intracisternal dose of rAAV.hGALC is approximately 1 × 10 11 ~7.0×10 14 GC (fixed dose) range, 10 9 ~5×10 10 GC / g patient brain mass. Alternatively, the following therapeutically effective fixed doses can be administered to patients in the indicated age groups: Newborn: Approx. 1 x 10 11 ~Approx. 3×10 14 G.C., 3 to 9 months: approx. 6 x 1012 ~Approx. 3×10 14 G.C., 9 months to 6 years: Approx. 6 x 10 12 ~Approx. 3×10 14 G.C., Ages 3 to 6: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C., Ages 6-12: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C., Ages 12 and up: Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C., 18 years old and over (adult): Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.

[0099] In certain embodiments, the dose is about 1x10 9 GC / g brain mass ~ approx. 1x10 12 GC / g brain mass. In certain embodiments, the dose is about 3x10 10 GC / g brain mass ~ approx. 3x10 11 GC / g brain mass. In certain embodiments, the dose is about 5x10 10 GC / g brain mass ~ approx. 1.85x10 11 For scaling between infants and adolescents / adults, brain mass is estimated to be about 600 g to about 800 g for 4-12 months of age, about 800 g to about 1000 g for 9-18 months of age, about 1000 g to about 1100 g for 18 months to 3 years of age, 1100 g to about 1300 g for adolescent or adult humans, or about 1300 g for adult humans, as the case may be.

[0100] In one embodiment, the viral construct comprises at least about 1×10 9 GC~approx. 1×10 15 , or approximately 1 × 10 11 ~5×10 13The GC may be delivered in doses of 1000 mg / kg / day. Appropriate volumes and concentrations for the delivery of these doses may be determined by one of skill in the art. For example, a volume of about 1 μL to 150 mL may be selected, although higher volumes may be selected for adults. Typically, a suitable volume for a newborn is about 0.5 mL to about 10 mL, and for older infants, about 0.5 mL to about 15 mL is selected. For infants, a volume of about 0.5 mL to about 20 mL is selected. A volume of up to about 30 mL may be selected for children. A volume of up to about 50 mL may be selected for preteens and teens. In yet other embodiments, patients may receive intrathecal administration at a selected volume of about 5 mL to about 15 mL, or about 7.5 mL to about 10 mL. Other suitable volumes and dosages may be determined. Such dosages may be adjusted to balance the therapeutic benefit against any side effects, and such dosages may vary depending on the therapeutic application for which the recombinant vector is utilized.

[0101] The recombinant vectors described above can be delivered to host cells according to published methods. The rAAV, preferably suspended in a physiologically compatible carrier, can be administered to a human or non-human mammalian patient. In certain embodiments, for administration to a human patient, the rAAV is suitably suspended in an aqueous solution containing saline, a surfactant, and a physiologically compatible salt or mixture of salts. Suitably, the formulation is adjusted to a physiologically acceptable pH, for example, in the range of pH 6-9, or pH 6.5-7.5, pH 7.0-7.7, or pH 7.2-7.8. The pH of cerebrospinal fluid is about 7.28 to about 7.32, so for intrathecal delivery, a pH within this range may be desirable, and for intravenous delivery, a pH of about 6.8 to about 7.2 may be desirable. However, other pHs within the broadest range, and subranges of these, may be selected for other delivery routes.

[0102] In another embodiment, the composition comprises a carrier, diluent, excipient and / or adjuvant. A suitable carrier can be easily selected by those skilled in the art, taking into consideration the indication for which the introduced virus is intended. For example, one suitable carrier comprises saline, and can be formulated with various buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The buffer / carrier should comprise a component that prevents rAAV from adhering to the injection tube, but does not interfere with rAAV binding activity in vivo.

[0103] In one example, the formulation can contain a buffered saline solution, for example, including one or more of sodium chloride, sodium bicarbonate, dextrose, magnesium sulfate (e.g., magnesium sulfate·7H2O), potassium chloride, calcium chloride (e.g., calcium chloride·2H2O), dibasic sodium phosphate, and mixtures thereof, in water. Suitably, for intrathecal delivery, the osmolality is within a range compatible with cerebrospinal fluid (e.g., about 275 to about 290), see, e.g., emedicine.medscape.com / article / 2093316-overview. Optionally, for intrathecal delivery, commercially available diluents can be used as suspending agents or in combination with another suspending agent and any other excipients. See, e.g., Elliotts B® solution [Lukare Medical].

[0104] In certain embodiments, the formulation may contain a buffered saline solution that does not contain sodium bicarbonate. Such formulation may contain a buffered saline solution, such as Harvard buffer, which contains one or more of sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and mixtures thereof in water. The aqueous solution may further contain the poloxamer Kolliphor® P188, which is commercially available from BASF and was previously sold under the trade name Lutrol® F68. The aqueous solution may have a pH of 7.2.

[0105] In other embodiments, the formulation may include one or more permeation enhancers. Examples of suitable permeation enhancers include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, It may also contain sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, or EDTA.

[0106] Optionally, the compositions of the present invention may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to rAAV and carriers.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.

[0107] The composition according to the present invention may comprise a pharma- ceutically acceptable carrier as defined above.Preferably, the composition described herein comprises one or more AAV suspended in an effective amount of a pharma- ceutically suitable carrier and / or mixed with a suitable excipient designed for delivery to a subject via injection, osmotic pump, intrathecal catheter, or by another device or route.In one embodiment, the composition is formulated for intrathecal delivery.

[0108] As used herein, the term "intracranial delivery" or "intracranial administration" refers to the route of administration of a drug via injection into the spinal canal, more specifically, by injection into the subarachnoid space to reach the cerebrospinal fluid (CSF). Intrathecal delivery may include lumbar puncture, intraventricular (including intraventricular ventricle (ICV)), suboccipital / intracisternal, and / or C1-2 puncture. For example, material may be introduced by lumbar puncture to diffuse throughout the subarachnoid space. In another example, injection may be intracisternal.

[0109] As used herein, the term "intracisternal delivery" or "intracisternal administration" refers to the administration of a drug directly into the cerebrospinal fluid of the cerebellum-medullary cisterna magna, more specifically, by suboccipital puncture or by direct injection into the cisterna magna, or by a permanently placed tube.

[0110] As used herein, the term computed tomography (CT) refers to radiography in which a three-dimensional image of a body structure is constructed by a computer from a series of planar cross-sectional images made along an axis.

[0111] The rAAV.GALC vectors and compositions provided herein are useful for correcting conditions associated with defective levels of GALC enzyme activity. In certain embodiments, the rAAV.GALC vectors and compositions provided herein are useful for treating peripheral nerve dysfunction caused by a deficiency in GALC, are useful for treating respiratory failure and / or loss of motor function caused by a deficiency in GALC, are useful for treating Krabbe disease, and / or are useful for treating symptoms associated with Krabbe disease in a patient.

[0112] In certain embodiments, a composition comprising an effective amount of rAAV.hGALC is administered to a patient under 6 months of age who has early infantile Krabbe disease (EIKD). In certain embodiments, the patient is under 6 months of age and has a GALC enzyme deficiency that is less severe than EIKD.

[0113] In certain embodiments, a composition comprising an effective amount of rAAV.hGALC is administered to a patient with late infantile Krabbe disease (LIKD) who is 6 months of age or older (e.g., 7 months to about 12 months of age). In certain embodiments, the patient is 6 months of age or older, or about 7 months to 12 months of age, and has a less severe GALC enzyme deficiency than LIKD.

[0114] In certain embodiments, the patient is over 1 year old (e.g., between 13 months and 10 years old) and has Juvenile Krabbe Disease (JKD). They have a less severe GALC enzyme deficiency than JKD.

[0115] In certain embodiments, the patient is over 10 years of age (eg, 10 to 12 years, or 10 to 18 years) and has adolescent or adult-onset Krabbe disease.

[0116] In any of the above-mentioned embodiments, the rAAV.hGALC therapy provided herein may be administered as a combination therapy with hematopoietic stem cell replacement therapy, bone marrow transplantation (BMT), and / or substrate synthesis inhibition therapy (SRT). In certain embodiments, the rAAV.hGALC therapy (e.g., EIKD) is followed by a combination therapy, such as HSCT or BMT, or enzyme replacement therapy. In certain embodiments, the therapy results in rapid enzyme production after administration of the vector (including within one week after treatment).

[0117] In certain embodiments, enzyme replacement therapy involves administration of human GALC protein of SEQ ID NO: 10. In other embodiments, other hGALC protein variants (e.g., canonical sequences identified herein, or engineered proteins) may be used in enzyme replacement therapy. Combinations of different hGALC proteins may be used in enzyme replacement therapy. In such embodiments, hGALC proteins may be produced in vitro using a suitable production system. See, for example, C. Lee et al, 2005 / 10 / 01. Enzyme replacement therapy results in substantial improvement of early clinical phenotypes in mouse models of globoid cell leukodystrophy (FASEB journal, The FASEB Journal 19(11):1549-51, October 2005). hGALC protein may be formulated for delivery by any suitable route, including but not limited to intravenous, intraperitoneal, or intrathecal routes (e.g., suspended in a physiologically compatible saline solution). Suitable doses may range from 1 mg / kg to 20 mg / kg, or 5 mg / kg to 10 mg / kg, and may be re-administered once a week, or more or less frequently (e.g., once every two days, once every two weeks, etc.) as needed. Using CSF administration of hAAVhu68.GALC vectors, GALC levels in the brain and serum may be supraphysiological without toxicity, and improved neuromotor function and myelination in the CNS and PNS may be observed. CSF administration to newborns followed by bone marrow transplantation in postnatal conditioning animal models may result in extended survival (e.g., to more than 300 days) in the absence of obvious symptoms. In pre-symptomatic Krabbe patients, a single cisternal injection of AAV.cGALC may provide phenotypic correction, increased survival, normalization of nerve conduction, and / or improved brain MRI.

[0118] In certain embodiments, rAAV.hGALC therapy is provided after HSCT or BMT (e.g., LIKD or JKD). However, in certain embodiments, rAAV.hGALC provides sufficient GALC levels such that HSCT or BMT is not required.

[0119] The goal of treatment is to functionally replace the defective GALC in patients via rAAV-based CNS- and PNS-directed gene therapy. The efficacy of therapy for patients with EIKD or LIKD can be measured by assessing improvements in one or more of the following EIKD or LIKD symptoms: crying and irritability, convulsions, fist clenching, loss of smiling, poor head control and feeding difficulties, mental and motor deterioration, hypertonia or hypotonia, seizures, blindness, hearing loss, and increased survival (EIKD, if untreated, typically results in death before age 2 years, while LIKD may extend survival to age 3-5 years). In addition, for these and other Krabbe patients, the efficacy of treatment can be monitored via imaging (e.g., magnetic resonance imaging (MRI)), reduction in hypomyelination and demyelination affecting both peripheral nerves and CNS white matter (deep cerebral white matter and dentate / cerebellar white matter), reduction in abnormal nerve conduction velocity (NCV) and / or brainstem auditory evoked potentials (BAEP), and cerebrospinal fluid (CSF) signaling. and / or may be assessed by an increase in GALC levels that can be observed in plasma, and / or a decrease in the accumulation of psychosine.

[0120] A composition is provided comprising a recombinant adeno-associated virus (rAAV), which targets cells of the central nervous system and comprises an AAV capsid into which is packaged a vector genome comprising a galactosylceramidase coding sequence encoding a mature galactosylceramidase protein having the amino acid sequence of SEQ ID NO: 10 under the control of regulatory sequences directing expression of the protein, the vector genome further comprising AAV inverted terminal sequences necessary for packaging the vector genome into the AAV capsid.

[0121] In certain embodiments, a composition useful for treating Krabbe disease is provided, comprising rAAVhu68 having a vector genome of CB7.CI.hGALC.rBG. In one embodiment, the vector genome has the coding sequence of SEQ ID NO:19.

[0122] In certain embodiments, the composition is used in the method for correcting peripheral nerve dysfunction caused by GALC deficiency, and / or the method for treating respiratory insufficiency and motor function loss caused by GALC deficiency.In certain embodiments, the method comprises administering a composition comprising a stock of recombinant adeno-associated virus (rAAV), the recombinant adeno-associated virus comprises (a) AAV capsid that targets cells of the central nervous system and (b) vector genome is packaged therein, and (b) vector genome comprises galactosylceramidase coding sequence that codes for mature galactosylceramidase protein having amino acid sequence of SEQ ID NO: 10 under the control of regulatory sequence that directs protein expression, and further comprises AAV inverted terminal sequence that is necessary for packaging vector genome into AAV capsid.

[0123] In certain embodiments, the rAAV.hGALC compositions provided herein are delivered intrathecally for the treatment of patients with early infantile Krabbe disease. In certain embodiments, the compositions provided herein are delivered intrathecally for the treatment of patients with late infantile Krabbe disease (LIKD). In certain embodiments, the rAAV.hGALC compositions provided herein are delivered intrathecally for the treatment of patients with juvenile Krabbe disease (JKD). In certain embodiments, the rAAV.hGALC compositions provided herein are delivered intrathecally for the treatment of patients with adolescent or adult-onset Krabbe disease. In certain embodiments, the rAAV.hGALC compositions are administered as a combination therapy with hematopoietic stem cell transplantation (HSCT), bone marrow transplantation, and / or matrix synthesis suppression therapy. In certain embodiments, the rAAV.hGALC compositions are administered as a single dose via computed tomography (CT)-guided (intracisternal) suboccipital injection into the cisterna magna.

[0124] Administration of rAAV.hGALC stabilizes disease progression (as measured by survival rate), prevents loss of developmental and motor milestones, potentially aids in the acquisition of new milestones, and reduces the onset and frequency of seizures. Thus, in certain embodiments, methods are provided for monitoring treatment, with endpoints measured, for example, at 30 days, 90 days, and / or 6 months, and then every 6 months during a short-term follow-up period, for example, 2 years. In certain embodiments, the frequency of measurements is reduced to once every 12 months during long-term extension. Given the severity of disease in the target population, subjects may have achieved motor skills by enrollment, developed other motor milestones that have since been lost, or may not yet show signs of motor milestone development. Evaluation tracks age-at-achievement and age-at-loss for all milestones. In certain embodiments, the milestones include, for example, one or more of sitting without support, crawling on all fours, standing with assistance, walking with assistance, standing independently, and / or walking independently. The treatment results in a delay in the onset of seizure activity and / or a reduction in the frequency of seizure events.

[0125] In certain embodiments, methods of monitoring treatment in a subject use clinical measures to quantitate the effect of treatment on development and changes in adaptive behavior, cognition, language, motor function, and / or health-related quality of life. Scales and domains include, for example, the Bayley Scales of Infant Development (assessing infant and toddler development across five domains: cognition, language, motor, social-emotional, and adaptive behavior), the Vineland Adaptive Behavior Scales (3rd Edition) (assessing adaptive behavior from birth to adulthood (0-90 years) across five domains: communication, daily living skills, socialization, motor skills, and maladaptive behavior), the Peabody Developmental Motor Scales-2nd Edition (measures interrelated motor function in children from birth to 5 years of age, with assessments focusing on six domains: reflexes, stillness, movement, object manipulation, grasping, and visual-motor integration), the Infant Quality of Life Questionnaire (ITQOL) (a parent-reported measure of health-related quality of life designed for infants aged 2 months to 5 years), and the Mullen Scales of Early Learning (assessing language, motor, and perceptual abilities in infants and toddlers up to 68 months of age). In certain embodiments, the effects of treatment are monitored or measured by assessing changes in myelination, functional outcomes related to myelination, and potential disease biomarkers. In certain embodiments, the progression of central and peripheral demyelination is delayed or stopped after the subject is treated.Central demyelination can be tracked by diffusion tensor magnetic resonance imaging (DT-MRI) anisotropy measurement of white matter area and fiber tracking of corticospinal motor tract, and their changes are indicators of disease state and progression.Peripheral demyelination can be indirectly measured through nerve conduction velocity (NCV) testing of motor nerve (deep peroneal nerve, tibial nerve, and ulnar nerve) and sensory nerve (sural nerve and median nerve) to monitor the fluctuations that indicate biologically active myelin changes (i.e., F-wave and distal latency, amplitude, or the presence or absence of response).

[0126] In certain embodiments, a method for monitoring treatment after administration of rAAV.hGALC is provided, and the subject is evaluated for delayed or absent vision loss for subjects who have not developed significant vision loss before treatment. Thus, measuring visual evoked potentials (VEP) is used to objectively measure response to visual stimuli as an indicator of central vision impairment or loss. In certain embodiments, the subject is monitored for hearing loss after treatment, for example, using brainstem auditory evoked response (BAER) testing. In certain embodiments, a method for monitoring treatment after administration of rAAV.hGALC is provided, and the subject's psychosine level is measured.

[0127] It should be noted that the terms "a" or "an" refer to one or more. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0128] The terms "comprise," "comprises," and "comprising" are to be interpreted inclusively rather than exclusively. The terms "consist," "consisting," and variations thereof are to be interpreted exclusively rather than inclusively. Although various embodiments herein are set forth using the word "comprising," it is also intended that in other circumstances the relevant embodiment should be construed and described using the word "consisting of" or "consisting essentially of."

[0129] As used herein, the term "about" means a variability of 10% (± 10%) from a given reference, unless otherwise specified.

[0130] As used herein, "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal condition in a subject.

[0131] The term "expression" is used herein in the broadest sense and includes the production of RNA or RNA and protein. With respect to RNA, the terms "expression" or "translation" particularly relate to the production of peptides or proteins. Expression can be transient or stable.

[0132] As used herein, "expression cassette" refers to a nucleic acid molecule that includes a coding sequence, a promoter, and may include other regulatory sequences therefor. In certain embodiments, a vector genome may include two or more expression cassettes. In other embodiments, the term "transgene" may be used interchangeably with "expression cassette." Typically, such expression cassettes for producing viral vectors include coding sequences for gene products described herein adjacent to the packaging signal of the viral genome, and other expression control sequences such as those described herein.

[0133] The abbreviation "sc" refers to self-complementary. "Self-complementary AAV" refers to constructs in which the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. During infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of the scAAV associate to form one double-stranded DNA (dsDNA) unit capable of immediate replication and transcription. See, for example, DM McCarty et al, "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis", Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated by reference herein in its entirety.

[0134] When used in reference to a protein or nucleic acid, the term "heterologous" indicates that the protein or nucleic acid contains two or more sequences or subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids with two or more sequences from unrelated genes arranged to create a new functional nucleic acid are typically produced recombinantly. For example, in one embodiment, a nucleic acid has a promoter from one gene arranged to direct the expression of a coding sequence from a different gene. Thus, with respect to the coding sequence, the promoter is heterologous.

[0135] "Replication-defective virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, and any viral genomic sequences packaged within the viral capsid or envelope are replication-defective, i.e., they cannot produce progeny virions, but can retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless," containing only the transgene of interest flanked by signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. It is therefore considered safe for use in gene therapy, since replication and infection by progeny virions cannot occur except in the presence of viral enzymes required for replication.

[0136] In many cases, rAAV particles are referred to as DNase-resistant. However, in addition to this endonuclease (DNase), other endonucleases and exonucleases can be used in the purification steps described herein to remove contaminating nucleic acids. Such nucleases can be selected to degrade single-stranded and / or double-stranded DNA, and RNA. Such steps can include a single nuclease, or a mixture of nucleases directed to different targets, which can be endonucleases or exonucleases.

[0137] The term "nuclease resistant" indicates that the AAV capsid is constructed entirely around an expression cassette designed to deliver genes into a host cell, and protects these packaged genomic sequences from degradation (digestion) during a nuclease incubation step designed to remove contaminating nucleic acids that may be present from the production process.

[0138] As used herein, "effective amount" refers to the amount of rAAV composition that delivers and expresses the amount of gene product from the vector genome in target cells. Effective amounts can be determined based on animal models rather than human patients. An example of a suitable mouse model is described herein.

[0139] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to published documents which provide general guidance to those of ordinary skill in the art for many of the terms used herein. EXAMPLES

[0140] The following examples are illustrative only and are not intended to limit the invention. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0141] Example 1 - Recombinant AAVhu68.hGALC rAAVhu68.hGALC is an AAV carrying an engineered sequence encoding human GALC. The AAVhu68 capsid of rAAVhu68.hGALC is 99% identical to AAV9 at the amino acid level. The two amino acids that differ between AAV9 capsid [SEQ ID NO: 4] and AAVhu68 capsid [SEQ ID NO: 2] are located in the VP1 (67 and 157) and VP2 (157) regions of the capsid and are identified in Figure 1. See, for example, WO2018 / 160852 (incorporated herein by reference).

[0142] rAAVhu68.hGALC is produced by triple plasmid transfection of HEK293 cells with an AAV cis plasmid encoding a transgene cassette flanked by AAV ITRs, an AAV trans plasmid (pAAV2 / hu68.KanR) encoding genes for AAV2 rep and AAVhu68 cap, and a helper adenovirus plasmid (pAdΔF6.KanR).

[0143] A. Sequence Elements of AAV Vector Genome Plasmids A linear map of the vector genome is shown in Figure 2. The vector genome contains the following sequence elements: Inverted Terminal Repeats (ITRs) ITRs are identical reverse complement sequences derived from AAV2 (130 bp, GenBank: NC_001401) and flank all elements of the vector genome. The ITR sequences function as both an origin of replication for the vector DNA and a packaging signal for the vector genome when AAV and adenovirus helper functions are provided in trans. Thus, the ITR sequences represent the only cis sequences required for replication and packaging of the vector genome.

[0144] Human cytomegalovirus immediate early enhancer (CMV IE): This enhancer sequence is derived from human-derived CMV (382 bp, GenBank: K03104.1) and increases the expression of downstream transgenes.

[0145] Chicken β-actin promoter (BA) This ubiquitous promoter (282 bp, GenBank: X00182.1) was selected to drive transgene expression in any CNS cell type.

[0146] Chimeric introns (CI) hybrid introns contain elements of the chicken β-actin splice donor (973 bp, GenBank: X00182.1) and rabbit β-globin splice acceptor. Introns are transcribed but, together with sequences at either end of them, are removed from the mature messenger RNA (mRNA) by splicing. The presence of an intron in an expression cassette regulates the expression of mRNA from the nucleus to the cytoplasm. It has been shown to facilitate RNA transport, thereby enhancing the accumulation of a constant level of mRNA for translation, a common feature in genetic vectors intended to increase gene expression levels.

[0147] Coding sequence: The engineered cDNA of the human GALC gene encodes the human galactosylceramidase protein, which is a lysosomal enzyme responsible for the hydrolysis and degradation of myelin galactolipids (2055 bp, 685 amino acids [aa], GenBank: EAW81361.1).

[0148] Rabbit β-globin polyadenylation signal (rBG PolyA): The rBG PolyA signal (127 bp, GenBank: V00882.1) promotes efficient polyadenylation of transgene mRNA in cis. This element functions as a signal for transcription termination, a specific cleavage event at the 3' end of the nascent transcript, and the addition of a long polyadenylation strand.

[0149] B. Transplasmid: pAAV2 / 1.KanR (p0068) The AAV2 / hu68 transformer plasmid pAAV2 / hu68.KanR(p0068) is presented in Figure 21.

[0150] The AAV2 / hu68 transplasmid is pAAV2 / hu68.KanR (p0068). The pAAV2 / hu68.KanR plasmid is 8030 bp in length and encodes the four wild-type AAV2 replicase (Rep) proteins required for replication and packaging of the AAV vector genome. The pAAV2 / hu68 transplasmid also encodes the three WT AAVhu68 virion protein capsid (Cap) proteins that assemble into the virion shell of AAV serotype hu68 to accommodate the AAV vector genome.

[0151] To generate the pAAV2 / hu68.KanR transplasmid, the AAV9 cap gene from plasmid pAAV2 / 9n(p0061-2) (encoding wild-type AAV2 rep and AAV9 cap genes on a plasmid backbone derived from the pBluescript KS vector) was removed and replaced with the AAVhu68 cap gene. The ampicillin resistance (AmpR) gene was replaced with the kanamycin resistance (KanR) gene to yield pAAV2 / hu68.KanR(p0068). In this cloning strategy, the AAV p5 promoter sequence (which normally drives expression of rep) is moved from the 5' end of rep to the 3' end of cap, leaving a truncated p5 promoter upstream of rep. This truncated promoter serves to downregulate expression of rep and, as a result, maximize vector production (Figure 21).

[0152] All component parts of the plasmid have been verified by direct sequencing.

[0153] C. Adenovirus helper plasmid: pAdDeltaF6(KanR) The adenovirus helper plasmid pAdDeltaF6(KanR) is presented in (Figure 22B). Plasmid pAdDeltaF6(KanR) is 15,770 bp in size. This plasmid contains the regions of the adenovirus genome important for AAV replication, namely E2A, E4, and VA RNAs (the function of adenovirus E1 is provided by HEK293 cells). However, this plasmid does not contain other adenovirus replication or structural genes. The plasmid does not contain cis elements important for replication, such as the adenovirus ITRs, and therefore, infectious adenovirus is not expected to be produced. The plasmid contains the E1, E3 deletion of Ad5. pAdeltaF6(KanR) was derived from a child clone (pBHG10, a pBR322-based plasmid). Deletions were introduced into Ad5 to eliminate expression of unnecessary adenoviral genes and reduce the amount of adenoviral DNA from 32 kb to 12 kb (Figure 22A). Finally, the ampicillin resistance gene was replaced with a kanamycin resistance gene to generate pAdeltaF6(KanR) (Figure 22B). The adenoviral genes E2, E4, and VAI remaining in this plasmid, along with E1 present in HEK293 cells, are necessary for AAV vector production. Vectors are produced and formulations are prepared according to the flow charts shown in Figures 30 and 31, respectively.

[0154] Example 2 - Delivery of AAVhu68.hGALC in the GALC-deficient Twitcher mouse model The studies described below used the Twitcher mouse model to establish the feasibility of delivering an rAAVhu68 vector (Figure 2) encoding an engineered human GALC sequence (SEQ ID NO: 9) to the CSF to achieve therapeutic levels of GALC expression levels and rescue several biomarkers of disease. An overview of the Twitcher mouse study is provided in Figure 4B.

[0155] The Twitcher mouse is a naturally occurring inbred model of Krabbe disease, identified as a spontaneous mutation in 1976 at the Jackson Laboratory (Kobayashi T., et al. (1980) Brain Research. 202(2):479-483). Affected mice are homozygous for a loss-of-function allele of Twitcher (twi), consisting of a G to A mutation in the Galc gene. This mutation results in a premature stop codon (W339X). The truncated GALC protein has close to 0% residual enzyme activity, which is similar to the GALC activity levels observed in patients with the infantile form of Krabbe disease. Heterozygous carrier mice (twi / +) are phenotypically normal.

[0156] Disease progression in Twitcher mice is well documented (Figure 4A) and phenocopies the various neuropathological and behavioral defects of infantile Krabbe disease presented in Table 5. Like infantile Krabbe patients, GALC deficiency in mice results in the accumulation of psychosine, a cytotoxic lipid intermediate. Twitcher mice similarly display massive infiltration of PNS and CNS white matter by phagocytic psychosine-filled globoid cells, thought to be derived from macrophage and / or microglial lineages (Tanaka K., et al. (1988) Brain Research. 454(1):340-346, Levine SM, et al. (1994) Intl J Neurosci. 1999, 103:1111-1123, 1999). Dev Neuro.12(4):275-288). This leads to demyelination, one of the major features of the disease in Krabbe patients. After an initial period of normal myelination, affected Twitcher mice lose myelin in the PNS after 10 days of age due to the death of myelinating Schwann cells (Jacobs JM,et al.(1982)J Neurol Sci.55(3):285-304) and in the CNS after 20 days of age due to the death of myelinating oligodendrocytes. Perhaps because of this delay, demyelination is more severe in the peripheral nerves than in the CNS of these mice (Suzuki K.&Suzuki K.(1983)The American journal of pathology.111(3):394-397). Finally, Twitcher mice show consistent and rapid neurological deterioration after the onset of symptoms, which is also observed in infantile Krabbe patients at the time of onset of symptoms. Behavioral symptoms in these mice include a motor phenotype reminiscent of that observed in human patients, including tremors, twitching, and hind limb weakness seen at approximately 20 days of age. Mice ultimately progress to a humane endpoint characterized by severe weight loss and paralysis by around 40 days of age (Wenger DA (2000) Molec Med Today. 6(11):449-451).

[0157] Patients with infantile Krabbe disease display similar clinical features as Twitcher mice. Therefore, the Twitcher mouse model is appropriate to evaluate the efficacy of rAAVhu68.hGALC (rescuing enzyme activity to improve survival, motor function, and brain and neuropathology) and support the indications of infantile Krabbe disease. Studies using Twitcher mice described below demonstrate the efficacy of rAAVhu68.hGALC to express active GALC enzyme in relevant tissues after a single ICV administration (the most effective route of administration in mouse models where ICM is not technically feasible) to rescue survival, improve motor function, and improve CNS and PNS histopathology.

[0158] Presymptomatic neonatal Twitcher mice The goal of this study was to establish the optimal ROA, capsid serotype, and dose range to achieve maximum efficacy in the Twitcher mouse model. For these studies, presymptomatic neonatal mice were selected to maximize the observed variability of disease rescue.

[0159] To establish the optimal ROA, the IV route by infusion into the temporal vein was compared to the ICV route, since both routes are capable of transducing the CNS and PNS in neonatal animals. At PND0, presymptomatic Twitcher mice (twi / twi) were administered rAAVhu68.hGALC at either 1.00 × 10 11 Administer IV at a dose of GC or 5-fold lower at 2.00 × 10 10 The GC was administered ICV at a dose of 1.00 × 10, the high dose required to achieve CNS transduction. 14The 5-fold lower ICV dose was chosen because it corresponds to GC / kg, and the 5-fold lower ICV dose was chosen because direct administration into the CSF facilitates CNS transduction even at lower doses. As controls, presymptomatic age-matched Twitcher mice were injected ICV with vehicle (PBS). Animals were euthanized when they reached a humane endpoint defined by weight loss >20% of maximum body weight and / or hind limb paralysis, and survival was recorded. A higher dose of rAAVhu68.hGALC (1.00 × 10 11 GC) provided some survival benefit compared to untreated controls. However, a 5-fold lower dose (2.00 × 10 10 rAAVhu68.hGALC administered ICV via the GC conferred a superior survival benefit (Figure 5). Therefore, the ICV route of administration (ROA) was chosen for subsequent studies.

[0160] To identify the best AAV capsid for nervous system delivery of vector genomes encoding human GALC, four different AAV capsids were tested. The capsids included AAV serotype 3b (AAV3b), AAV serotype 5 (AAV5), AAV serotype 1 (AAV1), and AAV serotype hu68 (rAAVhu68.hGALC). Each AAV vector was delivered at a concentration of 2.00 × 10 10 AAVhu68 capsid (rAAVhu68.hGALC) was administered ICV at a dose of 100 mg / kg / day of GC, a previously established low dose and ROA that allowed for a short study period while effectively extending survival in presymptomatic Twitcher mice (Figure 5). As a control, a group of presymptomatic Twitcher mice received vehicle only (PBS) ICV at PND0. Although all four capsids improved survival compared to vehicle-treated controls, the AAVhu68 capsid (rAAVhu68.hGALC) resulted in superior survival rates than AAV3b, AAV5, and AAV1 (Figure 6). Thus, the AAVhu68 capsid (rAAVhu68.hGALC) was selected for subsequent studies.

[0161] To determine the dose range, rAAVhu68.hGALC was administered at 2.00 × 10 10 GC, 5.00×10 10 GC, or 1.00 × 10 11 As a control, vehicle (PBS) was administered ICV to age-matched pre-symptomatic Twitcher (twi / twi) mice, as well as unaffected heterozygous (twi / +) and wild-type mice at PND0.

[0162] At PND35, mice were assessed for mobility and coordination using a rod assay (Figure 7). The rotarod is an accelerating rod on which mice run, and it is well established that the time from the start of the assay to the point at which the mouse falls off the rod correlates with motor coordination. The PND35 time point was chosen because it is the time point at which diseased Twitcher mice display measurable motor impairments before reaching the humane euthanasia endpoint. The rotarod assay revealed partial rescue of mobility and coordination following rAAVhu68.hGALC administration. The extent of rescue appeared to be dose-dependent, with a maximum rescue rate of 1.00 × 10 11 For the highest rAAVhu68.hGALC dose of GC, a significantly longer latency to fall was observed (p<0.01) (Figure 7).

[0163] Following the rotarod assay, survival was followed for all treatment groups. A dose-dependent increase in survival was observed in Twitcher (twi / twi) mice administered rAAVhu68.hGALC presymptomatically on PND0 compared to vehicle-treated Twitcher (twi / twi) controls. 11 The longest median survival time (130 days) was observed in mice receiving the highest rAAVhu68.hGALC dose of GC (Figure 8).

[0164] Together, these POC experiments to identify optimal ROA, capsid, and dose range demonstrate the efficacy of rAAVhu68.hGALC in preserving neuromotor function and extending survival when administered presymptomatically in Twitcher mice.

[0165] Symptomatic Twitcher mice The aim of this study was to examine the efficacy of rAAVhu68.hGALC when administered during early stages of disease pathology before the onset of behavioral symptoms (PND12, termed "early symptomatic"), or during later stages of disease pathology when mice show behavioral symptoms (PND21, termed "late symptomatic"), because we wanted to reproduce a similar context to patients enrolled after symptom onset. Furthermore, mice have brain maturity equivalent to preterm fetuses at PND0, but at PND12 and PND21, displaced to 2 and 9 months of age, respectively (www.translatingtime.org), which more closely reproduces the infant population subject to FIH.

[0166] Early symptomatic Twitcher mice were injected with rAAVhu68.hGALC at PND12 at 1.00 × 10 11 GC or 2.00 x 10 11 GC at either dose, while a separate cohort of late symptomatic Twitcher mice received rAAVhu68.hGALC at PND21, 2.00 × 10 11 The higher dose of GC was administered ICV. The lower dose was 1.00 × 10 11 GC was chosen for administration because it was found to be the most effective dose in increasing survival in Twitcher mice (see above). It was hypothesized that a higher dose may be required for mice with more severe demyelination, so a higher rAAVhu68.hGALC dose of 2.00 × 10 11GCs were also utilized to treat mice on PND 21. As controls, early symptomatic Twitcher mice (twi / twi) and unaffected heterozygous mice (twi / +) received vehicle only (PBS) ICV on PND 12 and, using historical data from Study 1, received 1.11 × 10 11 The results were compared with Twitcher mice (twi / twi) that received GC.

[0167] At PND35, the time point at which Twitcher mice (twi / twi) exhibit measurable motor impairment, mice were assessed for mobility and coordination using the rotarod assay. For the rotarod assay, rAAVhu68.hGALC was delivered to presymptomatic Twitcher mice at PND0 at 1.00 × 10 11 When administered at a dose of GC (p<0.01) or 1.00 × 10 11 GC (p<0.001) or 2.00×10 11 At PND21, late symptomatic Twitcher mice were administered 2.00 × 10 11 When GC doses of rAAVhu68.hGALC were administered, no significant rescue of motility and coordination was observed (Figure 9).

[0168] Following the rotarod assay, survival was followed for all treatment groups. Longer survival was observed in both early symptomatic Twitcher mice (administered at PND12) and late symptomatic Twitcher mice (administered at PND21) following administration of rAAVhu68.hGALC compared to vehicle-treated Twitcher controls. However, maximum survival was observed in early symptomatic Twitcher mice administered 2.00×10 rAAVhu68.hGALC at PND12. 11 This was obtained when high doses of GC were administered (Figure 10).

[0169] Taken together, the improved survival and neuromotor function of Twitcher mice suggested that rAAVhu68.hGALC may be more effective if administered at an early stage of the disease.

[0170] Pharmacological studies in symptomatic Twitcher mice The goal of this study is to evaluate pharmacological, functional, and histopathological readouts following administration of rAAVhu68.hGALC. In previous studies, animals were sacrificed at humane endpoints to facilitate survival analysis, which precluded the collection of pharmacological and histopathological readouts at age-matched time points in Twitcher mice and vehicle-treated controls. Therefore, in this study, we explored these endpoints.

[0171] Early symptomatic Twitcher mice (twi / twi) were injected with rAAVhu68.hGALC at PND12 at 2.00 × 10 11 As a control, PBS was administered ICV to age-matched unaffected Twitcher heterozygous (twi / +) and wild-type mice at PND12. To achieve maximal efficacy, a dose of 2.00 × 10 11 The rAAVhu68.hGALC dose of GC was selected for POC, and PND12 was chosen as the dosing date because it is shortly after the onset of PNS demyelination in animals with brain maturity equivalent to that of a 2-month-old infant (reflecting the infant population targeted in the FIH study) (www.translatingtime.org).

[0172] Beginning on PND22, all mice were monitored daily for clinical signs. PND22 was chosen as the initial time point for this evaluation as it is the earliest time at which a behavioral phenotype is observable in Twitcher mice. Clinical signs were scored using a confidential assessment of clasping ability, gait, tremor, kyphosis, and fur quality, as detailed in Table 1. These scales effectively assess the clinical status of Twitcher mice based on typically exhibited symptoms. A score of 0 or greater indicates clinical deterioration. [Table 4]

[0173] Using this assessment, early symptomatic Twitcher mice (twi / twi) administered rAAVhu68.hGALC on PND12 showed clinical scores approaching 0, which were comparable to those of wild-type and unaffected Twitcher heterozygotes (twi / +). In contrast, age-matched vehicle-treated Twitcher (twi / twi) mice showed higher assessment scores over most of the time course, indicating clinical deterioration (Figure 11A).

[0174] As a complementary functional assay, the rotarod test was performed to assess the neuromotor phenotype at PND 35. Early symptomatic Twitcher mice (twi / twi) administered rAAVhu68.hGALC at PND 12 showed fall latencies comparable to those of wild-type and unaffected Twitcher heterozygotes (twi / +), whereas age-matched vehicle-treated Twitcher mice (twi / twi) showed significantly shorter fall latencies (p<0.05), indicating impaired neuromotor function (Figure 11B).

[0175] To determine whether the observed benefits of rAAVhu68.hGALC administration on functional endpoints correlated with histological improvements, all mice were necropsied at PND40 and the sciatic nerves of the hind limbs were examined histologically. PND40 was chosen as the necropsy time point because it is the approximate age at which untreated or vehicle-treated Twitcher mice reach their humane endpoint and is the time point at which neuropathology is most severe. In Twitcher mice, the peripheral nerves are more affected by demyelination compared to the CNS, and therefore histological examination included In addition, since the lack of correction of the PNS by HSCT in patients remains a major unmet need in infants and young children, it will be interesting to examine the effect of rAAVhu68.hGALC administration on the PNS.

[0176] Sections of sciatic nerves were processed for visualization of myelin (dark staining) and globoid cells (light staining) (Figure 12). At PND40, sciatic nerves of vehicle-treated wild-type controls were rich in myelin and generally lacked infiltration of globoid cells. However, in vehicle-treated symptomatic Twitcher mice (twi / twi), severe subtotal demyelination was observed in the sciatic nerve, accompanied by nerve thickening and infiltration of globoid cells. In contrast, myelin was preserved in the sciatic nerves of symptomatic Twitcher mice (twi / twi) administered rAAVhu68.hGALC on PND12, but not to the extent typically observed in age-matched wild-type mice. Fewer globoid cells were also observed in the nerves of rAAVhu68.hGALC-treated Twitcher mice compared to vehicle-treated Twitcher mice. These data correlated with functional endpoints and suggest that the improvement in clinical scores and neuromotor function in symptomatic Twitcher mice administered rAAVhu68.hGALC early in disease progression may be associated with either a reversal or delayed onset of underlying disease neuropathology.

[0177] Finally, on the day of necropsy (PND40), brain, liver, and serum samples were obtained from wild-type and Twitcher mice (twi / twi) to quantify the activity levels of the transgene product, GALC. Quantification of GALC using a fluorophore-based GALC activity assay confirmed that the AAV vector was transduced and expressed functional enzyme after administration of rAAVhu68.hGALC. Wild-type animals were used as controls for this assay. Twitcher heterozygous (twi / +) mice were excluded because they have reduced levels of GALC activity despite having no observable phenotype. As the nervous system is the target tissue for GALC delivery, the brain was examined, and liver and serum were also examined to assess transduction in peripheral organ systems.

[0178] rAAVhu68.hGALC to 2.00 × 10 11Twenty-eight days after ICV administration of a dose of GC, supraphysiological levels of GALC activity were observed in the brain, liver, and serum of Twitcher (twi / twi) mice, which were higher than those observed in the same tissues of vehicle-treated Twitcher (twi / twi) mice and wild-type controls (Figures 13A-C).

[0179] Together, these data demonstrate that administration of rAAVhu68.hGALC to early symptomatic Twitcher mice resulted in supraphysiological levels of GALC activity that correlated with less severe clinical symptoms (neuromotor dysfunction, PNS demyelination, and globoid cell neuropathology).

[0180] Efficacy of bone marrow transplantation in combination with rAAVhu68.hGALC administration In this study, we investigated the potential benefits of a dual therapy of rAAVhu68.hGALC and bone marrow transplantation (BMT). We investigated this combination therapy due to the prominent neuroinflammatory component of Krabbe disease. In theory, hematopoietic stem cell transplantation (HSCT) would be synergistic because it would provide an additional source of GALC enzyme for the CNS (from macrophages / microglial cells derived from transplanted cells and neurons transduced with rAAVhu68.hGALC), while rAAVhu68.hGALC would provide correction to the PNS that is not affected by HSCT. Furthermore, this study explored different combination therapy designs to evaluate whether rAAVhu68.hGALC could be effective in 1) patients who first underwent HSCT through an NBS program and then gene therapy, and / or 2) patients who, if eligible, first underwent gene therapy and then HSCT.

[0181] Table 2 summarizes the combination therapy studies. [Table 5]

[0182] 1.00 × 10 because a better response to combination therapy was predicted 11A GC dose of rAAVhu68.hGALC was utilized (allowing for a lower dose of rAAVhu68.hGALC than that used in rAAVhu68.hGALC monotherapy in previous studies). Efficacy of rAAVhu68.hGALC will be assessed in terms of survival, body weight, and neurological observations (e.g., presence of tremors and abnormal clasping reflexes).

[0183] Survival data for groups 1-3 are shown in Figure 14A,B. To date, the best survival was achieved by treating presymptomatic Twitcher mice (twi / twi) (group 2) with a combination of ICV administration of rAAVhu68.hGALC on PND0 followed by BMT on PND10. Survival was extended to over 300 days in the absence of obvious signs. These mice appeared to be in better physical condition based on the clinical assessment described above (Table 1), exhibiting slight tremors but no significant gait abnormalities and no clasping (analysis still ongoing, data not shown). Mice that received BMT prior to rAAVhu68.hGALC (group 3) are currently still alive (N=3 / 7) but exhibit significant tremors, some gait abnormalities, and weight loss. However, the busulfan conditioning regimen in combination with BMT was toxic in mice younger than 10 days of age, with mice in both groups 2 and 3 showing increased mortality, regardless of the order of the combined therapies, either before or shortly after BMT. Group 4 is injected to mimic the clinically relevant situation in which early symptomatic patients are administered gene therapy followed by BMT.

[0184] Together, these data suggest that combining rAAVhu68.hGALC treatment with subsequent BMT may provide greater efficacy than each treatment alone in a mouse model of Krabbe disease.

[0185] Example 3 - Identification of the minimal effective dose (MED) of rAAVhu68.GALC in the Twitcher mouse model MED testing is performed in Twitcher mice using toxicological vector lots manufactured for non-human primate pharmacology-toxicology studies. The study includes at least two time points and evaluates four dose levels to determine MED, pharmacology, and histopathology (efficacy and safety). Dose levels were selected based on pilot dose-ranging studies and the maximum feasible dose when scaled to humans. Animals are injected ICV at PND12 to mimic early symptomatic patients. A portion of the animals are sacrificed one month post-injection (if vehicle treatment reached a humane endpoint) to obtain pharmacology and efficacy readouts compared to age-matched controls (similar design than in study 3). The remaining mice are followed until a humane endpoint to evaluate the effect of treatment on survival. Personnel performing in-life assessments (body weight, clinical scoring, and rotarod assay) are blinded to the treatment and genotype of the mice.

[0186] The MED will be determined based on analysis of survival benefit, clinical scoring, body weight, neuromotor function using the rotarod assay, GALC activity levels in target organs, and correction of neuropathology in the CNS and PNS (i.e., improved myelination, reduced globoid cell infiltration).

[0187] The study design and study schedule are shown in Tables 3 and 4 below. [Table 6] [Table 7]

[0188] Example 4 - Efficacy of AAV-mediated gene therapy to treat Crabbean fever - Injection of rAAVhu68.CB7.CI.cGALCco.rBG via the cisterna magna Although a useful disease model, the Twitcher mouse has some limitations. The mouse shows only mild CNS involvement, which is distinct from infantile Krabbe patients, who exhibit the more severe CNS characteristic of demyelination of brain atrophy. Furthermore, the small size of the mouse poses experimental challenges. The ICV route must be used in mice, because their small size makes it difficult to reliably inject AAV vectors via the intended clinical route (ICM). Sufficient amounts of serial samples of CSF and blood cannot be obtained from the mice for all of the desired pharmacological assays. Thus, treatment with rAAVhu68.GALC is more efficient than the canine model of Krabbe disease. It can be evaluated in large animals to overcome these technical limitations and confirm the scalability of the therapeutic approach.

[0189] Similar to the Twitcher mouse, the Krabbe mouse is a naturally occurring autosomal recessive disease model resulting from a spontaneous A to C mutation in the GALC gene causing a missense mutation (Y158S). The mutant GALC protein has close to 0% residual enzyme activity, which is similar to the GALC activity levels observed in patients with the infantile form of Krabbe disease. Heterozygous dogs are asymptomatic, while dogs homozygous for the mutation are affected.

[0190] Krabbe disease exhibits a severe phenotype similar to infantile Krabbe disease, as summarized in Table 5. Although the progression of the Krabbe disease phenotype is less well characterized than in Twitcher mice, affected Krabbe disease mice exhibit demyelination and accumulation of globoid cells affecting both the CNS and peripheral nerves. They develop hindlimb weakness, thoracic limb dysmetria, and tremors at approximately 4-6 weeks of age. Similar to infantile Krabbe disease patients, Krabbe disease mice exhibit consistent and rapid neurological deterioration following the onset of symptoms. Ultimately, these symptoms progress to a humane endpoint characterized by severe ataxia, pelvic limb paralysis, wasting, urinary incontinence, and sensory impairment by approximately 15 weeks of age (Fletcher TF & Kurtz HJ (1972) Am J Pathol. 66(2):375-8; Wenger DA (2000) Molec Med Today. 6(11):449-451; Bradbury AM, et al. (2018) Hum Gene Ther. 29(7):785-801). [Table 8]

[0191] The goal of this study is to evaluate the scalability of a therapeutic approach by assessing the efficacy of an AAV vector similar to rAAVhu68.hGALC in a large animal disease model. To accomplish this, we used a naturally occurring canine model of Krabbe disease. Utilizing the model, a vector similar to rAAVhu68.hGALC encoding an engineered canine version of GALC (AAVhu68.CB7.CI.cGALCco.rBG) was administered via the intended clinical route (ICM) (Figure 3). The canine version of GALC was selected as the transgene to limit the risk of an exaggerated immune response to the foreign transgene, but other elements of the vector are the same as rAAVhu68.hGALC (including the ubiquitous CB7 promoter and AAVhu68 capsid).

[0192] The study design is provided in Figure 23. Dogs (N=7 total) were randomly assigned to a 3.00 x 1013 Either a GC dose of GALC-expressing AAV vector (N=4 diseased dogs) or vehicle (artificial CSF, N=2 diseased dogs, N=1 healthy wild-type littermate) was injected ICM at 2-3 weeks of age. The age of the animals was chosen to ensure that the dogs were treated as early as possible, before the onset of behavioral symptoms, since rAAVhu68.hGALC was found to be more effective in pre-symptomatic Twitcher mice compared to symptomatic mice treated at later time points (see Example 2). [Table 9]

[0193] After dosing, animals were monitored daily (cageside observation), weighed weekly, and videotaped biweekly. They also underwent brain MRI and periodic complete physical examinations, neurological examinations, nerve conduction recordings, and BAER recordings. The purpose of these examinations was to evaluate the integrity of the CNS and PNS. Efficacy readouts included brain myelination (assessed by MRI 8 weeks after injection, BAER, and histology at terminal endpoint), peripheral nerve myelination (assessed by NCV and histology at terminal endpoint), neurological examinations, and physical examinations (weight, ambulation, reflexes, proprioception, videotaping of dogs playing in an open area, biweekly). In addition, pharmacology, safety, and vector biodistribution were evaluated, as the injected vector was equivalent to rAAVhu68.hGALC, and the intended clinical ROA (ICM administration) was used. Nerve conduction assessments measured nerve integrity, as an indirect measure of myelin integrity. BAER recordings are similar to NCV, except that they test conduction and myelin integrity in the auditory pathways of the CNS (i.e., brainstem). Safety readouts include periodic blood counts (CBC), serum chemistry, and coagulation on day 0 before infusion, and on days 14, 28, 56, 70, 120, and 180 (± 2 days each). CSF was also processed for lipidomic biomarker analysis and cell counts, when the volume allowed for disease correction (lipidomics, psychosine concentration) and WBC counts (CSF pleocytosis) to be examined as safety readouts. On day 56, dogs were examined by MRI (T1 and T2 weighted) to observe myelination in the CNS. GALC enzyme activity was assessed in both CSF and serum at baseline, at the time points indicated below, to assess the amount of active therapeutic enzyme secreted in the CNS and PNS. Testing days for each assay were selected to minimize animal sedation while collecting complete data at appropriate time points corresponding to the known progression of Krabbe disease in dogs (Figure 15).

[0194] Blood and CSF were collected for safety and biomarker analysis. A comprehensive list of tissues was sampled for histopathology to determine whether administration of rAAVhu68.hGALC reduced demyelination and neuroinflammation. Transduction and expression of rAAVhu68.hGALC was assessed by biodistribution. GALC enzyme activity readout (Figure 24B and Figure 24C) showed that there was rapid enzyme secretion into the CSF in all treated Crabbean mice.

[0195] In presymptomatic Crabbeinu, 3.00 × 10 13 A single cisternal injection of rAAVhu68.cGALC in the GC provided phenotypic correction (Figure 25E), increased survival (Figure 24A), normalization of nerve conduction (Figure 25A-D), normal blood tests, and improved brain MRI (Figure 29A and Figure 29B), demonstrating the scalability of the approach. Brain histology showed improved myelination (Figure 26A) and reduced neuroinflammation (IBA1 staining in cerebellar white matter) (Figure 26B) in rAAVhu68.cGALC crabbeines versus controls. None of the dogs initially administered either the GALC expression vector or vehicle in this study developed adverse events. Crabeines administered rAAVhu68.cGALC showed normal growth (Figure 27) and no toxicity was observed at 6 months based on the absence of CSF pleocytosis (Figure 28A) and histopathological lesions (Figure 28B). Due to progression of Krabbe disease, including severe peresis of the hind limbs, urinary incontinence, head tremors, and ataxia except for gait, vehicle-treated dogs had to be euthanized at weeks 8 and 12. Beyond 45 weeks post-injection, all vector-treated dogs appeared bright, alert, and symptom-free, and all dogs exceeded the maximum average lifespan of Krabbe disease.

[0196] The study schedule and endpoints are presented in Table 6 below. [Table 10-1] [Table 10-2]

[0197] Example 5 - Toxicity studies in non-human primates Toxicity studies were performed using the same vector lot of rAAVhu68.hGALC used in the mouse MED study. NHPs are performed because they better recapitulate human size and CNS anatomy and can be treated using clinical ROA (ICM). The similarity in size, anatomy, and ROA is expected to yield representative vector distribution and transduction profiles, allowing for more accurate assessment of toxicity than is possible in mice or dogs. In addition, more rigorous neurological evaluations can be performed in NHPs than in rodent or canine models, allowing for more sensitive detection of CNS toxicity.

[0198] ICM administration of vector results in immediate vector distribution within the CSF compartment. The dose is scaled by brain mass to provide an approximation of the size of the CSF compartment. Dose conversion is 0.15 g for neonatal mice (Gu Z., et al. (2012) PLoS One.7(7):e41542.), 0.4 g in juvenile-adult mice (Gu Z.,et al.(2012)PLoS One.7(7):e41542.), 90 g in juvenile and adult rhesus macaques (Herndon JG,et al.(1998)Neurobiol Aging.19(3):267-72), 60 g in dogs, 800 g in 4-12 month old infants, and 1300 g in adult humans (Dekaban AS(1978)Ann Neurol.4(4):3 45-56) based on brain mass. Doses for the NHP toxicity study, mouse MED study, and equivalent human doses are shown in Table 7. [Table 11]

[0199] Juvenile rhesus macaques are selected according to the disease target, to be anatomically similar to the proposed Phase 1 / 2 study population. Doses for NHP toxicity studies will reflect doses used in Phase 1 / 2 clinical trials and will be selected taking into account: 1) the results of pharmacology studies, and 2) the maximum feasible dose, taking into account transposition of doses from pharmacology studies to NHPs and humans.

[0200] Therefore, a 180-day GLP-compliant safety study will be conducted in adult rhesus macaques to examine the toxicity of rAAVhu68.CB7.CI.cGALCco.rBG (rAAVhu68.hGALC) following ICM administration. The 180-day evaluation period was chosen to allow sufficient time for the secreted transgene product to reach stable steady-state levels following ICM administration of AAV. Tables 8 and 9 outline the study design. Juvenile rhesus macaques (approximately 1.5 years of age) were administered a total of 4.50 × 10 12 GC or total 1.50 x 10 13 Subjects will receive either GC or GC+ (or vehicle). Dose levels were chosen to be equivalent to the dose evaluated in the MED study when scaled by brain mass (assuming 0.4 g for mice and 90 g for rhesus monkeys). The high dose will be equivalent to the dose evaluated in the Crabeyne model (assuming a brain weight of 60 g). Baseline neurological examinations, clinical pathology (cell counts with differential, clinical chemistry, and coagulation panel), CSF chemistry, and CSF cytology will be performed. After administration of vector (or vehicle), animals will be monitored daily for signs of distress and abnormal behavior.

[0201] Clinical pathology evaluation of blood and CSF and neurological examinations are performed for 30 days after vector or vehicle administration and every 30 days thereafter. At baseline and every 30 days thereafter, anti-AAVhu68 neutralizing antibody (NAb) and cytotoxic T lymphocyte (CTL) responses to AAVhu68 and GALC products are assessed by enzyme-linked immunospot (ELISpot) assay for interferon gamma (IFN-γ).

[0202] At either 90 or 180 days after rAAVhu68.hGALC or vehicle administration, animals are euthanized and tissues are harvested for comprehensive microscopic histopathological examination. Histopathological examination focuses on CNS tissues (brain, spinal cord, and dorsal root ganglia) and liver, as these are the most heavily transduced tissues after ICM administration of rAAVhu68 vectors. In addition, lymphocytes are harvested from the systemic circulation (PBMC), spleen, and CNS-draining lymph nodes to assess the presence of T cells reactive to both capsid and transgene product within these organs at necropsy. Tissues are harvested and stored if any findings warrant further analysis of vector biodistribution. [Table 12] [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4] [Table 13-5]

[0203] Example 6 - Treatment of Krabbe Disease with rAAVhu68.hGALC The FIH trial is a 1 / 2 dose escalation study of a single ICM administration of rAAVhu68.hGALC in pediatric patients with infantile forms of Krabbe disease, caused by homozygous or compound heterozygous mutations in the GALC gene. At least 12 subjects will be enrolled and treated in this FIH trial. These subjects will be followed up for 2 years, followed by a total of 5 years of long-term follow-up (LTFU) after administration, in line with the recommended LTFU for adenoviral vectors as outlined in the draft "FDA Guidance for Industry: Long Term Follow-Up after Administration of Human Gene Therapy Products" (July 2018). The primary objective is to evaluate the safety and tolerability of rAAVhu68.hGALC. Secondary objectives of the study are to evaluate the impact of rAAVhu68.hGALC on disease-related assessments, including survival, age-appropriate neurocognitive measures, and age-appropriate motor and / or linguistic assessments. These endpoints are selected in consultation with disease experts and clinicians and based on observations of disease progression in patients with untreated infantile Krabbe disease.

[0204] Optionally, combination therapy of HSCT and AAV gene therapy may be evaluated.

[0205] FIH is an open-label, multicenter, dose-escalation study of rAAVhu68.hGALC to evaluate safety, tolerability, and exploratory efficacy endpoints in pediatric subjects with infantile forms of Krabbe disease. The dose-escalation phase will evaluate the safety and tolerability of a single ICM administration of two dose levels of rAAVhu68.hGALC with staggered sequential administration to subjects. The rAAVhu68.hGALC dose levels will be determined based on data from GLP NHP toxicity studies and mouse (MED) studies and will consist of a low dose (administered to cohort 1) and a high dose (administered to cohort 2). Both dose levels are expected to confer therapeutic benefit, with the understanding that the higher dose is expected to be advantageous, if tolerated. Sequential evaluation of the low dose followed by the high dose will allow for identification of the maximum tolerated dose (MTD) of the doses tested. Finally, an expansion cohort (cohort 3) will receive rAAVhu68.hGALC at the MTD (Figure 16). The rapid production of GALC enzyme following administration of the vector (as early as one week after treatment) provides an extended therapeutic window.

[0206] Given that infantile Krabbe disease is characterized by a rapid disease course once symptoms appear, with some newborns exhibiting signs of disease at birth, the proposed study design allows for the simultaneous enrollment of subjects 30 days after dosing of the first patient in cohort 1 (low dose) and cohort 2 (high dose) based on the investigator's benefit-risk assessment for that subject. The rationale is that the risk of missing a therapeutic window because a patient has experienced disease progression outweighs the potential benefit of long-term safety follow-up before the next patient is dosed. Such a scenario, in which patients experience substantial disease progression within a matter of weeks, has been cited as a possible cause of poor transplant outcomes observed in EIKD patients identified through the New York NBS (Wasserstein MP, et al. (2016) Genet Med. 18 (12): 1235-1243), highlighting the need to promptly refer patients for treatment.

[0207] An independent safety committee will perform a safety review of all safety data accumulated between cohorts and after full enrollment of the second cohort and will make recommendations regarding further conduct of the trial. The safety review committee will also conduct a review whenever a safety review trigger (SRT) is observed. The 1-month dosing interval between the first and second subject in each cohort will allow for evaluation of AEs indicative of acute immune reactions, immunogenicity or other dose-limiting toxicities, as well as clinical review of any sensory neuropathy that may be consistent with the expected time course of development of sensory neuropathology following transduction of the DRG, which occurs within 2-4 weeks in nonclinical studies.

[0208] Additional subjects are enrolled in an expansion cohort that receives the MTD. Enrollment of these additional subjects does not require a 4-week observation period between subjects (FIG. 16). Optionally, this cohort receives combination therapy with HSCT and rAAVhu68.hGALC.

[0209] All treated subjects will be followed for two years to assess the safety profile and characterize the pharmacokinetic and efficacy properties of rAAVhu68.hGALC. Subjects will be followed for an additional three years (for a total of five years post-dose) during the LTFU period of the study to evaluate long-term clinical outcomes. This is in accordance with “FDA Guidance for Industry: Long Term Follow-Up after Administration of This is in accordance with the “Standards for Clinical Trials and Human Gene Therapy Products” (July 2018). [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5] [Table 14-6]

[0210] statistical methods No statistical comparisons were planned for safety evaluation. All results are descriptive only. Data will be listed and summary tables will be prepared.

[0211] Statistical comparisons will be performed for secondary and exploratory endpoints. Measurements at each time point will be compared to baseline values ​​for each subject, as well as data from age-matched healthy controls and natural history data from Krabbe disease patients with comparable cohort characteristics, when available for each endpoint.

[0212] All data will be presented in the subject data listing. Categorical variables will be summarized using frequencies and percentages and continuous variables will be summarized using descriptive statistics (number of non-missed observations, mean, standard deviation, median, minimum and maximum). Graphical representations will be presented as appropriate.

[0213] Basis of the group Study population - pediatric patients FIH focuses on infantile subjects with onset of symptoms before 9 months of age, representing a population with the highest unmet need, as HSCT is not indicated for these patients. Moreover, these patients have a highly destructive disease course with rapid and highly predictable decline that is homogenous in the presentation of both motor and cognitive disorders (Bascou N., et al. (2018) Orphanet J Rare Dis. 13(1):126). In fact, patients presenting before 9 months of age have a disease course similar to early infantile Krabbe disease, progressing to rapid and severe cognitive and motor impairments and failure to acquire any functional skills following the initial signs and symptoms of the disease. The majority of these patients are expected to die within the first few years of life (2-year survival rates range from 26 to 50%) (Duffner PK, et al. (2011) Pediatr Neurol. 45(3):141-8, Beltran-Quintero ML, et al. (2019) Orphanet J Rare Dis. 14(1):46). 9-12 The phenotype of infants with onset at 9-12 months of age is more diverse, some of whom show a severe early infantile Krabbe disease phenotype, while others have milder disease symptoms with (near) normal cognition and significantly better adaptive and fine motor skills, making it difficult to predict the phenotype of newly diagnosed patients with onset at 9-12 months of age (Bascou N., et al. (2018) Orphanet J Rare Dis. 13(1):126). As a result, the population is restricted to subjects with symptom onset <9 months of age, whose predictable and rapid decline within a reasonable follow-up period supports robust study design and evaluation of functional outcomes. In this group, treatment is expected to stabilize disease progression, prevent loss of acquired developmental and motor milestones and other skills, prolong survival, and delay or prevent the onset of seizures.

[0214] Despite the shared underlying pathophysiology, the adult Krabbe phenotype and disease course are remarkably milder than the devastating infantile Krabbe disease form, so the demonstration of disease stabilization in adults does not provide a reason to trust the treatment of infantile Krabbe disease. Importantly, the onset of adult Krabbe disease is highly variable, with slower and more variable progression, and decline occurring over years to decades (Jardim LB, et al. (1999) Arch Neurol. 56 (8): 1014-7, Debs R., et al. (2013) J Inherit Metab Dis. 36 (5): 859-68). It would be very difficult to design clinical trials that could clearly demonstrate the efficacy of investigational therapies in the long-term natural history. The fact that HSCT offers a therapeutic option that can stabilize or ameliorate disease manifestations is another important consideration (Sharp ME, et al. (2013) JIMD Rep. 10: 57-9, Laule C., et al. (2018) Journal of Neuroimaging. 28 (3): 252-255). Finally, NBS has not been widely adopted in the United States and is unavailable in Europe, and the vague and nonspecific clinical symptoms mean that adult Krabbe disease continues to be underdiagnosed, and therefore access to such patients is very rare (Wasserstein MP, et al. (2016) Genet Med. 18 (12): 1235-1243).

[0215] Study Population - Exclusion of Subjects with Serious Illnesses Given the nature of Krabbe disease with CNS damage that is believed to be largely irreversible, and the extremely rapid disease progression in the infant population, rAAVhu68.hGALC is expected to offer the greatest potential for benefit in patients with no disease or mild-to-moderate disease who do not exhibit signs inherently associated with later stages of the disease, including deafness, blindness, and severe weakness with loss of primitive reflexes (Escolar ML, et al. (2006) Pediatrics. 118(3):e879-89). Furthermore, abnormal pupillary reflexes, jerky eye movements, or visual tracking deficits are more common in very progressive disease than in patients with moderate signs and symptoms, and are typically not observed in the early stages of the disease (Escolar ML, et al. (2006) Pediatrics. 118(3):e879-89). Thus, evidence of one or more of these signs would be considered an indication of progressive disease and would exclude patients from clinical trials. Due to their severe disability, these patients are unlikely to derive substantial benefit from therapy beyond disease stabilization at a low level of clinical function, have an unfavorable benefit / risk profile, and exhibit floor effects for a variety of clinical and instrumental assessments that preclude evaluation of the efficacy of rAAVhu68.hGALC. This population also represents a high risk for non-treatment-related safety concerns due to the progressive nature of their disease sequelae, and was therefore excluded from the study.

[0216] Patients with clinical seizures will not be excluded from the trial unless, in the investigator's opinion, the child has other signs of progressive disease and is unlikely to benefit from treatment. This is because 1) the seizures are not uniquely associated with progressive disease and 2) seizures are not a trial endpoint. , because excluding patients with seizures may bias studies towards populations less likely to experience seizures.

[0217] Study Population - Inclusion of Pre-symptomatic Subjects Symptomatic infantile Krabbe disease patients are excluded from the dose escalation portion of the trial (cohort 1 and cohort 2), where only rAAVhu68.hGALC is evaluated. For these patients, at least in the United States, HSCT is considered a treatment option and the treatment of choice, even if it only delays disease progression. The general view of US KOLs is that it would be considered unethical to test unproven investigational therapies in this population, since an overly narrow therapeutic window would effectively deprive patients of access to a treatment that has been shown to provide at least partial benefit (i.e., if gene therapy proves unsuccessful, there will likely not be time to be "rescued" with HSCT). Thus, rAAVhu68.hGALC should be reserved for patients with the clearest unmet need (i.e., patients with infantile Krabbe disease who have signs and symptoms that make them ineligible for HSCT).

[0218] Our preclinical studies confirmed superior efficacy of gene therapy combined with HSCT over either approach alone, therefore, the combination of HSCT and rAAVhu68.hGALC will be evaluated as an expansion cohort of the FIH trial (cohort 3). This cohort will evaluate the safety and efficacy of rAAVhu68.hGALC in the context of HSCT and will progress only if preclinical data provide reason to trust improved efficacy over HSCT alone, so both symptomatic and presymptomatic patients who meet the criteria outlined herein will be eligible for enrollment.

[0219] Study population - justification for lower age limit Given that symptom onset can occur perinatally or even in utero, treatment is administered as early as possible to maximize potential benefit. Therefore, the minimum age for this study was chosen as 1 month of age at the time of dosing because current consensus guidelines recommend HSCT before 1 month of age in eligible patients (Kwon JM, et al. (2018) Orphanet J Rare Dis. 13(1):30). Requiring subjects to be 1 month or older allows subjects and families to consider other forms of standard treatment before qualifying for this trial.

[0220] Another consideration in selecting the lower age limit is ensuring that the treatment (specifically, the ICM procedure) can be performed safely in such young patients. After careful review of imaging scans from infants as young as 1 and 2 weeks of age, expert interventional radiologists at the University of Pennsylvania confirmed that there were no specific anatomical concerns with performing CT-guided ICM administration in infants as young as 1 month of age, as long as the rationale for the treatment was supported.

[0221] Endpoints In addition to measuring safety and tolerability as the primary endpoints, secondary and exploratory pharmacodynamic and efficacy endpoints were selected for this study based on the current literature and in consultation with leading clinicians specializing in Krabbe disease. These endpoints are expected to demonstrate meaningful functional and clinical outcomes in this population. Endpoints will be measured at 30 days, 90 days, and 6 months, except for those requiring sedation and / or lumbar puncture, and then every 6 months during the 2-year short-term follow-up period, as shown in Figures 18A-C. In the long-term follow-up phase, the frequency of measurements will be reduced to once every 12 months. These time points were selected to facilitate a complete evaluation of the safety and tolerability of rAAVhu68.hGALC. Early time points and 6-month intervals were also selected given the rapid disease progression in untreated infant Krabbe patients. This will allow for a full evaluation of pharmacodynamic and clinical efficacy measures in treated subjects over a follow-up period during which untreated comparator data exist. Following administration of rAAVhu68.hGALC, subjects will continue to be monitored for safety and efficacy for a total of 5 years in accordance with the draft “FDA Guidance for Industry: Long Term Follow-Up After Administration of Human Gene Therapy Products” (July 2018).

[0222] Disease progression and clinical outcomes Considering the rapid and uniform rate of disease progression in the infant population (Duffner PK,et al.(2011)Pediatr Neurol..45(3):141-8, Bascou N.,et al.(2018)Orphanet J Rare Dis.13(1):126), a 2-year follow-up for assessment of primary outcomes is considered sufficient to evaluate the impact of rAAVhu68.hGALC over time. Furthermore, LTFU up to 5 years after treatment would be highly informative to assess long-term outcomes and whether the treatment is effective in prolonging survival and stabilizing patients with a functional level similar to or better than outcomes observed in presymptomatic patients after HSCT.

[0223] Administration of rAAVhu68.hGALC stabilizes disease progression as measured by survival rate, prevents loss of developmental and motor milestones, potentially aids in the acquisition of new milestones, and reduces onset and frequency of seizures. Death typically occurs within the first 3 years of life for most patients diagnosed with early infantile Krabbe disease, with median mortality extending to 5 years in the later infantile population, incorporating patients with symptom onset from 7-12 months (Duffner PK,et al.(2012)Pediatr Neurol.46(5):298-306). By limiting selection criteria to patients with onset before 9 months of age, the population has a more severe infantile-like phenotype and disease course (Bascou N.,et al.(2018)Orphanet J Rare Dis.13(1):126). Given the rapid decline seen in untreated infantile Krabbe disease cases, treatment with rAAVhu68.hGALC increases life expectancy during the follow-up period. Development of motor milestones depends on the age and stage of disease at the time of enrollment of the subject (Bascou N.,et al.(2018)Orphanet J Rare Dis.13(1):126, Beltran-Quintero ML,et al.(2019)Orphanet J Rare Dis.14(1):46). Given the severity of the disease in the target population, subjects may have achieved motor skills by enrollment, developed other motor milestones and subsequently lost them, or may not yet show signs of motor milestone development. Evaluation tracks age-at-achievement and age-at-loss for all milestones. Motor milestone achievement was defined for six gross milestones based on WHO criteria and is outlined in Table 11 . [Table 15]

[0224] Considering that subjects with infantile Krabbe disease may develop symptoms within weeks or months of birth and acquisition of the first WHO motor milestone (sitting unsupported) typically does not appear before 4 months of age (median age: 5.9 months), this endpoint may lack sensitivity for assessing the degree of therapeutic benefit, especially in subjects who had more obvious symptoms at the time of treatment. For this reason, assessment of age-appropriate developmental milestones that may be applicable to infants is also included (Sharp ME, et al. (2013) JIMD Rep. 10:57-9). One drawback is that the published tool is intended for use by clinicians and parents and organizes skills around the typical age of milestone acquisition without reference to normal ranges. However, the data may be informative for summarizing the retention, gain, or loss of developmental milestones over time compared to typical acquisition times in untreated children with infantile Krabbe disease or neurotypical children.

[0225] Although seizures are not a presenting symptom in the infant population, approximately 30-60% of infantile patients ultimately develop seizures in the later stages of the disease (Duffner PK, et al. (2011) Pediatr Neurol. 45(3):141-8). The delayed onset of seizure activity allows us to determine whether treatment with rAAVhu68.hGALC can prevent or delay the onset of seizures or reduce the frequency of seizure events in this population. Parents are asked to keep a seizure diary, tracking the onset, frequency, duration, and type of seizures. These entries are discussed and interpreted with a clinician at each visit.

[0226] As exploratory measures, clinical scales will be used to quantify the impact of rAAVhu68.hGALC on development and change in adaptive behavior, cognition, language, motor function, and health-related quality of life. Each proposed measure has been used in either Krabbe or related populations.

[0227] The scales and associated domains are briefly described below. Bayley Scales of Infant Development (3rd Edition): Evaluates infant and toddler development across five domains: cognition, language, motor, social-emotional, and adaptive behavior. All domains are assessed in the trial. Vineland Adaptive Behavior Scales (3rd edition): assesses adaptive behavior from birth to adulthood (ages 0-90) across five domains: communication, daily living skills, socialization, motor skills, and maladaptive behaviors. Improvements from the 2nd edition to the 3rd edition include questions that allow for a better understanding of developmental disorders. Peabody Developmental Motor Scales-2nd Edition: Measures interrelated motor function from birth to age 5 years. Assessment focuses on six areas: reflexes, poise, locomotion, manipulation, grasping, and visual-motor integration. · Infant Quality of Life Questionnaire (ITQOL): a parent-reported health-related quality of life measure designed for infants aged 2 months to toddlers aged 5 years. · Mullen Scales of Early Learning: assesses language, motor, and perceptual abilities in infants up to 68 months of age.

[0228] Disease Biomarkers To evaluate the effect of rAAVhu68.hGALC on disease pathology, we measure changes in myelination, functional outcomes related to myelination, and potential disease biomarkers. Central and peripheral demyelination, a major feature of the disease, is slowed or halted by administration of rAAVhu68.hGALC. Central demyelination is tracked by diffusion tensor magnetic resonance imaging (DT-MRI) anisotropy measurements of white matter regions and fiber tracking of corticospinal motor tracts, and these changes are indicative of disease status and progression (McGraw P.,et al.(2005)Radiology.236(1):221-30,Escolar ML,et al.(2009)AJNR Am J Neuroradiol.30(5):1017-21). Peripheral demyelination is measured indirectly via nerve conduction velocity (NCV) testing of motor (deep peroneal, tibial, and ulnar) and sensory (sural and median) nerves, monitoring fluctuations indicative of changes in biologically active myelin (i.e., F-wave and distal latency, amplitude, or presence or absence of response).

[0229] According to one study, the onset of visual impairment is common in early infantile Krabbe disease, with 61.2% of that population developing vision loss at some point during the disease (Duffner PK, et al. (2011) Pediatr Neurol. 45(3):141-8). Like seizures, vision loss is not a common symptom. This provides an opportunity to evaluate the ability of rAAVhu68.hGALC to delay or prevent vision loss in subjects who have not developed significant vision loss prior to treatment. Thus, measurements of visual evoked potentials (VEPs) are used to objectively measure responses to visual stimuli as an indicator of impairment or loss of central vision. Hearing loss is also common during disease progression, with early signs of hearing abnormalities measured via brainstem auditory evoked response (BAER) testing.

[0230] GALC is responsible for the hydrolysis of psychosine. The deficiency of GALC in Krabbe disease is It results in the accumulation of psychosine both centrally and peripherally. Increased psychosine levels have been proposed as an indicator of Krabbe disease (Escolar ML, et al. (2017) Mol Genet Metab. 121 (3): 271-278). Although evidence exists to support its use in detecting early severe cases of infantile Krabbe disease, later disease may also result in decreased psychosine levels, making the interpretation of fluctuations in psychosine levels over time after treatment difficult. Thus, evidence of decreased psychosine levels alone is not sufficient evidence of treatment efficacy unless it is accompanied by stabilization of clinical disease.

[0231] (Sequence Listing Free Text) The following information is a numeric identifier <223> Below we provide an array containing free text. [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4]

[0232] All documents cited herein are incorporated herein by reference, and the text of the sequences and sequence listing (labeled "18-8584PCT_ST25.txt") filed herewith are also incorporated herein by reference. U.S. Provisional Patent Application No. 62 / 810,708, filed February 26, 2019, U.S. Provisional Patent Application No. 62 / 817,482, filed March 12, 2019, U.S. Provisional Patent Application No. 62 / 877,707, filed July 23, 2019, and U.S. Provisional Patent Application No. 62 / 916,652, filed October 17, 2019, are incorporated herein by reference in their entirety, together with their sequence listings. The present invention has been described with reference to certain embodiments, but it will be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to be within the scope of the appended claims.

Claims

1. 1. A composition comprising a recombinant adeno-associated virus (rAAV), the rAAV comprising: (a) an AAV capsid that targets cells of the central nervous system; (b)(ii) a vector genome comprising (i) a signal peptide and a coding sequence for galactosylceramidase, the coding sequence being under the control of regulatory sequences that encode a mature human galactosylceramidase protein having at least the amino acid sequence of aa 43 to 685 of SEQ ID NO: 10, and directing expression of said protein, and (ii) an AAV inverted terminal end sequence necessary for packaging of said vector genome into said AAV capsid, wherein said vector genome is packaged into said AAV capsid.

2. The composition of claim 1 , wherein the AAV capsid is an AAVhu68 capsid.

3. The composition of claim 1 or claim 2, wherein the coding sequence encodes the full-length human galactosylceramidase signal peptide of SEQ ID NO: 10 and the mature human galactosylceramidase protein (amino acids 1 to 685).

4. 4. The composition of any one of claims 1 to 3, wherein the coding sequence has a coding sequence for an exogenous peptide and a nucleic acid sequence of nucleotides 127 to 2055 of SEQ ID NO:9 or a sequence that is 95% to 99.9% identical thereto, or a nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO:9 or a sequence that is 95% to 99.9% identical thereto.

5. The composition of claim 1 or claim 2, wherein the coding sequence encodes the mature human galactosylceramidase protein of SEQ ID NO: 10 (amino acids 43-685) and an exogenous signal peptide suitable for human cells of the central nervous system.

6. The composition of any one of claims 1 to 5, wherein the regulatory sequences include a beta-actin promoter, an intron, and a rabbit globin polyA.

7. The composition of any one of claims 1 to 6, wherein the regulatory sequence comprises SEQ ID NO:

13.

8. The composition of any one of claims 1 to 7, wherein the regulatory sequence comprises SEQ ID NO:

15.

9. The composition of any one of claims 1 to 8, wherein the regulatory sequence comprises SEQ ID NO:

16.

10. 3. The composition of claim 1 or 2, wherein the vector genome comprises CB7.CI.hGALC.RBG having the sequence of nt 198 to 4168 of SEQ ID NO:

19.

11. The composition of any one of claims 1 to 10, wherein the vector genome further comprises the 5' ITR of AAV2, the coding sequence, and the regulatory sequence, and the 3' ITR of AAV2.

12. The composition of any one of claims 1 to 11, wherein the composition further comprises an aqueous liquid suitable for intrathecal delivery to a patient.

13. The composition according to any one of claims 1 to 12, wherein the composition comprises an artificial cerebrospinal fluid and a surfactant.

14. The composition is formulated for intrathecal delivery and contains 1.4×10 １３ ~4×10 １４ In front of the GC The composition according to any one of claims 1 to 13, comprising the rAAV.

15. The composition is formulated for intracisternal delivery and contains 1.4×10 １３ ~4×10 １４ The composition according to any one of claims 1 to 13, comprising the rAAV of GC.

16. 1. A recombinant adeno-associated virus comprising: (a) an AAV capsid that targets a cell of the central nervous system; and (b) a vector genome, the vector genome comprising: (i) a signal peptide, and a coding sequence for galactosylceramidase, the coding sequence being under the control of a regulatory sequence that directs expression of the signal peptide and the mature human galactosylceramidase protein, the coding sequence being under the control of a regulatory sequence that directs expression of the signal peptide and the mature human galactosylceramidase protein, and (ii) an AAV inverted terminal sequence necessary for packaging the vector genome into the AAV capsid, wherein the vector genome is packaged into the AAV capsid.

17. The rAAV of claim 16, wherein the AAV capsid is an AAVhu68 capsid.

18. 18. The rAAV of claim 16 or 17, wherein the coding sequence has a coding sequence for an exogenous signal peptide and nucleotides 127-2055 of SEQ ID NO:9, or a sequence that is 95% to 99.9% identical thereto, or the nucleotide sequence of nucleotides 1-2055 of SEQ ID NO:9, or a sequence that is 95% to 99.9% identical thereto.

19. 19. The rAAV of any one of claims 16 to 18, wherein the coding sequence encodes the mature protein from amino acids 43 to 685 (SEQ ID NO: 10) and an exogenous signal peptide.

20. The rAAV of any one of claims 16 to 19, wherein the regulatory sequences include a chicken beta actin promoter, an intron, and a rabbit globin polyA.

21. The rAAV of any one of claims 16 to 20, wherein the regulatory sequence comprises SEQ ID NO:

13.

22. The rAAV of any one of claims 16 to 21, wherein the regulatory sequence comprises SEQ ID NO:

15.

23. The rAAV of any one of claims 16 to 22, wherein the regulatory sequence comprises SEQ ID NO:

16.

24. 24. The rAAV of any one of claims 16 to 23, wherein the vector genome comprises CB7.CI.hGALC.RBG having the sequence of nt 198 to 4168 of SEQ ID NO:

19.

25. The rAAV of any one of claims 16 to 24, wherein the vector genome comprises the 5' ITR, the coding sequence, and the regulatory sequence of AAV2, and the 3' ITR of AAV2.

26. A recombinant adeno-associated virus comprising an AAVhu68 capsid and a CB7.CI.hGALC.RBG vector genome.

27. A composition comprising a stock of the recombinant adeno-associated virus (rAAV) of any one of claims 16 to 26, the composition being useful for the treatment of Krabbe disease.

28. Use of a composition comprising a stock of recombinant adeno-associated virus (rAAV) according to any one of claims 16 to 26 in the preparation of a medicament.

29. 29. The composition of claim 27 or the use of claim 28, wherein the composition is useful for treating peripheral nerve dysfunction and / or for treating Krabbe's disease.

30. 29. The composition of claim 27 or the use of claim 28, wherein the rAAV is capable of being administered as a combination therapy with hematopoietic stem cell therapy or bone marrow transplantation.

31. 29. The composition of claim 27 or the use of claim 28, wherein the rAAV can be administered as a combination therapy with substrate synthesis inhibition therapy.

32. A plasmid comprising a coding sequence for galactosylceramidase that encodes a signal peptide and a mature human galactosylceramidase protein having the amino acid sequence of amino acids 43 to 685 of SEQ ID NO:

10.

33. 33. The plasmid of claim 32, wherein the coding sequence has the nucleic acid sequence of SEQ ID NO:9, or a sequence that is 95% to 99.9% identical thereto.

34. 1. A method for treating Krabbe disease, comprising administering to a patient in need thereof a composition comprising a stock of recombinant adeno-associated virus (rAAV), wherein the rAAV comprises: (a) an AAV capsid that targets cells of the central nervous system; and (b) a vector genome comprising a signal peptide and a coding sequence for galactosylceramidase, the coding sequence being under the control of regulatory sequences that direct expression of a mature human galactosylceramidase protein having an amino acid sequence of amino acids 43-685 of SEQ ID NO:10, wherein the vector genome further comprises AAV inverted terminal end sequences necessary for packaging the vector genome into the AAV capsid, and wherein the vector genome is packaged into the AAV capsid.

35. 1. A method for correcting peripheral nerve dysfunction causing respiratory failure and loss of motor function caused by Krabbe disease, comprising administering to a patient a composition comprising a stock of recombinant adeno-associated virus (rAAV), said rAAV comprising: (a) an AAV capsid that targets cells of the central nervous system; and (b) a vector genome comprising a signal peptide and a coding sequence for galactosylceramidase, said coding sequence being under the control of regulatory sequences that encode a mature human galactosylceramidase protein having the amino acid sequence of amino acids 43-685 of SEQ ID NO:10, said vector genome further comprising AAV inverted terminal end sequences necessary for packaging of said vector genome into said AAV capsid, and said vector genome being packaged into said AAV capsid.

36. 1. A method of delaying the onset or frequency of attacks caused by Krabbe disease, comprising administering to a patient a composition comprising a stock of recombinant adeno-associated virus (rAAV), said rAAV comprising: (a) an AAV capsid that targets cells of the central nervous system; and (b) a vector genome comprising a signal peptide and a coding sequence for galactosylceramidase, said coding sequence being under the control of regulatory sequences that encode a mature human galactosylceramidase protein having an amino acid sequence of amino acids 43-685 of SEQ ID NO: 10, said vector genome further comprising AAV inverted terminal end sequences necessary for packaging of said vector genome into said AAV capsid, and said vector genome being packaged into said AAV capsid. 。

37. The method of any one of claims 34 to 36, wherein the patient has early infantile Krabbe disease (EIKD).

38. The method of any one of claims 34 to 36, wherein the patient has late infantile Krabbe disease (LIKD).

39. The method of any one of claims 34 to 36, wherein the patient has Juvenile Krabbe Disease (JKD).

40. 37. The method of any one of claims 34 to 36, wherein the patient has Juvenile / Adult Krabbe Disease.

41. The method of any one of claims 34 to 40, wherein the AAV capsid is an AAVhu68 capsid.

42. 42. The method of any one of claims 34 to 41, wherein the coding sequence has the coding sequence of an exogenous peptide from nucleotides 127 to 2055 of SEQ ID NO:9 or a sequence that is 95% to 99.9% identical thereto, or has the nucleotide sequence from nucleotides 1 to 2055 of SEQ ID NO:9 or a sequence that is 95% to 99.9% identical thereto.

43. The method according to any one of claims 34 to 41, wherein the coding sequence encodes the mature human galactosylceramidase protein of amino acids 43 to 685 of SEQ ID NO: 10 and an exogenous signal peptide.

44. The method of any one of claims 34 to 43, wherein the regulatory sequences comprise a beta-actin promoter, an intron, and rabbit globin polyA.

45. The method of any one of claims 34-44, wherein the rAAV is administered as a combination therapy with hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation.

46. 46. ​​The method of claim 45, wherein the HSCT or bone marrow transplant is performed prior to administration of the rAAV vaccine.

47. The method of any one of claims 34 to 46, wherein the rAAV is administered as a combination therapy with substrate synthesis inhibition therapy.

48. The composition of any one of claims 1 to 15, wherein the composition is formulated for intrathecal, intraventricular, or intraparenchymal administration.

49. 48. The method of any one of claims 34-47, wherein the rAAV is delivered via intrathecal, intracerebroventricular, or intraparenchymal administration.

50. 16. The composition of any one of claims 1 to 15, wherein the composition is formulated for administration as a single dose via computed tomography (CT)-guided suboccipital injection into the cisterna magna (intracisternal).

51. The method of any one of claims 34 to 47, wherein the composition is administered as a single dose via computed tomography (CT)-guided (intracisternal) suboccipital injection into the cisterna magna. Law.

52. The composition is 1×10 １０ GC / g brain mass ~5×10 １１ 15. The composition of any one of claims 1 to 14, wherein the rAAV is formulated for intrathecal administration at a dose of GC / g brain mass.

53. The composition comprises 1×10 １０ GC / g brain mass ~5×10 １１ 48. The method of any one of claims 34 to 47, administered intrathecally at a dose of GC / g brain mass.

54. The composition is 1.4×10 １３ ~4×10 １４ The composition of any one of claims 1 to 13, formulated for administration of a GC dose of the rAAV.

55. The composition is administered to a human patient and １３ ~4×10 １４ 48. The method of any one of claims 34 to 47, wherein a GC dose of the rAAV is administered.

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