Compositions useful for treating Krabbe disease

Abstract

A pharmaceutical composition formulated for delivery of a recombinant adeno-associated virus (rAAV) vector is provided, the pharmaceutical composition comprising an AAV capsid and a vector genome having a sequence encoding human galactosylceramidase (GALC). Also provided are methods and uses of the pharmaceutical composition comprising rAAV for treating Krabbe disease.

Description

[Background technology]

[0001] Adeno-associated viruses (AAVs), members of the Parvoviridae family, are small, non-enveloped, icosahedral viruses 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 consists of four duplicate genes encoding the rep protein necessary for the AAV life cycle, while cap contains the duplicate nucleotide sequences VP1, VP2, and VP3 of the capsid protein, which self-assemble to form an icosahedral symmetrical capsid.

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

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

[0004] Several different AAV9 variants have been manipulated to detarget or target different tissues. 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 See (2011). The development of AAV9 variants for gene delivery across the blood-brain barrier has also been reported. For example, see BEDeverman et al, Nature Biotech, Vol.34, No.2, pp. 204-211 (published online 1 Feb 2016) and Caltech press release A. Weatherston, www.neurology-central.com / 2016 / 02 / 10 / successful-delivery-of-genes-through-the-blood-brain-barrier / , accessed See 10 / 05 / 2016. Also see WO2016 / 0492301 and US8,734,809.

[0005] AAVhu68 was identified after amplification of a naturally occurring capsid gene, and recently, a new A It was identified as an AV capsid. See also WO2018 / 160582. This AAV, like AAV9, is within lineage group F.

[0006] Krabbe disease (globoid cell leukodystrophy, GLD) is an autosomal recessive lysosomal storage disorder (LSD) caused by mutations in the gene encoding the hydrolytic enzyme 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 myelin sheaths. In Krabbe disease, GALC deficiency leads to the toxic accumulation of psychosine (but not galactosylceramide) within lysosomes (Svennerholm). (et al., 1980). Psychosine accumulation is particularly toxic to myelin-producing oligodendrocytes in the central nervous system (CNS) and Schwann cells in the posterior nervous system (PNS), leading to 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 the death of oligodendrocytes that 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 delivered via umbilical cord blood transplantation (UCBT), allogeneic peripheral blood stem cells, or allogeneic bone marrow. Treatment of patients with infantile Krabbe disease with HSCT has been only slightly successful, and symptoms typically appear before the first birthday. When performed after the onset of apparent symptoms in infantile Krabbe disease, HSCT provides only minimal neurological improvement and substantially no improvement in survival (Escolar ML, et al. (2005) N Engl J Med. 352(20):2069-81). HSCT may be effective when performed in pre-symptomatic patients, but even then, motor outcomes are poor (Escolar ML, et al. (2005) N Engl J 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 rates and functional outcomes compared to infants who received transplants later (Allewelt H., et al. (2018) Biol Blood Marrow Transplant. 24(11):2233-2238). Pre-symptomatic transplantation has been reported to result in significantly better outcomes compared to untreated or treated infantile Krabbe disease patients after the onset of symptoms, accompanied by 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 treated before the onset of symptoms remain well below average in height and weight, and have progressive gross motor delay ranging from mild seizures 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 develop acquired microcephaly and require gastrostomy. Patients also have the need for this, as well as residual impairments including dysarthria (Duffner PK, et al. (2009) Genet Med. 11(6):450-4). Furthermore, HSCT appears to affect only the pathology of CNS-specific diseases. Clinical features associated with PNS pathology, such as peripheral neuropathy, remain unaffected by HSCT. These results highlight the limitations of HSCT, particularly in early-onset forms, where the rapid progression of the disease outpaces the time required for hematopoietic stem cells to be transplanted, migrate to the CNS, differentiate, and for therapeutic effects to be achieved through GALC secretion and cross-correction (i.e., the process by which enzymes secreted by corrective cells are taken up by GALC-deficient cells).

[0008] In this technological field, there is still a need for improved treatments for patients with Krabbe disease. [Overview of the Initiative]

[0009] These and other aspects of the present invention will become apparent from the following detailed description of the invention.

[0010] In one embodiment, a pharmaceutical composition is provided comprising a stock of recombinant AAV (rAAV) having an AAV capsid and a packaged vector genome. The vector genome comprises (a) a 5' inverted terminal repeat (ITR), (b) a CB7 promoter, (c) an intron, (d) a galactosylceramidase (GALC) coding sequence containing nucleotides 1-2055 of SEQ ID NO: 9 or a sequence at least 95% identical to that which codes for amino acids 1-685 of SEQ ID NO: 10, (e) poly(A), and (f) a 3'ITR. In a particular embodiment, the composition comprises about 1.7 × 10⁻⁶ 10 Genome copy (GC) / g: Brain mass ~ approximately 5.0 × 10⁻⁶ 11 It is formulated for administration of GC / g brain mass doses. In certain embodiments, the AAV capsid is the AAVhu68 capsid. In further embodiments, the vector genome contains nucleotides 198-4168 of SEQ ID NO: 19.

[0011] In one embodiment, a method for treating Krabbe disease in a patient requiring treatment of Krabbe disease, the method comprising intracisternal magna (ICM) administration of the pharmaceutical composition described herein. In certain embodiments, the method comprises hematopoietic stem cell transplantation before or after administration of the pharmaceutical composition. Hematopoietic stem cell transplantation may enable the patient to receive a reduced dose of rAAV.

[0012] One embodiment provides a method for increasing the expression and enzyme activity of GALC in the serum and / or cerebrospinal fluid (CSF) of a patient having Krabbe disease, comprising administering to the patient a pharmaceutical composition described herein.

[0013] In one embodiment, a method for reducing neuroinflammation in the peripheral nerves of a patient with Krabbe disease comprises administering to the patient a pharmaceutical composition described herein.

[0014] One embodiment provides a method for increasing the expression and activity of GALC in cortical and / or hippocampal neurons of a patient having Krabbe disease, comprising administering to the patient a pharmaceutical composition described herein.

[0015] In one embodiment, a pharmaceutical composition is provided for use in the treatment of patients with Krabbe disease. In a particular embodiment, the treatment involves i) increasing the expression and enzyme activity of GALC in serum and / or cerebrospinal fluid (CSF), ii) increasing the expression and activity of GALC in neurons of the cortex and / or hippocampus, and / or iii) increasing psychosine in serum and / or cerebrospinal fluid (CSF).

[0016] In one embodiment, the use of the pharmaceutical compositions described herein for treating Krabbe disease in a patient requiring treatment for Krabbe disease is provided, followed optionally by a bone marrow transplant. [Brief explanation of the drawing]

[0017] [Figure 1] This provides alignment of the capsid sequences for AAV9 (SEQ ID NO: 4) and AAVhu68 (SEQ ID NO: 2). The two amino acids that differ between the 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. [Figure 2] A schematic diagram of the CB7.CI.hGALC.rBG vector genome is shown. The linear map shows the vector genome, designed to express human GALC under the control of the ubiquitous CB7 promoter. CB7 is a hybrid between the CMV IE enhancer and the chicken β-actin (CB) promoter. Abbreviations: CMV IE: Itomegalovirus initial stage, GALC: Galkutosylceramidase, ITR: Inverted terminal sequence, PolyA: Polyadenylation, rBG: Rabbit β-globin. [Figure 3]This shows the vector map of pENN AAV.CB7.CI.RBG(p1044) with the manipulated cGALC gene (cGALCco) inserted. [Figure 4] This shows the linear vector map of the transplasmid pAAV2 / hu68.KanR(p0068). Abbreviations: AAV2: adeno-associated virus serotype 2, AAVhu68: adeno-associated virus serotype hu68, bp: base pair, Cap: capsid, KanR: kanamycin resistance, Ori: origin of replication, Rep: replicase. [Figure 5A-5B] The adenovirus helper plasmid pAdDeltaF6(KanR) is shown. (Figure 5A) Induction of the helper plasmid pAdΔF6 from the parent plasmid pBHG10 via intermediates pAdΔF1 and pAdΔF5. (Figure 5B) The ampicillin resistance gene of pAdΔF6 was replaced with the kanamycin resistance gene to generate pAdΔF6(Kan). [Figure 6A] This describes the progression of the neuropathological and behavioral phenotype in Twitcher mice (twi / twi). The mice exhibit accumulation of cytotoxic psychosine, followed by infiltration of the PNS and CNS white matter by phagocytic globoid cells. After an initial period of myelination, demyelination is observed to a lesser extent in the PNS, followed by the CNS, and is attributed to the death of myelin-forming Schwann cells and oligodendrocytes, respectively. The behavioral phenotype appears around PND20, consisting of tremors, convulsions, hindlimb weakness, followed by paralysis and weight loss, and requires euthanasia around PND40. (Excerpted from Nicaise AM, et al. (2016) J Neurosci Res. 94(11):1049-61). Abbreviations: CNS: Central Nervous System, PND: Days After Birth, PNS: Peripheral Nervous System, twi: Twitcher loss-of-function allele. [Figure 6B] This document presents a study design for evaluating AAV.CB7.cGALCco.rBG gene therapy using a Twitcher mouse model. [Figure 7]This study shows the survival rates of twi / twi mice administered with rAAVhu68.hGALC or a vehicle. In PND0, twi / twi mice were intravenously administered rAAVhu68.hGALC at a dose of 1.0 × 10¹¹ GC. Age-matched twi / twi mice and WT mice were intravenously administered PBS as a control. Survival rates were monitored. Based on comparisons between each group and the vehicle-treated twi / twi control group using the log-rank (Mantel-Cox) test, P=p=0.0006. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 8] This shows the expression of the transgene (GALC activity) in the brains of twi / twi mice administered with rAAVhu68.hGALC or vehicle. In PND0, twi / twi mice were intravenously administered rAAVhu68.hGALC at a dose of 1.0 × 10¹¹ GC. Twi / twi and WT mice of the same age were intravenously administered PBS as a control. Brain GALC enzyme activity was measured. One-way ANOVA and Tukey post-hoc multiple comparisons were performed to compare each group with twi / twi PBS. The dashed line shows the mean of the wild-type PBS group. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 9] This study shows the survival rates of twi / twi mice administered with rAAVhu68.hGALC or a vehicle. In PND0, twi / twi mice were administered ICV with one of three doses of rAAVhu68.hGALC (2.0 × 10¹⁰, 5.0 × 10¹⁰, or 1.0 × 10¹¹ GC). Same-age twi / twi mice and WT mice were administered ICV with PBS as a control. Survival rates were monitored. Based on comparisons between each group and the vehicle-treated twi / twi control group using the log-rank (Mantel-Cox) test, p=0.0001. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 10]This table shows the body weight of twi / twi mice administered with rAAVhu68.hGALC or vehicle. In PND0, twi / twi mice were administered ICV with one of three doses of rAAVhu68.hGALC: 2.0 × 10¹⁰, 5.0 × 10¹⁰, or 1.0 × 10¹¹ GC. Same-age twi / twi and WT mice were administered ICV with PBS as a control. After weaning, animals were weighed three times per week. Error bars represent standard deviation. p=0.0001 is based on statistical analysis of longitudinal data using linear mixed-effects modeling to compare each group with the PBS-treated twi / twi control group. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 11] This study describes the neuromotor function of twi / twi mice administered rAAVhu68.hGALC or a vehicle. In PND0, twi / twi mice were administered one of three doses of rAAVhu68.hGALC (2.0 × 10¹⁰, 5.0 × 10¹⁰, and 1.0 × 10¹¹ GC) via ICV. Same-age twi / twi and WT mice were administered PBS as a control via ICV. In PND35, neuromotor function was assessed by the time (seconds) until a mouse fell while running on an acceleration rod that rotated initially at 5 RPM and increased to 40 RPM over 120 seconds. Error bars represent standard deviation. Based on comparisons between each group and the PBS-treated twi / twi control group using one-way ANOVA and post-hoc Dunn's multiple comparison tests, **p<0.007, ****p<0.0001. Abbreviations: GC: Genome copy, PBS: Phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 12]This shows the expression of the transgene (GALC activity) in the brains of twi / twi mice administered with rAAVhu68.hGALC or vehicle. In PND0, twi / twi mice were administered ICV with one of three doses of rAAVhu68.hGALC: 2.0 × 10¹⁰, 5.0 × 10¹⁰, and 1.0 × 10¹¹GC. Twi / twi and WT mice of the same age were administered ICV with PBS as a control. At necropsy, brains were collected for GALC enzyme activity assays and transgene expression was evaluated. Error bars represent standard deviation. The dashed line shows the mean of the wild-type PBS-treated group. Based on comparisons between each group and the PBS-treated twi / twi control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, ****p < 0.0001. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 13] This study shows the expression of the transgene (GALC activity) in the liver of twi / twi mice administered with rAAVhu68.hGALC or vehicle. In PND0, twi / twi mice were administered one of three doses of rAAVhu68.hGALC (2.0 × 10¹⁰, 5.0 × 10¹⁰, and 1.0 × 10¹¹ GC) via ICV. Same-age twi / twi mice and WT mice were administered PBS as a control via ICV. At necropsy, liver samples were collected for GALC enzyme activity assays, and transgene expression was evaluated. Error bars represent standard deviation. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 14]This shows the expression of the transgene (GALC activity) in the serum of twi / twi mice administered with rAAVhu68.hGALC or vehicle. In PND0, twi / twi mice were administered one of three doses of rAAVhu68.hGALC (2.0 × 10¹⁰, 5.0 × 10¹⁰, and 1.0 × 10¹¹ GC) via ICV. Twi / twi mice of the same age and WT mice were administered PBS as a control via ICV. In PND28, blood was collected for a GALC enzyme activity assay, and transgene expression was evaluated. Error bars represent the standard deviation. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of a loss-of-function mutation in the Galc gene). [Figure 15] This shows brain myelination in twi / twi mice administered with rAAVhu68.hGALC or vehicle. In PND0, twi / twi mice were administered ICV with one of three doses of rAAVhu68.hGALC: 2.0 × 10¹⁰, 5.0 × 10¹⁰, and 1.0 × 10¹¹GC. Same-age twi / twi mice and WT mice were administered ICV with PBS as a control. At necropsy, brains were collected, processed, and stained with LFB / PAS to evaluate myelination and globoid cell infiltration. Photographs were taken at low magnification. Arrows indicate the corpus callosum. Scale bar, 2 mm. Abbreviations: GC: Genome copy, LFB: Luxol Fast Blue, PAS: Periodate Schiff, PBS: Phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 16]This image shows brain myelination (high magnification) in twi / twi mice administered with rAAVhu68.hGALC or vehicle. In PND0, twi / twi mice were administered one of three doses of rAAVhu68.hGALC (2.0 × 10¹⁰, 5.0 × 10¹⁰, or 1.0 × 10¹¹ GC) via ICV. Same-age twi / twi and WT mice were administered ICV with PBS as a control. At necropsy, brains were collected, processed, and stained with LFB / PAS to evaluate myelination and globoid cell infiltration. Photographs were taken at high magnification (20x). Yellow arrows indicate globoid cells. Star: Central white matter cerebellum. Scale bar 100 μm. Abbreviations: GC: Genome copy, LFB: Luxol Fast Blue, PAS: Periodate Schiff, PBS: Phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 17] This image shows myelin formation in the sciatic nerve of twi / twi mice administered with rAAVhu68.hGALC or vehicle. In PND0, twi / twi mice were administered one of three doses of rAAVhu68.hGALC (2.0 × 10¹⁰, 5.0 × 10¹⁰, and 1.0 × 10¹¹GC) via ICV. Same-age twi / twi and WT mice were administered PBS as a control via ICV. At necropsy, the sciatic nerve was collected, processed, and stained with toluidine blue to evaluate myelin formation and globoid cell infiltration. Photographs were taken at 40x magnification. Arrows indicate myelinated nerve fibers. Abbreviations: GC: genome copy, phosphate-buffered saline, twi: Twitcher allele (consisting of a loss-of-function mutation in the Galc gene). [Figure 18]This image shows neuroinflammation (IBA1) in the brains of twi / twi mice administered with rAAVhu68.hGALC or vehicle. In PND0, twi / twi mice were administered one of three doses of rAAVhu68.hGALC (2.0 × 10¹⁰, 5.0 × 10¹⁰, or 1.0 × 10¹¹ GC) via ICV. Same-age twi / twi and WT mice were administered PBS as a control via ICV. At necropsy, brain tissue was collected, processed, and stained for neuroinflammation (IBA1 staining). Dark staining indicates activated microglia and globoid cells (arrows). The central cerebellar white matter is indicated by a black star, and globoid cells are absent. Globoid cells were present in the cerebellar gyri (cerebellum folia) and brainstem of rAAVhu68.hGALC-treated mice. Scale bar is 100 μm. Abbreviations: GC: Genome copy, PBS: Phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 19] This study shows hGALC expression in the brains of twi / twi mice administered with rAAVhu68.hGALC or a vehicle. In PND0, twi / twi mice were administered one of three doses of rAAVhu68.hGALC (2.0 × 10¹⁰, 5.0 × 10¹⁰, or 1.0 × 10¹¹ GC) via ICV. Same-age twi / twi and WT mice were administered ICV with PBS as a control. At necropsy, brains were collected, processed, and stained for hGALC detection. Scale bar is 200 μm. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figures 20A-20B] This study shows the survival rate of twi / twi mice. In PND0, twi / twi mice were administered ICV at a dose of 2.0 × 10¹⁰ of either AAVhu68.hGALC, AAV1.hGALC, AAV3B.hGALC, or AAV5.hGALC. Survival rates were monitored. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 21]This shows the body weight of twi / twi mice. In PND0, twi / twi mice were administered either AAV1.hGALC, AAV3B.hGALC, or AAV5.hGALC via ICV at a dose of 2.0 × 10¹⁰ GC. After weaning, the animals' body weight was measured three times a week. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 22] This study demonstrates the neuromotor function of twi / twi mice. In PND0, twi / twi mice were administered either AAV1.hGALC, AAV3B.hGALC, or AAV5.hGALC via ICV at a dose of 2.0 × 10¹⁰. In PND35, neuromotor function was assessed by the time (in seconds) it took for a mouse running on an acceleration rod, which initially rotated at 5 RPM and increased to 40 RPM over 120 seconds, to fall. Error bars represent the standard deviation. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 23] This figure shows the expression of transgenes (GALC activity) in the brains of twi / twi mice. In PND0, twi / twi mice were administered either AAV1.hGALC, AAV3B.hGALC, or AAV5.hGALC via ICV at a dose of 2.0 × 10¹⁰. At necropsy, brain samples were collected for GALC enzyme activity assays, and transgene expression was evaluated. Error bars represent standard deviation. The dashed line shows the mean of the wild-type PBS-treated group. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 24]This shows the expression of transgenes (GALC activity) in the liver of twi / twi mice. In PND0, twi / twi mice were administered either AAV1.hGALC, AAV3B.hGALC, or AAV5.hGALC via ICV at a dose of 2.0 × 10¹⁰. At necropsy, liver samples were collected for GALC enzyme activity assays, and transgene expression was evaluated. Error bars represent standard deviation. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 25] This shows the expression of transgenes (GALC activity) in the serum of twi / twi mice. In PND0, twi / twi mice were administered either AAV1.hGALC, AAV3B.hGALC, or AAV5.hGALC via ICV at a dose of 2.0 × 10¹⁰. In PND28, blood was collected for a GALC enzyme activity assay, and transgene expression was evaluated. Error bars represent the standard deviation. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of a loss-of-function mutation in the Galc gene). [Figure 26] This study demonstrates myelin formation of the sciatic nerve in twi / twi mice. In PND0 mice, twi / twi mice were administered either AAV1.hGALC, AAV3B.hGALC, or AAV5.hGALC via ICV at a dose of 2.0 × 10¹⁰. At necropsy, the sciatic nerve was collected, processed, and stained with toluidine blue to evaluate myelin formation and globoid cell infiltration. Photographs were taken at 40x magnification. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 27]This image shows neuroinflammation (IBA1) in the brains of twi / twi mice. In PND0, twi / twi mice were administered either AAV1.hGALC, AAV3B.hGALC, or AAV5.hGALC via ICV at a dose of 2.0 × 10¹⁰. At necropsy, brain tissue was collected, processed, and stained for neuroinflammation (IBA1 staining). Dark staining indicated activated microglia and globoid cells. Scale bar is 100 μm. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 28] This shows hGALC expression in the brains of twi / twi mice. In PND0, twi / twi mice were administered either AAV1.hGALC, AAV3B.hGALC, or AAV5.hGALC via ICV at a dose of 2.0 × 10¹⁰. At autopsy, brains were collected, processed, and stained for hGALC detection. Scale bar is 200 μm. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figures 29A-29B] This report describes the survival rates of twi / twi mice administered rAAVhu68.hGALC or a vehicle in PND12 or PND21. In PND12 or PND21, twi / twi mice were administered rAAVhu68.hGALC via ICV (intracellular delivery) at two doses: 1.0 × 10¹¹ GC or 2.0 × 10¹¹ GC. Same-age twi / twi mice and WT mice were administered PBS as a control via ICV. Survival rates were monitored. In PND12, the comparison between the rAAVhu68.hGALC group and the PBS group showed p<0.0001. Based on comparisons between each group and the vehicle-treated twi / twi control group using the log-rank (Mantel-Cox) test, the comparison between the PND21 group and the PBS group showed p=0.0008. Abbreviations: GC: Genome copy, PBS: Phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 30]This study shows the survival rates of twi / twi mice administered rAAVhu68.hGALC at a dose of 2.0 × 10¹¹ GC in PND12 or PND21 mice. Twi / twi mice in PND12 or PND21 mice received ICV administration of one of two doses of rAAVhu68.hGALC, either 1.0 × 10¹¹ GC or 2.0 × 10¹¹ GC. Same-age twi / twi mice and WT mice received ICV administration with PBS as a control. Survival rates were monitored. Based on comparisons between each group and the vehicle-treated twi / twi control group using the log-rank (Mantel-Cox) test, p < 0.0001. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 31] This figure shows the body weight of twi / twi mice administered rAAVhu68.hGALC or vehicle in PND12. In PND12, twi / twi mice were administered rAAVhu68.hGALC via ICV at one of two doses: 1.0 × 10¹¹ GC or 2.0 × 10¹¹ GC. Same-age twi / twi mice and WT mice were administered PBS as a control via ICV. Animal body weight was measured three times per week. Error bars represent standard deviation. Based on group comparisons using one-way ANOVA and post-hoc Dunn multiple comparison tests, p=0.0001. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 32] This figure shows the body weight of twi / twi mice administered rAAVhu68.hGALC or vehicle at PND21. Twi / twi mice at PND12 or PND21 were administered rAAVhu68.hGALC via ICV at one of two doses: 1.0 × 10¹¹ GC or 2.0 × 10¹¹ GC. Same-age twi / twi mice and WT mice were administered PBS as a control via ICV. Animal body weight was measured three times per week. Error bars represent standard deviation. Based on group comparisons using one-way ANOVA and post-hoc Dunn multiple comparison tests, p=0.0001. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 33] Neuromotor function in twi / twi mice administered rAAVhu68.hGALC or vehicle in PND12 is shown. In PND12, twi / twi mice were administered rAAVhu68.hGALC via ICV at one of two doses: 1.0 × 10¹¹ GC or 2.0 × 10¹¹ GC. Same-age twi / twi mice and WT mice were administered PBS as a control via ICV. In PND35, neuromotor function was assessed by the time (seconds) until a mouse fell off an acceleration rod that rotated initially at 5 RPM and increased to 40 RPM over 120 seconds. Error bars represent standard deviation. Based on comparisons between each group and the PBS-treated twi / twi control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, **p=0.0004** for low doses and ***p=0.0006*** for high doses. Abbreviations: GC: Genome copy, PBS: Phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 34] This paper shows the neuromotor function of twi / twi mice administered rAAVhu68.hGALC or vehicle at PND12 or PND21. At PND12 or PND21, twi / twi mice were administered rAAVhu68.hGALC via ICV at one of two doses: 1.0 × 10¹¹ GC or 2.0 × 10¹¹ GC. Same-age twi / twi mice and WT mice were administered PBS as a control via ICV. At PND35, neuromotor function was assessed by the time (seconds) it took for a mouse running on an acceleration rod, initially rotating at 5 RPM and increasing to 40 RPM over 120 seconds, to fall. Error bars represent the standard deviation. Abbreviations: GC: genome copy, PBS: phosphate-buffered saline, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 35]This shows the expression of the transgene (GALC activity) in the brains of twi / twi mice administered with rAAVhu68.hGALC or vehicle on PND12 or PND21. Twi / twi mice were administered via ICV (intracellular transfusion) with one of two doses of rAAVhu68.hGALC, either 1.0 × 10¹¹ GC or 2.0 × 10¹¹ GC, to twi / twi mice on PND12 or PND21. Same-age twi / twi and WT mice were administered via ICV with PBS as a control. At necropsy, brains were collected for GALC enzyme activity assays to evaluate transgene expression. Error bars represent standard deviation. The dashed line shows the mean value from PBS-treated wild-type mice. Based on comparisons between each group and the PBS-treated twi / twi control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, **p=0.002**. [Figure 36] This shows the expression of the transgene (GALC activity) in the liver of twi / twi mice administered with rAAVhu68.hGALC or vehicle in PND12 or PND21. In PND12 or PND21, twi / twi mice were administered rAAVhu68.hGALC via ICV at one of two doses: 1.0 × 10¹¹ GC or 2.0 × 10¹¹ GC. Same-age twi / twi mice and WT mice were administered PBS as a control via ICV. At necropsy, liver samples were collected for GALC enzyme activity assays, and transgene expression was evaluated. Error bars represent standard deviation. The dashed line shows the mean value from PBS-treated wild-type mice. Based on comparisons between each group and the PBS-treated twi / twi control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, ****p < 0.0001. [Figure 37]This shows transgene expression (GALC activity) in serum from twi / twi mice administered rAAVhu68.hGALC or vehicle in PND12 or PND21. In PND12 or PND21, twi / twi mice were administered rAAVhu68.hGALC in one of two doses, 1.0 × 10¹¹ GC or 2.0 × 10¹¹ GC, via ICV. Same-age twi / twi mice and WT mice were administered PBS as a control via ICV. In PND18, blood was collected and used for GALC enzyme activity assays to evaluate transgene expression. Error bars represent standard deviation. The dashed line shows the mean value from PBS-treated wild-type mice. Based on comparisons between each group and the PBS-treated twi / twi control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, **p<0.01, ****p<0.0001. [Figure 38] This shows brain myelination in twi / twi mice administered rAAVhu68.hGALC or vehicle via ICV in PND12 or PND21. Twi / twi mice were administered rAAVhu68.hGALC via ICV in PND12 or PND21 at two doses: 1.0 × 10¹¹ GC or 2.0 × 10¹¹ GC. Same-age twi / twi and WT mice were administered ICV with PBS as a control. At necropsy, brains were collected, processed, and stained with LFB / PAS to assess myelination and globoid cell infiltration. High-magnification photographs were taken. Arrows point to globoid cells in the cerebellar gyrus, and asterisks indicate the corpus callosum. Scale bar 100 μm. [Figure 39]This shows myelination of the sciatic nerve in twi / twi mice administered rAAVhu68.hGALC in PND12 or PND21 mice. Twi / twi mice in PND12 or PND21 were administered rAAVhu68.hGALC via ICV at one of two doses: 1.0 × 10¹¹ GC or 2.0 × 10¹¹ GC. Same-age twi / twi mice and WT mice were administered PBS as a control via ICV. At necropsy, the sciatic nerve was harvested, processed, and stained with toluidine blue to assess myelination and globoid cell infiltration. The longest-surviving mice showed more myelinated fibers (center image). Photographs were taken at 40x magnification. Arrows indicate myelinated nerve fibers. [Figure 40] This shows neuroinflammation (IBA1) in the brains of twi / twi mice administered rAAVhu68.hGALC or vehicle via ICV in PND12 or PND21. Twi / twi mice were administered rAAVhu68.hGALC via ICV in PND12 or PND21 at two doses: 1.0 × 10¹¹ GC or 2.0 × 10¹¹ GC. Same-age twi / twi and WT mice were administered PBS as a control via ICV. At necropsy, brains were collected, processed, and stained for neuroinflammation (IBA1 staining). Dark staining indicates activated microglia and globoid cells (arrows). The central cerebellar white matter, indicated by black asterisks, is free of globoid cells. Globoid cells were present in the cerebellar gyri and brainstem of rAAVhu68.hGALC-treated mice. Scale bar is 100 μm. [Figure 41] This study shows hGALC expression in the brains of twi / twi mice administered with rAAVhu68.hGALC or a vehicle via PND12 or PND21. Twi / twi mice were administered via ICV (intracellular catheter) with either PND12 or PND21, receiving one of two doses of rAAVhu68.hGALC: 1.0 × 10¹¹ GC or 2.0 × 10¹¹ GC. Same-age twi / twi mice and WT mice were administered via ICV with PBS as a control. At necropsy, brains were collected, processed, and stained for hGALC detection. Scale bar is 200 μm. [Figure 42]This table shows the body weight of twi / twi mice administered rAAVhu68.hGALC or vehicle. In PND40, twi / twi mice were administered rAAVhu68.hGALC via ICV at a dose of 2.0 × 10¹¹ GC. Same-age twi / twi mice and WT mice were administered PBS as a control via ICV. Animal body weight was measured three times per week. Error bars represent the standard deviation. Due to the limited number of male and female mice per group, male and female body weight data were combined. [Figure 43] This study shows the neuromotor function of twi / twi mice administered with rAAVhu68.hGALC or a vehicle. At PND40, twi / twi mice were administered rAAVhu68.hGALC via ICV at a dose of 2.0 × 10¹¹ GC. Same-age twi / twi mice and WT mice were administered PBS as a control via ICV. At PND35, neuromotor function was assessed by the time (seconds) until a mouse fell off an acceleration rod that rotated initially at 5 RPM and increased to 40 RPM over 120 seconds. Error bars represent the standard deviation. Based on comparisons between each group and the PBS-treated twi / twi control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, **p=0.0001**. [Figure 44] This report presents the clinical scoring evaluation of twi / twi mice administered with rAAVhu68.hGALC or a vehicle. PND40 twi / twi mice were administered rAAVhu68.hGALC via ICV at a dose of 2.0 × 10¹¹ GC. Same-age twi / twi mice and WT mice were administered PBS via ICV as controls. Standardized clinical evaluations were performed three times a week. Error bars represent the standard deviation. [Figure 45] This shows the expression of the transgene (GALC activity) in the brains of twi / twi mice administered with rAAVhu68.hGALC or vehicle. twi / twi mice were administered rAAVhu68.hGALC via ICV at a dose of 2.0 × 10¹¹ GC in PND40. Twi / twi mice and WT mice of the same age were administered PBS as a control via ICV. At necropsy, brain samples were collected for GALC enzyme activity assays, and transgene expression was evaluated. Error bars represent the standard deviation. [Figure 46] This shows the expression of the transgene (GALC activity) in the liver of twi / twi mice administered with rAAVhu68.hGALC or vehicle. In PND40, twi / twi mice were administered rAAVhu68.hGALC via ICV at a dose of 2.0 × 10¹¹ GC. Twi / twi mice and WT mice of the same age were administered PBS as a control via ICV. At necropsy, liver samples were collected for GALC enzyme activity assays, and transgene expression was evaluated. Error bars represent the standard deviation. [Figures 47A-47B] This shows the expression of the transgene (GALC activity) in the serum of twi / twi mice administered with rAAVhu68.hGALC or vehicle. At PND40, twi / twi mice were administered rAAVhu68.hGALC via ICV at a dose of 2.0 × 10¹¹ GC. Twi / twi mice and WT mice of the same age were administered via ICV with PBS as a control. At PND28 (Figure 47A) and autopsy (PND40) (Figure 47B), blood was collected for GALC enzyme activity assays and transgene expression was evaluated. Error bars represent standard deviation. Based on comparisons between each group and the PBS-treated twi / twi control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, **p<0.01, ****p<0.0001. [Figure 48A-48C] This shows brain myelination in twi / twi mice administered with rAAVhu68.hGALC or vehicle. twi / twi mice were administered rAAVhu68.hGALC via ICV at a dose of 2.0 × 10¹¹ GC in PND40 (results shown in Figure 48C). Same-age twi / twi mice (Figure 48B) and WT mice (Figure 48A) were administered PBS as a control via ICV. At necropsy, brains were collected, processed, and stained with LFB / PAS to evaluate myelination and globoid cell infiltration. Photographs were taken at low magnification. Stain intensity represents the degree of myelination. Lighter myelin can be seen in the corpus callosum of twi / twi PBS mice. The rAAVhu68.hGALC-treated group shows normal WT-like corpus callosum myelin intensity. Scale bar is 2 mm. Abbreviations are as above. LFB: Luxol Fast Blue, PAS: Periodate Schiff. [Figure 49]These are a series of nine high-magnification images showing brain myelination in twi / twi mice administered with rAAVhu68.hGALC (results in the third column) or vehicle (results shown in the second column). twi / twi mice were administered rAAVhu68.hGALC via ICV at a dose of 2.0 × 10¹¹ GC in PND40. Same-age twi / twi and WT mice (results in the first column) were administered PBS as a control via ICV. At necropsy, brains were collected, processed, and stained with LFB / PAS to assess myelination and globoid cell infiltration. Photographs were taken at high magnification (20x). Arrows indicate globoid cells. Scale bar 100 μm. The first row shows images of the brainstem. The second row shows images of the cerebellum. The third row shows images of the corpus callosum. [Figure 50] These are a series of 12 photographs showing myelin formation of peripheral nerves in twi / twi mice administered with rAAVhu68.hGALC (results in the 3rd column) or vehicle (results in the 2nd column). rAAVhu68.hGALC was administered via ICV to twi / twi mice at a dose of 2.0 × 10¹¹ GC in PND40. Same-age twi / twi mice and WT mice (results in the 1st column) were administered via ICV with PBS as a control. At necropsy, nerves were collected, processed, and stained with LFB / PAS or toluidine blue to evaluate myelin formation and globoid cell infiltration. The first and second rows provide results for the sciatic and axillary nerves, respectively, using LBS staining. The third row shows a high-magnification image of the sciatic nerve stained with toluidine blue. The fourth row shows a high-magnification image of the sciatic nerve stained with IBA1 IHC. Scale bar is 100 nm. [Figure 51]These are a series of nine photographs showing spinal cord myelination in twi / twi mice administered with rAAVhu68.hGALC or vehicle. twi / twi mice were administered rAAVhu68.hGALC via ICV at a dose of 2.0 × 10¹¹ GC in PND40 (results in the third column). Same-age twi / twi mice and WT mice were administered via ICV with PBS as a control (results in the second and first columns, respectively). At necropsy, spinal cord was collected, processed, and stained with LFB / PAS for evaluation of myelination and globoid cell infiltration. Photographs were taken at low magnification. Globoid cells are indicated by arrows. Scale bar is 100 nm. The first row provides samples from the cervical (C) spine, the second row provides samples from the thoracic (T) spine, and the third row provides results from the lumbar (L) spine. [Figures 52A-52C] This shows neuroinflammation (IBA1) in the brains of twi / twi mice administered with rAAVhu68.hGALC (Figure 52C) or vehicle (Figure 52B). rAAVhu68.hGALC was administered via ICV to twi / twi mice at a dose of 2.0 × 10¹¹ GC in PND40. Same-age twi / twi mice and WT mice (Figure 52A) were administered via ICV with PBS as a control. At necropsy, brains were collected, processed, and stained for neuroinflammation (IBA1 staining). Brown staining indicated activated microglia and globoid cells. Microglia were large and gave a patchy, coarse stain, particularly in the cortical cortex, corpus callosum, brainstem, and cerebellum. In rAAVhu68.hGALC-treated twi / twi mice, patchy IBA1 staining was not observed in the cortex and corpus callosum, but remained in the cerebellum and brainstem. The photo was taken at low magnification. Scale bar: 2mm. [Figure 53]These are a series of 15 high-magnification images showing neuroinflammation (IBA1) in the brains of twi / twi mice administered with rAAVhu68.hGALC (3rd column) or vehicle (2nd column). rAAVhu68.hGALC was administered via ICV to twi / twi mice at a dose of 2.0 × 10¹¹ GC in PND40. Same-age twi / twi mice and WT mice (1st column) were administered via ICV with PBS as a control. At necropsy, brains were collected, processed, and stained for neuroinflammation (IBA1 staining). Dark staining indicated activated microglia and globoid cells. High-magnification photographs were taken. The first row shows images of the cortex. The second row shows images of the hippocampus. The third row shows images of the corpus callosum. The fourth row shows images of the cerebellum. The fifth row shows images of the brainstem. Scale bar 300 μm. [Figure 54] These are a series of nine photographs showing neuroinflammation (IBA1) in the spinal cord of twi / twi mice administered with rAAVhu68.hGALC (3rd column) or vehicle (2nd column). rAAVhu68.hGALC was administered via ICV to twi / twi mice at a dose of 2.0 × 10¹¹ GC in PND40. Same-age twi / twi mice and WT mice (1st column) were administered via ICV with PBS as a control. At necropsy, brain tissue was collected, processed, and stained for neuroinflammation (IBA1 staining). Dark staining indicated activated microglia and globoid cells. Photographs were taken at high magnification. The first row shows the C vertebra. The second row shows the T vertebra. The third row shows the L vertebra. Scale bar 200 μm. Abbreviations: C: Cervical spinal cord, GC: Genome copy, L: Lumbar spinal cord, PBS: Phosphate-buffered saline, T: Lumbar spinal cord, Twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene). [Figure 55]These are a series of 12 photographs showing hGALC expression in the brains of twi / twi mice administered with rAAVhu68.hGALC (3rd column) or vehicle (2nd column). rAAVhu68.hGALC was administered via ICV to twi / twi mice at a dose of 2.0 × 10¹¹ GC in PND40. Same-age twi / twi mice and WT mice (1st column) were administered via ICV with PBS as a control. At necropsy, brains were collected, processed, and stained for hGALC expression. Dark staining indicated activated microglia and globoid cells. Photographs were taken at high magnification. Scale bar 300 μm. The first row shows images of the cortex. The second row shows images of the hippocampus. The third row shows images of the cerebellum. The fourth row shows images of the brainstem. [Figures 56A-56C] The results of combination therapy with rAAVhu68.hGALC and bone marrow transplantation are shown. Twitcher mice (twi / twi) were treated with BMT alone (N=13, PND10), rAAVhu68.hGALC alone (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) administered PBS served only as an existing control (N=8, Study 1, PND0; N=4, Study 2, PND12). Interim results for survival rates are shown, and the experiment is still ongoing. Abbreviations: BMT: Bone marrow transplant, GC: Genome copy, ICV: Intraventricular, N: Number of animals, PBS: Phosphate-buffered saline, PND: Days after birth. [Figure 56C] Brain engraftment of GFP+ donor cells in the cerebellum of wild-type and Twitcher (Krabbe) mice 8 weeks after HSCT. [Figure 57]This study describes the survival rates of twi / twi mice administered with rAAVhu68.hGALC or a vehicle. In PND12-14, twi / twi mice were administered rAAVhu68.hGALC via ICV at doses of 6.8×10⁹GC, 2.0×10¹⁰GC, 6.8×10¹⁰GC, or 2.0×10¹¹GC. Twi / twi and WT mice of the same age were administered via ICV with a vehicle (ITFFB) as a control. Survival rates were monitored. Data from both the PND40 necropsy cohort and survival cohort were combined by treatment and genotype. Based on comparisons between each group and the vehicle-treated twi / twi control group using the log-rank (Mantel-Cox) test, *p<0.05, **p<0.01, ***p<0.001. Abbreviations: Galc: Galactosylceramidase (gene, mouse), GC: Genome copy, ICV: Intraventricular, ITFFB: Intrathecal final preparation buffer, N: Number of animals, PND: Days after birth, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: Wild type. [Figures 58A-58B]Figure 58A shows the body weight of male or female twi / twi mice administered rAAVhu68.hGALC or vehicle (PND40 and survival cohort). Figure 58B shows the results for males and females, respectively. In PND12–14, twi / twi mice were administered rAAVhu68.hGALC via ICV at doses of 6.8 × 10⁹ GC, 2.0 × 10¹⁰ GC, 6.8 × 10¹⁰ GC, or 2.0 × 10¹¹ GC. Twi / twi and WT mice of the same age were administered via ICV with vehicle (ITFFB) as a control. Animals were weighed three times a week. Data from both the necropsy and survival cohorts of PND40 are combined by treatment and genotype, and mean body weight represents the period from weaning to necropsy in the PND40 cohort. Error bars represent the standard deviation. Based on statistical analysis of longitudinal data using linear mixed-effects modeling to compare each group with the vehicle-treated twi / twi control group, **p<0.01, ****p<0.0001. Abbreviations: Galc: galactosylceramidase (gene, mouse), GC: genome copy, ICV: intraventricular, ITFFB: intrathecal final preparation buffer, N: number of animals, PND: days after birth, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: wild type. [Figures 59A-59B]Figure 59A shows the body weight of male or female twi / twi mice administered with rAAVhu68.hGALC or vehicle (PND40 and survival cohort). Figure 59B shows the results for males and females, respectively. In PND12-14, twi / twi mice were administered rAAVhu68.hGALC via ICV at doses of 6.8×10⁹GC, 2.0×10¹⁰GC, 6.8×10¹⁰GC, or 2.0×10¹¹GC. Same-age twi / twi mice were administered rAAVhu68.hGALC via ICV at doses of 6.8×10⁹GC, 2.0×10¹⁰GC, 6.8×10¹⁰GC, or 2.0×10¹¹GC. Twi / twi mice and WT mice were administered a vehicle (ITFFB) via ICV as a control. After weaning, animals were weighed three times a week. Data from both the PND40 necropsy cohort and the survival cohort are combined by treatment and genotype to present the mean body weight from weaning to necropsy of the last surviving twi / twi mouse. Error bars represent the standard deviation. Abbreviations: Galc: galactosylceramidase (gene, mouse), GC: genome copy, ICV: intraventricular, ITFFB: intrathecal final preparation buffer, N: number of animals, PND: days after birth, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: wild type. [Figure 60]This report presents clinical scoring evaluations of twi / twi mice administered with rAAVhu68.hGALC or a vehicle (PND40 and survival cohort). PND12–14 twi / twi mice were administered rAAVhu68.hGALC via ICV at doses of 6.8 × 10⁹ GC, 2.0 × 10¹⁰ GC, 6.8 × 10¹⁰ GC, or 2.0 × 10¹¹ GC. Same-age twi / twi mice and WT mice were administered via ICV with a vehicle (ITFFB) as a control. Standardized clinical evaluations were performed at specified postnatal days. Data from both the PND40 necropsy cohort and the survival cohort are combined by treatment and genotype to present the mean total clinical score from weaning to necropsy in the PND40 cohort. Error bars represent standard deviation. Based on statistical analysis of longitudinal data using linear mixed-effects modeling to compare each group with the vehicle-treated twi / twi control group, **p<0.01, ****p<0.0001. Abbreviations: Galc: galactosylceramidase (gene, mouse), GC: genome copy, ICV: intraventricular, ITFFB: intrathecal final preparation buffer, N: number of animals, PND: days after birth, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: wild type. [Figure 61]This report presents clinical scoring evaluations of twi / twi mice administered with rAAVhu68.hGALC or a vehicle (PND40 and survival cohort). For PND12–14, twi / twi mice were administered rAAVhu68.hGALC via ICV at doses of 6.8 × 10⁹ GC, 2.0 × 10¹⁰ GC, 6.8 × 10¹⁰ GC, or 2.0 × 10¹¹ GC. Same-age twi / twi and WT mice were administered via ICV with a vehicle (ITFFB) as a control. Standardized clinical evaluations were performed at specified postnatal days. Data from both the PND40 necropsy cohort and the survival cohort are combined by treatment and genotype, and the mean total clinical severity score is presented from weaning to the necropsy of the last surviving twi / twi mouse. Error bars represent standard deviation. Based on statistical analysis of longitudinal data using linear mixed-effects modeling to compare each group with the vehicle-treated twi / twi control group, **p<0.01, ****p<0.0001. Abbreviations: Galc: galactosylceramidase (gene, mouse), GC: genome copy, ICV: intraventricular, ITFFB: intrathecal final preparation buffer, N: number of animals, PND: days after birth, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: wild type. [Figure 62]This report shows the neuromotor function of twi / twi mice administered with rAAVhu68.hGALC or a vehicle (PND40 and survival cohort). In PND12–14, twi / twi mice were administered rAAVhu68.hGALC via ICV at doses of 6.8 × 10⁹ GC, 2.0 × 10¹⁰ GC, 6.8 × 10¹⁰ GC, or 2.0 × 10¹¹ GC. Same-age twi / twi and WT mice were administered via ICV with a vehicle (ITFFB) as a control. In PND35, neuromotor function was assessed by the time (seconds) until a mouse fell off an acceleration rod that rotated initially at 5 RPM and increased to 40 RPM over 120 seconds. Data from both the PND40 necropsy cohort and survival cohort are combined by treatment and genotype. Error bars represent the standard deviation. Based on comparisons between each group and the vehicle-treated twi / twi control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, **p<0.01, ****p<0.0001. Abbreviations: Galc: galactosylceramidase (gene, mouse), GC: genome copy, ICV: intraventricular, ITFFB: intrathecal final preparation buffer, N: number of animals, PND: days after birth, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: wild type. [Figure 63]This report shows the expression of transgenes in the serum of twi / twi mice administered with rAAVhu68.hGALC or a vehicle (PND40 and survival cohort). In PND12-14, twi / twi mice were administered rAAVhu68.hGALC via ICV at doses of 6.8×10⁹GC, 2.0×10¹⁰GC, 6.8×10¹⁰GC, or 2.0×10¹¹GC. Same-age twi / twi mice and WT mice were administered via ICV with a vehicle (ITFFB) as a control. In PND35-37, serum was collected for GALC enzyme activity assays, and transgene expression was evaluated. Data from both the PND40 necropsy cohort and survival cohort are combined by treatment and genotype. The Y-axis is split to show the difference between data points within the range of 0-1000 RFU. Error bars represent the standard deviation. Based on comparisons between each group and the vehicle-treated twi / twi control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, **p<0.01, ****p<0.0001. Abbreviations: Galc: galactosylceramidase (gene, mouse), GALC: galactosylceramidase (protein, mouse), GC: genome copy, ICV: intraventricular, ITFFB: intrathecal final preparation buffer, N: number of animals, PND: days after birth, RFU: relative fluorescence unit, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: wild type. [Figure 64]This shows the expression of transgenes in the brains of twi / twi mice administered with rAAVhu68.hGALC or a vehicle (PND40 and survival cohort). In PND12-14, twi / twi mice were administered rAAVhu68.hGALC via ICV at doses of 6.8×10⁹GC, 2.0×10¹⁰GC, 6.8×10¹⁰GC, or 2.0×10¹¹GC. Twi / twi mice and WT mice of the same age were administered via ICV with a vehicle (ITFFB) as a control. At necropsy, brains were collected for GALC enzyme activity assays, and transgene expression was evaluated. Data from both the PND40 necropsy cohort and the survival cohort are combined by treatment and genotype. Error bars represent the standard deviation. Based on comparisons between each group and the vehicle-treated twi / twi control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, **p<0.01, ****p<0.0001. Abbreviations: Galc: galactosylceramidase (gene, mouse), GALC: galactosylceramidase (protein, mouse), GC: genome copy, ICV: intraventricular, ITFFB: intrathecal final preparation buffer, N: number of animals, PND: days after birth, RFU: relative fluorescence unit, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: wild type. [Figures 65A-65D]This report shows the expression of transgenes in the heart, kidney, liver, and spleen of twi / twi mice administered with rAAVhu68.hGALC or a vehicle (PND40 and survival cohort). In PND12-14, twi / twi mice were administered rAAVhu68.hGALC via ICV at doses of 6.8×10⁹GC, 2.0×10¹⁰GC, 6.8×10¹⁰GC, or 2.0×10¹¹GC. Same-age twi / twi mice and WT mice were administered via ICV with a vehicle (ITFFB) as a control. At necropsy, the heart (Figure 65A), kidney (Figure 65B), liver (Figure 65C), and spleen (Figure 65D) were collected for GALC enzyme activity assays, and transgene expression was evaluated. Data from both the PND40 necropsy cohort and the survival cohort are combined by treatment and genotype. Error bars represent standard deviation. Based on comparisons between each group and the vehicle-treated twi / twi control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, *p<0.05, ****p<0.0001. Abbreviations: Galc: galactosylceramidase (gene, mouse), GALC: galactosylceramidase (protein, mouse), GC: genome copy, ICV: intraventricular, ITFFB: intrathecal final preparation buffer, N: number of animals, PND: days after birth, RFU: relative fluorescence unit, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: wild type. [Figures 66A-66C]This report shows the expression of the transgene in the lungs, quadriceps muscles, and diaphragm of twi / twi mice administered with rAAVhu68.hGALC or a vehicle (PND40 and survival cohort). In PND12-14, twi / twi mice were administered rAAVhu68.hGALC via ICV at doses of 6.8×10⁹GC, 2.0×10¹⁰GC, 6.8×10¹⁰GC, or 2.0×10¹¹GC. Same-age twi / twi mice and WT mice were administered via ICV with a vehicle (ITFFB) as a control. At necropsy, the lungs (Figure 66A), quadriceps muscles (Figure 66B), and diaphragm (Figure 66C) were collected for GALC enzyme activity assays, and transgene expression was evaluated. Data from both the PND40 necropsy cohort and the survival cohort are combined by treatment and genotype. Error bars represent the standard deviation. Based on comparisons between each group and the vehicle-treated twi / twi control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, **p<0.01, ****p<0.0001. Abbreviations: Galc: galactosylceramidase (gene, mouse), GALC: galactosylceramidase (protein, mouse), GC: genome copy, ICV: intraventricular, ITFFB: intrathecal final preparation buffer, N: number of animals, PND: days after birth, RFU: relative fluorescence unit, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: wild type. [Figures 67A-67B]This figure shows the lymphocyte count in twi / twi mice administered rAAVhu68.hGALC or vehicle. rAAVhu68.hGALC was administered via ICV to twi / twi mice at doses of 6.8 × 10⁹ GC, 2.0 × 10¹⁰ GC, 6.8 × 10¹⁰ GC, or 2.0 × 10¹¹ GC at PND12–14. Vehicle (ITFFB) was administered via ICV to twi / twi and WT mice of the same age as a control. Blood samples were collected at necropsy at PND40–42 (Figure 67A) and at humane euthanasia of the survival cohort (Figure 67B), and lymphocytes were quantified. Blood samples collected from untreated twi / twi and WT mice on the day of administration served as baseline controls. Error bars represent the standard deviation. Based on comparisons between each group and the vehicle-treated WT control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, *p<0.05, **p<0.01, ***p<0.001. Abbreviations: Galc: galactosylceramidase (gene, mouse), GC: genome copy, ICV: intraventricular, ITFFB: intrathecal final preparation buffer, N: number of animals, PND: days after birth, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: wild type. [Figures 68A-68D]Figures 68A and 68B and 68C and 68D show aspartate aminotransferase levels (Figures 68A and 68B) and bilirubin levels (Figures 68C and 68D) in twi / twi mice administered rAAVhu68.hGALC or vehicle. rAAVhu68.hGALC was administered via ICV to twi / twi mice at doses of 6.8 × 10⁹ GC, 2.0 × 10¹⁰ GC, 6.8 × 10¹⁰ GC, or 2.0 × 10¹¹ GC during PND12-14. Vehicle (ITFFB) was administered via ICV to twi / twi mice and WT mice of the same age as controls. Serum was collected at necropsy during PND40-42 and at humane euthanasia of the survival cohort, and AST and bilirubin levels were evaluated as part of a serum chemistry panel. Figures 68A and 68B show AST levels in the PND cohort and survival cohort, respectively. Figures 68C and 68D show the total bilirubin levels of the PND cohort and the survival cohort, respectively. Serum collected from untreated twi / twi and WT mice on the day of administration served as baseline controls. Error bars represent standard deviation. Based on comparisons between each group and the vehicle-treated WT control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, **p<0.01, ***p<0.001. Untreated WT mice (baseline cohort, group 2) and twi / twi mice administered rAAVhu68.hGALC at a dose of 6.8 × 10⁹ GC (PND40 cohort, group 8a) were excluded from statistical analysis due to insufficient sample size. Abbreviations: AST: Aspartate aminotransferase, Galc: Galactosylceramidase (gene, mouse), GC: Genome copy, ICV: Intraventricular, ITFFB: Intrathecal final preparation buffer, N: Number of animals, PND: Days after birth, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: Wild type. [Figures 69A-69B]This figure shows alanine aminotransferase levels in twi / twi mice administered rAAVhu68.hGALC or vehicle. At PND12–14, twi / twi mice were administered rAAVhu68.hGALC via ICV at doses of 6.8 × 10⁹ GC, 2.0 × 10¹⁰ GC, 6.8 × 10¹⁰ GC, or 2.0 × 10¹¹ GC. Twi / twi and WT mice of the same age were administered vehicle (ITFFB) via ICV as a control. Serum was collected at necropsy at PND40–42 (Figure 69A) and at humane euthanasia of the survival cohort (Figure 69B), and ALT levels were evaluated as part of a serum chemistry panel. Serum collected from untreated twi / twi and WT mice on the day of administration served as a baseline control. Error bars represent standard deviation. Based on comparisons between each group and the vehicle-treated WT control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, *p<0.05, **p<0.01, and ***p<0.001 were observed. Untreated WT mice (baseline cohort, group 2) and twi / twi mice administered rAAVhu68.hGALC at a dose of 6.8 × 10⁹ GC (PND40 cohort, group 8a) were excluded from statistical analysis due to insufficient sample size. Abbreviations: ALT: Alanine aminotransferase, Galc: Galactosylceramidase (gene, mouse), GC: Genome copy, ICV: Intraventricular, ITFFB: Intrathecal final preparation buffer, N: Number of animals, PND: Days after birth, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: Wild type. [Figures 70A-70D]Glucose levels (Figures 70A and 70B) and amylase levels (Figures 70C and 70D) in twi / twi mice administered rAAVhu68.hGALC or vehicle are shown. rAAVhu68.hGALC was administered via ICV to twi / twi mice at doses of 6.8 × 10⁹ GC, 2.0 × 10¹⁰ GC, 6.8 × 10¹⁰ GC, or 2.0 × 10¹¹ GC at PND12–14. Vehicle (ITFFB) was administered via ICV to twi / twi and WT mice of the same age as a control. Serum was collected as part of a serum chemistry panel at necropsy at PND40–42 and at humane euthanasia of the survival cohort, and glucose and amylase levels were evaluated. Serum collected from untreated twi / twi and WT mice on the day of administration served as a baseline control. Error bars represent standard deviation. Based on comparisons between each group and the vehicle-treated WT control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, *p<0.05, **p<0.01. Untreated WT mice (baseline cohort, group 2) and twi / twi mice administered rAAVhu68.hGALC at a dose of 6.8 × 10⁹ GC (PND40 cohort, group 8a) were excluded from statistical analysis due to insufficient sample size. Abbreviations: Galc: galactosylceramidase (gene, mouse), GC: genome copy, ICV: intraventricular, ITFFB: intrathecal final preparation buffer, N: number of animals, PND: days after birth, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: wild-type. [Figure 71]This study presents a semi-quantitative scoring of hepatic microvacuolation in twi / twi mice administered with rAAVhu68.hGALC or vehicle. twi / twi mice with PND12-14 were administered rAAVhu68.hGALC via ICV at doses of 6.8×10⁹GC, 2.0×10¹⁰GC, 6.8×10¹⁰GC, or 2.0×10¹¹GC. Twi / twi and WT mice of the same age were administered via ICV with vehicle (ITFFB) as a control. The surviving cohort was autopsied at the time of humane euthanasia, and livers were collected for histopathology. Hepatocyte vacuolation was semi-quantitatively scored as follows: Grade 0: No vacuoles, Grade 1: Minimal vacuoles, Grade 2: Mild vacuoles, Grade 3: Moderate vacuoles. Abbreviations: Galc: Galactosylceramidase (gene, mouse), GC: Genome copy, ICV: Intraventricular, ITFFB: Intrathecal final preparation buffer, N: Number of animals, PND: Days after birth, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: Wild type. [Figures 72A-72F]This report quantifies the size of IBA1-positive cells in the cortex and cerebellum of twi / twi mice administered rAAVhu68.hGALC or a vehicle. At PND12–14, twi / twi mice were administered rAAVhu68.hGALC via ICV at doses of 6.8×10⁹GC, 2.0×10¹⁰GC, 6.8×10¹⁰GC, or 2.0×10¹¹GC. Twi / twi and WT mice of the same age were administered a vehicle (ITFFB) via ICV as a control. Brains were collected at necropsy at PND40–42 and at humane euthanasia of the surviving cohort. Brains collected from untreated twi / twi and WT mice on the day of administration served as baseline controls. IBA1 immunohistochemistry was performed on tissue sections from the cortex—Figure 72A (baseline), Figure 72B (PND40 cohort), Figure 72C (survivors)—and on tissue sections from the cerebellum—Figure 72D (baseline), Figure 72E (PND40 cohort), Figure 72F (survivors). The size (mean area of ​​study) of individual IBA1-positive globoid cells was quantified using image analysis software. Error bars represent standard deviation. Based on comparisons between each group and the vehicle-treated twi / twi control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Abbreviations: Galc: Galactosylceramidase (gene, mouse), GC: Genome copy, IBA1: Ionized calcium-binding adapter molecule 1, ICV: Intraventricular, ITFFB: Intrathecal final preparation buffer, N: Number of animals, PND: Days after birth, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: Wild type. [Figures 73A-73F]This figure shows the quantification of IBA1-positive cell size in the brainstem and spinal cord of twi / twi mice administered with rAAVhu68.hGALC or vehicle. Figures 73A, 73B, and 73C provide baseline, PND40 cohort, and survival cohort for the brainstem, respectively. Figures 73D, 73E, and 73F provide baseline, PND40 cohort, and survival cohort for the spinal cord, respectively. In PND12-14, twi / twi mice were administered rAAVhu68.hGALC via ICV at doses of 6.8 × 10⁹ GC, 2.0 × 10¹⁰ GC, 6.8 × 10¹⁰ GC, or 2.0 × 10¹¹ GC. Twi / twi mice and WT mice of the same age were administered via ICV with vehicle (ITFFB) as a control. Brains and spinal cords were collected at autopsy of PND40-42 mice and at humane euthanasia of the survival cohort. Brains and spinal cords collected from untreated twi / twi and WT mice on the day of drug administration served as baseline controls. IBA1 immunohistochemistry was performed on tissue sections from the brainstem and spinal cord (cervical, lumbar, and thoracic). The size (mean area of ​​reference) of individual IBA1-positive globoid cells was quantified using image analysis software. Error bars represent standard deviation. Based on comparisons between each group and the vehicle-treated twi / twi control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, **p<0.01, ***p<0.001, ****p<0.0001. Abbreviations: Galc: Galactosylceramidase (gene, mouse), GC: Genome copy, IBA1: Ionized calcium-binding adapter molecule 1, ICV: Intraventricular, ITFFB: Intrathecal final preparation buffer, N: Number of animals, PND: Days after birth, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: Wild type. [Figures 74A-74C]This figure shows the quantification of IBA1-positive cell size in the sciatic nerve of twi / twi mice administered rAAVhu68.hGALC or vehicle. Figure 74A provides baseline results. Figure 74B provides results from the PND40 cohort. Figure 74C provides results from the survival cohort. In PND12–14, twi / twi mice were administered rAAVhu68.hGALC via ICV at doses of 6.8 × 10⁹ GC, 2.0 × 10¹⁰ GC, 6.8 × 10¹⁰ GC, or 2.0 × 10¹¹ GC. Twi / twi and WT mice of the same age were administered vehicle (ITFFB) via ICV as a control. Sciatic nerves were harvested at autopsy in PND40–42 and by humane euthanasia for the survival cohort. Sciatic nerves harvested from untreated twi / twi and WT mice on the day of administration served as baseline controls. IBA1 immunohistochemistry was performed on tissue sections from the sciatic nerve. The size (mean area of ​​study) of individual IBA1-positive globoid cells was quantified using image analysis software. Error bars represent the standard deviation. Based on comparisons between each group and the vehicle-treated twi / twi control group using one-way ANOVA and post-hoc Dunn multiple comparison tests, **p<0.01, ****p<0.0001. Abbreviations: Galc: galactosylceramidase (gene, mouse), GC: genome copy, IBA1: ionized calcium-binding adapter molecule 1, ICV: intraventricular, ITFFB: intrathecal final preparation buffer, N: number of animals, PND: days after birth, twi: Twitcher allele (consisting of loss-of-function mutations in the Galc gene), WT: wild type. [Figure 75] This report compares serum GALC activity in twitcher mice administered rAAVhu68 containing either a manipulated GALC (cGALCco) or a natural canine GALC (cGALnat) sequence. Improved survival was observed in twitcher mice administered rAAVhu.cGALCco compared to rAAVhu68 containing the natural sequence. [Figure 76]The progression of Krabbaine's neuropathology and behavioral phenotype are 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). A dashed line indicates that data for the previous point in time for the specified phenotype is not available. *An asterisk indicates demyelination observed histologically. Abbreviations: BAER: Brainstem auditory evoked response, CNS: Central nervous system, MRI: Magnetic resonance imaging, NCV: Nerve conduction velocity, PNS: Peripheral nervous system. [Figure 77] This paper outlines a study design for evaluating AAV.CB7.cGALCco.rBG gene therapy in Krabbaneu. [Figure 78] This shows the body weight of Krabbain dogs after ICM administration of rAAVhu68.cGALCco. At 2-3 weeks of age, Krabbain dogs received a single ICM dose of rAAVhu68.cGALCco (AAV) at either 3.0 × 10¹³ GC (N=4) or vehicle (ITFFB; N=2). Healthy wild-type littermates were administered vehicle as a control (N=1). Animal body weight was measured weekly. Abbreviations: AAVhu68.cGALCco: AAVhu68.CB7.CI.cGALCco.rBG, GC: genome copy, ICM: intracisional, ITFB: intrathecal final preparation buffer, N: number of animals, NCV: nerve conduction velocity. [Figures 79A-79D]This figure shows nerve conduction velocities in Krabbaine dogs after ICM administration of rAAVhu68.cGALCco. Figures 79A and 79B provide radial sensory NSV and ischial motor NCV. Figures 79C and 79D provide ulnar motor NCV and tibial motor NCV. At 2-3 weeks of age, Krabbaine dogs received a single ICM dose of rAAVhu68.cGALCco (AAV) at either 3.0 × 10¹³ GC (N=4) or vehicle (ITFFB; N=2). Healthy wild-type littermates were administered vehicle as a control (N=1). Nerve conduction studies were performed at the indicated ages. NCVs for the radial nerve (sensory nerve) and the sciatic, ulnar, and tibial nerves (motor nerves) are presented. Abbreviations: AAVhu68.cGALCco:AAVhu68.CB7.CI.cGALCco.rBG, GC: Genome copy, ICM: Intraciscana, ITFFB: Intrathecal final preparation buffer, N: Number of animals, NCV: Nerve conduction velocity. [Figure 80] This shows the BAER peak latency in Crabbeinu dogs after ICM administration of rAAVhu68.cGALCco. At 2-3 weeks of age, Crabbeinu dogs received a single ICM dose of rAAVhu68.cGALCco (AAV) at either 3.0 × 10¹³ GC (N=4) or vehicle (ITFFB; N=2). Healthy wild-type littermates were administered vehicle as a control (N=1). BAER was performed at the indicated ages. Central conduction time was defined as the time between the first and fifth peaks. A value of 0 indicates that no response was measured. Abbreviations: AAVhu68.cGALCco: AAVhu68.CB7.CI.cGALCco.rBG, BAER: Brainstem Auditory Evoked Response, GC: Genome Copy, ICM: Intraciscana Magnifica, ITFB: Intrathecal Final Form Buffer, N: Number of Animals [Figure 81]This report shows the BAER auditory threshold in Crabbeinu dogs after ICM administration of rAAVhu68.cGALCco. At 2-3 weeks of age, Crabbeinu dogs received a single ICM dose of rAAVhu68.cGALCco (AAV) at either 3.0 × 10¹³ GC (N=4) or vehicle (ITFFB; N=2). Healthy wild-type littermates were administered vehicle as a control (N=1). BAER auditory assessment was performed at the indicated ages. The auditory threshold was defined as the intensity of the sound at which the evoked waveform first appeared. A value of 0 indicates that no response was measured. Abbreviations: AAVhu68.cGALCco: AAVhu68.CB7.CI.cGALCco.rBG, BAER: Brainstem Auditory Evoked Response, GC: Genome Copy, ICM: Intraciscana Magnificent, ITFB: Intrathecal Final Form Buffer, N: Number of Animals. [Figures 82A-82D]This report presents the results of a brain MRI study in Krabbaine dogs after ICM administration of rAAVhu68.cGALCco. At 2-3 weeks of age, Krabbaine dogs received a single ICM dose of rAAVhu68.cGALCco (AAV) at either 3.0 × 10¹³ GC (N=4) or via vehicle (ITFFB; N=2). Healthy wild-type littermates were administered vehicle as a control (N=1). Brain MRI was performed when all animals were 8-10 weeks of age, and at 61 weeks of age in the long-term cohorts (K933 and K928). Semi-quantitative brain white matter intensity scores were assigned to the white matter of the internal capsule, corona radiata, corpus callosum, and occipital and cerebellar regions as follows: 0 = normal myelination (low intensity signal), 1 = suboptimal myelination (isointensity signal), 2 = demyelination (high intensity signal). Figure 82A shows brain MRI scores—high signal intensity in the white matter of individual treated animals and vehicle controls. Figure 82B shows MRI images from vehicle-treated krabbe animals showing high signal intensity white matter. Figure 82C shows MRI images from rAAVhu68.cGalCco-treated krabbe animals showing isointensity white matter. For each animal, the cumulative white matter intensity score across all brain regions is presented. Figure 82D shows measurements of the mass intermedia (PSC) diameter. Abbreviations: AAVhu68.cGALCco: AAVhu68.CB7.CI.cGALCco.rBG, GC: genome copy, ICM: cisterna magna, ITFFB: intrathecal final formulation buffer, MRI: magnetic resonance imaging, N: number of animals. [Figure 83] This study shows psychosine levels in CSF in Crabbeinu dogs after ICM administration of rAAVhu68.cGALCco. At 2-3 weeks of age, Crabbeinu dogs received a single ICM dose of rAAVhu68.cGALCco (AAV) at either 3.0 × 10¹³ GC (N=4) or vehicle (ITFFB; N=2). Healthy wild-type littermates were administered vehicle as a control (N=1). CSF samples were collected throughout the study for quantification of sphingoglycolipids. Abbreviations: AAVhu68.cGALCco: AAVhu68.CB7.CI.cGALCco.rBG, GC: genome copy, ICM: intracisional endothelial cell, ITFB: intrathecal final preparation buffer, MRI: magnetic resonance imaging, N: number of animals. [Figure 84] This document shows 12 stained images of myelin formation and globoid cells in the brain and peripheral nerves of Crabbine after ICM administration of rAAVhu68.cGALCco. The first column shows stained images from Crabbine treated with vehicle at 2 months of age. The second column shows stained images from Crabbine treated at 9 months of age. The third column shows stained images from Crabbine treated at 6 months of age. The first row shows stained images from the corpus callosum. The second row shows the cerebellum. The third row shows the spinal cord. The fourth row shows the peripheral nerves. At 2-3 weeks of age, Crabbine received a single ICM dose of AAVhu68.cGALCco (AAV) at either 3.0 × 10¹³ GC (animal K937, K938) or vehicle (ITFFB; animal K930). During autopsy, CNS and PNS tissues were collected for LFB / PAS staining to visualize myelin (blue staining) and globoid cells. Representative images of the brain (corpus callosum), cerebellar gyrus, spinal cord, and sciatic nerve from one vehicle-treated animal (animal K930, 35 days post-necropsy) and two AAV-treated animals (animal K938, 181 days post-necropsy; animal K937, 261 days post-necropsy) are presented. Abbreviations: AAVhu68.cGALCco:AAVhu68.CB7.CI.cGALCco.rBG, CNS: central nervous system, GC: genome copy, ICM: cisterna magna, ITFFB: intrathecal final preparation buffer, N: number of animals, PAS: periodate Schiff, PNS: peripheral nervous system. [Figures 85A-85F]This shows semi-quantitative scoring of demyelination and globoid cell infiltration in the nervous system of Krabbine after ICM administration of rAAVhu68.cGALCco. Figures 85A, 85B, and 85C show demyelination in the brain, spinal cord, and peripheral nerves, respectively. Figures 85D, 85E, and 85F show globoid cell infiltration in the brain, spinal cord, and peripheral nerves, respectively. At 2-3 weeks of age, Krabbine received a single ICM dose of rAAVhu68.cGALCco (AAV) at either a dose of 3.0 × 10¹³ GC (animals K937, K938, K939) or on a vehicle (ITFFB, animals K930, K948). Necropsies of vehicle-treated animals were performed on day 35 (animal K930) and day 66 (animal K948). Autopsies of AAV-treated animals were performed on day 180±3 (animals K938, K939) and day 261 (animal K937). During necropsy, the brain, spinal cord (cervical, lumbar, and thoracic), and peripheral nerves (optic nerve, sciatic nerve, median nerve, peroneal nerve, radial nerve, tibial nerve, and ulnar nerve) were collected for LFB / PAS staining. Histology was scored on a 4-point graded severity scale ranging from 1 (normal myelin formation and absence of globoid cells) to 4 (minimal to no myelin formation and diffuse globoid cell infiltration). Abbreviations: AAVhu68.cGALCco:AAVhu68.CB7.CI.cGALCco.rBG, GC: genome copy, ICM: cisterna magna, ITFFB: intrathecal final preparation buffer, N: number of animals, PAS: periodate schiff. [Figures 86A-86B]This study shows neuroinflammation and globoid cell accumulation in the nervous system of Krabbine after ICM administration of rAAVhu68.cGALCco. At 2-3 weeks of age, Krabbine received a single ICM dose of AAVhu68.cGALCco (AAV) at either 3.0 × 10¹³ GC (animals K937, K938, K939) or vehicle (ITFFB, animals K930, K948). Necropsies of vehicle-treated animals were performed on day 35 (animal K930) and day 66 (animal K948). Necropsies of AAV-treated animals were performed on day 180±3 (animals K938, K939) and day 261 (animal K937). Brains were collected for IBA1 immunohistochemistry during necropsies. Globoid cell size was measured using image analysis software. Abbreviations: AAVhu68.cGALCco:AAVhu68.CB7.CI.cGALCco.rBG, GC: genome copy, ICM: intracisional cisternae, ITFFB: intrathecal final preparation buffer, N: number of animals, PAS: periodate schiff. [Figures 87A-87B] This study shows neuroinflammation and globoid cell accumulation in the spinal cord of Crabbine after ICM administration of rAAVhu68.cGALCco. At 2-3 weeks of age, Crabbine received a single ICM dose of rAAVhu68.cGALCco (AAV) at either 3.0 × 10¹³ GC (animals K937, K938, K939) or vehicle (ITFFB, animals K930, K948). Necropsies of vehicle-treated animals were performed on day 35 (animal K930) and day 66 (animal K948). Necropsies of AAV-treated animals were performed on day 180±3 (animals K938, K939) and day 261 (animal K937). At necropsy, spinal cord (cervical, lumbar, and thoracic) was collected for LFB / PAS staining. Globoid cell size was measured using image analysis software. Abbreviations: AAVhu68.cGALCco:AAVhu68.CB7.CI.cGALCco.rBG, GC: genome copy, ICM: intracisional cisternae, ITFFB: intrathecal final preparation buffer, N: number of animals, PAS: periodate schiff. [Figure 88A-88D]Figures 88A and 88B show the GALC activity in CSF and serum of Krabbaine dogs after ICM administration of rAAVhu68.cGALCco. Figures 88C and 88D show the GALC activity in CSF of AAV-treated animals and vehicle-treated animals, respectively. At 2-3 weeks of age, Krabbaine dogs received a single ICM dose of rAAVhu68.cGALCco (AAV) at either a dose of 3.0 × 10¹³ GC (N=4) or vehicle (ITFFB; N=2). Healthy wild-type littermates were administered vehicle as a control (N=1). At the instructed post-treatment time, CSF and serum were collected from all animals, and fluorescence-based GALC activity assays were performed to evaluate the expression of the transgene product. The dotted lines in each graph represent the mean activity level of wild-type GALC in CSF (upper graph - Figures 88A and 88B) or serum (lower graph - Figures 88C and 88D) of animals K928 during 18 months of follow-up. Abbreviations: AAVhu68.cGALCco:AAVhu68.CB7.CI.cGALCco.rBG, CSF: cerebrospinal fluid, FU: fluorescent unit, GALC: galactosylceramidase (protein), GC: genome copy, ICM: cisterna magna, ITFFB: intrathecal final preparation buffer, N: number of animals. [Figures 89A-89G]Figures 89A-89D show GALC activity in the central nervous system of Krabbaine after ICM administration of rAAVhu68.cGALCco. Brain: Figures 89A-89D provide results of GALC activity in the cerebellum (Figure 89A), frontal cortex (Figure 89B), medulla oblongata (Figure 89C), or occipital cortex (Figure 89D). Spinal cord: Figures 89E-89G provide GALC activity in the cervical spinal cord (Figure 89E), thoracic spinal cord (Figure 89F), or lumbar spinal cord (Figure 89G). At 2-3 weeks of age, Krabbaine received a single ICM dose of rAAVhu68.cGALCco (AAV) at either a dose of 3.0 × 10¹³ GC (animal K937, K938, K939) or vehicle (ITFFB, animal K930, K948). Autopsies of vehicle-treated animals were performed on day 35 (animal K930) and day 66 (animal K948). Autopsies of AAV-treated animals were performed on day 180±3 (animals K938, K939) and day 261 (animal K937). During autopsies, brain and spinal cord tissue as specified was collected for fluorescence-based GALC activity assays, and the expression of the transgene product was evaluated. Abbreviations: AAVhu68.cGALCco:AAVhu68.CB7.CI.cGALCco.rBG, FU: fluorescence unit, GALC: galactosylceramidase (protein), GC: genome copy, ICM: intracisional, ITFFB: intrathecal final preparation buffer, N: number of animals. [Figures 90A-90D]Figure 90A shows GALC activity in the peripheral nervous system of Krabbaine after ICM administration of rAAVhu68.cGALCco, measured by fluorescence units (FU) / 50 μg. Figure 90B shows GALC activity in the cervical vertebrae of the dorsal root ganglia (DRG). Figure 90C shows GALC activity in the lumbar vertebrae of the DRG. Figure 90C shows GALC activity in the sciatic nerve. Figure 90D shows GALC activity in the median nerve. At 2-3 weeks of age, Krabbaine received a single ICM dose of rAAVhu68.cGALCco (AAV) at either a dose of 3.0 × 10¹³ GC (animals K937, K938, K939) or on a vehicle (ITFFB, animals K930, K948). Necropsies of vehicle-treated animals were performed on day 35 (animal K930) and day 66 (animal K948). Autopsies of AAV-treated animals were performed on day 180±3 (animals K938, K939) and day 261 (animal K937). During autopsy, specified peripheral nervous system tissue was collected for fluorescence-based GALC activity assays, and the expression of the transgene product was evaluated. Abbreviations: AAVhu68.cGALCco:AAVhu68.CB7.CI.cGALCco.rBG, DRG: dorsal root ganglion, FU: fluorescence unit, GALC: galactosylceramidase (protein), GC: genome copy, ICM: cisterna magna, ITFFB: intrathecal final preparation buffer, N: number of animals. [Figure 91A-91E]The GALC activity in peripheral organs of Krabbaine after ICM administration of rAAVhu68.cGALCco, measured by fluorescence units (FU) / 50 μg, is shown. Figure 91A provides results in the heart. Figure 91B provides results in the kidney. Figure 91C provides results in the liver. Figure 91D provides results in the diaphragm. Figure 91E provides results in skeletal muscle (quadriceps femoris). At 2-3 weeks of age, Krabbaine received a single ICM dose of rAAVhu68.cGALCco (AAV) at either a dose of 3.0 × 10¹³ GC (animals K937, K938, K939) or on a vehicle (ITFFB, animals K930, K948). Necropsies of vehicle-treated animals were performed on day 35 (animal K930) and day 66 (animal K948). Autopsies of AAV-treated animals were performed on day 180±3 (animals K938, K939) and day 261 (animal K937). During autopsy, specified peripheral nervous system tissue was collected for fluorescence-based GALC activity assays, and the expression of the transgene product was evaluated. Abbreviations: AAVhu68.cGALCco:AAVhu68.CB7.CI.cGALCco.rBG, DRG: dorsal root ganglion, FU: fluorescence unit, GALC: galactosylceramidase (protein), GC: genome copy, ICM: cisterna magna, ITFFB: intrathecal final preparation buffer, N: number of animals. [Figure 92]This shows the in vivo tissue distribution of rAAVhu68.cGALCco after ICM administration in Krabbaine. At 2-3 weeks of age, Krabbaine received a single ICM dose of rAAVhu68.cGALCco (AAV) at either 3.0 × 10¹³ GC (animals K937, K938, K939) or via vehicle (ITFFB, animals K930, K948). Necropsies of vehicles treated with vehicle were performed on day 35 (animal K930) and day 66 (animal K948). Necropsies of AAV-treated animals were performed on day 180±3 (animals K938, K939) and day 261 (animal K937). Tissue samples were collected during necropsies to determine the in vivo distribution. Abbreviations: AAVhu68.cGALCco:AAVhu68.CB7.CI.cGALCco.rBG, DRG: Dorsal root ganglion, FU: Fluorescent unit, GALC: Galactosylceramidase (protein), GC: Genome copy, ICM: Intraciscana, ITFFB: Intrathecal final preparation buffer, N: Number of animals. [Figure 93] This shows a typical median nerve SNAP recorded from the second finger of a healthy NHP. Sensory nerve conduction velocity was calculated by dividing the physical distance between the stimulus cathode and the recording site on the second finger by the onset latency (i.e., the time between the stimulus and the onset of the SNAP). SNAP amplitude was calculated as the difference between the potential at the start of the SNAP and the SNAP peak. Abbreviations: NHP: Non-human primate, SNAP: Sensory nerve action potential. [Figures 94A-94B]Figure 94A shows SNAP amplitude and nerve conduction velocity after ICM administration of rAAVhu68.hGALC to NHP (90-day cohort), with results presented as graphs of μV against study days. Figure 94A provides SNAP amplitude results graphed from the right median nerve (left side) and left median nerve (right side). Figure 94B provides nerve conduction velocity graphs from the right median nerve (left side) and the left median nerve (right side). Young NHPs received a single ICM dose of either the vehicle (ITFFB, N=1) or rAAVhu68.hGALC at doses of 4.5 × 10¹² GC (low dose), 1.5 × 10¹³ GC (medium dose), or 4.5 × 10¹³ GC (high dose) (N=3 / group). Sensory nerve conduction studies were performed on days BL and 28±3, 60±3, 90±4, and 120±4. The SNAP amplitude and conduction velocity of the left and right median nerves are presented. For SNAP amplitude, the shaded area (17.1–92.3 μV) represents values ​​within two standard deviations of the baseline mean of all animals in the study. Abbreviations: BL: baseline, GC: genome copy, ICM: cisterna magna, ITFFB: intrathecal final preparation buffer, N: animal number, NHP: non-human primate, SNAP: sensory nerve action potential. [Figures 95A-95B]Figure 95A shows SNAP amplitude and nerve conduction velocity after ICM administration of rAAVhu68.hGALC to NHP patients (180-day cohort), with results presented as a graph of μV against study days. Figure 95A provides SNAP amplitude results from graphs of the right median nerve (left side) and left median nerve (right side). Figure 95B provides nerve conduction velocity graphs from the right median nerve (left side) and the left median nerve (right side). Young NHP patients received a single ICM dose of either the vehicle (ITFFB, N=1) or rAAVhu68.hGALC at doses of 4.5 × 10¹² GC (low dose), 1.5 × 10¹³ GC (medium dose), or 4.5 × 10¹³ GC (high dose) (N=3 / group). Sensory nerve conduction studies were performed at BL and on days 28±3, 60±3, 90±4, and 120±4. The SNAP amplitude and conduction velocity of the left and right median nerves are presented. For SNAP amplitude, the shaded area (17.1–92.3 μV) represents values ​​within two standard deviations of the baseline mean of all animals in the study. Abbreviations: BL: baseline, GC: genome copy, ICM: cisterna magna, ITFFB: intrathecal final preparation buffer, N: animal number, NHP: non-human primate, SNAP: sensory nerve action potential. [Figures 96A-96B] This shows the white blood cell count in the cerebrospinal fluid of NHP patients after ICM administration of rAAVhu68.hGALC or vehicle. Young NHP patients received a single ICM dose of either vehicle (ITFFB, N=1 / group) or rAAVhu68.hGALC at doses of 4.5 × 10¹² GC (low dose), 1.5 × 10¹³ GC (medium dose), or 4.5 × 10¹³ GC (high dose) (N=3 / group). Cerebrospinal fluid (CSF) was collected on days 0, 7±1, 14±2, 28±3, 60±3, 90±4, 120±4, 150±4, and 180±5. White blood cell count was quantified as the number of WBCs per μl of CSF. Abbreviations: CSF: Cerebrospinal fluid, GC: Genome copy, ID: Identification number, ICM: Intracerebral cisterna magna, ITFFB: Intrathecal final preparation buffer, N: Animal count, NHP: Non-human primate, WBC: White blood cell. [Figures 96C-96D]Safety monitoring of CSF and sensory neurons in sham-treated and wild-type dogs, as well as in CSF-treated [Figures 97A-97B] The figures show the body weight of NHP patients after ICM administration of rAAVhu68.hGALC on day 90 (Figure 97A) or day 180 (Figure 97B). Young NHP patients received a single ICM dose of either vehicle (ITFFB, N=1 / group) or rAAVhu68.hGALC at a dose of 4.5 × 10¹² GC (low dose), 1.5 × 10¹³ GC (medium dose), or 4.5 × 10¹³ GC (high dose) (N=3 / group). Body weight was monitored at BL on days 0, 7±1, 14±2, 28±3, 60±3, 90±4, 120±4, 150±4, and 180±5. Abbreviations: BL: Baseline, GC: Genome copy, ICM: Intraciscanal, ITFFB: Intrathecal final preparation buffer, N: Animal number, NHP: Non-human primate, SNAP: Sensory nerve action potential. [Figures 98A-98C]The DRG neurodegeneration severity scores are shown for NHP after ICM administration of rAAVhu68.hGALC to NHP on day 90 (Figure 98A), day 180 (Figure 98B), or a combination of these (Figure 98C). Young NHPs received a single ICM dose of either vehicle (ITFFB, N=1 / group) or rAAVhu68.hGALC at doses of 4.5 × 10¹² GC (low dose), 1.5 × 10¹³ GC (medium dose), or 4.5 × 10¹³ GC (high dose) (N=3 / group). Severity grade scores for all ITFFB-treated and rAAVhu68.hGALC-treated animals necropped on days 90 and 180 are presented for each DRG segment (cervical, thoracic, and lumbar) for findings of neuronal cell body degeneration with mononuclear cell infiltration. For each DRG segment, the following scores were assigned: Severity Grade 1 = Minimum, Severity Grade 2 = Mild, Severity Grade 3 = Moderate, Severity Grade 4 = Severe (marked), Severity Grade 5 = Severe. Abbreviations: DRG: Dorsal root ganglion, GC: Genome copy, ICM: Intraciscana, ITFFB: Intrathecal final preparation buffer, N: Animal number, NHP: Non-human primate, TRG: Trigeminal ganglion. [Figures 99A-99C]The severity scores for spinal cord axonal damage after ICM administration of rAAVhu68.hGALC to NHPs are shown on day 90 (Figure 99A), day 180 (Figure 99B), or a combination of all scores (Figure 99C). Young NHPs received a single ICM dose of either vehicle (ITFFB, N=1 / group) or rAAVhu68.hGALC at doses of 4.5 × 10¹² GC (low dose), 1.5 × 10¹³ GC (medium dose), or 4.5 × 10¹³ GC (high dose) (N=3 / group). Severity scores for all ITFFB-treated and rAAVhu68.hGALC-treated animals necropped on days 90 and 180 are presented for axonal damage in the dorsal white matter tract of the spinal cord (cervical, thoracic, and lumbar segments). The following scores were assigned to each finding. Severity grade 1 = minimum, severity grade 2 = mild, severity grade 3 = moderate, severity grade 4 = severe, severity grade 5 = severe. Based on the Kriskall-Wallis test, followed by the Dunn test comparing each group with the vehicle-treated control group, *p<0.05. Abbreviations: GC: genome copy, ICM: cisterna magna, ITFFB: intrathecal final preparation buffer, N: animal number, NHP: non-human primates. [Figures 100A-100D]Figure 100A shows GALC enzyme activity in serum and CSF of NHP treated with rAAVhu68.hGALC or vehicle. Figure 100B shows a magnified view of day 14 for various doses. Figure 100C shows GALC CSF levels over the 180-day study. Figure 100D shows a magnified view of day 7 for various doses. Young NHP patients received a single ICM dose of either vehicle (ITFFB, N=1 / group) or rAAVhu68.hGALC at doses of 4.5 × 10¹² GC (low dose), 1.5 × 10¹³ GC (medium dose), or 4.5 × 10¹³ GC (high dose) (N=3 / group). CSF and serum were collected on the instructed day and analyzed for transgene product expression (GALC enzyme activity). In the graphs for serum on day 14 (Figure 100B) and for CSF on day 7 (Figure 100D), hollow shapes indicate animals that were negative for circulating serum NAb against the vector capsid at the time of treatment. White shapes indicate animals that were negative for circulating serum NAb against the vector capsid at the time of treatment. Error bars represent the standard deviation. Abbreviations: BL: Baseline, GALC: Galactosylceramidase (protein), GC: Genome copy, ICM: Intraciscanal, ITFFB: Intrathecal final preparation buffer, N: Number of animals, NAb: Neutral antibody, NHP: Non-human primate. [Figure 101A] This shows anti-human GALC antibodies in the CSF of NHP after ICM administration of rAAVhu68.hGALC. [Figure 101B]This shows anti-human GALC antibodies in the serum of NHPs after ICM administration of rAAVhu68.hGALC. Young NHPs received a single ICM dose of either a vehicle (ITFFB, N=1 / group) or rAAVhu68.hGALC at doses of 4.5 × 10¹² GC (low dose), 1.5 × 10¹³ GC (medium dose), or 4.5 × 10¹³ GC (high dose) (N=3 / group). CSF and serum were collected on the specified days, and antibodies against the transgene product (anti-human GALC antibodies) were measured by ELISA. Error bars represent standard deviation. Abbreviations: BL: baseline, ELISA: enzyme-linked immunosorbent assay, GALC: galactosylceramidase (protein), GC: genome copy, ICM: intracisional, ITFB: intrathecal final preparation buffer, N: number of animals, NHP: non-human primate. [Modes for carrying out the invention]

[0018] Provided are recombinant adeno-associated viruses (rAAV) expressing the human galactosylceramidase (GALC) protein, as well as compositions containing rAAV and their uses. In certain embodiments, rAAV.hGALC provides a disease-modifying treatment for symptomatic infantile Krabbe disease (EIKD). In certain embodiments, rAAV.hGALC provides a treatment for pre-symptomatic infantile patients. In certain embodiments, rAAV.hGALC provides a therapy that can correct peripheral nerve damage causing respiratory failure and loss of motor function. In certain embodiments, rAAV.hGALC provides an additional treatment option for late-onset patients for whom the benefit-risk ratio is not favorable for hematopoietic stem cell transplantation (HSCT), which is currently the only disease-modifying treatment.

[0019] As used herein, "rAAV.GALC" refers to an rAAV having an AAV capsid and a vector genome containing a coding sequence for at least a galactosylceramidase protein (enzyme) packaged therein. rAAVhu68.GALC refers to an rAAV whose AAV capsid is the AAVhu68 capsid, as defined herein. The following examples also illustrate other AAV capsids.

[0020] The term "cGALC" refers to the coding sequence that expresses canine GALC and is used in the following examples for research in dogs. Canine GALC has a 26 bp signal peptide and a protein with a full length of 669 amino acids.

[0021] The term "hGALC" refers to the code sequence of the human GALC.

[0022] Isoform 1 of hGALC is the canonical sequence, 685 amino acids long. Its amino acid sequence is reproduced in SEQ ID NO: 6. The mature protein is located approximately 43–685 amino acids, and the signal peptide is located 1–42, although some suggest that the start Met is at position 17, not position 1. Multiple isoforms of GALC (isoforms 1–5) are known, and over 36 native variants have been described. However, the inventors have found that a variant with 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 a protein sequence encoded by the coding sequence of human galactosylceramidase (hGALC), and is illustrated 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.

[0023] As used herein, “CB7.CI.hGALC.rBG” refers to a vector genome (e.g., shown in Figure 2) containing the coding sequence for human GALC under the control of a ubiquitous CB7 promoter, and containing at least the CMV IE (cytomegalovirus IE) enhancer, chimeric intron, and rabbit β-globin (rBG) poly(A) sequence, all of which are adjacent to the 5'ITR and 3'ITR. In certain embodiments, CB7.CI.hGALC.rBG contains the coding sequence for GALC encoding a mature GALC protein having the amino acid sequence of SEQ ID NO: 10. In certain embodiments, CB7.CI.hGALC.rBG contains the coding sequence for GALC containing the nucleic acid sequence of SEQ ID NO: 9, or a sequence that is 95% to 99.9% identical thereto. In yet another embodiment, the CB7.CI.hGALC.rBG vector genome contains SEQ ID NO: 19. In certain embodiments, CB7.CI .hGALC.rBG contains the coding sequence for the mature protein of SEQ ID NO: 10 and an exogenous signal peptide.

[0024] In certain embodiments, a fusion protein is envisioned that includes at least mature GALC (aa1-17, or aa1-42) in which all or part of the native signal peptide has been removed and replaced with an exogenous signal peptide. Such a fusion protein may also include the exogenous signal peptide and at least 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 suitable for human cells in the CNS (i.e., a signal peptide in which the native signal peptide has been replaced to improve the production, intracellular transport, and / or secretion of the protein (i.e., hGALC) in cells present in the human CNS). Suitable exogenous signal peptides for human CNS cells include, but are not limited to, naturally occurring secretory signal peptides of immunoglobulins (e.g., IgG), cytokines (e.g., IL-2, IL-12, IL-18, etc.), insulin, albumin, β-glucuronidase insulin, alkaline proteases, von Willebrand factor (VWF), or fibronectin (see, for example, www.signalpeptide.de / index.php?m=listspdb_mammalia).

[0025] Furthermore, the present invention also includes nucleic acid sequences encoding GALC proteins provided herein (e.g., SEQ ID NO: 6, SEQ ID NO: 10, or fusion proteins containing mature GALC). In certain embodiments, the coding sequence is a cDNA sequence encoding a protein. However, the corresponding RNA sequence is also included.

[0026] In certain embodiments, the nucleic acid coding sequence has the cDNA sequence of SEQ ID NO: 5, or a sequence that is 95% to 99.9% identical thereto, or a fragment thereof. Preferred fragments include the coding sequence of a mature protein (approximately nt127 to nt2058), or the coding sequence of a mature protein having a fragment of a signal peptide (e.g., approximately nt54 to nt2058). In certain embodiments, the coding sequence has a nucleic acid sequence encoding a fusion protein including the mature hGALC (nt127 to 2058) of SEQ ID NO: 5, or its leader or an exogenous leader, or a sequence that is 95% to 99.9% identical thereto. In certain embodiments, the coding sequence has a nucleic acid sequence (nt127 to 2058) encoding the mature hGALC of SEQ ID NO: 5, or a sequence that is 95% to 99.9% identical thereto, or a fragment thereof (including the leader sequence and a fragment of the 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 certain embodiments, the coding sequence encodes the hGALC reader (nucleic acids 1-126) and the mature protein (encoded by nucleic acids 127-2058) of sequence number 5.

[0027] In certain embodiments, the expression cassette further comprises one or more miRNA target sequences that suppress the expression of hGALC in the dorsal root ganglion (DRG). Such miRNA target sequences can be operably ligated to the hGALC coding sequence. Preferred miRNA target sequences are described in PCT / US19 / 67872, filed on December 20, 2019, entitled "Compositions for DRG-specific reduction of transgene expression." International Patent Application No. PCT / US19 / 67872 is incorporated herein by reference.

[0028] 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), an 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. Dependent. The enzyme deficiencies range from the most severe to the least severe, including early infantile Krabbe disease (EIKD), defined as onset before 6 months of age; late infantile Krabbe disease (LIKD), defined as onset between 7 and 12 months of age; juvenile Krabbe disease (JKD), defined as onset between 13 months and 10 years of age; and adolescent / adult-onset Krabbe disease.

[0029] In certain embodiments, an effective amount of rAAV.GALC vector increases the level of GALC enzyme in CSF to within approximately 30% to 100% of normal levels. In other embodiments, an effective amount of rAAV.GALC vector increases the level of GALC enzyme in plasma to within approximately 30% to 100% of normal levels. In certain embodiments, a lower amount of increased GALC in CSF or plasma levels is observed, but improvement is observed in one or more of the symptoms associated with Krabbe disease, as described herein.

[0030] "Recombinant AAV" or "rAAV" is a DNAse-resistant viral particle comprising two elements: an AAV capsid and a vector genome containing at least a non-AAV coding sequence packaged within the AAV capsid. Unless otherwise specified, this term may be used interchangeably with the phrase "rAAV vector." rAAV is a "replica-deficient virus" or "viral vector" because it lacks any functional AAV rep gene or functional AAV cap gene and is unable to produce offspring. In certain embodiments, the sole AAV sequence is an AAV inverted terminal repeat (ITR), typically located at the 5' and 3' ends of the vector genome to allow genes and regulatory sequences located between the ITRs to be packaged within the AAV capsid.

[0031] As used herein, “vector genome” refers to a nucleic acid sequence packaged inside the rAAV capsid that forms a viral particle. Such a nucleic acid sequence includes an AAV inverted terminal repeat (ITR). In the examples herein, the vector genome includes, at least, an AAV 5'ITR, a coding sequence, and an AAV 3'ITR from 5' to 3'. ITRs other than those from AAV2, AAV from a different source than the capsid, or full-length ITRs may be selected. In certain embodiments, the ITR is from the same AAV source as the AAV that provides rep function or trans-complementary AAV during production. Further other ITRs may be used. In addition, the vector genome includes regulatory sequences that direct the expression of gene products. Preferred components of the vector genome are discussed in more detail herein.

[0032] AAVhu68 As described in the following examples, the rAAV provided in the present invention comprises the AAVhu68 capsid. See, for example, WO2018 / 160582 (incorporated herein by reference). AAVhu68 is in lineage F. AAVhu68 (SEQ ID NO: 2) is distinct from AAV9 (SEQ ID NO: 4), a virus of another lineage F, by two encoded amino acids at positions 67 and 157 of vp1. In contrast, AAVs of other lineage F (AAV9, hu31, hu31) have Ala at position 67 and Ala at position 157.

[0033] rAAVhu68 consists of the AAVhu68 capsid and vector genome. In one embodiment, a composition comprising rAAVhu68 includes a collection of heterogeneous populations of vp1 protein, vp2 protein, and vp3 protein. Where used herein to refer to vp capsid proteins, the term “heterogeneous” or any grammatical variation thereof refers to a collection of non-identical elements having, for example, vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences. Sequence ID No. 2 provides the encoded amino acid sequence of the AAVhu68 vp1 protein. The AAVhu68 capsid contains subpopulations within the vp1 protein, vp2 protein, and vp3 protein, having modifications from the amino acid residues predicted in Sequence ID No. 2. These subpopulations contain at least one specific deamidated asparagine (N or Asn) residue. For example, a particular subpopulation contains at least one, two, three, or four highly deamidated asparagine (N) positions in the asparagine-glycine pair of SEQ ID NO: 2, and optionally further contains other deamidated amino acids, where deamidation results in amino acid changes and other optional modifications. Various combinations of these and other modifications are described herein.

[0034] As used herein, a “subpopulation” of vp proteins means, unless otherwise specified, a group of vp proteins having at least one defined common feature and consisting of at least one group member and fewer members than all members of the reference group. For example, a “subpopulation” of vp1 proteins is, unless otherwise specified, at least one (1)vp1 protein in an assembled AAV capsid and fewer than all vp1 proteins. A “subpopulation” of vp3 proteins may be, unless otherwise specified, one (1)vp3 protein less than all vp3 proteins in an assembled AAV capsid. For example, vp1 proteins may be a subpopulation of vp proteins, vp2 proteins may be a separate subpopulation of vp proteins, and vp3 may be a further subpopulation of vp proteins in an assembled AAV capsid. In another example, the vp1, vp2, and vp3 proteins may comprise subpopulations having different modifications, for example, at least one, two, three, or four highly deamidated asparagines, such as asparagine-glycine pairs.

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

[0036] As provided herein, each deamidated N in SEQ ID NO: 2 may independently be aspartic acid (Asp), isoaspartic acid (isoAsp), aspartate, and / or an interconverted blend of Asp and isoAsp, or a combination thereof. Any preferred ratio of α- and isoaspartic acid may exist. For example, in certain embodiments, the ratio may 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) in SEQ ID NO: 2 may 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 may be interconverted via a common glutarimide intermediate). Any preferred ratio of α- and γ-glutamic acid may exist. For example, in certain embodiments, the ratio may be α:γ in an α:γ ratio of 10:1 to 1:10, α:γ in an α:γ ratio of approximately 50:50, or α:γ in an α:γ ratio of approximately 1:3, or another selected ratio.

[0037] Therefore, rAAVhu68 comprises subpopulations within the rAAVhu68 capsid of vp1, vp2, and / or vp3 proteins having deamidated amino acids, and at a minimum, at least one subpopulation containing at least one highly deamidated asparagine. In addition, other modifications are particularly distinct at selected aspartic acid (D or Asp) residue positions. This may include merization. In yet another embodiment, the modification may include amidation at the Asp position.

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

[0039] In certain embodiments, the AAVhu68 capsid is further characterized by one or more of the following: AAVhu68 capsid protein is produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 1-736, 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 SEQ ID NO: 1-736, an AAVhu68 vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 2-736, a vp2 protein produced from a sequence containing 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 SEQ ID NO: 2-736, and / or AAVhu68 is produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 2-736. The present invention includes a vp3 protein, a vp3 protein produced from a sequence containing 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, encoding the predicted amino acid sequence of at least approximately amino acids 203-736 of SEQ ID NO: 2.

[0040] Additionally, or alternatively, an AAV capsid is provided comprising, optionally, a heterogeneous population of vp1 protein containing valine at position 157, optionally, a heterogeneous population of vp2 protein containing valine at position 157, and optionally, a heterogeneous population of vp3 protein, wherein at least the vp1 and vp2 protein subpopulations contain valine at position 157 and optionally further contain glutamic acid at position 67, based on the vp1 capsid numbering of SEQ ID NO: 2. Additionally, or alternatively, AAVhu68 capsid is provided, which comprises a heterogeneous population of vp1 protein, which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2; a heterogeneous population of vp2 protein, which is the product of a nucleic acid sequence encoding at least approximately amino acids 138-736 of SEQ ID NO: 2; and a heterogeneous population of vp3 protein, which is the product of a nucleic acid sequence encoding at least amino acids 203-736 of SEQ ID NO: 2, wherein the vp1, vp2, and vp3 proteins include subpopulations having amino acid modifications.

[0041] The vp1, vp2, and vp3 proteins of AAVhu68 are typically expressed as alternative splicing 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 can be used alone to express the vp1, vp2, and vp3 proteins. Alternatively, this sequence may be co-expressed with one or more of the following: a nucleic acid sequence or complementary strand encoding the AAVhu68 vp3 amino acid sequence (approximately aa203-736) of SEQ ID NO: 2, which lacks the vp1 specific region (approximately aa1-aa137) and / or the vp2 specific region (approximately aa1-aa202); the corresponding mRNA or tRNA (approximately nt607-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 coding sequence and / or vp2 coding sequence may be co-expressed with one or more of the following: The code sequence can be co-expressed with a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence (approximately aa138-736) of SEQ ID NO: 2, which does not have the vp1 specific region (approximately aa1-137), or a complementary strand thereof, the corresponding mRNA or tRNA (e.g., nt412-22121 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 approximately aa138-736 of SEQ ID NO: 2.

[0042] As described herein, rAAVhu68 comprises an rAAVhu68 capsid produced in a production system that expresses the AAVhu68 nucleic acid sequence encoding the vp1 amino acid sequence of SEQ ID NO: 2, and optionally an additional nucleic acid sequence (e.g., a sequence encoding the vp3 protein that does not contain the vp1 and / or vp2 specific regions). rAAVhu68 obtained from production using a single nucleic acid sequence vp1 produces heterogeneous populations of vp1, vp2, and vp3 proteins. The rAAVhu68 capsid contains subpopulations within the vp1, vp2, and vp3 proteins and has modifications from the predicted amino acid residues of SEQ ID NO: 2. These subpopulations include at least a deamidated asparagine (N or Asn) residues. For example, the asparagine in the 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, for example, the corresponding mRNA or tRNA. In certain embodiments, the vp2 and / or vp3 proteins may be expressed from nucleic acid sequences different from vp1, additionally or alternatively, for example, to alter the ratio of vp proteins in a selected expression system. In certain embodiments, nucleic acid sequences are also provided that encode the AAVhu68 vp3 amino acid sequence of SEQ ID NO: 2 (approximately aa203 to 736) or a complementary strand, the corresponding mRNA or tRNA (approximately nt607 to nt2211 of SEQ ID NO: 1), or a complementary strand, without having the vp1-specific region (approximately aa1 to approximately aa137) and / or the vp2-specific region (approximately aa1 to approximately aa202). In certain embodiments, nucleic acid sequences are also provided that encode the AAVhu68 vp2 amino acid sequence (approximately aa138-736) of SEQ ID NO: 2, which does not have a vp1 specific region (approximately aa1-137), or a complementary strand, the corresponding mRNA, or tRNA (nt412-2211 of SEQ ID NO: 1).

[0044] However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 2 may be selected for use in the production of the rAAVhu68 capsid. In certain embodiments, the nucleic acid sequence has at least 70% to 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 99% identical to the nucleic acid sequence of SEQ ID NO: 1, or at least 70% to 99%,5%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 99% identical to the vp2 capsid protein (at least aa138 to 736) of SEQ ID NO: 2. In certain embodiments, the nucleic acid sequence is the nucleic acid sequence of approximately nt607 to approximately nt2211 of SEQ ID NO: 1, or has a sequence that is at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 99% identical to the sequence of approximately nt607 to approximately nt2211 of SEQ ID NO: 1, and encodes the vp3 capsid protein (approximately aa203 to 736) of SEQ ID NO: 2.

[0045] Designing nucleic acid sequences encoding this rAAVhu68 capsid, including DNA (genome or cDNA) or RNA (e.g., mRNA), is within the capabilities of those skilled in the art. In certain embodiments, nucleic acids encoding the AAVhu68 vp1 capsid protein The sequence is provided in Sequence ID No. 1. In other embodiments, a nucleic acid sequence 70% to 99.9% identical to Sequence 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%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% to 99.9% identical to Sequence ID No. 1. Such a nucleic acid sequence may be codon-optimized for expression in a selected system (i.e., cell type) and can be designed by various methods. This optimization may be performed using methods available online (e.g., GeneArt), published methods, or by companies providing 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. 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 fragments of the ORF may be modified. By using one of these methods, frequencies can be applied to any given polypeptide sequence to produce nucleic acid fragments of codon-optimized coding regions encoding the polypeptide. Several options are available for performing actual modifications to codons or for synthesizing codon-optimized coding regions designed as described herein. Such modifications or synthesis can be carried out using standard and routine molecular biological operations well known to those skilled in the art. In one approach, a series of complementary oligonucleotide pairs, each 80-90 nucleotides long and spanning the length of the desired sequence, are synthesized by standard methods. These oligonucleotide pairs are synthesized such that, upon annealing, a double-stranded fragment of 80–90 base pairs including the attached end is formed, for example, each oligonucleotide in the pair is synthesized to extend by 3, 4, 5, 6, 7, 8, 9, 10 or more bases beyond the region complementary to the other oligonucleotide in the pair.Each pair of oligonucleotides is designed to anneal to 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 aggregated single-stranded ends, then they are ligated together and cloned into a standard bacterial cloning vector, e.g., the TOPO® vector available from Invitrogen Corporation, Carlsbad, Calif. The constructs are then sequenced using standard methods. Several of these constructs, consisting of 5-6 fragments of the ligated 80-90 base pair fragments, i.e., fragments of approximately 500 base pairs, are prepared so that the entire desired sequence is represented in a series of plasmid constructs. The insertions in these plasmids are then cleaved with appropriate restriction enzymes and ligated together to form the final construct. The final constructs are then cloned into a standard bacterial cloning vector and sequenced. Additional methods will be readily apparent to those skilled in the art. Furthermore, gene synthesis is commercially readily available.

[0046] In certain embodiments, the asparagine (N) residues of the NG pairs in 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) routinely exhibit deamidation levels of over 70% across various lots, and in most cases, over 90%. Further asparagine residues (N94, N253, N270, N304, N409, N477, and Q599) also exhibit deamidation levels of up to approximately 20% across various lots. Deamidation levels were initially identified using trypsin digestion and validated with chymotrypsin digestion.

[0047] In certain embodiments, the rAAVhu68 capsid comprises a subpopulation of vp1, vp2, and / or vp3 capsid proteins of AAV having at least four asparagine (N) positions in the highly deamidated rAAVhu68 capsid protein. Morphologically, approximately 20–50% of NN pairs (excluding NNN triplets) exhibit deamidation. In certain embodiments, the first N is deamidated. In certain embodiments, the second N is deamidated. In certain embodiments, deamidation is approximately 15%–25%. Deamidation at position 259 Q of SEQ ID NO: 2 is approximately 8%–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, based on the numbering in Sequence ID No. 2, about 70% to about 75% of the D at position 297 of the vp1, vp2, and / or vp3 proteins of the AAVhu68 capsid is amidated. In certain embodiments, at least one Asp of vp1, vp2, and / or vp3 of the capsid isomerized to D-Asp. Such isomers are generally present in amounts of less than 1% of the Asp at one or more residue positions 97, 107, and 384, based on the numbering in Sequence ID No. 2.

[0049] In certain embodiments, rAAVhu68 has an AAVhu68 capsid containing vp1, vp2, and vp3 proteins and has subpopulations containing combinations of one, two, three, four or more deamidated residues at the positions shown in the table below. Deamidation of rAAV 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 coupled to a Q Exactive HF with a NanoFlex source (Thermo Fisher Scientific) and a Thermo UltiMate 3000 RSLC system (Thermo Fisher Scientific). MS data are acquired using the data-dependent top-20 method of the Q Exactive HF, dynamically selecting the most abundant and yet-to-be-sequenced precursor ions from a survey scan (200–2000 m / z). Sequencing was performed via higher-energy collisional dissociation fragmentation at a target value of 1e5 ions determined by predictive auto-increase control, and precursor isolation was performed with a 4 m / z window. Investigational scans were acquired at a resolution of 120,000 at m / z 200. The HCD spectral resolution may be set to 30,000 at m / z 200 with a maximum ion injection time of 50 ms and a normalized collision energy of 30. The S-lens RF level can be set to 50 to optimize the transmittance in the m / z region occupied by peptides from the digest. Precursor ions can be excluded as single, unassigned, or in six or more charge states from fragmentation selection. BioPharma Finder 1.0 software (Thermo Fischer Scientific) can be used for analysis of acquired data. For peptide mapping, searches are performed using a single-entry protein FASTA database with carbamide methylation set as the fixed modification, oxidation, deamide, and phosphorylation set as variable modifications, 10 ppm mass precision, high protease specificity, and a confidence level of 0.8 for MS / MS spectra. Suitable proteases may include, for example, trypsin or chymotrypsin.Since deamidation adds 0.984 Da (the mass difference between the -OH and -NH2 groups) to the mass of the intact molecule, the mass spectrometric identification of deamidated peptides is relatively straightforward. 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 peptide and the native peptide. Considering the number of possible deamidation sites, isobars deamidated at different sites may co-migrate in a single peak. Therefore, multiple deamidation sites can be identified or distinguished using fragment ions derived from peptides with multiple potential deamidation sites. In these cases, the relative intensities within the observed isotopic pattern can be used to specifically determine the relative abundance of different deamidated peptide isomers. This method assumes that the fragmentation efficiency is the same for all isomer species and is independent of the deamidation site. It is established that several variations of these exemplary methods may be used, as will be understood by those skilled in the art. For example, suitable mass spectrometers may include, for example, quadruple-to-flight mass spectrometers (QTOF) such as the Waters Xevo or Agilent 6530, or orbitrap devices such as Orbitrap Fusion or Orbitrap Velos (Thermo Fisher). Preferably, 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), and Peaks DB (Bioinformatics Solutions). Further techniques can 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 by 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 amino acid sequence numbering 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 amino acid sequence numbering of SEQ ID NO: 2). In these and other embodiments, the AAVhu68 capsid is further characterized by having a population of proteins in which, based on the amino acid sequence numbering of SEQ ID NO: 2, about 1% to about 20% of N residues have deamidation at one or more of the positions N94, N253, N270, N304, N409, N477, and / or Q599. In certain embodiments, AAVhu68 comprises at least subpopulations of vp1, vp2, and / or vp3 proteins that are deamidated at one or more of the following positions, or combinations thereof, based on the amino acid sequence numbering of SEQ ID NO: 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, based on the amino acid sequence numbering of SEQ ID NO: 2. In certain embodiments, the capsid protein may have one or more amidated amino acids.

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

[0052] In certain embodiments, the rAAVhu68 capsid is a heterogeneous population of vp1 proteins, which are the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2, wherein the vp1 protein comprises glutamic acid (Glu) at position 67 and / or valine (Val) at position 157; optionally, a heterogeneous population of vp2 proteins comprising valine (Val) at position 157; and a heterogeneous population of vp3 proteins. Based on the residue numbering of the amino acid sequence of SEQ ID NO: 2, the AAVhu68 capsid comprises at least one subpopulation in which at least 65% of the asparagine (N) in the asparagine-glycine pair located at position 57 of the vp1 protein and at least 70% of the asparagine (N) in the asparagine-glycine pairs at positions 329, 452, and / or 512 of the vp1, v2, and vp3 proteins are deamidated, and deamidation results in an amino acid change.

[0053] As will be discussed in more detail herein, deamidated asparagine can be deamidated to aspartic acid, isoaspartic acid, interconverted aspartic acid / isoaspartic acid pairs, or combinations thereof. In certain embodiments, rAAVhu68 is further characterized by one or more of the following: (a) each of the vp2 proteins is independently the 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 the 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 a sequence identical to SEQ ID NO: 1, or 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%), and encoding the amino acid sequence of SEQ ID NO: 2. Optionally, the sequence is used alone to express the vp1, vp2, and vp3 proteins. Alternatively, this sequence may be co-expressed with one or more of the following: a nucleic acid sequence or complementary strand encoding the amino acid sequence (approximately aa203-736) of SEQ ID NO: 2 AAVhu68 vp3 that lacks the vp1-specific region (approximately aa1-aa137) and / or the vp2-specific region (approximately aa1-aa202); the corresponding mRNA or tRNA (approximately nt607-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 coding sequence and / or vp2 coding sequence may be co-expressed with a nucleic acid sequence or complementary strand encoding the AAVhu68 vp2 amino acid sequence (approximately aa138-736) of SEQ ID NO: 2 without the vp1 specific region (approximately aa1-137), the corresponding mRNA or tRNA (nt412-2211 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 approximately aa138-736 of SEQ ID NO: 2.

[0054] Additionally, or alternatively, the rAAVhu68 capsid contains at least a subpopulation of vp1, vp2, and / or vp3 proteins that are deamidated at one or more of the following positions, or combinations thereof, based on the numbering in SEQ ID NO: 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, N663, N709. (e)rAAVhu68 capsid contains 1% to 20% deamination at one or more of the following positions, or combinations thereof, based on the numbering of SEQ ID NO: N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N336, N409, N410, N477, N515, N598, Q599, N628, N651, N663, N709, or vp1, vp2, and / or vp3. (f) The rAAVhu68 capsid contains a subpopulation of the vp1 protein in which 65% to 100% of the N at position 57 of the vp1 protein is deamidated, based on the numbering in SEQ ID NO: 2; (g) The rAAVhu68 capsid contains a subpopulation of the vp1 protein in which 75% to 100% of the N at position 57 of the vp1 protein is deamidated; (h) The rAAVhu68 capsid contains a subpopulation of the vp1 protein in which 65% to 100% of the N at position 329 is deamidated, based on the numbering in SEQ ID NO: 2 (i) The rAAVhu68 capsid contains a subpopulation of vp1, vp2, and / or vp3 proteins in which 80% to 100% of N at position 452 is deaminated, and (j) The rAAVhu68 capsid contains a subpopulation of vp1, vp2, and / or vp3 proteins in which 80% to 100% of N at position 512 is deaminated, based on the numbering in SEQ ID NO: 2 (k)rAAV contains subpopulations of vp1, vp2, and / or vp3 proteins that are 0% to 100% deaminated, with (k)rAAV containing approximately 60 total capsid proteins in a ratio of approximately 1 vp1 protein: approximately 1 to 1.5 vp2 proteins: 3 to 10 vp3 proteins, and (l)rAAV containing approximately 60 total capsid proteins in a ratio of approximately 1 vp1 protein: approximately 1 vp2 protein: 3 to 9 vp3 proteins.

[0055] In certain embodiments, AAVhu68 is modified to reduce deamidation by changing the glycine in the asparagine-glycine pair. In other embodiments, asparagine is changed to a different amino acid, such as glutamine, which deamidates more slowly, 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, amino acids lacking an amide or amine side group refer to, for example, glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, phenylalanine, tyrosine, or tryptophan, and / or proline. The modifications described may be on one, two, or three of the asparagine-glycine pairs found in the amino acid sequence of the encoded AAVhu68. In certain embodiments, such modifications are not made on all four asparagine-glycine pairs. Therefore, a method is provided for reducing the deamidation of rAAVhu68 and / or modified rAAVhu68 variants having a lower deamidation rate. In addition, or alternatively, the deamidation of rAAVhu68 can be reduced by changing one or more other amide amino acids to non-amide amino acids.

[0056] These amino acid modifications can be performed by conventional genetic engineering techniques. For example, a nucleic acid sequence containing modified AAVhu68 vp codons may be generated, in which 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 containing modified asparagine codons may have one to three of the asparagine-glycine pairs located at positions 57, 329, 452, and / or 512 of SEQ ID NO: 2 manipulated to encode an amino acid other than asparagine. Each modified codon may encode a different amino acid. Alternatively, one or more of the modified codons may encode the same amino acid. In certain embodiments, these modified AAVhu68 nucleic acid sequences may be used to generate mutant rAAVhu68 having a capsid with lower deamidation than the natural hu68 capsid. Such mutant rAAVhu68 may reduce immunogenicity and / or increase stability during storage (especially in suspension form). As used herein, "codon" refers to a sequence encoding an amino acid. It refers to three nucleotides.

[0057] As used herein, “encoded amino acid sequence” refers to the amino acids predicted based on the translation of known DNA codons in a reference nucleic acid sequence that are translated into amino acids. The following table illustrates DNA codons and 20 common amino acids, showing both single-letter codes (SLC) and three-letter codes (3LC). [Table 2]

[0058] 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 patients undergoing gene therapy. Techniques for generating useful anti-AAVhu68 antibodies, labeling of such antibodies or empty capsids, and preferred assay formats are known to those skilled in the art.

[0059] In certain embodiments, the nucleic acid sequence of Sequence ID No. 1 is provided herein, or the details of this specification. The sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 99% of the sequence encoding the amino acid sequence of vp1 of SEQ ID NO: 2 having the modifications described in the document (e.g., deamidated amino acids). In certain embodiments, the vp1 amino acid sequence is reproduced in SEQ ID NO: 2.

[0060] As used herein, the term “clade” in relation to a group of AAVs refers to a group of AAVs that are systematically related to one another and are determined using a neighbor-joining algorithm based on the alignment of AAV vp1 amino acid sequences, with at least 75% bootstrap values ​​(out of at least 1000 replicates) and Poisson-corrected distance measurements of 0.05 or less. The neighbor-joining algorithm is described in the literature. For example, see Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York (2000)). Computer programs that can be used to implement this algorithm are available. For example, the MEGA v2.1 program implements the modified Nei-Gojobori method. Using these techniques and computer programs, as well as the sequences of AAV vp1 capsid proteins, those skilled in the art can easily determine whether a selected AAV belongs to one lineage group identified herein, another lineage group, or outside of these lineage groups. For example, see G Gao, et al, J Virol, 2004 Jun;78 (10:6381-6388), which identifies lineage groups A, B, C, D, E, and F (GenBank acceptance numbers AY530553~AY530629). Also see WO2005 / 033321.

[0061] As used herein, “AAV9 capsid” is a self-assembled AAV capsid consisting of multiple AAV9 vp proteins. The AAV9 vp proteins typically encode the vp1 amino acid sequence of SEQ ID NO: 4 (GenBank accession number AAS99264). These splice variants result in proteins of different lengths of SEQ ID NO: 4. In certain embodiments, “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. “AAV9 variants” as used herein include, for example, the variants described in WO2016 / 049230, US8,927,514, US2015 / 0344911, and US8,734,809.

[0062] Methods for generating capsids and therefore coding sequences, as well as methods for producing rAAV viral vectors, are described. See, for example, Gao, et al, Proc. Natl. Acad. Sci. USA100(10), 6081-6086(2003) and US2013 / 0045186A1.

[0063] The terms "substantial homology" or "substantial similarity," when referring to a nucleic acid or a fragment thereof, indicate that when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, the aligned sequence has at least about 95–99% nucleotide sequence identity. Preferably, the homology extends over the full-length sequence, or its open reading frame, or another suitable fragment of at least 15 nucleotides in length. Examples of suitable fragments are described herein.

[0064] In the context of nucleic acid sequences, the terms “sequence identity,” “sequence identity percentage,” or “identical percentage” refer to residues in two sequences that are identical when aligned to the greatest extent possible. The length of the sequence identity comparison can span the entire genome, the entire gene coding sequence, or, preferably, a fragment of at least approximately 500–5000 nucleotides. However, identity between smaller fragments of, for example, at least about 9 nucleotides, typically at least about 20-24 nucleotides, at least about 28-32 nucleotides, and at least about 36 or more nucleotides may also be desired. Similarly, the “sequence identity percentage” can be readily determined for the amino acid sequence over the entire length of the protein or for fragments thereof. Preferably, the fragments are at least about 8 amino acids long and may be up to about 700 amino acids long. Examples of suitable fragments are described herein.

[0065] The terms "substantial homology" or "substantial similarity," when referring to a nucleic acid or a fragment thereof, indicate that when optimally aligned with another nucleic acid (or its complementary chain) by appropriate amino acid insertions or deletions, the aligned sequence has at least about 95–99% amino acid sequence identity. Preferably, the homology extends over the full-length sequence or its protein, e.g., a cap protein, a rep protein, or a fragment thereof of at least 8 amino acids in length, or more preferably, at least 15 amino acids in length. Examples of suitable fragments are described herein.

[0066] The term "highly preserved" means at least 80% identity, preferably at least 90% identity, and more preferably more than 97% identity. Identity can be readily determined by those skilled in the art using algorithms and computer programs known to those skilled in the art.

[0067] Generally, when referring to “identity,” “homology,” or “similarity” between two different adeno-associated viruses, “identity,” “homology,” or “similarity” is determined by referring to “aligned” sequences. “Aligned” sequences or “alignment” refer to multiple nucleic acid sequences or protein (amino acid) sequences that, compared to a reference sequence, often include corrections for missing or additional bases or amino acids. In the examples, AAV alignment is performed using a publicly available AAV9 sequence as a reference point. Alignment is performed using various multiple sequence alignment programs, either public or commercial. 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 skilled in the art. Alternatively, the Vector NTI utility can also be used. Furthermore, several algorithms known in the art, including those included in the programs described above, can be used to measure nucleotide sequence identity. As another example, polynucleotide sequences can be compared using Fasta®, a program from GCG version 6.1. Fasta® provides the best alignment of overlapping regions and sequence identity percentage between query sequences and search sequences. For example, the sequence identity percentage between nucleic acid sequences can be determined using Fasta® with its default parameters (word size 6 and NOPAM factor for the scoring matrix), as provided in GCG version 6.1 (incorporated herein by reference). Multiple sequence alignment programs such as "Clustal Omega," "Clustal X," "MAP," "PIMA," "MSA," "BLOCKMAKER," "MEME," and "Match-Box" are also available for amino acid sequences. Generally, one of these programs is used with its default settings, but those skilled in the art can modify these settings as needed.Alternatively, those skilled in the art may utilize other algorithms or computer programs that provide at least a level of identity or alignment, as provided by the algorithms and programs of reference. See, for example, JDThomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999). .

[0068] rAAV vector As described above, the AAVhu68 sequence and protein are useful for rAAV production and also for recombinant AAV vectors (which may be antisense delivery vectors, gene therapy vectors, or vaccine vectors). Furthermore, the engineered AAV capsids described herein (e.g., AAV capsids having mutant 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 the delivery of several suitable nucleic acid molecules to target cells and tissues.

[0069] The genomic sequence packaged in the AAV capsid and delivered to the host cell typically consists of at least a transgene, its regulatory sequence, and an AAV inverted terminal repeat (ITR). Both single-stranded AAV and self-complementary (sc)AAV are included in rAAV. The transgene is a nucleic acid coding sequence heterogeneous to the vector sequence encoding the target polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor), or other gene product.

[0070] In particular, this disclosure provides rAAVs comprising a coding sequence for 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 the GALC gene (GALCco) of SEQ ID NO: 9. In certain embodiments, the GALC coding sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 9.

[0071] The nucleic acid coding sequence is operably linked to regulatory elements in a manner that allows for transcription, translation, and / or expression of the transgene in the cells of the target tissue. In some embodiments, the regulatory sequences include a beta-actin promoter, an intron, and a rabbit globin polyA. In some embodiments, the regulatory sequence has SEQ ID NO: 13. In some embodiments, the regulatory sequence has SEQ ID NO: 15. In some embodiments, the regulatory sequence has SEQ ID NO: 16.

[0072] The AAV sequences of the vector typically include cis - acting 5' and 3' inverted terminal repeat sequences (see, e.g., B.J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 - 168 (1990)). The ITR sequences are about 145 bp in length. Preferably, the entire sequences that substantially encode the ITRs are used within the molecule, although some minor modifications of these sequences are tolerated. The ability to modify these ITR sequences is within the ability of those skilled in 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)). An example of such a molecule utilized in the present invention is a “cis - acting” plasmid that contains a transgene in which the selected transgene sequence and associated regulatory elements are adjacent to 5' and 3' AAV ITR sequences. In one embodiment, the ITR is derived from AAV different from that which supplies the capsid. In one embodiment, it is an ITR sequence derived from AAV2. A shortened version of the 5' ITR, designated ΔITR, in which the D sequence and the terminal resolution site (trs) are deleted, has been described. In other embodiments, full - length AAV 5' and 3' ITRs are used. However, ITRs from other AAV sources may be selected. If the source of the ITR is from AAV2 and the AAV capsid is from another AAV source, the resulting vector can be referred to as pseudotyped. However, other configurations of these elements may also be suitable. In certain embodiments, the vector genome contains a 130 - base - pair shortened AAV2 ITR in which the external “a” element is deleted. The shortened ITR is used as a template for the internal A element and is restored to the wild - type length of 145 base pairs during vector DNA amplification. In other embodiments, full - length AAV 5' and 3' ITRs are used.

[0073] In addition to the key elements described above for recombinant AAV vectors, the vector also includes necessary conventional regulatory elements that are operably linked to the transgene in such a manner that they enable its transcription, translation, and / or expression in cells transfected with a plasmid vector or infected with a virus produced by the present invention. As used herein, “operably linked” sequences include both expression regulatory sequences adjacent to the gene of interest and expression regulatory sequences that act trans or asynchronously to control the gene of interest.

[0074] The regulatory element typically includes, for example, a promoter sequence located between a selected 5'ITR sequence and a coding sequence as part of the expression control sequence. Constitutive promoters, regulated promoters [see, e.g., WO2011 / 126808 and WO2013 / 04943], tissue-specific promoters, or promoters responsive to physiological hints may be used in the vectors described herein. The promoter can be selected from different sources, such as the human cytomegalovirus (CMV) early enhancer / promoter, SV40 early enhancer / promoter, JC polymomavirus promoter, myelin basic protein (MBP) or collagen fiber acidic protein (GFAP) promoter, herpes simplex virus (HSV-1) latent-associated promoter (LAP), Roussarcoma virus (RSV) long-terminal repeat (LTR) promoter, neuron-specific promoter (NSE), platelet-derived growth factor (PDGF) promoter, hSYN, melanin-concentrating hormone (MCH) promoter, CBA, matrix metalloprotein promoter (MPP), and chicken beta-actin promoter. In addition to the promoter, the vector may include one or more other suitable transcription start, termination, and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals, sequences that stabilize cytoplasmic mRNA, e.g., WPRE, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and, optionally, sequences that enhance the 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 one another. For example, the enhancer may include a CMV initial enhancer. This enhancer may be present in two copies located adjacent to each other. Alternatively, the duplicate copies of the enhancer are separated by one or more sequences.In yet another embodiment, the expression cassette further comprises introns, such as chicken beta-actin introns. Other suitable introns include those known in the art, described, for example, 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. An example of such a sequence is a modified WPRE sequence, which can be manipulated 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).

[0075] These rAAVs are particularly suitable for gene delivery for therapeutic and immunization purposes, including inducing protective immunity. Furthermore, the compositions of the present invention can also be used to produce a desired gene product in vitro. For in vitro production, the desired product (e.g., a protein) is transfected with host cells using rAAVs containing molecules encoding the desired product. After transfection and culturing the cell culture under conditions that enable expression, the desired product can be obtained from the culture. 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.

[0076] rAAV vector production For use in the production of AAV virus vectors (e.g., recombinant (r)AAV), the expression cassette can be supported on any suitable vector, e.g., a plasmid, to be 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 cells, insect cells, and mammalian cells, among others. Suitable transfection techniques and packaging host cells are known and / or can be readily designed by those skilled in the art.

[0077] Methods for generating and isolating AAVs suitable for use as vectors are known in the art. Generally, for example, see Grieger & Samulski, 2005, “Adeno-associated virus as a gene therapy vector: Vector development, production See “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 these is incorporated herein by reference in its entirety). To package the gene into a virion, the ITR is the only AAV component required in cis in the same construct as the nucleic acid molecule containing the expression cassette. The cap and rep genes may be supplied trans.

[0078] In one embodiment, the expression cassette described herein is engineered into a genetic element (e.g., a shuttle plasmid) that introduces an immunoglobulin construct sequence carried thereon into a packaging host cell in order to produce a viral vector. In one embodiment, the selected genetic element may be delivered to AAV packaging cells by any preferred method, including transfection, electroporation, liposome delivery, membrane fusion techniques, fast DNA-coated pellets, viral infection, and protoplast fusion. Stable AAV packaging cells can also be produced. Alternatively, the expression cassette may be used to generate viral vectors other than AAV, or for the production of antibody mixtures in vitro. Methods used to construct such constructs are known to technicians 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).

[0079] The term “AAV intermediate” or “AAV vector intermediate” refers to an assembled rAAV capsid that lacks the desired genomic sequence packaged within it. These may also be referred to as “empty” capsids. Such capsids may contain no detectable genomic sequence at all for the expression cassette, or only a partially packaged genomic sequence that is insufficient to achieve gene product expression. These empty capsids are nonfunctional for introducing the gene of interest into host cells.

[0080] The recombinant adeno-associated viruses (AAVs) described herein may be produced using known techniques. See, for example, WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, and US7588772B2. Such methods include a nucleic acid sequence encoding the AAV capsid protein, a functional rep gene, and at a minimum, the AAV itself. The method involves culturing an expression cassette consisting of an inverted terminal repeat (ITR) and a transgene, and host cells having sufficient helper function to allow packaging of the expression cassette into an AAV capsid protein. Methods for generating the capsid, coding sequences therefor, and methods for producing an rAAV viral vector are described. For example, Gao, et al. See al., Proc.Natl.Acad.Sci.USA100(10),6081-6086(2003) and US2013 / 0045186A1.

[0081] In one embodiment, a production cell culture useful for producing recombinant rAAVhu68 is provided. Such a cell culture comprises a nucleic acid expressing the rAAVhu68 capsid protein in a host cell, a nucleic acid molecule suitable for packaging into the rAAVhu68 capsid (e.g., a vector genome containing AAV ITR), and a non-AAV nucleic acid sequence encoding a gene product operably linked to a sequence that directs the expression of the product in the host cell, as well as sufficient AAV rep and adenovirus helper functions to enable the packaging of the nucleic acid molecule into the recombinant AAVhu68 capsid. In one embodiment, the cell culture consists of mammalian cells (e.g., human embryonic kidney 293 cells in particular) or insect cells (e.g., baculovirus).

[0082] Preferably, the rep function is provided by an AAV derived from the same source as the ITR present in the vector genome, or from another source (e.g., AAVhu68) that packages the vector genome into the AAV capsid. In certain embodiments, the rep protein is derived from AAV2. In other embodiments, the rep protein is a heterologous rep protein other than AAVhu68rep, and may be, for example, 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 sequences that direct their expression in the producing cells.

[0083] In one embodiment, the cells are produced in a suitable cell culture (e.g., HEK293) of cells. The methods for producing the gene therapy vectors described herein include methods well known in the art, such as generating plasmid DNA used for the production of the gene therapy vector, generating the vector, and purifying the vector. In some embodiments, the gene therapy vector is an AAV vector, and the produced plasmids are an AAV cis-plasmid encoding the AAV genome and the gene of interest, an AAV trans-plasmid containing the rep and cap genes of AAV, and an adenovirus helper plasmid. The vector production process may include method steps such as initiating cell culture, subculturing cells, seeding cells, transfection of cells with plasmid DNA, changing the medium to serum-free medium after transfection, and recovering the cells and culture medium containing the vector. The recovered vector-containing cells and culture medium are referred to herein as crude cell recoveries. 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, for example, Zhang et al., 2009, “Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production,” Human Gene Therapy 20:922-929 (each of which is incorporated herein by reference in its entirety). Methods for constructing and using these and other AAV production systems are also referred to in the following U.S. Patents No. 5,139,941, 5,741,683, 6,057,152, 6,204,059, and 6, respectively, each of which is incorporated herein by reference in its entirety. It is described in publications No. 268,213, No. 6,491,907, No. 6,660,514, No. 6,951,753, No. 7,094,604, No. 7,172,893, No. 7,201,898, No. 7,229,823, and No. 7,439,065.

[0084] In certain embodiments, the production process for rAAV.hGALC involves transient transfection of HEK293 cells with plasmid DNA. Single or multiple batches are produced in a PALL iCELLis bioreactor by PEI-mediated triple transfection of HEK293 cells. The recovered AAV material is sequentially purified, where possible, in a disposable, closed bioprocessing system by clarification, TFF, affinity chromatography, and anion exchange chromatography.

[0085] The crude cell recovery can then be subjected to method steps such as concentration of the vector recovery, dialysfiltration of the vector recovery, microfluidization of the vector recovery, nuclease digestion of the vector recovery, filtration of the microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration for preparing a bulk vector.

[0086] The vector drug product is purified using a two-step high-salt affinity chromatography process, followed by anion exchange resin chromatography to remove empty capsids. These methods are described in detail in International Patent Application PCT / US2016 / 065970, filed on 9 December 2016, and its priority document, U.S. Patent Application No. 62 / 322,071, filed on 13 April 2016, and U.S. Patent Application No. 62 / 226,357, filed on 11 December 2015, entitled “Scalable Purification Method for AAV9,” which are incorporated herein by reference. For AAV8, see International Patent Application PCT / US2016 / 065976 filed on December 9, 2016, and its priority document, U.S. Patent Application No. 62 / 322,098 filed on April 13, 2016, and No. 62 / 266,341 filed on December 11, 2015, and for rh10, see International Patent Application PCT / US16 / 66013 filed on December 9, 2016, and its priority document, U.S. Patent Application No. 62 / 322,055 filed on April 13, 2016, and "Scalable Purification Method for Patent No. 62 / 266,347, titled "AAVrh10," and AAV1 are described in International Patent Application PCT / US2016 / 065974, filed on 9 December 2016, and its priority document, U.S. Patent Application No. 62 / 322,083, filed on 13 April 2016, and Patent No. 62 / 26,351, titled "Scalable Purification Method for AAV1," filed on 11 December 2015, all of which are incorporated herein by reference.

[0087] To calculate the content of empty and filled particles, the VP3 band volume for a selected sample (e.g., in the examples herein, a preparation purified with an iodixanol gradient, where GC = number of particles) is plotted against the loaded GC particles. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volume of the test peak. The number of particles per 20 μL loaded (pt) is then multiplied by 50 to obtain particles (pt) / mL. Divide pt / mL by GC / mL to obtain the ratio of particles to genome copies (pt / GC). Pt / mL ~ GC / mL gives the empty pt / mL. Divide empty pt / mL by pt / mL and then multiply by 100 to obtain the percentage of empty particles.

[0088] Generally, AAV vector particles containing an empty capsid and a packaged genome are used. Methods for performing this task are known in the relevant art. For example, Grimm et al. See al., Gene Therapy (1999) 6:1322-1330 and Sommer et al., Molec. Ther. (2003) 7:122-128. To test for denatured capsids, the method involves subjecting a treated AAV stock to SDS-polyacrylamide gel electrophoresis using any gel capable of separating three capsid proteins (e.g., a gradient gel containing 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). The anti-AAV capsid antibody is then used as a primary antibody that binds to the denatured capsid protein, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293). Next, a secondary antibody is used that binds to the primary antibody and includes means for detecting binding to an anti-IgG antibody containing a detection molecule covalently bound to the primary antibody, more preferably to the antibody, and most preferably to a sheep anti-mouse IgG antibody covalently bound to horseradish peroxidase. To semi-quantitatively determine the binding between the primary and secondary antibodies, a method for detecting binding is used, preferably a detection method capable of detecting radioisotope radiation, electromagnetic radiation, or a colorimetric change, most preferably a chemiluminescence detection kit. For example, in SDS-PAGE, a sample can be taken from a column fraction and heated in an SDS-PAGE packing buffer containing a reducing agent (e.g., DTT), and the capsid protein was degraded 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, namely SYPRO ruby ​​or Coomassie staining, may be performed. In one embodiment, the concentration of the AAV vector genome (vg) in the column fraction can be measured by quantitative real-time PCR (Q-PCR). The sample is diluted and DNase The sample is digested with I (or another suitable nuclease) to remove exogenous DNA. After nuclease inactivation, the sample is further diluted and amplified using a TaqMan® fluorescence-generating probe specific to the DNA sequences between primers and primers. The number of cycles required to reach a specified level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 sequence detection system. A standard curve for the Q-PCR reaction is created using plasmid DNA containing the same sequence as that contained in the AAV vector. The vector genome titer was determined by normalizing the cycle threshold (Ct) value obtained from the sample against the Ct value of the plasmid standard curve. Digital PCR-based endpoint assays may also be used.

[0089] 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 genome titer assay is similar to the standard assay, except that after DNase I digestion, the sample is diluted with protease K buffer, treated with protease K, and subsequently inactivated by heat. Preferably, the sample is diluted with an amount of protease K buffer equal to the sample size. The protease K buffer can be concentrated more than 2-fold. 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 processing step is generally carried out at approximately 55°C for approximately 15 minutes, but may be carried out at a lower temperature (e.g., approximately 37°C to approximately 50°C) for a longer period (e.g., approximately 20 minutes to approximately 30 minutes), or at a higher temperature (e.g., up to approximately 60°C) for a shorter period (e.g., approximately 5 to 10 minutes). Similarly, thermal inactivation is generally carried out at approximately 95°C for approximately 15 minutes, but may be performed at a lower temperature (e.g., approximately 70°C to approximately 90°C) and for a longer period (e.g., approximately 20 minutes to approximately 30 minutes). The sample is then diluted (e.g., 1000-fold) and performed as described in a standard assay. This will be subjected to TaqMan analysis.

[0090] Additionally, or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for measuring single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g., M. Lock et al, Hu Gene Therapy Methods. 2014 Apr;25(2):115-25. doi:10.1089 / hgtb.2013.131. Epub 2014 Feb See 14.

[0091] Briefly, a method for separating rAAVhu68 particles having a packaged genomic sequence from a genome-deficient AAVhu68 intermediate comprises subjecting a suspension comprising recombinant AAVhu68 virus particles and AAVhu68 capsid intermediates to high performance liquid chromatography, binding the AAVhu68 virus particles and AAVhu68 intermediates to a strong anion exchange resin equilibrated at a pH of 10.2, and subjecting to a salt gradient while monitoring the eluate for ultraviolet absorbance at about 260 and about 280. For rAAV9hu68, although less optimal, the pH can be in the range of about 10.0 - 10.4. In this method, the AAVhu68 full capsids are recovered from the fraction that elutes when the A260 / A280 ratio reaches the inflection point. In one embodiment, in the affinity chromatography step, the diafiltered product can 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 protein pass through the column while the AAV particles are efficiently captured.

[0092] Compositions and Uses This specification provides compositions comprising at least one rAAV.hGALC stock (e.g., rAAVhu68 stock or mutant rAAV stock) and an optional carrier, excipient, and / or preservative. An rAAV stock refers to multiple identical rAAV vectors, e.g., in the amounts described below in the consideration of concentration and dose units. Despite the heterogeneity of the capsid protein due to deamidation, the rAAVs in the stock are expected to share the same vector genome. A stock may, for example, comprise a selected AAV capsid protein and rAAVs having capsids with heterogeneous deamidation patterns characteristic of a selected production system. A stock may be produced from a single production system or pooled from multiple runs of a production system. A variety of production systems may be selected, including, but not limited to, those described herein.

[0093] As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antimicrobial and antifungal agents, isotonic and absorption retardants, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients may also be incorporated into the composition. The phrase “pharmaceutically 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, and vesicles may be used to introduce the compositions of the present invention into suitable host cells. In particular, the rAAV vector delivery vector genome may be formulated for delivery encapsulated in any of the following: lipid particles, liposomes, vesicles, nanospheres, or nanoparticles.

[0094] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, for example, an aqueous liquid suspension buffered to a physiologically suitable pH and salt concentration. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition is diluted for administration to a subject. It can be transported as a concentrated solution. In other embodiments, the composition may be lyophilized and reconstituted at the time of administration.

[0095] A suitable surfactant or combination of surfactants may be selected from among non-toxic nonionic surfactants. In one embodiment, for example, a primary hydroxyl-terminated bifunctional block copolymer surfactant such as Pluronic® F68 [BASF], also known as poloxamer 188, which has a neutral pH and an average molecular weight of 8400, is selected. Other surfactants and other poloxamers, namely nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) adjacent to two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycaprylic acid glyceride), polyoxy-10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol may be selected. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named using the letter "P" (for poloxamer) followed by a three-digit number, where the first two digits multiplied by 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit multiplied by 10 gives the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. A surfactant may be present in an amount of up to about 0.0005% to about 0.001% of the suspension.

[0096] The vector is administered in an amount sufficient to transfect cells and provide a sufficient level of gene transfer and expression to provide a therapeutic effect without undue adverse effects or with a medically acceptable physiological effect, which can be determined by one of ordinary skill in the art. Conventional and pharmaceutically acceptable routes of administration include delivery directly to the desired organ (e.g., liver (optionally via the hepatic artery), lung, heart, eye, kidney), oral, inhalation, intranasal, intrathecal, intratracheal, intraarterial, intravitreal, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration, but are not limited thereto. Routes of administration may be combined if desired.

[0097] The dosage of the viral vector depends primarily on factors such as the condition being treated, the age, weight, and health status of the patient, and thus may vary between patients. For example, a therapeutically effective human dosage of a viral vector generally ranges from about 25 to about 1000 microliters to about 100 mL of a solution containing from about 1×10 9 ~1×10 16 genomic viral vector concentration. The dosage may be adjusted to balance the therapeutic effect against any side effects, and such dosage may vary depending on the therapeutic use for which the recombinant vector is utilized. The expression level of the transgene product can be monitored to determine the dosing frequency that results in a viral vector (preferably an AAV vector containing a minigene). Optionally, a dosing regimen similar to that described for therapeutic purposes can be utilized for immunization using the compositions of the present invention.

[0098] The replication-deficient viral composition is formulated in dosage units and, for a human patient, contains an amount of replication-deficient virus in the range of about 1.0×10 9 GC to about 1.0×10 16 GC (including all integers or fractional amounts within that range), preferably in the range of 1.0×10 12 GC to 4.0×10 14 GC. In some embodiments, the composition contains from 1.4×10 13 ~4×10 14The formulation is designed to contain GC replication-defective viruses. In some embodiments, the composition is 4 × 10 13 ~4×10 14 The formulation contains GC replication-defective viruses. In one embodiment, the composition contains all integers or fractional quantities within the range, with at least 1 × 10 per dose. 9 , 2×10 9 , 3 x 10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , or 9×10 9 The formulation is to include GC. In another embodiment, the composition contains at least 1 × 10 per dose, including all integers or fractional quantities within the range. 10 , 2×10 10 , 3 x 10 10 , 4×10 10 , 5 ×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , or 9×10 10 The formulation is to include GC. In another embodiment, the composition contains at least 1 × 10 per dose, including all integers or fractional quantities within the range. 11 , 2×10 11 , 3 x 10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , or 9×10 11 The formulation is to include GC. In another embodiment, the composition contains at least 1 × 10 per dose, including all integers or fractional quantities within the range. 12 , 2×10 12 , 3 x 10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , or 9×10 12The formulation is to include GC. In another embodiment, the composition contains at least 1 × 10 per dose, including all integers or fractional quantities within the range. 13 , 2×10 13 , 3 x 10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , or 9×10 13 The formulation is to include GC. In another embodiment, the composition contains at least 1 × 10 per dose, including all integers or fractional quantities within the range. 14 , 2×10 14 , 3 x 10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , or 9×10 14 The formulation is to include GC. In another embodiment, the composition contains at least 1 × 10 per dose, including all integers or fractional quantities within the range. 15 , 2×10 15 , 3 x 10 15 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 , or 9×10 15 The formulation includes GC. In one embodiment, for human application, the dose is 1 × 10 per dose, including all integers or fractions within the range. 10 ~Approx. 1×10 12 It may be within the GC range. In one embodiment, for human application, the dose is 1.4 × 10 per dose, including all integers or fractional quantities within the range. 13 ~Approx. 4×10 14 It could be within the scope of garbage collection.

[0099] These above doses may be administered in various volumes of carrier, excipient, or buffer formulations ranging from about 25 to about 1000 microliters, or in higher volumes containing all of the above, 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 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 approximately 325 μL. In yet another embodiment, the volume is approximately 350 μL. In yet another embodiment, the volume is approximately 375 μL. In yet another embodiment, the volume is approximately 400 μL. In yet another embodiment, the volume is approximately 450 μL. In yet another embodiment, the volume is approximately 500 μL. In yet another embodiment, the volume is approximately 550 μL. In yet another embodiment, the volume is approximately 600 μL. In yet another embodiment, the volume is approximately 650 μL. In yet another embodiment, the volume is approximately 700 μL. In yet another embodiment, the volume is approximately 700 to 1000 μL.

[0100] The therapeutically effective intrathecal / intracisional dose of rAAV.hGALC is approximately 1 × 10⁻⁶. 11 ~7.0×10 14 GC (constant dose) (10 8 ~7×10 11 This is within the range of brain mass equivalent to that of GC / g patients. Alternatively, the following therapeutically effective constant doses may be administered to patients in the indicated age groups. ●Newborn: Approximately 1 x 10 11 ~Approx. 3×10 14 GC, ● 3-9 months: Approximately 6 x 10 12 ~Approx. 3×1014 GC, ● 9 months to 6 years old: about 6×10 12 ~ about 3×10 14 GC, ● 3 to 6 years old: about 1.2×10 13 ~ about 6×10 14 GC, ● 6 to 12 years old: about 1.2×10 13 ~ about 6×10 14 GC, ● Over 12 years old: about 1.4×10 13 ~ about 7.0×10 14 GC, ● Over 18 years old (adult): about 1.4×10 13 ~ about 7.0×10 14 GC.

[0101] In certain embodiments, the dosage can be in the range of about 1×10 9 GC / g brain mass ~ about 1×10 12 GC / g brain mass. In certain embodiments, the dosage can be in the range of about 3×10 10 GC / g brain mass ~ about 3×10 11 GC / g brain mass. In certain embodiments, the dosage can be in the range of about 1.70×10 10 GC / g brain mass ~ about 5×10 11 GC / g brain mass. In certain embodiments, the dosage can be in the range of about 5×10 10 GC / g brain mass ~ about 5×10 11 GC / g brain mass. In certain embodiments, the dosage can be in the range of about 5×10 10 GC / g brain mass ~ about 1.85×10 11 GC / g brain mass. In certain embodiments, the dosage can be in the range of about 1.70×10 10 GC / g brain mass ~ about 1.70×10 11 GC / g brain mass. In certain embodiments, the dosage can be in the range of about 5.00×10 10 GC / g brain mass ~ about 1.70×10 11 GC / g brain mass. In certain embodiments, the dosage can be in the range of about 1.70×10 11 GC / g brain mass ~ about 5×10 11 GC / g brain mass. In certain embodiments, the dosage can be in the range of about 5×1010 GC / g brain mass ~ approx. 10 12 The GC / g brain mass may be in the range. In certain embodiments, the dose is about 10 10 GC / g brain mass ~ approx. 10 12 The GC / g brain mass may be within the range. In certain embodiments, the dose is at least 1.70 × 10⁻⁶ 10 This is GC / g brain mass. In certain embodiments, the dose is at least 5 × 10⁻⁶ 10 This is GC / g brain mass. In a particular embodiment, the dose is 1.70 × 10⁻⁶ 11 This is GC / g brain mass.

[0102] Regarding scaling between infants and adolescents / adults, brain mass is estimated to be approximately 600g to 800g at 4-12 months of age, 800g to 1000g at 9-18 months of age, 1000g to 1100g at 18 months to 3 years of age, 1100g to 1300g in adolescents or adult humans, or approximately 1300g in adult humans.

[0103] In one embodiment, the virus construct is at least about 1 × 10 9 GC~approx. 1×10 15 , or approximately 1 x 10 11 ~5×10 13GC can be delivered in doses. Suitable volumes for delivering these doses and concentrations can be determined by those skilled in the art. For example, volumes of about 1 μL to 150 mL may be selected, but higher volumes may be selected for adults. In certain embodiments, the delivered volumes are about 2.0 mL, about 2.5 mL, about 3.0 mL, about 3.5 mL, about 4.0 mL, about 4.5 mL, about 5.0 mL, about 5.5 mL, about 6.0 mL, about 6.5 mL, about 7.0 mL, about 7.5 mL, about 8.0 mL, about 8.5 mL, about 9.0 mL, about 9.5 mL, about 10.0 mL, about 10.5 mL, about 11.0 mL, about 11.5 mL, about 12.0 mL, about 12.5 mL, about 13.0 mL, about 13.5 mL, about 14.0 mL, about 14.5 mL, or about 15.0 mL. Typically, for neonates, the preferred volume is about 0.5 mL to about 10 mL, and for older infants, about 0.5 mL to about 15 mL. For toddlers, a volume of about 0.5 mL to about 20 mL may be selected. For children, a volume of up to about 30 mL may be selected. For preteens and teens, a volume of up to about 50 mL may be selected. In certain embodiments, the patient receives intrathecal administration in volumes of about 2 mL to about 4 mL, about 3 mL to about 5 mL, about 4 mL to about 6 mL, about 5 mL to about 7 mL, about 6 mL to about 8 mL, about 7 mL to about 9 mL, or about 8 mL to about 10 mL. In yet another embodiment, the patient may receive intrathecal administration in volumes of about 5 mL to about 15 mL, or about 7.5 mL to about 10 mL. In certain embodiments, the volume administered intrathecal is about 5.0 mL. In certain embodiments, the volume administered intrathecal is about 5.6 mL. Other suitable volumes and dosages may be determined. The dosage may be adjusted to balance the therapeutic effect with any side effects, and such dosages may vary depending on the therapeutic application in which the recombinant vector is used.

[0104] The recombinant vectors described above can be delivered to host cells according to the published methods. Preferably, rAAV suspended on a physiologically compatible carrier is administered to human or non-human mammalian patients. It can be administered. In certain embodiments, for administration to human patients, rAAV is preferably suspended in an aqueous solution containing saline, a surfactant, and a physiologically compatible salt, or a mixture of salts. Preferably, the formulation is adjusted to a physiologically acceptable pH range, for example, pH 6 to 9, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. Since the pH of cerebrospinal fluid is approximately 7.28 to approximately 7.32, a pH within this range is desirable for intrathecal delivery, and a pH of approximately 6.8 to approximately 7.2 may be desirable for intravenous delivery. However, other pH ranges within the broadest range, and sub-ranges of these, may be selected for other delivery routes.

[0105] In another embodiment, the composition comprises a carrier, a diluent, an excipient and / or an adjuvant. A suitable carrier can be readily selected by those skilled in the art, taking into account the indication to which the introduced virus is directed. 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 contain components that prevent rAAV from adhering to the infusion tube but do not interfere with rAAV binding activity in vivo.

[0106] In one embodiment, the formulation may contain, for example, a buffered saline solution containing one or more of the following in water: 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. Preferably, for intrathecal delivery, the molar osmotic concentration is within a range compatible with cerebrospinal fluid (e.g., about 275 to about 290). See, for example, emedicine.medscape.com / article / 2093316-overview. Optionally, for intrathecal delivery, commercially available diluents may be used as suspending agents or in combination with other suspending agents and any other excipients. See, for example, Elliotts B® solution [Lukare Medical].

[0107] In certain embodiments, the formulation may contain a buffered saline solution that does not contain sodium bicarbonate. Such a formulation may contain a buffered saline solution such as Harvard buffer, which contains one or more of the following in water: sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and mixtures thereof. 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.

[0108] In other embodiments, the formulation may contain one or more permeation enhancers. Examples of suitable permeation enhancers include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, or EDTA.

[0109] Optionally, the compositions of the present invention may include 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.

[0110] The compositions according to the present invention may include pharmaceutically acceptable carriers as defined above. In particular, the compositions described herein comprise one or more AAVs suspended in an effective amount of a pharmaceutically suitable carrier and / or mixed with a suitable excipient designed for delivery to a subject via infusion, permeation pump, intrathecal catheter, or by another device or route. In one example, the composition is formulated for intrathecal delivery.

[0111] As used herein, the term “intrathecal delivery” or “intrathecal administration” refers to the route of drug administration 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 lateral ventricles (ICV)), suboccipital / cisternal, and / or C1-2 puncture. For example, a material may be introduced by lumbar puncture to diffuse across the subarachnoid space. In another embodiment, the injection may be into the cisterna magna.

[0112] As used herein, the terms “intracisternal delivery” or “intracisternal administration” refer to a direct route of drug administration into the cerebrospinal fluid of the cisterna magna cerebellomedularis, more specifically, a route of drug administration by suboccipital puncture, direct injection into the cisterna magna, or by a permanently placed tube. In certain embodiments, the composition is delivered using the Ommaya reservoir for CNS-targeted administration.

[0113] As used herein, the term computed tomography (CT) refers to radiography in which a computer constructs a three-dimensional image of a body structure from a series of planar cross-sectional images created along an axis.

[0114] The rAAV.GALC vectors and compositions provided herein are useful for correcting conditions associated with deficiency 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 GALC deficiency, for treating respiratory failure and / or motor function loss caused by GALC deficiency, for treating Krabbe disease, and / or for treating symptoms associated with Krabbe disease in patients.

[0115] In certain embodiments, a composition containing an effective amount of rAAV.hGALC is administered to a patient under 6 months of age with early infantile Krabbe disease (EIKD). In certain embodiments, the patient is under 6 months of age and has GALC enzyme deficiency that is not more severe than EIKD.

[0116] In certain embodiments, a composition containing an effective amount of rAAV.hGALC is administered to patients with late infantile Krabbe disease (LIKD) who are over 6 months of age (e.g., 7 to about 12 months of age). In certain embodiments, the patient is over 6 months of age, or about 7 to 12 months of age, and has a GALC enzyme deficiency that is not more severe than LIKD.

[0117] In certain embodiments, the patient is over 1 year old (e.g., 13 months to 10 years old) and has juvenile Krabbe disease (JKD). In certain embodiments, the patient is 13 months to 10 years old and has GALC enzyme deficiency that is less severe than JKD.

[0118] In certain embodiments, the patient is over 10 years of age (for example, between 10 and 12 years of age, or between 10 and 18 years of age or older) and has adolescent or adult-onset Krabbe disease.

[0119] In any of the embodiments described above, the rAAV.hGALC therapy provided herein It may be administered as a combination therapy with hematopoietic stem cell replacement therapy, bone marrow transplantation (BMT), and / or substrate synthesis suppression therapy (SRT). In certain embodiments, 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 vector administration (including within one week post-treatment).

[0120] In certain embodiments, enzyme replacement therapy includes administration of the human GALC protein of Sequence ID No. 10. In other embodiments, other hGALC protein variants (e.g., canonical sequences or engineered proteins as specified herein) may be used in enzyme replacement therapy. Combinations of different hGALC proteins may be used in enzyme replacement therapy. In such embodiments, the hGALC protein 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 improvements in early clinical phenotype in a mouse model of globoid cell leukodystrophy (FASEB journal, The FASEB Journal 19(11):1549-51, October 2005). The 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 physiologically suitable saline). The preferred dose may be in the range of 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 as needed (e.g., every two days, every two weeks, etc.). Using CSF administration of the hAAVhu68.GALC vector, GALC levels in the brain and serum may be non-toxic and paraphysiological, and improvements in neuromotor function and myelin formation in the CNS and PNS may be observed. When CSF is administered to neonates and then bone marrow transplantation is performed in postnatal conditioned animal models, survival may be extended without apparent signs (e.g., to more than 300 days). In pre-symptomatic Krabbe patients, a single cisterna magna infusion of AAV.cGALC may provide phenotypic correction, increased survival rate, normalization of nerve conduction, and / or improved brain MRI.

[0121] In certain embodiments, a combination therapy or regimen is provided, involving treatment of the patient with rAAVhu68.hGALC and BMT. 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 without requiring HSCT or BMT. In certain embodiments, gene therapy treatment precedes the use of BMT. In certain embodiments, this combination therapy provides enhanced transgene expression in skeletal muscle (e.g., quadriceps, lungs, diaphragm, heart, liver, kidneys) or in specific cells of the peripheral or central nervous system. In other embodiments, BMT may precede the use of gene therapy treatment as described herein.

[0122] The goal of treatment is to functionally replace the patient's defective GALC through rAAV-based CNS and PNS-targeted gene therapy. The effectiveness of the therapy for patients with EIKD or LIKD can be measured by evaluating improvement in one or more of the following symptoms of EIKD or LIKD: crying and irritability, seizures, fist-clenching, loss of smile, poor head control and feeding difficulties, worsening mental and motor conditions, hypertonia or hypotonia, seizures, blindness, hearing loss, and increased survival (in the case of EIKD, without treatment, death is typically before the age of 2, while in the case of LIKD, survival can be extended to 3-5 years). In addition, for these and other Krabbe patients, the effectiveness of the therapy can be monitored via imaging (e.g., magnetic resonance imaging (MRI)) to reduce hypomyelination and demyelination affecting both peripheral nerves and CNS white matter (deep brain white matter and dentate / cerebellar white matter), abnormal nerve conduction velocity (NCV), and Alternatively, it can be assessed by a decrease in brainstem auditory evoked potentials (BAEPs), an increase in GALC levels that may be observed in cerebrospinal fluid and / or plasma, and / or a decrease in psychosine accumulation.

[0123] A composition comprising recombinant adeno-associated virus (rAAV) is provided, comprising an AAV capsid in which a vector genome is packaged, the vector genome comprising 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 the expression of the protein, the vector genome further comprising AAV inverted terminal repeats necessary for packaging the vector genome within the AAV capsid.

[0124] In certain embodiments, a composition useful for treating Krabbe disease is provided, comprising rAAVhu68 having the vector genome CB7.CI.hGALC.rBG. In one embodiment, the vector genome has the coding sequence of SEQ ID NO: 19.

[0125] In certain embodiments, the use of the composition is provided in a method for correcting peripheral nerve dysfunction caused by GALC deficiency, and / or a method for treating respiratory failure 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), wherein the recombinant adeno-associated virus comprises (a) an AAV capsid in which a vector genome is packaged, and (b) 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 a regulatory sequence that directs protein expression, and further comprising AAV inverted terminal repeats necessary for packaging the vector genome in the AAV capsid.

[0126] In certain embodiments, the rAAV.hGALC composition provided herein is delivered intrathecally for the treatment of patients with early infantile Krabbe disease. In certain embodiments, the composition provided herein is delivered intrathecally for the treatment of patients with late infantile Krabbe disease (LIKD). In certain embodiments, the rAAV.hGALC composition provided herein is delivered intrathecally for the treatment of patients with juvenile Krabbe disease (JKD). In certain embodiments, the rAAV.hGALC composition provided herein is delivered intrathecally for the treatment of patients with adolescent or adult-onset Krabbe disease. In certain embodiments, the rAAV.hGALC composition is administered as a combination therapy with hematopoietic stem cell transplantation (HSCT), bone marrow transplantation, and / or substrate synthesis suppression therapy. In certain embodiments, the rAAV.hGALC composition is administered as a single dose via computed tomography (CT)-guided suboccipital injection into the cisterna magna.

[0127] Administration of rAAV.hGALC stabilizes disease progression (measured by survival rate), prevents the loss of developmental and motor milestones, potentially supports the acquisition of new milestones, and reduces seizure onset and frequency. Therefore, in certain embodiments, methods are provided for monitoring treatment, and endpoints are measured, for example, at 30 days, 90 days, and / or 6 months, and then every 6 months during a short-term follow-up period of, for example, 2 years. In certain embodiments, the measurement frequency is reduced to once every 12 months during long-term extension. 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 have not yet shown signs of development of motor milestones. Assessment tracks age-at-achievement and age-at-loss for all milestones. In certain embodiments, milestones include, for example, sitting without support, crawling on all fours, standing with assistance, walking with assistance, standing on one's own, and / This includes one or more of the following: or walking alone. In certain embodiments, the treatment results in a delay in the onset of seizure activity and / or a reduction in the frequency of seizure events.

[0128] In certain embodiments, the method for monitoring treatment in a subject involves using clinical scales to quantify the effects 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 Bailey Scale for Infant Development (assessing infant and toddler development across five domains: cognition, language, motor, social-emotion, and adaptive behavior), the Vineland Adaptive Behavior Scale (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 Scale - 2nd edition (measuring interrelated motor functions in children from birth to 5 years, with assessment focusing on six domains: reflexes, static, movement, object manipulation, grasping, and visual-motor integration), the Infant Quality of Life Questionnaire (ITQOL) (a parent-reported scale of health-related quality of life designed for children from 2 months to 5 years), and the Mullen Scale for Early Learning (assessing language, motor, and perceptual abilities in infants and toddlers up to 68 months). In certain embodiments, the effects of treatment are monitored or measured by assessing myelination, myelination-related functional outcomes, and changes in potential disease biomarkers. In certain embodiments, central and peripheral demyelination may be delayed or halted after treatment of the subject. Central demyelination can be tracked by diffusion tensor magnetic resonance imaging (DT-MRI) anisotropy measurements of the white matter region and fiber tracing of the corticospinal motor pathway, and these changes serve as indicators of disease state and progression. Peripheral demyelination can be indirectly measured via nerve conduction velocity (NCV) testing of motor nerves (deep peroneal, tibial, and ulnar nerves) and sensory nerves (sural and median nerves) to monitor changes in biologically active myelin (i.e., F-waves and distal latency, amplitude, or presence or absence of response).

[0129] In certain embodiments, a method is provided for monitoring treatment after rAAV.hGALC administration, evaluating subjects for delayed or absent vision loss in subjects who have not developed significant vision loss prior to treatment. Thus, measurement of visual evoked potentials (VEPs) is used to objectively measure the response to visual stimuli as an indicator of central vision impairment or loss. In certain embodiments, subjects are monitored for post-treatment hearing loss, for example, using brainstem auditory evoked response (BAER) testing. In certain embodiments, a method is provided for monitoring treatment after rAAV.hGALC administration, measuring the psychosine levels of subjects.

[0130] Please note that the terms "a" or "an" refer to one or more. Therefore, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably in this specification.

[0131] The terms “comprise,” “comprises,” and “comprising” should be interpreted comprehensively, not exclusively. The terms “consist,” “consisting,” and their variations should be interpreted exclusively, not comprehensively. While various embodiments herein are shown using the word “comprising,” in other contexts, the relevant embodiments are also intended to be interpreted and described using the words “consisting of” or “consisting essentially of.”

[0132] As used herein, the term “about” means a variability of 10% (±10%) from the given reference, unless otherwise specified.

[0133] As used herein, “disease,” “disorder,” and “condition” are used interchangeably to describe an abnormal condition in the subject.

[0134] The term “expression” is used herein in its broadest sense and includes RNA products, or RNA and protein products. With respect to RNA, the terms “expression” or “translation” particularly relate to the production of peptides or proteins. Expression can be transient or stable.

[0135] As used herein, “expression cassette” refers to a nucleic acid molecule including a coding sequence, a promoter, and other regulatory sequences for them. 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 an expression cassette for generating a viral vector includes a coding sequence for a gene product described herein, adjacent to the packaging signal of the viral genome, and other expression regulatory sequences such as those described herein.

[0136] The abbreviation "sc" stands for self-complementary. "Self-complementary AAV" refers to a construct in which the coding region supported by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. During infection, instead of waiting for cell-mediated synthesis of the second strand, the two complementary halves of scAAV associate to form a single double-stranded DNA (dsDNA) unit that is immediately capable of 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. Patents No. 6,596,535, No. 7,125,717, and No. 7,456,683 (each of which is incorporated herein by reference in whole).

[0137] When used in reference to proteins or nucleic acids, the term “heterogeneity” indicates that a 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 having two or more sequences from unrelated genes arranged to create a novel functional nucleic acid are typically produced by recombination. For example, in one embodiment, a nucleic acid has a promoter derived from one gene and is arranged to direct the expression of coding sequences derived from different genes. Thus, with respect to the coding sequences, the promoter is heterogeneous.

[0138] A “replication-deficient virus” or “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing the gene of interest is packaged within a viral capsid or envelope, and any viral genome sequence packaged within the viral capsid or envelope is replication-deficient, i.e., it cannot produce progeny 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 target transgene adjacent to the signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. Therefore, it is considered safe for use in gene therapy because replication and infection by progeny cannot occur without the presence of the viral enzymes required for replication.

[0139] In many cases, rAAV particles are referred to as DNase-resistant. However, in addition to this endonuclease (DNase), other endonucleases and exonucleases may also be used in the purification steps described herein to remove contaminating nucleic acids. Such nucleases may be selected to degrade single-stranded DNA and / or double-stranded DNA, and RNA. Such steps may comprise a single nuclease or a mixture of nucleases targeted to different targets, which may be endonucleases or exonucleases.

[0140] The term "nuclease resistance" indicates that the AAV capsid is fully constructed around the expression cassette designed to deliver the gene to the host cell, protecting these packaged genomic sequences from degradation (digestion) during the nuclease incubation step, which is designed to remove any contaminating nucleic acids that may be present in the production process.

[0141] As used herein, “effective dose” refers to the amount of rAAV composition that delivers and expresses a quantity of gene products from the vector genome in target cells. The effective dose may be determined based on an animal model, rather than a human patient. Examples of suitable mouse models are described herein.

[0142] Unless otherwise defined herein, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art and by referring to published literature that provides a general guide to those skilled in the art for the many terms used herein. [Examples]

[0143] The following examples are for illustrative purposes only and are not intended to limit the present invention. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0144] 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 the AAV9 capsid [SEQ ID NO: 4] and the 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).

[0145] rAAVhu68.hGALC is produced by triple plasmid transfection of HEK293 cells using an AAV cis-plasmid encoding an AAV transgene cassette adjacent to the AAV ITR, an AAV transplasmid (pAAV2 / hu68.KanR) encoding the genes for AAV2 rep and AAVhu68 cap, and a helper adenovirus plasmid (pAdΔF6.KanR).

[0146] 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:

[0147] Inverted Terminal Repeats (ITRs): ITRs are identical reverse complementary sequences derived from AAV2 (130 bp, GenBank: NC_001401) and adjacent to all components of the vector genome. When AAV and adenovirus helper functions are provided in trans, ITR sequences function as both the origin of replication for vector DNA and the packaging signal for the vector genome. Therefore, ITR sequences represent only the cis sequences required for replication and packaging of the vector genome.

[0148] Human cytomegalovirus early enhancer (CMV IE): This enhancer sequence is derived from human CMV (382 bp, GenBank: K03104.1) and increases the expression of downstream transgenes.

[0149] Chicken β-actin (CB) promoter: This ubiquitous promoter (282 bp, GenBank: X00182.1) was selected to drive the expression of transgenes in any CNS cell type.

[0150] Chimeric introns (CI): Hybrid introns contain elements from chicken β-actin splice donor (973 bp, GenBank: X00182.1) and rabbit β-globin splice acceptor. Introns are transcribed but, along with the sequences at either end, are removed from mature mRNA by splicing. The presence of introns in expression cassettes has been shown to promote mRNA transport from the nucleus to the cytoplasm, thereby increasing the accumulation of a certain level of mRNA for translation. This is a common feature in gene vectors intended to increase gene expression levels.

[0151] Code sequence: The manipulated cDNA of the human GALC gene codes for the human galactosylceramidase protein, which is responsible for the hydrolysis and degradation of myelin galactolipids. It is a lysosomal enzyme (2055 bp, 685 amino acids [aa], GenBank: EAW81361.1).

[0152] Rabbit β-globin polyadenylation signal (rBG PolyA): The rBG PolyA signal (127 bp, GenBank: V00882.1) promotes efficient polyadenylation of transgene mRNA in cis form. This element functions as a signal for transcription termination, a specific 3' cleavage event in the nascent transcript, and the addition of a long polyadenyl tail.

[0153] B. Transplasmid: pAAV2 / 1.KanR(p0068) The AAV2 / hu68 transplasmide pAAV2 / hu68.KanR(p0068) is shown in Figure 21.

[0154] The AAV2 / hu68 transplasmid is pAAV2 / hu68.KanR(p0068). The pAAV2 / hu68.KanR plasmid is 8030 bp long and encodes four wild-type AAV2 replicase (Rep) proteins required for the replication and packaging of the AAV vector genome. The pAAV2 / hu68 transplasmid also encodes three WT AAVhu68 virion protein capsid (Cap) proteins that are assembled into a virion shell of AAV serotype hu68 to accommodate the AAV vector genome.

[0155] To construct the AAV2 / hu68.KanR transplasmid, the AAV9 cap gene from plasmid pAAV2 / 9n(p0061-2), which encodes the wild-type AAV2 rep and AAV9 cap genes on the 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 obtain pAAV2 / hu68.KanR(p0068). In this cloning strategy, AAV The p5 promoter sequence (which normally drives rep expression) is moved from the 5' end of rep to the 3' end of cap, leaving a cleaved p5 promoter upstream of rep. This cleaved promoter downregulates rep expression, thereby maximizing vector production (Figure 4).

[0156] All components of the plasmid have been verified by direct sequencing.

[0157] C. Adenovirus Helper Plasmid: pAdDeltaF6(KanR) The adenovirus helper plasmid pAdDeltaF6 (KanR) is shown in (Figure 5B).

[0158] Plasmid pAdDeltaF6(KanR) has a size of 15,770 bp. This plasmid contains regions of the adenovirus genome important for AAV replication, namely E2A, E4, and VA RNA (the function of adenovirus E1 is provided by HEK293 cells). However, this plasmid does not contain any other adenovirus replication or structural genes. The plasmid does not contain cis elements important for replication, such as adenovirus ITR, and therefore, the generation of infectious adenovirus is not expected. The plasmid was derived from an E1, E3 deletion molecular clone of Ad5 (pBHG10, pBR322-based plasmid). Deletions were introduced into Ad5 to remove the expression of unwanted adenovirus genes and reduce the amount of adenovirus DNA from 32kb to 12kb (Figure 5A). Finally, the ampicillin resistance gene was replaced with the kanamycin resistance gene to create pAdeltaF6(KanR) (Figure 5B). The adenovirus genes E2, E4, and VAI remaining in this plasmid were HE Along with E1 present in K293 cells, it is necessary for the production of AAV vectors.

[0159] Example 2 - Materials and Methods Assessment of neuromotor function (RotaRod) Coordination and balance were measured using the rotorod test (Ugo Basile; Gemonio, Italy) by a blinded operator for each treatment group. Briefly, mice were first trained to the rotorod by placing up to five mice in a lane of rotorod devices facing a wall in each trial. Mice were stabilized on a stationary (non-rotating) rod for 2 minutes. Then, two training trials were performed, with the rod rotating at a constant speed of 5 revolutions per minute (RPM) for 1 minute. Between each training trial, mice were allowed to rest in a rotorod retrieval box for approximately 1 minute. If a mouse fell during the training phase, it was immediately returned to the rod.

[0160] Immediately after training, test trials were conducted to measure how long each mouse could remain on the rotating rod while it was accelerating. Mice were placed in a lane of the rotor rod device facing the wall and allowed to balance and firmly grasp the stationary (non-rotating) rod. The rod was then rotated at a constant speed of 5 RPM for several seconds to allow the mouse to balance. Once balanced, the rod was set to accelerate from 5 RPM to 40 RPM over 120 seconds. For each animal, the test trial was considered complete when the mouse fell from the rod, when two passive rotations were completed, or when 120 seconds had elapsed. Fall latency (defined as the time from the start of rod acceleration to the end of the trial) was recorded. A total of three consecutive test replications were performed for each mouse in each trial, with a 1-3 minute break between runs to allow the animals to rest in the retrieval box.

[0161] Histological treatment and evaluation brain The entire brain was removed from the skull of the necropped animals and cut in half along the sagittal midline using a razor blade. For evaluation of transgene expression (GALC enzyme activity assay), the left hemisphere of the brain was frozen on dry ice and stored at -60°C or below, and for histology, the right hemisphere of the brain was placed in 10% NBF.

[0162] Sciatic nerve The sciatic nerve was collected and immediately fixed in 2.5% glutaraldehyde + 2% paraformaldehyde in PBS, and collected at room temperature for approximately 24 hours. After fixation, the sample was washed in PBS, fixed in 1% osmium tetroxide aqueous solution for 2 hours, washed with water, dehydrated with an increasing concentration of ethanol series, and subsequently dehydrated through propylene oxide, a mixture of propylene oxide and embedding resin, and the embedding resin alone. The embedding resin was prepared by mixing its components (LX-122, DDSA, NMA, and DMP-30) (Ladd Research Industries) and used according to the manufacturer's instructions. The sample was then placed in a mold using the embedding medium, cured at 70°C for 48 hours, sectioned to a thickness of 1 μm using an ultramicrotome, stained with toluidine blue, dried, and covered with a coverslip using Permount mounting medium.

[0163] Paraffin embedding and sectioning Tissue samples were embedded in paraffin and sectioned according to SOP4004 and SOP4006. Briefly, the cassette containing the fixed tissue was placed in a Leica ASP300 tissue processor and the processor was run overnight. The tissue was embedded with proper orientation using an embedding center, and the sample was cured on a cooling plate. The block was then trimmed, cooled on ice for at least 10 minutes, and then sectioned. Sections 5–7 μm thick were prepared using a microtome. The sections were transferred to a water bath preheated to 38–52°C, floated, and wrinkles removed. The material was transferred to a slide and dried at room temperature. The slide was then deparaffinized by heating at 60–65°C for at least 15 minutes (or until the paraffin melted). The slide was incubated in xylene (3 times for 3–5 minutes), rehydrated through an ethanol series, and washed twice with distilled water. Slides containing at least three serial sections were then stained with LFB, PAS (for myelin formation and globoid cell infiltration, respectively), or IBA1 IHC (for globoid cell sizing).

[0164] LFB / PAS staining (evaluation of myelin formation and globoid cell infiltration) After deparaffinization, sections of the brain, spinal cord, and sciatic nerve were stained with LFB / PAS stain. Briefly, the slides were incubated overnight at 65°C in LFB solution (SLMP, LLC; catalog number: STLFBPT). The sections were differentiated in 0.05% lithium carbonate solution and 70% ethanol, and monitored under a microscope until differentiation was complete. The slides were then placed in 0.5% periodic acid for 5 minutes (Sigma; catalog number: 395B-1Kit). After washing the slides under running tap water, they were transferred to Schiff's reagent (Sigma; catalog number: 395B-1Kit) for 15 minutes. The slides were washed under running tap water for 5 minutes, lightly counterstained with hematoxylin to identify the nuclei, and covered with coverslips. Histopathological evaluation was performed.

[0165] IBA1 immunohistochemistry (neuroinflammation) After deparaffinization, sections of the brain, spinal cord, and sciatic nerve were immunohistochemically stained for IBA1. Antigen retrieval was briefly performed using a citrate-based antigen unmasking solution (Vector Laboratories; catalog number: H-3300) in a pressure cooker at 100°C for 20 minutes. Slides were incubated with 3% hydrogen peroxide for 10 minutes and then stained with avidin / biotin reagent (Vector Using Laboratory (catalog number: SP-2001), the slides were incubated with 1% donkey serum and 0.2% Triton-X for 15 minutes at room temperature, with blocking for 15 minutes each. The slides were then incubated overnight at 4°C with rabbit anti-IBA1 primary antibody (Abcam; catalog number: ab178846) diluted 1:2000. The slides were incubated with biotinylated donkey anti-rabbit IgG secondary antibody (Jackson; catalog number: 711-065-152) diluted 1:1000 at room temperature for 30 minutes. The slides were washed and then treated with Vectastain ABC reagent (Vector The samples were incubated with (Vector Laboratories; catalog number: PK-6100). Stain development was performed using a DAB kit (Vector Laboratories; catalog number: SK-4100), followed by counterstaining with hematoxylin, and then the samples were covered with coverslips.

[0166] GALC immunohistochemistry Tissue samples were fixed in formalin for at least 24 hours and then embedded in paraffin. Sections (6 μm) were then deparaffinized with xylene and ethanol series and returned to water. Antigen retrieval was performed using a pressure cooker in a citrate-buffered (pH 6.0)-based antigen unmasking solution (Vector Laboratories). Next, the sections were sequentially treated with 2% H2O2 (15 minutes), avidin / biotin blocking reagent (15 minutes each, Vector Laboratories), and blocking buffer (1% donkey serum + 0.2% Triton in PBS, 1 hour). Next, the sections were incubated with primary antibody (rabbit anti-human GALC, Thermo Fisher PA5-72315, 1:100, overnight at 4°C), washed in PBS / 0.02% Tween-20, and then incubated with donkey-derived biotinylated secondary antibody (30 minutes, Jackson ImmunoResearch, 1:500). All antibodies were diluted in blocking buffer. After washing, DAB was used as a substrate using the Vectastain Elite ABC kit (Vector Laboratories) according to the manufacturer's instructions. The bound antibody was stained as a brown precipitate using a staining agent (with a 5-minute development time). The section was counterstained with hematoxylin to reveal the nuclei, and then covered with a coverslip.

[0167] Evaluation of transgene expression (GALC activity assay) Processing of tissue samples Frozen brains were homogenized using a Qiagen TissueLyzer in 0.9% NaCl (pH 4.0) containing 0.05% Triton-X100 for 2 minutes and 30 seconds at 30 Hz. The samples were frozen on dry ice for 20 minutes, thawed at room temperature, and gently vortexed. The lysates were clarified by centrifugation at 10,000 RPM on a benchtop centrifuge for 10 minutes. The clarified lysates were transferred to new tubes for analysis. Protein content was measured by a bicinchoninic acid (BCA) assay.

[0168] Measurement of GALC enzyme activity GALC activity in brain and serum was measured using a commercially available kit from Marker Gene Technologies, Inc. (catalog number: M2774). In this assay, 50 μg of total protein from brain or 10 μL of serum was mixed with the reaction buffer provided in the kit to a final volume of 100 μL. A tube containing 100 μL of reaction buffer was provided as a blank. The sample was incubated at 37°C for 2 hours, and the reaction was stopped by adding 1 mL of stop solution supplied in the kit. Finally, 300 μL of each reactant was transferred to a 96-well black plate, and fluorescence was measured using a plate reader at an emission wavelength of 454 nm when excited at 365 nm.

[0169] Example 3 - Delivery of AAVhu68.hGALC in a GALC-deficient twitcher mouse model The study described below, using the Twitcher mouse model, established the potential for delivery of AAVhu68.CB7.CI.GALC.rBG (an AAVhu68 vector containing engineered human GALC cDNA (SEQ ID NO: 9) under the control of the CB7 promoter and a BG polyA sequence adjacent to the AAV2 ITR (also known as rAAVhu68.hGALC)) to achieve therapeutic levels of GALC expression and restore several biomarkers of the disease. An overview of the Twitcher mouse study is provided in Figure 6B.

[0170] The Twitcher mouse is a natural inbred model of Krabbe disease, identified as a spontaneous mutation in the Jackson Laboratory in 1976 (Kobayashi T., et al. (1980) Brain Research. 202(2):479-483). Affected mice are homozygous for the twister loss-of-function allele (twi), which consists of a G-to-A mutation in the Galc gene. This mutation results in an early stop codon (W339X). The cleaved GALC protein has near-zero residual enzyme activity, which is similar to the GALC activity levels observed in infants with Krabbe disease. Heterozygous carrier mice (twi / +) are phenotypically normal.

[0171] The progression of the disease in Twitcher mice has been well documented (Figure 6A), and various neuropathological and behavioral defects mimic infantile Krabbe disease. Similar to infantile Krabbe patients, GALC deficiency in mice leads to the accumulation of psychosine, a cytotoxic lipid intermediate. Twitcher mice also exhibit extensive infiltration of the PNS and CNS white matter by phagocytic psychosine-filled globoid cells (thought to originate from macrophage and / or microglial lineages) (Tanaka K., et al. (1988) Brain Research. 454(1):340-346, Levine SM, et al. (1994) Intl J Dev Neuro. 1). 2(4):275-288). This results in demyelination, one of the main features of Krabbe's disease. After an initial period of normal myelin formation, affected Twitcher mice in PNS lose their myelin 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), in the central nervous system (CNS), myelin loss occurs after 20 days of age due to the death of myelinating oligodendrocytes. Perhaps due to this delay, demyelination is more severe in the peripheral nervous system than in the CNS of these mice (Suzuki K. & Suzuki K. (1983) The American journal of pathology. 111(3):394-397). Ultimately, Twitcher mice exhibit a consistent and rapid neurological deterioration after the onset of symptoms, which is also observed in infantile Krabbe patients at the time of symptom onset. Behavioral symptoms in these mice include motor phenotypes reminiscent of those observed in human patients, including tremors, spasms, and hindlimb weakness present around 20 days of age. The mice eventually progressed 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). The humane endpoint was selected as the end of the study, and the effectiveness of treatments to save the mice was evaluated. At the humane endpoint, the CNS and PNS were collected for histopathology, and demyelination and globoid cell infiltration, hGALC expression, and transgene expression (GALC enzyme activity), which are characteristic of Krabbe disease in mice and humans, were observed.

[0172] Patients with infantile krabbe exhibit similar clinical features to those of Twitcher mice. Therefore, the Twitcher mouse model is suitable for evaluating the efficacy of rAAVhu68.hGALC (rescue of enzyme activity to improve survival, motor function, and brain and neurological pathology) and supports the indication for infantile krabbe. The studies using Twitcher mice described below demonstrate the efficacy of rAAVhu68.hGALC, in which, after a single ICV administration (the most effective route of administration in mouse models where ICM is not technically feasible), active GALC enzyme was expressed in relevant tissues, rescuing survival, improving motor function, and improving CNS and PNS histopathology.

[0173] Newborn Twitcher (twi / twi) mice (PND0) are 1.0 × 10 11 GC(6.7×10 11 Mice received a single intravenous dose of rAAVhu68.hGALC in either GC / g brain weight or phosphate-buffered saline (PBS). Wild-type, heterozygous (twi / +), and homozygous (twi / twi) (PND0) mice were administered PBS intravenously to serve as a control group (see table below). In-life assessment included daily survival checks and monitoring. Autopsies were performed on animals at the humane endpoint. Brain samples were taken at autopsy to evaluate transgene expression (GALC enzyme activity). [Table 4]

[0174] In rAAVhu68.hGALC twi / twi treated mice, a minimal increase in cerebral GALC activity was observed. All twi / twi animals were euthanized upon reaching the humane endpoint. Three twi / twi animals were found dead and included in the survival analysis. 1 × 10 11 Intravenous administration of GC-derived rAAVhu68.hGALC resulted in a small but statistically significant increase in survival time, from a median survival time of 40.5 days in twi / twi PBS mice to a median survival time of 49 days in twi / twi mice (Figure 7). Intravenous administration of rAAVhu68.hGALC resulted in a minimal increase in cerebral GALC activity, which remained below wild-type levels. Mean GALC activity in mice treated with rAAVhu68.hGALC was 44% of wild-type levels (Figure 8).

[0175] Next, we conducted a study to determine the efficacy of rAAVhu68.hGALC (AAVhu68.CB7.hGALCco.rBG) in a Twitcher mouse model after intraventricular (ICV) administration. Since direct administration to the CSF is known to facilitate CNS transduction at lower doses, rAAVhu68.hGALC ICV was administered to pre-symptomatic Twitcher (twi / twi) mice. Twitcher (twi / twi) mice (PND0) received one of three dose levels (2.0 × 10⁶). 10 GC, 5.0×10 10 GC, or 1.0 × 10 11 The mice received a single ICV dose of rAAVhu68.hGALC (GC) or phosphate-buffered saline (PBS). Wild-type, heterozygous (twi / +), and homozygous (twi / twi) (PND0) mice were administered PBS via ICV and used as a control group. In-life assessment included survival checks, weight monitoring, neuromotor assessment (rotarod), and survival monitoring. Autopsies were performed on the animals at the humane endpoint. At the humane endpoint, CNS and PNS were collected for histopathology and observed for demyelination and globoid cell infiltration, hGALC expression, and transgene expression (GALC enzyme activity). [Table 5]

[0176] rAAVhu68.hGALC showed that in PBS-treated twi / twi mice, the median survival time was 43 days, compared to 2 × 10⁻¹⁰. 10 The GC dosage is 5 x 10 for 62 days. 10 GC doses last 99 days, and 1 x 10 11 At GC doses, a statistically significant dose-dependent increase in survival time was observed up to 130 days (Figure 9). Administration of rAAVhu68.hGALC resulted in a statistically significant recovery of body weight loss in twi / twi mice at all dose levels compared to body weight in PBS-treated twi / twi mice. Body weight of mice given rAAVhu68.hGALC was higher in the two highest dose groups (5.0 × 10⁴). 10GC and 1.0 × 10 11 The results were the same in GC (Figure 10). Neuromotor function was assessed by the rotorod test. This assesses coordination and balance by measuring the time it takes for a mouse running on a gradually accelerating rotating rod to fall. A decrease in fall latency indicates neuromotor impairment, while an increase in fall latency indicates improvement in neuromotor function. Compared to PBS-treated twi / twi mice, ICV administration of rAAVhu68.hGALC to twi / twi mice resulted in a dose-dependent increase in fall latency. The increase in fall latency was observed at two peak doses (5.0 × 10⁻¹⁰). 10 and 1.0 × 10 11 GC) was statistically significant (Figure 11). Brain GALC enzyme activity increased in a dose-dependent manner compared to wild-type PBS-treated mice. GALC activity in the rAAVhu68.hGALC-treated group was 2.0 × 10⁻⁶ compared to the wild-type PBS-treated group. 10 GC group: 117%, 5.0 × 10 10 173% in the GC group, and 1.0 × 10⁻⁶ 11 The GC group showed a 210% improvement. Compared to PBS-treated twi / twi mice, rA AVhu68.hGALC-treated mice also showed increased GALC activity (Figure 12).

[0177] The brains of PBS-treated twi / twi animals showed minimal to moderate demyelination, and all treatment groups exhibited normal WT-like corpus callosum myelin strength (Figure 15). Reduced myelin staining and infiltration were observed in the white matter of the corpus callosum and cerebellum of PBS-treated twi / twi mice. Treatment at all dose levels reduced demyelination and suppressed globoid cells in the corpus callosum (Figure 16). In the cerebellum, rAAVhu68.hGALC was less effective, and demyelination and globoid cell infiltration were present in all rAAVhu68.hGALC treatment groups. Similar demyelination and globoid cell infiltration were observed in PBS-treated twi / twi animals, as well as in 2.0 × 10⁶ mice. 10 GC and 5.0×10 10 Observed in rAAVhu68.hGALC-treated animal groups with GC doses. The white matter in the most central part of the cerebellar gyrus was almost always 1.0 × 10⁶. 11Animals treated with rAAVhu68.hGALC at GC doses appeared normal, exhibiting dark blue myelin staining and lacking globoid cells, although the apex was rich in globoid cells (Figure 16). Wild-type mice showed abundant myelinated nerve fibers that were densely packed and lacked inflammatory cells, while twi / twi mice showed a reduced number of myelinated nerve fibers and numerous mononuclear giant cells (globoid cells) were observed between nerve fibers (Figure 17). Sciatic nerves from all rAAVhu68.hGALC-treated twi / twi mice showed similar pathology to the PBS-treated twi / twi group, exhibiting severe demyelination and globoid cell infiltration (Figure 17). This finding is likely due to the fact that the humane endpoint was defined as hindlimb paralysis (which would occur if the sciatic nerve were severely affected).

[0178] IBA1-positive cells in the corpus callosum and cortex were normal in PBS-treated wild-type mice and all rAAVhu68.hGALC-treated mice. Neuroinflammation persisted in the cerebellum and brainstem where globoid cells were enlarged (Figure 18). 5.0 × 10 10 GC and 1.0 × 10 11 In animals administered rAAVhu68.hGALC at GC doses, a partial therapeutic effect was observed in the cerebellum, as evidenced by fewer and smaller IBA1-positive cells. Dose-dependent expression of hGALC in neurons of the cerebral cortex, hippocampus, cerebellum, and choroid plexus (but not in the brainstem) (Figure 19).

[0179] Example 4 - Evaluation of AAV1.CB7.CI.hGALCco.rBG, AAV3B.CB7.CI.hGALCco.rBG, and AAV5.CB7.CI.hGALCco.rBG in neonatal Twitcher(twi / twi) mice after intracerebroventricular administration. To evaluate three additional clinical vectors (AAV1.CB7.CI.hGALCco.rBG, AAV3B.CB7.CI.hGALCco.rBG, AAV5.CB7.CI.hGALCco.rBG) and determine their efficacy in a mouse model of infantile Krabbe disease after intravascular (ICV) administration, we conducted a study. The potential candidates had different serotypes but all contained engineered human GALC cDNA under the control of the CB7 promoter and a BG polyA sequence adjacent to the AAV2 ITR.

[0180] Newborn Twitcher (twi / twi) mice (PND0) are 2.0 x 10 10 GC(1.3 × 10 11 Animals received a single ICV (intracytoplasmic coagulation) dose of AAV1.CB7.CI.hGALCco.rBG, AAV3B.CB7.CI.hGALCco.rBG, or AAV5.CB7.CI.hGALCco.rBG at a dose of GC / g brain weight. The ICV route (involving direct administration of the AAV vector to the ventricular CSF) was selected to evaluate the potential for delivering GALC enzymes to the cerebral neuropathy (CNS) and postnatal neuropathy (PNS) (these are targets for the treatment of infantile Krabbe disease). In-life assessments included survival checks, weight monitoring, neuromotor assessment (rotarod), and survival monitoring. Autopsies were performed on animals at the humane endpoint. At the humane endpoint, the CNS and PNS were collected for histopathology and observed for demyelination and globoid cell infiltration, hGALC expression, and transgene expression (GALC enzyme activity). [Table 6]

[0181] All twi / twi animals were euthanized upon reaching the humane endpoint. One twi / twi animal was found dead (AAV3B.hGALC group) and included in the survival analysis. The median survival time was 57 days after administration of AAV1.hGALC in twi / twi animals, and 51 days in the AAV3B.hGALC and AAV5.hGALC groups (Figures 20A and 20B).

[0182] Administration of AAV5.hGALC led to the recovery of weight loss in twi / twi mice. Weight recovery was statistically significant in AAV5.hGALC compared to PBS-treated twi / twi mice (Figure 21).

[0183] Neuromotor function was assessed using the rotarod test. This test evaluates coordination and balance by measuring the time it takes for a mouse running on a gradually accelerating rotating rod to fall. A decrease in fall latency indicates neuromotor impairment, while an increase in fall latency indicates improvement in neuromotor function (Figure 22). No significant changes in fall latency were observed with the capsids tested.

[0184] GALC enzyme activity levels in the brain increased after administration of AAV1.hGALC compared to other capsids and twi / twi PBS-treated groups (Figure 23). GALC activity was similar between AAV3B.hGALC and AAV5.hGALC, and twi / twi It was higher than in the PBS-treated group. In the liver, GALC enzyme activity levels were twi / twi Compared to the PBS-treated group, the AAV-treated group showed lower levels (Figure 24). In serum, GALC enzyme activity levels were lower in the AAV-treated group compared to the twi / twi PBS-treated group (Figure 25).

[0185] Following ICV administration of AAV1.hGALC, sciatic nerve (Figure 26) IBA1-positive cells normalized in the cerebral cortex and hippocampus (Figure 27). Numerous IBA1-positive globoid cells were observed in the corpus callosum of animals treated with AAV3B.hGALC and AAV5.hGALC, indicating the lower efficacy of these capsids. AAV1.hGALC administration resulted in robust transduction of neurons in the hippocampus and cerebral cortex, cerebellar Purkinje cells, choroid plexus cells, and medulla oblongata adjacent to the pontine cistern (Figure 28). The stronger transduction of choroid plexus and some medullary neurons may explain the higher GALC activity measured in the brains of animals injected with AAV1.hGALC. AAV3B.hGALC primarily transduced cerebellar neurons, with very few neurons transduced elsewhere. AAV5.hGALC transduced overall This resulted in low neuronal transduction, primarily affecting cells in the choroid plexus (Figure 28).

[0186] Example 5 - Evaluation of rAAVhu68.hGALC in juvenile Twitcher (twi / twi) mice after intracerebroventricular administration. The efficacy of rAAVhu68.hGALC (AAVhu68.CB7.hGALCco.rBG) was determined in a Twitcher mouse model of infantile Krabbe disease after intraventricular (ICV) administration. rAAVhu68.hGALC is a recombinant adeno-associated virus (AAV) serotype hu68 vector expressing the human galactocerebrosidase (GALC) gene. Young Twitcher (twi / twi) mice at PND12 measured 1.0 × 10⁶ mice. 11 GC or 2.0 × 10 11 GC (each, 2.5 × 10) 11 or 5.0 × 10 11 A single ICV dose of rAAVhu68.hGALC was administered at a dose of GC / g brain weight. Alternatively, 2.0 × 10⁶ doses were administered via PND21. 11 GC(5.0×10 11rAAVhu68.hGALC was administered at a dose of GC / g brain weight. Animal ages were selected as follows: PND12 represented the age before the onset of behavioral symptoms ("early symptomatic"), and PND21 represented the age at which the mice exhibited behavioral symptoms ("late symptomatic"). Furthermore, PND12 and PND21 correspond to 2 months and 9 months of age in humans, respectively (www.translatingtime.org), which is similar to the infant population targeted in the FIH trial.

[0187] In-life assessment included daily survival checks, weight monitoring, neuromotor assessment (rotarod), and survival monitoring. Autopsies were performed on animals at the humane endpoint. At the humane endpoint, CNS and PNS were collected for histopathology and observed for demyelination and globoid cell infiltration, hGALC expression, and transgene expression (GALC enzyme activity). [Table 7]

[0188] All twi / twi animals were euthanized upon reaching the humane endpoint. In twi / twi mice, administration of rAAVhu68.hGALC to PND12 and PND21 mice resulted in a statistically significant increase in survival rate (Figures 29A and 29B). The median survival time for mice treated with PND12 was 41.5 days for PBS-treated mice and 1.0 × 10⁶ days for rAAVhu68.hGALC doses. 11 GC-treated mice for 71 days, and rAAVhu68.hGALC dose 2.0 × 10 11 The life expectancy for mice treated with GC was 81 days. PND21 measured 2.0 × 10⁶. 11 In mice treated with rAAVhu68.hGALC at GC doses, the median survival time was 52 days. Compared to PND21, PND12 showed a 2.0 × 10⁶ difference. 11In animals treated with rAAVhu68.hGALC at GC doses, survival rates were statistically significantly improved (Figure 30). The median survival times for mice treated with PND12 and PND21 were 81 days and 52 days, respectively. On PND12, 1.0 × 10 11 GC or 2.0 × 10 11 Administration of rAAVhu68.hGALC at GC doses resulted in a statistically significant recovery of weight loss in twi / twi mice compared to PBS-treated mice (Figure 31). 2.0 × 10⁶ in PND21 11 Treatment with rAAVhu68.hGALC at GC doses did not significantly improve weight loss compared to vehicle-treated animals (Figure 32).

[0189] Neuromotor function was assessed by the rotorod test. This test evaluates coordination and balance by measuring the time it takes for a mouse running on a gradually accelerating rotating rod to fall. A decrease in fall latency indicates neuromotor impairment, while an increase in fall latency indicates improvement in neuromotor function. ICV administration of rAAVhu68.hGALC to twi / twi mice in PND12 resulted in a statistically significant increase in fall latency compared to PBS-treated twi / twi mice (Figure 33). Fall latency was similar in PBS-treated wild-type and twi / + mice. 2.0 × 10⁶ twi / twi mice in PND21 11 Administering rAAVhu68.hGALC at the GC dose did not improve fall latency (Figure 34).

[0190] In twi / twi mice administered rAAVhu68.hGALC to PND12, brain GALC enzyme activity increased in a dose-dependent manner (Figure 35). Mice were then subjected to 2.0 × 10⁶ doses in PND21. 11 When treated with rAAVhu68.hGALC at GC doses, the increase in GALC enzyme activity was not very significant. PND21 showed 2.0 × 10⁶ 11In twi / twi mice treated with rAAVhu68.hGALC at GC doses, hepatic and serum GALC enzyme activity was higher compared to mice treated with PND12 (Figures 36 and 37). The higher GALC activity in PND21-treated mice may be a result of a shorter post-treatment survival period, leading to reduced transgene loss after liver growth in juvenile animals. The brains of twi / twi PBS-treated animals showed reduced myelin staining in the white matter of the corpus callosum and cerebellum, and abundant globoid cells stained with PAS were observed in the cerebellar gyri (Figure 38). rAAVhu68.hGALC-treated mice reduced demyelination in the corpus callosum in all dose groups administered with PND12 and PND21, but were less effective in correcting demyelination in the cerebellum where globoid cell infiltration was observed.

[0191] Wild-type mice exhibited abundant myelinated nerve fibers that were densely packed and lacked inflammatory cells, while twi / twi mice showed a reduced number of myelinated nerve fibers, with numerous mononuclear giant cells (globoid cells) observed between the nerve fibers. In PND12 or PND21, sciatic nerves from all twi / twi mice treated with rAAVhu68.hGALC showed moderate to severe demyelination of myelin fibers (Figure 39). This finding is likely due to the fact that the humane endpoint was defined as hindlimb paralysis (which would occur if the sciatic nerve were severely affected). The longest-surviving rAAVhu68.hGALC-treated mice had more myelin fibers.

[0192] IBA1-positive cells were similar to wild-type cells in the cortex of twi / twi mice treated with rAAVhu68.hGALC on PND12, but mice treated on PND21 showed enlargement of globoid cells and activated microglia in all analyzed brain regions (Figure 40). Neuroinflammation and globoid cell hypertrophy persisted in the corpus callosum, cerebellum (Figure 40), and brainstem (data not shown). Positive expression of hGALC in neurons of the cerebral cortex and hippocampus (near the injection site) was observed in twi / twi mice treated with rAAVhu68.hGALC on PND12 or PND21 (Figure 41). Cerebellar Purkinje cells were hGALC-positive only in animals treated with rAAVhu68.hGALC on PND12. The brainstem was not transduced by rAAVhu68.hGALC with respect to age at administration.

[0193] Example 6 - Evaluation of delivery of rAAVhu68.hGALC to juvenile Twitcher (twi / twi) mice Further research was conducted to determine the efficacy of rAAVhu68.hGALC (AAVhu68.CB7.hGALCco.rBG) in Twitcher mice after intraventricular (ICV) administration. Young Twitcher (twi / twi) mice in PND12 had a 2.0 × 10⁶ gestational age. 11 GC(1.3 × 10 12 At a dose of GC / g brain weight, rAAVhu68.h The animals received a single ICV dose of GALC. PND12 was chosen for the age of the animals before the onset of behavioral symptoms ("early symptomatic"), which corresponds to a 2-month-old human (www.translatingtime.org) and is similar to the infant population targeted for FIH trials.

[0194] In-life assessment included daily survival checks, weight monitoring, and neuromotor assessment (rotarod). Autopsies were performed on animals using PND40. During autopsies, the central nervous system (CNS) and primary nervous system (PNS) were collected for histopathology to observe demyelination and globoid cell infiltration, hGALC expression, and transgene expression (GALC enzyme activity). [Table 8]

[0195] Clinical signs were scored three times a week by blinded personnel in the treatment group using unpublished assessments of clasping ability, gait, tremor, kyphosis, and fur quality (see table below). These scales were selected to assess clinical status based on symptoms typically exhibited by twi / twi mice. Scores greater than 0 indicate clinical deterioration. [Table 9]

[0196] 2.0×10 11 Administering rAAVhu68.hGALC at the GC dose restored weight loss to levels comparable to wild-type mice treated with the vehicle (Figure 42).

[0197] Neuromotor function was assessed by the rotorod test. This test evaluates coordination and balance by measuring the time it takes for a mouse running on a gradually accelerating rotating rod to fall. A decrease in fall latency indicates neuromotor impairment, while an increase in fall latency indicates improvement in neuromotor function. Administration of rAAVhu68.hGALC to twi / twi mice resulted in a statistically significant increase in fall latency compared to PBS-treated twi / twi mice (Figure 43). Administration of rAAVhu68.hGALC to twi / twi mice resulted in a decrease in total clinical score compared to PBS-treated twi / twi mice (Figure 44). rAAVhu68.hGALC-treated twi / twi mice had similar clinical scores to wild-type PBS-treated mice.

[0198] Brain GALC enzyme activity was increased in rAAVhu68.hGALC-treated mice compared to wild-type or twi / twi PBS-treated mice (Figure 45). Liver GALC enzyme activity was increased in rAAVhu68.hGALC-treated mice compared to wild-type or twi / twi PBS-treated mice (Figure 46). Serum GALC enzyme activity in PND28 and PND40 was increased in rAAVhu68.hGALC-treated mice compared to wild-type or twi / twi PBS-treated mice (Figures 47A and 47B).

[0199] Administration of rAAVhu68.hGALC to twi / twi mice did not result in signs of demyelination (Figure 48). The level of demyelination in rAAVhu68.hGALC-treated mice was similar to that of wild-type PBS-treated mice. Decreased myelin staining and infiltration (due to abundant globoid cells stained with PAS) were observed in the white matter of the brainstem, corpus callosum, and cerebellum of twi / twi PBS-treated mice (Figure 49). Treatment of twi / twi mice with rAAVhu68.hGALC reduced demyelination and suppressed globoid cells in these myelin-rich areas (Figure 48). In PBS-treated twi / twi mice, peripheral nerves showed a significant loss of myelin fiber volume, and the nerve fiber structure appeared disordered (Figure 50). In contrast, peripheral nerves from twi / twi mice treated with rAAVhu68.hGALC showed some degree of demyelination, but the number and structure of nerve fibers were not disordered compared to PBS-treated mice (Figure 50). Similar findings were observed in the sciatic nerve (Figure 50).

[0200] Administration of rAAVhu68.hGALC reduced demyelination in the posterior columns of the spinal cord compared to PBS-treated twi / twi mice exhibiting severe demyelination (Figure 51). IBA1-positive cells in wild-type PBS-treated mice were small and uniformly distributed throughout the brain, whereas in PBS-treated twi / twi mice, microglia were larger and gave a patchy, coarse staining appearance in the cortex, corpus callosum, brainstem, and cerebellum (Figures 52A-52C). In twi / twi mice treated with rAAVhu68.hGALC, more consistent staining of IBA1-positive cells was present in the cortex and corpus callosum, but patchy staining, indicative of neuroinflammation, persisted in the cerebellum and brainstem (Figures 52A-52C). Administration of rAAVhu68.hGALC dramatically reduced IBA1-positive cells in the cortex, hippocampus, and corpus callosum (Figure 53). However, levels remained the same in the cerebellum and brainstem.

[0201] Administration of rAAVhu68.hGALC reduced neuroinflammation in peripheral nerves (Figure 50). However, rAAVhu68.hGALC administration did not inhibit neuroinflammation in the spinal cord compared to PBS-treated twi / twi mice (Figure 54). hGALC was strongly expressed in cortical and hippocampal neurons, and some positive staining was observed in cerebellar Purkinje neurons. No significant staining was observed in the brainstem of rAAVhu68.hGALC-treated twi / twi mice (Figure 55). No positive staining was observed in the brains of wild-type and twi / twi PBS-treated mice.

[0202] Example 7 - Efficacy of bone marrow transplantation in combination with rAAVhu68.hGALC administration This study investigated the potential benefits of dual therapy with rAAVhu68.hGALC and bone marrow transplantation (BMT). The inventors investigated this combination therapy due to the prominent neuroinflammatory component of Krabbe disease. Theoretically, hematopoietic stem cell transplantation (HSCT) provides a further source of GALC enzymes to the CNS (derived from transplanted cells and macrophage / microglia cells derived from neurons transduced with rAAVhu68.hGALC), while rAAVhu68.hGALC provides modification to the PNS, which is unaffected by HSCT, thus creating a synergistic effect. Furthermore, this study examined different combination therapy designs to evaluate whether rAAVhu68.hGALC may be effective in 1) patients who initially received HSCT through an NBS program and subsequently received gene therapy, and / or 2) patients who, if eligible, initially received gene therapy and subsequently received HSCT.

[0203] The following table summarizes the studies on combination therapy. [Table 10-1] [Table 10-2]

[0204] A better response was predicted with combination therapy, therefore, 1.00 × 10 11 We utilized GC-dose rAAVhu68.hGALC (allowing for lower doses of rAAVhu68.hGALC than those used in previous studies with rAAVhu68.hGALC monotherapy). The efficacy of rAAVhu68.hGALC was evaluated in terms of survival, body weight, and neurological observations (e.g., presence of tremor and abnormal clasping reflexes).

[0205] Figures 56A and 56B show survival data for groups 1-3. To date, the best survival has been achieved by treating pre-symptomatic Twitcher mice (twi / twi) (group 2) with a combination of ICV administration of rAAVhu68.hGALC at PND0 followed by BMT administration at PND10. In the absence of obvious signs, survival time extended to over 300 days. These mice appeared to be in better physical condition based on the aforementioned clinical evaluation, exhibiting slight tremors but no significant gait abnormalities or clasping (analysis is still in progress, data not yet described). Mice that received BMT before rAAVhu68.hGALC (group 3) are still alive (N=3 / 7) but exhibit significant tremors, some gait abnormalities, and weight loss. However, the busulfan conditioning regimen combined with BMT was toxic in mice younger than 10 days of age, and in both groups 2 and 3, increased mortality was observed either before or immediately after BMT, regardless of the order of combination therapy. Group 4 was infused to mimic a clinically relevant situation in which gene therapy is administered to early symptomatic patients, followed by BMT.

[0206] 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.

[0207] Example 8 - Efficacy of rAAVhu68.hGALC after intraventricular administration in Twitcher(twi / twi) mice for determining the minimum effective dose (MED). The purpose of this pharmacological study is to determine the minimum effective dose (MED) and transgene expression levels of AAVhu68.CB7.CI.GALC.rBG (rAAVhu68.hGALC) after intraventricular (ICV) administration in a Twitcher mouse model of infantile Krabbe disease. That was the task.

[0208] In-life assessment included daily observations, survival monitoring, weight measurement, neurological examination, neuromotor function assessment (rotarod), and evaluation of serum transgene expression (GALC enzyme activity). Autopsies were performed on untreated mice on the day of administration (PND12-14 [untreated baseline cohort]), 4 weeks after administration (PND40-42 [PND40 cohort]), and at a humane endpoint to assess survival (up to 10 weeks after administration in twi / twi mice [survival cohort]). A comprehensive list of tissues was collected at autopsies for histopathological evaluation. Samples of the brain, spinal cord, and sciatic nerve were collected and evaluated for myelin formation (Luxor Fast Blue [LFB] staining), along with globoid cell infiltration and neuroinflammation (periodate Schiff [PAS] staining and IBA1 immunohistochemistry). Brain, peripheral organs, and serum were collected for transgene expression assays (GALC enzyme activity). Blood was collected for whole blood cell count (CBC) testing and serum clinical chemistry analysis.

[0209] At enrollment (PND12-14), litters of mice were randomly assigned to treatment groups. Animal ages were selected to model the disease stage of early symptomatic patients. Due to the size of the study and the unpredictable birth dates of litters, litters were injected as soon as they became available. The randomization process was as follows: as litters became available, the mating cage card number was entered into a random list generator. The randomized list was then assigned to study groups in ascending order (the first number was assigned to group 1, the second to group 2, and so on, until all groups were enrolled). The randomized lists were stored in a study binder. Animals enrolled earlier were assigned to survival cohorts within each group, and mice enrolled later were assigned to the PND40 cohort. Animals that died before weaning (for appropriate and justifiable reasons) were replaced at the discretion of the principal investigator.

[0210] After group assignment, each animal (excluding the untreated baseline control) received a single ICV infusion from one of the following treatments: ●6.8×10 9GC / Animal Dose rAAVhu68.hGALC (Test Substance) ●2.0×10 10 GC / Animal Dose rAAVhu68.hGALC (Test Substance) ●6.8×10 10 GC / Animal Dose rAAVhu68.hGALC (Test Substance) ●2.0×10 11 GC / Animal Dose rAAVhu68.hGALC (Test Substance) ●ITFFB (Control Object)

[0211] The group designation, dose level, and route of administration (ROA) are shown in the table below. [Table 11]

[0212] In the PND40 cohort, one twi / twi mouse (animal 1074, group 3a, N=1 / 9) administered with vehicle was euthanized at PND38 due to disease progression resulting in a 20% weight loss, according to the study-defined euthanasia criteria. The animals survived until the scheduled autopsy.

[0213] In the survival cohort, all vehicle-treated wild-type mice (N=8 / 8) survived until the end of the study, while most rAAVhu68.hGALC-treated twi / twi mice (N=34 / 36) and vehicle-treated twi / twi controls (N=8 / 9) were euthanized according to the euthanasia criteria defined in the study. All euthanized twi / twi mice showed signs of disease progression. Of the remaining mice, 2 out of 36 rAAVhu68.hGALC-treated twi / twi mice and 1 out of 9 vehicle-treated twi / twi mice died. Animal 1071 (rAAVhu68.hGALC, 2.0 × 10⁻⁶) 10GC (group 7b) was found to have died at PND23 (11 days post-treatment), which is likely due to failure to thrive after weaning. Animal 1053 (rAAVhu68.hGALC, 2.0×10) 11 Animal GC (group 5b) was found to have died at PND23 (11 days post-treatment), and animal 1062 (ITFFB, group 3b) was found to have died at PND21 (7 days post-treatment), but the cause of death for both of these mice could not be determined.

[0214] In summary, there are three maximum doses (2.0 x 10 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 Administration of rAAVhu68.hGALC in GC resulted in a significant dose-dependent increase in survival rate in twi / twi mice compared to administration in vehicle-treated twi / twi controls (Figure 57). The median survival time was 40.5 days in the vehicle-treated twi / twi control group, but a dose-dependent increase in survival time was observed in rAAVhu68.hGALC-treated mice. In rAAVhu68.hGALC-treated animals, the median survival age was 44.5 days (6.8 × 10⁻⁶). 9 GC), 48 days (2.0×10 10 GC), 56.5 days (6.8×10 10 GC), or 70 days (2.0 x 10 11 The mice were GC (Gross Classification). All vehicle-treated WT mice survived to the study endpoint and were euthanized at a median age of 125 days.

[0215] Throughout the study, no clinical abnormalities associated with rAAVhu68.hGALC were observed.

[0216] The following three twi / twi mice were found to have died during the study: ●Animal 1071 (rAAVhu68.hGALC, 2.0×10 10GC (group 7b) was found to have died on PND23 (11 days after administration). One day before PND22 (10 days after administration), this animal was observed to be small in size (less than 5g) compared to its cagemate, lethargic, and kyphosis. No disease-related neurological signs (e.g., tremor or ataxia) were observed, and no significant findings were apparent in histopathology, so the cause of death was considered to be due to post-weaning growth failure. ●Animal 1053 (rAAVhu68.hGALC, 2.0×10 11 GC (group 5b) was found to have died on PND23 (11 days post-treatment) after exhibiting seizure-like behavior (tonic-clonic activity accompanied by hyperactivity) the previous day. For animal 1053, there were no macroscopic or microscopic histopathological findings other than the expected disease-related demyelination and globoid cell infiltration in the sciatic nerve and cerebellar white matter. Since seizure-like activity is not a phenotype demonstrated in twi / twi mice, the cause of death for animal 1053 remained undetermined, but the possibility of an ICV procedure-related etiology could not be ruled out. ● Animal 1062 (ITFFB, group 3b, identified as M34-3F in the histopathological report because it died before microchip implantation) was found dead on PND21 (7 days post-procedure). No clinical abnormalities were observed, and histopathologically, no significant findings other than disease-related demyelination expected from the CNS were found. The cause of death of animal 1062 was unknown.

[0217] The remaining rAAVhu68.hGALC-treated mice (N=67 / 69) and vehicle-treated mice (N=17 / 18) in the PND40 and survival cohort exhibited clinical signs associated with the Krabbe disease phenotype, including tremor, ataxia, hindlimb weakness, hindlimb paralysis, kyphosis, and / or weight loss. These animals were euthanized at scheduled autopsies in PND40 or for humane reasons due to disease progression when they met the study-defined euthanasia criteria.

[0218] Three maximum doses (2.0 x 10 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶11 Administration of rAAVhu68.hGALC in GC resulted in a significant dose-dependent recovery of weight loss in both male and female twi / twi mice, compared to the body weight of sex-matched vehicle-treated twi / twi mice from PND21-22 (weaning) to PND41-42 (Figures 58A and 58B).

[0219] In the survival cohort, all groups of twi / twi mice showed weight loss from PND41-42 onward. However, the rate of weight loss was inversely correlated with the dose; generally, higher doses of rAAVhu68.hGALC were associated with slower weight loss in twi / twi mice. No sex differences related to weight were observed in this study (Figures 59A and 59B).

[0220] Three maximum doses (2.0 × 10) for twi / twi mice 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 Administration of rAAVhu68.hGALC to GC mice resulted in a significant dose-dependent reduction in total clinical score compared to vehicle-treated twi / twi mice from PND21-22 (weaning) to PND41-42. The significant reduction in clinical severity score indicated improvement in Krabbe disease-related clinical phenotype after rAAVhu68.hGALC administration (Figure 60).

[0221] In the survival cohort, all groups of twi / twi mice showed a gradual increase in clinical severity scores from PND41-42 before humane euthanasia, but the rate of increase was inversely correlated with the dose, and generally, higher doses of rAAVhu68.hGALC resulted in a slower increase in clinical severity scores in twi / twi mice. (Highest dose of rAAVhu68.hGALC (2.0 × 10) 11 In GC, twi / twi mice achieved significantly lower peak clinical severity scores at the time of humane euthanasia compared to vehicle-treated twi / twi mice, suggesting a better overall clinical status at the humane endpoint (Figure 61).

[0222] Neuromotor function was assessed using the rotorod test. This test evaluates coordination and balance by measuring the time it takes for a mouse running on a gradually accelerating rotating rod to fall. A decrease in fall latency indicates neuromotor impairment, while an increase in fall latency indicates improvement in neuromotor function. 6.8 × 10 10 GC or 2.0 × 10 11 Administration of rAAVhu68.hGALC at GC doses to twi / twi mice resulted in a dose-dependent increase in fall latency at PND35-37 (3 weeks post-treatment) compared to vehicle-treated twi / twi mice, indicating improvement in neuromotor function after rAAVhu68.hGALC administration (Figure 62).

[0223] In serum, three maximum doses (2.0 × 10) were used in twi / twi mice. 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 Administration of rAAVhu68.hGALC in GC mice resulted in a significant dose-dependent increase in GALC enzyme activity compared to vehicle-treated twi / twi mice at PND35-37 (3 weeks post-treatment). Furthermore, all doses (6.8 × 10⁻¹⁰) were significantly increased. 9 GC, 2.0×10 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 The GC-derived rAAVhu68.hGALC increased GALC enzyme activity to levels above those of vehicle-treated wild-type (Figure 63).

[0224] In the brain, three optimal doses (2.0 × 10) were used in twi / twi mice. 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 Administration of rAAVhu68.hGALC in GC mice resulted in a significant dose-dependent increase in GALC enzyme activity compared to vehicle-treated twi / twi mice. Furthermore, these doses (2.0 × 10⁻¹⁰) 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11The GC-derived rAAVhu68.hGALC increased the mean GALC enzyme activity in twi / twi mice to a level higher than that of vehicle-treated wild-type controls (Figure 64).

[0225] In the heart, all doses (6.8 × 10) 9 GC, 2.0×10 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 Administration of rAAVhu68.hGALC to GC mice resulted in a significant dose-dependent increase in GALC enzyme activity compared to vehicle-treated twi / twi mice (Figure 65A).

[0226] For the quadriceps, three maximum doses (2.0 × 10) were used for twi / twi mice. 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 Administration of rAAVhu68.hGALC to GC mice resulted in a significant dose-dependent increase in GALC enzyme activity compared to vehicle-treated twi / twi mice (Figures 67A and 67B). In the liver (Figure 65C), lungs, and diaphragm (Figure 66C), two highest doses (6.8 × 10⁶) were administered to twi / twi mice. 10 GC or 2.0 × 10 11 Administration of rAAVhu68.hGALC to GC mice resulted in a significant dose-dependent increase in GALC enzyme activity compared to vehicle-treated twi / twi mice.

[0227] In the kidneys, the highest dose for twi / twi mice was (2.0 × 10⁻¹⁰). 11 Administration of rAAVhu68.hGALC to GC mice resulted in a significant increase in GALC enzyme activity compared to vehicle-treated twi / twi mice (Figure 65B).

[0228] In the spleens of twi / twi mice treated with rAAVhu68.hGALC at arbitrary doses, no increase in GALC enzyme activity was observed compared to that of vehicle-treated twi / twi mice (Figure 65D). However, it should be noted that the artificial fluorescence-generating substrate used in this assay is known to react with other lysosomal enzymes, such as β-galactosidase. Since background GALC enzyme activity was elevated in both the spleen and kidneys of vehicle-treated twi / twi mice, non-specific activity may be present in these organs.

[0229] When the expression of the transgene product in rAAVhu68.hGALC-treated twi / twi mice was compared with that of vehicle-treated wild-type controls, the average GALC enzyme activity in twi / twi mice was compared to that of all doses of rAAVhu68.hGALC (6.8 × 10⁻¹⁰) in the heart and quadriceps muscles. 9 GC, 2.0×10 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 In GC, recovery was achieved at or above wild-type levels, and in the brain and diaphragm, the three highest doses (2.0 × 10) were observed. 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 In GC, recovery was achieved at or above wild-type levels, and in the spleen, the two highest doses (6.8 × 10) were observed. 10 GC or 2.0 × 10 11 In GC, recovery was observed at or above wild-type levels, and in the liver and lungs, the maximum dose (2.0 × 10) was achieved. 11 In GC, its condition recovered to a level higher than that of the wild type (Figures 65C and 66A).

[0230] In clinicopathology, no toxicity associated with rAAVhu68.hGALC treatment was observed. Due to the twi / twi mouse model phenotype, which results in severe difficulty walking due to hindlimb paralysis (paralysis / dragging of both hind limbs), these animals were unable to access food and water, resulting in physical debilitation and growth retardation. Several abnormalities associated with the twi / twi phenotype included lymphocyte count, aspartate aminotransferase (AST), and biotin. Clinicopathological parameters, including rubin, alanine aminotransferase (ALT), glucose, amylase, and triglycerides, were observed. In addition, some abnormalities were corrected by rAAVhu68.hGALC administration. Only phenotypic or rAAVhu68.hGALC-corrected parameters are discussed in this section.

[0231] At baseline, untreated twi / twi mice showed similar lymphocyte counts to untreated WT controls. However, in PND40 and the humane endpoint, vehicle-treated twi / twi mice showed a significant decrease in lymphocytes (lymphopenia) compared to vehicle-treated wild-type controls, as expected. In contrast, 2.0 × 10⁶ twi / twi mice 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 Administration of rAAVhu68.hGALC at GC doses normalized lymphocyte counts to levels similar to vehicle-treated wild-type controls at PND40-42 (4 weeks post-treatment). In the humanitarian endpoint, all doses (6.8 × 10) normalized lymphocyte counts to levels similar to vehicle-treated wild-type controls. 9 GC, 2.0×10 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 The GC-derived rAAVhu68.hGALC normalized lymphocyte counts in twi / twi mice to levels similar to vehicle-treated wild-type controls (Figures 67A and 67B).

[0232] At baseline, serum AST levels appeared similar between untreated twi / twi mice and untreated WT controls, but statistical analysis was not possible due to the small sample size in the untreated twi / twi group. In PND40, AST levels were elevated in vehicle-treated twi / twi mice compared to vehicle-treated WT controls, but all rAAVhu68.hGALC-treated groups showed similar AST levels to vehicle-treated WT controls. In the humanitarian endpoint, AST levels in vehicle-treated twi / twi mice were comparable to those in vehicle-treated WT controls, but 6.8 × 10⁻⁶ 9GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 AST levels in twi / twi mice administered GC doses of rAAVhu68.hGALC were elevated compared to vehicle-treated WT controls (Figures 68A and 68B). The elevated AST levels observed in both PND40 and the humane endpoint appear to be primarily due to a small number of outliers. While the study animals containing the outliers with elevated AST levels did not exhibit histopathological liver lesions that would explain these elevations, treatment-related effects could not be ruled out. However, since vehicle-treated twi / twi mice also showed elevated AST levels in PND40, the most likely explanation is that this is a phenotypic abnormality.

[0233] At baseline, total bilirubin levels appeared similar between untreated twi / twi mice and untreated WT controls, but statistical analysis was not possible due to the small sample size in the untreated twi / twi group. In PND40, bilirubin levels were 2.0 × 10⁶ in vehicle-treated twi / twi mice and 2.0 × 10⁶ in vehicle-treated WT controls compared to vehicle-treated WT controls. 10 The dose of GC increased in rAAVhu68.hGALC-treated twi / twi mice. In the humane endpoint, bilirubin levels were increased in vehicle-treated twi / twi mice and the three highest doses (2.0 × 10⁻¹⁰) compared to vehicle-treated WT controls. 10 GC, 6.8×10 10 GC, and 2.0 × 10 11 Total bilirubin levels were increased in GC) rAAVhu68.hGALC-treated twi / twi mice (Figures 68C and 68D). In this study, the animals did not show any histopathological liver lesions that could explain the elevated total bilirubin levels. Therefore, the lowest dose (6.8 × 10⁶) was observed. 9 Note that statistical analysis could not be performed due to insufficient sample size in the PND40 cohort of the rAAVhu68.hGALC group of GC. However, it is possible that elevated bilirubin levels are associated with the twi / twi mouse phenotype, and the two highest doses (6.8 × 10) 10 GC and 2.0 × 10 11The normalization of bilirubin levels in PND40 in the rAAVhu68.hGALC group (GC) was a dose-dependent therapeutic effect.

[0234] At baseline, alanine aminotransferase (ALT) levels appeared similar between untreated twi / twi mice and untreated WT controls, but statistical analysis was not possible due to the small sample size in the untreated twi / twi group. In PND40, all vehicle-treated and rAAVhu68.hGALC-treated twi / twi mice showed similar ALT levels to vehicle-treated WT controls. At the humanitarian endpoint, ALT levels were 6.8 × 10⁶ in vehicle-treated twi / twi mice and 6.8 × 10⁶ in vehicle-treated WT controls compared to vehicle-treated WT controls. 9 GC, 6.8×10 10 GC, and 2.0 × 10 11 ALT levels were elevated in twi / twi mice treated with rAAVhu68.hGALC at doses of GC (Figures 69A and 69B). In this study, the animals did not exhibit any histopathological liver lesions that could explain the elevated ALT levels. Therefore, the elevated ALT levels observed in twi / twi mice at the humane endpoint appear to be related to the phenotype of twi / twi mice and not affected by rAAVhu68.hGALC administration.

[0235] At baseline, glucose levels appeared similar between untreated twi / twi mice and untreated WT controls, but statistical analysis was not possible due to the small sample size in the untreated twi / twi group. In PND40, vehicle-treated twi / twi mice and 2.0 × 10⁶ mice were compared. 10 Twi / twi mice administered GC doses of rAAVhu68.hGALC showed decreased glucose levels compared to vehicle-treated WT controls, but the two highest doses (6.8×) were also lower. 10 GC and 2.0 × 10 11 Glucose levels in the rAAVhu68.hGALC group (GC) appeared similar to those of vehicle-treated WT controls. In the humanitarian endpoint, vehicle-treated twi / twi mice and the highest dose (2.0 × 10⁶) were observed. 11twi / twi mice administered rAAVhu68.hGALC (GC) showed a decrease in glucose levels compared to vehicle-treated WT controls, while the remaining rAAVhu68.hGALC dose group (6.8 × 10) showed a decrease in glucose levels. 9 GC, 2.0×10 10 GC, 6.8×10 10 The GC group showed glucose levels similar to those of the vehicle-treated WT control group (Figures 70A and 70B).

[0236] The significance of this finding is unclear because the mice were not fasted before blood sampling during necropsy. Since vehicle-treated twi / twi mice showed decreased glucose levels compared to WT controls in both PND40 and the humane endpoint, this finding may be related to the feeding difficulties exhibited by twi / twi mice as they approach the humane endpoint due to progressive ataxia. Therefore, the minimum dose (6.8 × 10) 9 Note that statistical analysis could not be performed due to insufficient sample size in the PND40 cohort of the rAAVhu68.hGALC group of GC. 10 GC and 2.0 × 10 11 Normalization of glucose levels in rAAVhu68.hGALC (GC) may represent a dose-dependent therapeutic effect in PND40. Since a dose-dependent effect was not evident in twi / twi mice, it is unclear whether this therapeutic effect persists to the humane endpoint.

[0237] At baseline, amylase levels appeared similar between untreated twi / twi mice and untreated WT controls, but statistical analysis was not possible due to the small sample size in the untreated twi / twi group. In PND40, similar amylase levels were observed in vehicle-treated twi / twi mice and all rAAVhu68.hGALC dose groups compared to vehicle-treated WT controls. In the humanitarian endpoint, vehicle-treated twi / twi mice and 6.8 × 10⁶ 10Twi / twi mice administered GC doses of rAAVhu68.hGALC showed elevated amylase levels compared to vehicle-treated WT controls (6.8 × 10⁶). 9 GC, 2.0×10 10 GC, and 2.0 × 10 11 The GC mice showed similar amylase levels to the vehicle-treated WT controls (Figures 70C and 70D). This finding was significant because the vehicle-treated twi / twi mice showed elevated amylase levels exceeding WT levels at the humane endpoint. This may be related to feeding difficulties in twi / twi mice, which are exhibited as they approach the humane endpoint due to progressive ataxia. Most rAAVhu68.hGALC dose groups (6.8 × 10) 9 GC, 2.0×10 10 GC, or 2.0 × 10⁻⁶ 11 Normalization of amylase levels at the humane endpoint in GC (Gross Classification) is likely to reflect the treatment-related effect.

[0238] At baseline, triglyceride levels appeared similar between untreated twi / twi mice and untreated WT controls, but statistical analysis was not possible due to the small sample size in the untreated twi / twi group. PND40 showed 2.0 × 10⁶ 10 In twi / twi mice administered rAAVhu68.hGALC at GC doses, a decrease in triglyceride levels was observed compared to vehicle-treated WT controls. In the humanitarian endpoint, vehicle-treated twi / twi mice and the two highest doses (6.8 × 10⁶) were compared. 10 GC and 2.0 × 10 11A decrease in triglyceride levels was observed in twi / twi mice administered rAAVhu68.hGALC (GC). The significance of this finding is unclear because the mice were not fasted before blood collection at necropsy. The decrease in triglyceride levels observed in vehicle-treated twi / twi mice at the humane endpoint, compared to WT mice, may be related to feeding difficulties exhibited by twi / twi mice as they approached the humane endpoint due to progressive ataxia. No clear treatment-related effect of rAAVhu68.hGALC on triglyceride levels was observed in either PND40 or the humane endpoint, and the decrease in triglycerides was not correlated with the dose.

[0239] No toxicity related to rAAVhu68.hGALC was observed in any of the brain, spinal cord, sciatic nerve, or internal organs evaluated in this study.

[0240] The efficacy of rAAVhu68.hGALC in treating Krabbe disease-related pathologies in twi / twi mice was demonstrated across all rAAVhu68.hGALC-treated cohorts by a significant or trending reduction (correction) of myelin loss and globoid cell infiltration in the nervous system compared to vehicle-treated twi / twi mice. The highest dose (2.0 ×) was more effective than the lowest dose. 11 Mice treated with rAAVhu68.hGALC (GC) showed no clear dose-response effect, but the second highest dose (6.8 × 10) was observed. 10 rAAVhu68.hGALC (GC) microscopically showed, to a lesser extent, a more pronounced modification of the twi / twi disease phenotype overall.

[0241] At baseline (PND12-14), globoid cells were present in untreated twi / twi mice. Severity was generally minimal to mild, and more pronounced in the spinal cord and peripheral nerves compared to the brain. This finding suggested that disease-related pathology was already present at the time of rAAVhu68.hGALC administration. At PND40 (4 weeks post-treatment) and the humane endpoint in survival cohort euthanasia (up to 10 weeks post-treatment for twi / twi mice), rAAVhu68.hGALC-treated twi / twi mice showed reduced demyelination and globoid cell infiltration in several neuroanatomical regions (brain, spinal cord, and / or sciatic nerve) compared to vehicle-treated twi / twi controls. Notably, all doses (6.8 × 10⁶) 9 GC, 2.0×10 10 GC, 6.8×10 10 GC, and 2.0 × 10 11 rAAVhu68.hGALC (GC) reduced demyelination and globoid cell infiltration in the brain white matter in both PND40 and the humane endpoint compared to vehicle-treated twi / twi controls. Two highest doses (6.8 × 10⁻¹⁰) 10 GC and 2.0 × 10 11 GC)rAAVhu68.hGALC reduced demyelination and globoid cell infiltration in the sciatic nerve at PND40 compared to vehicle-treated twi / twi control. Maximum dose (2.0 × 10) 11 rAAVhu68.hGALC (GC) showed improved spinal cord activity in PND40 compared to vehicle-treated twi / twi controls. It reduced demyelination and globoid cell infiltration.

[0242] Histopathology did not reveal any significant lesions in the mouse liver that could explain the observed changes in liver-related clinicopathological parameters. The only significant finding in the liver was improvement in hepatic microvacuole formation after administration of rAAVhu68.hGALC. In the survival cohort, vehicle-treated twi / twi mice did not show hepatocyte vacuolation (grade 0), while all vehicle-treated WT mice showed mild to moderate microvacuole formation (grade 2 to grade 3). All doses (6.8 × 10⁴) to twi / twi mice 9 GC, 2.0×10 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 Administration of rAAVhu68.hGALC (GC) resulted in increased microvacuole formation in most animals to levels comparable to those of vehicle-treated WT controls (Figure 71). Hepatocyte vacuolation is typically attributed to the presence of physiological triglyceride and glycogen reserves. The absence of vacuolation in vehicle-treated twi / twi mice may be attributed to the absence of triglyceride and / or glycogen reserves as a result of a debilitating phenotype at the humane endpoint. The increase in vacuolation after rAAVhu68.hGALC treatment was observed at all doses (6.8 × 10⁻⁶). 9 GC, 2.0×10 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 This likely reflects the improvement in the wasting phenotype of twi / twi mice in rAAVhu68.hGALC (GC). Improvement in hepatic microvacuole formation does not appear to be dose-dependent.

[0243] Animal 1071 (rAAVhu68.hGALC, 2.0×10 10 GC; group 7b; PND23, 11 days after treatment), animal 1053 (rAAVhu68.hGALC, 2.0 × 10 11Three mice were found dead, including animal GC (group 5b; PND23, 11 days post-treatment) and animal 1062 (ITFFB; group 3b; PND21, 7 days post-treatment). Aside from typical CNS and PNS demyelination and globoid cell infiltration in the twi / twi mouse phenotype, no other macroscopic or microscopic abnormalities were observed. The cause of death was undetermined, but animal 1053 (rAAVhu68.hGALC, 2.0×10⁻¹⁰) exhibited a seizure one day prior to death. 11 For GC (group 5b), we were unable to rule out procedure-related causes.

[0244] IBA1 immunohistochemistry was performed on the brain (cortex, cerebellum, brainstem), spinal cord (cervical, lumbar, thoracic), and sciatic nerve to visualize globoid cells in the central nervous system (CNS) and primary nervous system (PNS). The size (average target area) of individual IBA1-positive cells was measured using image analysis software. A decrease in the size of IBA1-positive cells was expected with improvement in the disease phenotype.

[0245] In the cortex, IBA1-positive cells in untreated twi / twi mice were similar in size to those in baseline untreated WT mice. In PND40, vehicle-treated twi / twi mice showed significantly larger IBA-1-positive cells compared to vehicle-treated WT controls. Three highest doses (2.0 × 10⁶) were administered to twi / twi mice. 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 Administration of rAAVhu68.hGALC to GC mice resulted in a significant dose-dependent reduction in IBA-1 positive cell size compared to vehicle-treated twi / twi mice. Notably, the two highest doses (6.8 × 10⁶) 10 GC or 2.0 × 10 11 In rAAVhu68.hGALC (GC), the size of IBA-1-positive cells was similar to that of vehicle-treated WT controls. In the humanitarian endpoint (survival cohort), vehicle-treated twi / twi mice showed significantly larger IBA-1-positive cells compared to vehicle-treated WT controls. All doses (6.8 × 10⁶) to twi / twi mice9 GC, 2.0×10 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 Administration of rAAVhu68.hGALC in GC mice resulted in a significant and generally dose-dependent reduction in the size of IBA-1-positive cells compared to vehicle-treated twi / twi mice (Figures 72A-72C).

[0246] In the cerebellum, IBA1-positive cells in untreated twi / twi mice were similar in size to those in baseline untreated WT mice. In PND40, vehicle-treated twi / twi mice showed significantly larger IBA-1-positive cells compared to vehicle-treated WT controls. Administration of rAAVhu68.hGALC to twi / twi mice did not reduce the size of IBA-1-positive cells at any dose compared to vehicle-treated twi / twi mice. In the humanitarian endpoint (survival cohort), vehicle-treated twi / twi mice showed significantly larger IBA-1-positive cells compared to vehicle-treated WT controls. The highest dose to twi / twi mice was 2.0 × 10⁻¹⁴. 11 Administration of rAAVhu68.hGALC to GC mice significantly increased the size of IBA-1-positive cells compared to vehicle-treated twi / twi mice (Figures 72D-72F).

[0247] In the brainstem, IBA1-positive cells in untreated twi / twi mice were similar in size to those in baseline untreated WT mice. In PND40, vehicle-treated twi / twi mice showed significantly larger IBA-1-positive cells compared to vehicle-treated WT controls. Two highest doses (6.8 × 10⁶) were administered to twi / twi mice. 10 GC or 2.0 × 10 11Administration of rAAVhu68.hGALC to GC mice resulted in a significant reduction in the size of IBA-1-positive cells compared to vehicle-treated twi / twi mice. In humane euthanasia, vehicle-treated twi / twi mice showed significantly larger IBA-1-positive cells compared to vehicle-treated WT controls. Administration of rAAVhu68.hGALC to twi / twi mice, at any dose, did not reduce the size of IBA-1-positive cells compared to vehicle-treated twi / twi mice (Figures 73A-73C).

[0248] In the spinal cord, IBA1-positive cells in untreated twi / twi mice were similar in size to those in baseline untreated WT mice. In PND40, vehicle-treated twi / twi mice showed significantly larger IBA-1-positive cells compared to vehicle-treated WT controls. Three highest doses (2.0 × 10⁶) were administered to twi / twi mice. 10 GC, 6.8×10 10 GC, or 2.0 × 10⁻⁶ 11 Administration of rAAVhu68.hGALC to GC mice resulted in a significant reduction in IBA-1-positive cell size compared to vehicle-treated twi / twi mice. In the humanitarian endpoint (survival cohort), vehicle-treated twi / twi mice showed significantly larger IBA-1-positive cells compared to vehicle-treated WT controls. Administration of rAAVhu68.hGALC to twi / twi mice, at any dose, did not reduce the size of IBA-1-positive cells compared to vehicle-treated twi / twi mice (Figures 73D-73F).

[0249] In the sciatic nerve, IBA1-positive cells in untreated twi / twi mice were significantly larger than those in baseline untreated WT controls. In PND40, vehicle-treated twi / twi mice still showed significantly larger IBA-1-positive cells compared to vehicle-treated WT controls. Two highest doses (6.8 × 10⁶) were administered to twi / twi mice. 10 GC or 2.0 × 10 11Administration of rAAVhu68.hGALC (GC) resulted in a significant reduction in the size of IBA-1 positive cells compared to vehicle-treated twi / twi mice. In humane euthanasia, vehicle-treated twi / twi mice showed significantly larger IBA-1 positive cells compared to vehicle-treated WT controls. The highest dose to twi / twi mice was (2.0 × 10⁶). 11 Administration of rAAVhu68.hGALC (GC) significantly reduced the size of IBA-1 positive cells compared to vehicle-treated twi / twi mice. Furthermore, the highest dose (2.0 × 10) 11 In rAAVhu68.hGALC (GC), IBA-1-positive cells approached the size of vehicle-treated WT cells in both PND40 and humane euthanasia (Figures 74A-74C).

[0250] In summary, MED is 2.0 × 10 10 GC(5.0×10 10 GC / g brain) This was determined because this dose resulted in significant improvements in survival, physical weakness / growth impairment (weight loss), Krabbe disease-related clinical symptoms (clinical assessment scoring), and lymphopenic phenotype (potentially indicating a reduction in autonomic degeneration). This dose reduced demyelination, globoid cell infiltration, and neuroinflammation in the brain (LFB / PAS semi-quantitative scoring), and reduced globoid cell size in the brain and spinal cord (IBA-1). (IHC quantification). This dose was also the minimum dose that led to a significant increase in the expression of the transgene product (GALC activity) in the brain, a key target tissue.

[0251] Example 9 - Efficacy of AAV-mediated gene therapy for treating Clavus latus - Injection of rAAVhu68.CB7.CI.cGALCco.rBG via Cisterna Magna While a beneficial disease model, the Twitcher mouse has several limitations. The mouse exhibits only mild CNS involvement, unlike infantile Krabbe patients who present with more severe CNS features such as demyelination of brain atrophy. Furthermore, the small size of the mouse presents experimental challenges. The ICV pathway must be used in mice because their small size makes it difficult to reliably inject the AAV vector via the intended clinical pathway (ICM). For all desired pharmacological assays, sufficient quantities of CSF and serial blood samples cannot be obtained from the mouse. Therefore, treatment with rAAVhu68.GALC should be evaluated in larger animals, such as a canine model of Krabbe disease, to overcome these technical limitations and confirm the scalability of the therapeutic approach.

[0252] Similar to the Twitcher mouse, Krabbe's dog is a naturally occurring autosomal recessive disease model stemming from a spontaneous A-to-C mutation in the GALC gene, which causes a missense mutation (Y158S). The mutant GALC protein has near-zero residual enzyme activity, which is similar to the GALC activity levels observed in patients with Krabbe's disease in infantile form. Heterozygous dogs are asymptomatic, while homozygous dogs are affected by the mutation.

[0253] The phenotypic progression of Krabbe's disease, along with associated behavioral phenotypes, includes elevated psychosine levels in both the CNS and PNS, demyelination, and globoid cell infiltration. Krabbe's disease develops hindlimb weakness, thoracic limb dysmetria, and tremors at approximately 4–6 weeks of age. Similar to patients with infantile Krabbe's disease, Krabbe's disease presents with a consistent and rapid neurological deterioration after the onset of symptoms. Ultimately, these symptoms progress by around 8–15 weeks of age to a humane endpoint characterized by severe ataxia, pelvic limb paralysis, wasting, urinary incontinence, and sensory impairment (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 12]

[0254] The purpose of this study was to evaluate the efficacy, pharmacology, safety, and biodistribution of AAVhu68.CB7.CI.cGALCco.rBG, a recombinant adeno-associated virus (AAV) serotype hu68 vector expressing the canine galactocerebrosidase (GALC) enzyme, after intracisional administration (ICM) in a canine model of infantile krabbe. At 2-3 weeks of age, krabbe-affected dogs had a distribution of 3.0 × 10⁶ 13 AAVhu68.cGALCco received a single ICM dose of either GC or vehicle (intrathecal final preparation buffer [ITFFB]). Wild-type littermates were also administered vehicle.

[0255] In-life assessment included cage-side observation, weight monitoring, bi-weekly monitoring of behavior and motor function, physical examination, standardized neurological examination, assessment of brain myelination (assessed by magnetic resonance imaging [MRI] and brainstem auditory evoked responses [BAER]) and peripheral nerve myelination (assessed by nerve conduction studies [NCS]), quantification of CSF sphingolipids to analyze psychosine (disease biomarker) accumulation, and expression of transgene products in serum and CSF (GALC activity assay). Except for healthy wild-type untreated littermates controls euthanized simultaneously with the last treated animal, necropsies were performed at 6 months post-treatment (n=2) or at the humane endpoint (n=4). Tissues were obtained from each animal at necropsy for comprehensive histopathological examination and in vivo distribution analysis. Brain tissue and peripheral nerve samples were collected and myelination and storage (Luxor blue / periodate Schiff [PAS] staining) were assessed. Brain and spinal cord tissue samples were also collected, and microglia were activated and globoid cells were infiltrated. We quantified Jun (IBA1 IHC).

[0256] Several relevant biomarkers are accessible in this large animal model, and the intended clinical administration route (ICM) can be used, making it an attractive model for studying the efficacy of gene therapy. To prevent confusion of results with exaggerated immune responses to exogenous proteins, a vector encoding the canine version of GALC (AAVhu68.CB7.CI.cGALCco.rBG) was administered. Although the administered transgene differed, the vector utilized the same ubiquitous CB7 promoter, AAVhu68 capsid, as AAVhu68.CB7.CI.hGALCco.rBG. The animals' age was selected to ensure that the dogs were treated before the onset of behavioral symptoms. Furthermore, this age reflects the age of the intended infant patient population.

[0257] After group assignment, each animal is 3 × 10 13A single ICM infusion of AAVhu68.CB7.cGALCco or vehicle (ITFFB) was administered at GC / animal doses. The following table shows the group designation, dose level, and route of administration (ROA). Figure 77 provides the study design. [Table 13]

[0258] We selected the scheduled 180-day autopsy time, collected tissue at fixation, and searched for disease biomarkers 6 months after treatment and 4 months after the survival of the last vehicle-treated Krabbey dogs. This timing was considered to provide sufficient duration to compare with untreated young dogs that reached the humane endpoint before 12 weeks of age, while measuring the final progression of significant disease-related phenotypes and biomarkers. To obtain longer-term follow-up and assess the durability of the therapy, two dogs were enrolled in long-term follow-up (up to 19 months post-treatment).

[0259] Progression of behavioral and motor function was assessed using bi-weekly video recordings, periodic neurological examinations, brain MRI, and electrophysiology (NCV and BAER). The initial time point was designed to capture the onset and progression of the disease in the vehicle-type Crabbei dogs. Periodic examinations were performed every 2-3 months in the treated Crabbei dogs. One wild-type vehicle-type treated dog was compared to the treated animals. For comparison purposes, it was included in the long-term follow-up.

[0260] Two vehicle-treated Krabbaine dogs reached a predetermined humane endpoint at 8 weeks of age (animal K930) and 12 weeks of age (animal K948), characterized by severe hind limb weakness and inability to stand and walk, consistent with the natural history of the disease.

[0261] One vector-treated Krabbaine K937 was found lying on its side with a severely elevated fever (106.4°C) during a morning observation 9 months (38 weeks) after treatment. The veterinarian suspected a seizure episode and administered 0.5 mg / kg of barium intravenously. Blood was taken for CBC, chemistry, and culture, and CSF was collected for cytology. Rectal temperature returned to normal, but the animal remained lying on its side, so euthanasia was chosen. Significant findings in blood tests included neutrophilia (16,647 / μl, a four-fold increase compared to previous measurements), mild lymphopenia (337 / μL), elevated D-dimer (>5,400 ng / mL, range <250), mild elevated fibrinogen (455 mg / dl, range 150-400 mg / dL), AST (184 IU / L, range 15-66 IU / L), and BUN (37 mg / dL, range 6-31 mg / dL). Blood cultures revealed methicillin-resistant Staphylococcus. The presence of epidermidis was confirmed. CSF showed a mild elevation in protein levels (69 mg / dL), a small number of WBCs (2 per μL), and no infectious pathogens were observed. Pathological reports revealed lesions of Krabbe-related demyelination and globoid cell infiltration, which were less pronounced than in vehicle-treated Krabbe dog controls (animals K930 and K948) and similar to two treated dogs from the scheduled 6-month autopsy time (animals K938 and K939). The spinal cord and peripheral nerves did not show demyelination prior to the acute hyperthermic episode, suggesting that therapeutic efficacy was maintained and consistent with the animal's normal motor function.

[0262] The clinical symptoms of animal K937 (acute lying down, high fever, suspected seizures) are not typical of Krabbe disease. The clinical pathology and pathological report are compatible with the causative infection. Blood cultures revealed proliferation of antibiotic-resistant Staphylococcus epidermidis. According to the canine records, the dog had been treated with topical antibiotic ointment for abrasion lesions on the right anterior P4 paw pad for 8 days prior to the fever episode. However, the suspicion of seizures associated with cerebral demyelination and globoid cell infiltration in Krabbe disease cannot be ruled out.

[0263] Animal K933 (treated at AAVhu68.cGALC.co) was euthanized at 19.5 months of age due to weight loss. The weight loss was a result of recurrent vomiting and regurgitation that did not respond to symptomatic treatment. An X-ray performed approximately 4 days before euthanasia, when the dog had lost up to 28% of its body weight, revealed a gas-distended esophagus corresponding to megaesophagus. Autopsy revealed that the dog had severe bilateral salivary gland hypertrophy, which may have explained the difficulty swallowing and regurgitation. Electromyography of the esophagus did not show signs of terminal denervation (lack of spontaneous activity). Behavioral and motor function, as well as neurological examination, were normal at the time of euthanasia.

[0264] Two vehicle-treated Krabbey dogs reached a predetermined humane endpoint at day 35 (8 weeks old; animal K930) or day 66 (12 weeks old; animal K948), characterized by severe hind limb weakness and inability to stand and walk, consistent with the natural history of the disease. In contrast, all AAVhu68.cGALCco-treated dogs (N=4 / 4) maintained normal motor function and did not reach the predetermined humane endpoint associated with hind limb paralysis.

[0265] All animals administered AAVhu68.cGALCco or vehicle (ITFFB) via ICM tolerated the procedure well and recovered successfully from the sedative. No adverse events related to the test substance occurred in animals administered AAVhu68.cGALCco during the study period.

[0266] Growth and weight gain were normal in all treated dogs (Figure 78). One treated dog (K933) was euthanized at 19.5 months of age (85 weeks) due to weight loss.

[0267] All animals were recorded playing in open space to assess their behavior and motor function. Assessments began at 12 weeks of age for animals K928, ​​K930, and K933, and at 3-4 weeks of age for animals K937, K938, K939, and K948. Two vehicle-treated Krabbeinu (animals K930 and K948) exhibited expected Krabbein-related abnormal motor function. At 8 weeks of age, animal K930 developed head and limb tremors, intentional tremors, and severe hind limb weakness, accompanied by muscle atrophy and joint laxity that prevented the animal from standing and walking (humane endpoint criterion). Animal K948 began showing hind limb weakness at 7 weeks of age and progressed to ataxia, severe weakness, and inability to stand and walk by 12 weeks of age (humane endpoint).

[0268] All animals treated with AAVhu68.cGALCco exhibited normal motor function during open play, similar to vehicle-treated wild-type controls, demonstrating the ability to walk, run, jump, and stand on their hind limbs. All AAVhu68.cGALCco-treated Krabbeinu (4 / 4) exhibited normal behaviors such as playing with caregivers and looking at / fetching toys, suggesting that this treatment prevented Krabbe-related phenotypes in all animals during the study period.

[0269] One of the vehicle-treated Krabba dogs (animal K948) exhibited abnormal neurological findings beginning at 11 weeks of age, including general proprioceptive deficits, ataxia, head tremor / trunk sway, muscle atrophy, absence of menace reflex (indicating suspected blindness), and wide-based posture. This animal was euthanized immediately after observation. The other vehicle-treated Krabba dogs (animal K930) reached humane endpoints before the first scheduled neurological scoring point. All AAVhu68.cGALC.co treated dogs (4 / 4) presented similar neurological findings throughout the study period compared to wild-type vehicle-treated animals.

[0270] NCVs in vehicle-treated Krabbain dogs at 6 weeks of age (animals K930 and K948) and 12 weeks of age (animal K948) were generally lower than those of vehicle-treated wild-type controls in all four nerves evaluated (Figures 79A–79D). Both vehicle-treated Krabbain dogs showed complete loss of NCV, particularly in the radial sensory nerve. In contrast, AAVhu68.cGALCco-treated Krabbain dogs necropped at 180 days (animals K938, K939), emergency necropped at 261 days (animal K937), or in the ongoing long-term cohort (animal K933) showed similar NCVs throughout the study to those of vehicle-treated wild-type controls. All treated dogs (4 / 4) had normal NCVs similar to those of the WT controls.

[0271] The interpeak latency (IPL) between wave I and wave V in BAER recordings indicates conduction latency within the brainstem auditory pathway and therefore central nervous system conduction. One of the vehicle-treated Krabbey dogs (animal K948) did not evoke any evoked potentials (auditory threshold > 90 dB; Figure 80). Another vehicle-treated Krabbey dog ​​(animal K930) showed increased IV IPL (mean 3.275 ms compared to mean 2.275 ms in vehicle-treated wild-type dogs). All treated Krabbey dogs (4 / 4) had normal IV IPL similar to that of vehicle-treated wild-type dogs.

[0272] The auditory threshold could not be determined in one vehicle-treated Krabbey dog ​​(animal K948) (>90 dB), but was similar to that of a vehicle-treated wild-type dog (animal K928) in another vehicle-treated Krabbey dog ​​(animal K...

Claims

1. A pharmaceutical composition comprising a stock of recombinant AAV (rAAV) having an AAVhu68 capsid and a vector genome packaged therein, wherein the vector genome is (a) 5' inverted terminal repeat (ITR), (b) The CB7 promoter, which is a hybrid between the CMV IE enhancer and the chicken β-actin promoter. (c) Intron, (d) A galactosylceramidase (GALC) coding sequence containing nucleotides 1 to 2055 of SEQ ID NO: 9, or a sequence that is at least 95% identical to the one coding amino acids 1 to 685 of SEQ ID NO: 10, (e) Poly A, and (f) Including 3'ITR, The composition is approximately 1.7 × 10 10 Genome copy (GC) / g brain mass ~ approximately 5.0 × 10⁻⁶ 11 A pharmaceutical composition for administering a dose of GC / g brain mass, wherein the pharmaceutical composition is administered to a human patient by intracisional administration (ICM), and the treatment is such that i) the expression and enzyme activity of GALC in serum and cerebrospinal fluid (CSF), or ii) the expression and activity of GALC in neurons of the cortex and hippocampus.

2. The pharmaceutical composition according to claim 1, wherein the GALC coding sequence comprises nucleotides 1 to 2055 of sequence number 9.

3. The composition is 1.4 × 10 13 GC ~ 4.0 x 10 14 GC or 4.0 x 10 13 GC ~ 4.0 x 10 14 A pharmaceutical composition according to claim 1 or 2, formulated for administering a dose of recombinant AAV of GC.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the total volume of the pharmaceutical composition is 4.5 mL to 5.5 mL.

5. A pharmaceutical composition according to any one of claims 1 to 4, for use in a method of treating Krabbe disease in human patients requiring treatment for Krabbe disease.

6. The pharmaceutical composition according to claim 5, wherein the method further comprises hematopoietic stem cell transplantation or bone marrow transplantation before or after administration of the pharmaceutical composition.

7. The pharmaceutical composition according to claim 6, wherein the hematopoietic stem cell transplant or bone marrow transplant enables a reduced dose of the recombinant AAV to be administered.

8. The pharmaceutical composition according to any one of claims 1 to 4 for use in a method for reducing neuroinflammation in the peripheral nerves of a human patient.

9. The pharmaceutical composition according to any one of claims 5 to 8, wherein the method comprises the step of measuring psychosine in the serum and / or CSF.

10. The pharmaceutical composition according to any one of claims 5 to 9, wherein the patient is less than 2 months old, less than 6 months old, or less than 12 months old.

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