Compositions useful for treating GM1 gangliosidosis

Abstract

This provides a useful treatment regimen for GM1 gangliosidosis in human patients. [Solution] The administration regimen comprises administering a recombinant adeno-associated virus (rAAV) vector having an AAV capsid and a vector genome containing a sequence encoding human β-galactosidase under the control of a regulatory sequence that directs its expression in target cells, and includes a single dose intracision macromolecular (ICM) injection of a genome copy number appropriate for the patient's age in months. The regimen may further include administering at least one immunosuppressive combination therapy to the patient at least one day before or on the day of rAAV delivery.

Description

[Background technology]

[0001] GM1 gangliosidosis (hereinafter referred to as GM1) is an autosomal recessive lysosomal storage disorder caused by mutations in the GLB1 gene, which encodes lysosomal acid beta-galactosidase (β-gal), an enzyme that catalyzes the first step in the degradation of GM1 ganglioside and keratan sulfate (Brunetti-Pierri and Scaglia, 2008, GM1 gangliosidosis: Review of clinical, molecular, and therapeutic). aspects, Molecular Genetics and Metabolism, 94:391-96). The GLB1 gene is located on chromosome 3 and yields two alternatively spliced ​​mRNAs: a 2.5kb transcript encoding a β-gal lysosomal enzyme and a 2.0kb transcript encoding elastin-binding protein (EBP) (Oshima et al. 1988, Cloning, sequencing, and expression of cDNA for human). β-galactosidase,Biochemical and Biophysical Research Communications,157:238-44,Moreau et al.1989,Alternative splicing of beta-galactosidase mRNA generates the (Classic lysosomal enzyme and a beta-galactosidase-related protein, Journal of Biological Chemistry, 264:20655-63). β-gal is synthesized as an 85 kDa precursor, post-translation glycosylated to an 88 kDa form, and processed into a mature 64 kDa lysosomal enzyme (D'Azzo et al. 1982, Molecular defect in combined beta-galactosidase and neuraminidase deficiency in man, Proceedings of the National Academy of Sciences, 79:4535-39). Within the lysosome, the enzyme complexes with the protective protein cathepsin A (PPCA) and neuraminidase hydrolase.

[0002] In patients carrying a GLB1 allele that produces little to no residual β-gal, GM1 gangliosides accumulate in neurons throughout the brain, causing rapidly progressive neurodegenerative disease (Brunetti-Pierri and Scaglia 2008). The molecular mechanisms leading to the pathogenesis of the disease are not yet well understood, but hypotheses include astrogliosis and microgliosis in areas of significant neuronal vacuolar degeneration, neuronal cell death and demyelination accompanied by neuronal apoptosis (Tessitore et al.). al.2004, GM1-Ganglioside-Mediated Activation of the Unfolded Protein Response Causes Neuronal Death in a Neurodegenerative Gangliosidosis, Molecular Cell, 15:753-66, abnormal axonal transport causing myelin deficiency (van der Voorn et al.2004, The leukoencephalopathy of infantile GM1 gangliosidosis: oligodendrocytic loss and axonal dysfunction, Acta Neuropathologica, 107:539-45), disruption of neuronal-oligodendritic glial cell interactions (Folkerth 1999, Abnormalities of Developing White Matter in Lysosomal Storage Diseases, Journal of Neuropathology and Experimental Neurology,58:887-902, Kaye This includes 'Dysmyelinogenesis in animal model of GM1 gangliosidosis' (et al. 1992, Pediatric Neurology, 8:255-61), as well as inflammatory responses (Jeyakumar et al. 2003, Central nervous system inflammation is a hallmark of pathogenesis in mouse models of GM1 and GM2 gangliosidosis, Brain, 126:974-87).

[0003] Currently, there are no therapies that modify the disease of GM1. Supportive care and symptomatic treatment, including feeding tube placement, respiratory therapy, and antiepileptic drugs, are the current treatment approaches (Jarnes Utz et al. 2017, Infantile gangliosidoses: Mapping a timeline of clinical changes, Molecular Genetics and Metabolism, 121:170-79). Substrate reduction therapy (SRT) with the glucosylceramide synthase inhibitor miglustat is being evaluated in GM1 and GM2 patients. Miglustat is generally well-tolerated but does not result in significant improvement in symptom management or disease progression, and some patients experience dose-limiting gastrointestinal side effects (Shapiro et al., 2009, Regier et al., 2016b). When used in combination with a ketogenic diet, miglustat is well-tolerated and has been shown to increase survival rates in some patients (Jarnes Utz et al., 2017). However, it should be noted that no randomized controlled studies using miglustat have been conducted, and miglustat is not approved for the treatment of GM1 gangliosidosis. Hematopoietic stem cell transplantation (HSCT) using bone marrow or umbilical cord blood has limited experience in this disease. Bone marrow transplantation performed in patients with type 2 GM1 normalized leukocyte β-galactosidase levels in patients with juvenile GM1-gangliosidosis before symptom onset, but did not improve long-term clinical outcomes (Shield et al., 2005, Bone marrow transplantation correcting β-galactosidase activity does not influence neurological outcome in juvenile GM1-gangliosidosis. Journal of Inherited Metabolic Disease.28(5):797-798). Due to the slow effect of HSCT, it is not suitable for rapidly progressive type 1 GM1 disease (Peters and Steward, 2003, Hematopoietic cell transplantation for inherited metabolic diseases: an overview of outcomes and practice guidelines. Bone Marrow Transplantation.31:229). Adeno-associated virus (AAV), a member of the parvovirus family, is a small, non-enveloped, icosahedral virus with a single-stranded linear DNA (ssDNA) genome approximately 4.7 kilobases (kb) long. The wild-type genome contains inverted end 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, and cap contains the duplicate nucleotide sequences VP1, VP2, and VP3 of the capsid protein, which self-assemble to form a dodecahedral-symmetric capsid.

[0004] AAV is assigned to the genus Dependovirus because it was discovered as a contaminant in purified adenovirus stocks. The life cycle of AAV includes a latent phase, in which the AAV genome is specifically integrated into the host chromosome after infection, and a subsequent integration after infection with either an adenovirus or herpes simplex virus. The process includes an infection phase in which the extracted genome is subsequently rescued, replicated, and packaged into an infectious virus. Due to its infectivity across a broad range of non-pathogenic hosts, including non-dividing cells, and its potential for site-specific chromosomal integration, AAVs are an attractive tool for gene transfer.

[0005] Alternative therapies are needed to treat conditions associated with abnormal GLB1 genes. [Overview of the project]

[0006] A therapeutic recombinant(r) replication-deficient adeno-associated virus (AAV) is provided that is useful for treating and / or reducing symptoms associated with GM1 gangliosidosis in human patients. The rAAV is preferably replication-deficient and possesses a vector genome containing the GLB1 gene encoding human(h)β-galactosidase under the control of a regulatory sequence that directs its expression in targeted human cells, and may be referred to as rAAV.GLB1 as used herein. In certain embodiments, the rAAV comprises an AAVhu68 capsid. This rAAV is referred to herein as rAAVhu68.GLB1, but in certain cases, the terms rAAVhu68.GLB1 vector, rAAVhu68.hGLB1, rAAVhu68.hGLB1 vector, AAVhu68.GLB1, or AAVhu68.GLB1 vector are used interchangeably to refer to the same construct.

[0007] In one embodiment, the Specified Publicly Provided is a therapeutic regimen useful for the treatment of GM1 gangliosidosis in human patients, comprising the administration of a recombinant adeno-associated virus (rAAV) vector having an AAV capsid and a vector genome containing a sequence encoding human β-galactosidase under the control of a regulatory sequence that directs its expression in target cells, wherein the administration is (i) in patients approximately 1 month to approximately 4 months old, at a dose of approximately 1.6 × 10⁻¹⁴ 13 ~Approx. 1.6×10 14 GC, (ii) Patients are at least about 4 months to less than 8 months old, approximately 2.1 × 10 13 ~Approx. 2.1×10 14 GC, (iii) Patients are at least 8 months to a maximum of 12 months old, approximately 2.6 × 10 13 ~Approx. 2.6×10 14 GC, or (iv) the patient is at least 12 months old, approximately 3.2 × 10 13 ~Approx. 3.2×10 14The treatment regimen includes single-dose large-volume (ICM) injection containing GC. In certain embodiments, the human β-galactosidase coding sequence comprises a nucleotide sequence set forth in SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 5, or a sequence that is at least 95% identical to any one of SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 5 and encodes the mature β-galactosidase of amino acids 24 to 677 of SEQ ID NO: 4. In certain embodiments, the encoded human β-galactosidase has a sequence selected from synthetic human enzymes comprising (a) approximately amino acids 1 to 677 of SEQ ID NO: 4, and (b) a heterologous leader sequence fused to approximately amino acids 24 to 677 of SEQ ID NO: 4. In further embodiments, the vector genome also includes a 5' inverted terminal repeat (ITR) sequence, regulatory elements derived from the human ubiquitin C (UbC) promoter, a chimeric intron, a polyA signal, and / or a 3' ITR sequence. In certain embodiments, the patient is identified as having type 1 (infantile) GM1 or type 2a (late infantile) GM1. In certain embodiments, the regimen includes administration to the patient of at least one immunosuppressive combination therapy on at least 1 day before or on the day of delivery of rAAV. The immunosuppressive combination therapy may include one or more corticosteroids, optionally oral prednisolone. In certain embodiments, the immunosuppressive combination therapy continues for at least 3 to 4 weeks after administration of rAAV. In certain embodiments, the effectiveness of the treatment is evaluated by one or more of delay in onset of seizures, reduction in seizure frequency, β-galactosidase in serum and / or cerebrospinal fluid, and volume changes in brain tissue measured by magnetic resonance imaging (MRI).

[0008] In one aspect, provided herein is a composition comprising an AAV capsid, a vector genome comprising a human β -galactosidase coding sequence, and an expression control sequence that directs its expression in a target cell, the recombinant AAV (rAAV) vector, wherein the rAAV vector is administered to a human subject who needs it, (i) the patient is from about 1 month old to about 4 months old, about 1.6×10 13 ~about 1.6×10 14GC, (ii) Patients are at least about 4 months to less than 8 months old, approximately 2.1 × 10 13 ~Approx. 2.1×10 14 GC, (iii) Patients are at least 8 months to a maximum of 12 months old, approximately 2.6 × 10 13 ~Approx. 2.6×10 14 GC, or (iv) the patient is at least 12 months old, approximately 3.2 × 10 13 ~Approx. 3.2×10 14 It is formulated for intracisional cisterna magna (ICM) injection to administer a dose of GC. In certain embodiments, the human β-galactosidase coding sequence includes the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 5, or a sequence encoding mature β-galactosidase from amino acids 24-677 of SEQ ID NO: 4, which is at least 95% identical to any one of SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 5. In further embodiments, the vector genome also includes a 5' inverted terminal repeat (ITR) sequence, a regulatory element derived from the human ubiquitin C (UbC) promoter, a chimeric intron, a polyA signal, and / or a 3' ITR sequence. In certain embodiments, rAAV is 3.33 × 10¹⁶ per gram of brain mass. 10 GC ~ 3.33 × 10⁻¹⁴ of brain mass per gram 11 The GC is formulated in suspension form for delivery, with the dose volume optionally being approximately 3.0 mL to approximately 5.0 mL. In certain embodiments, the rAAV is in a formulation buffer having a pH of 6 to 9, optionally having a pH of approximately 7.2. In certain embodiments, the composition is intended for use in combination therapy, which includes administration to the patient of at least one immunosuppressant at least one day before or on the day of delivery of the rAAV. The immunosuppressant may be a corticosteroid, or optionally prednisolone delivered orally.

[0009] In one embodiment, the foregoing provides a method for treating a patient having GM1 gangliosidosis, the method comprising administering a single dose of recombinant adeno-associated virus (rAAV) to the patient by intracisional cisterna macrocephaly (ICM), wherein the rAAV comprises an AAV capsid and a vector genome containing a sequence encoding human β-galactosidase under the control of a regulatory sequence that directs its expression in target cells, and the single dose is 1 × 10⁶ per gram of the patient's estimated brain mass. 10 GC~3.4×10 11 This is a GC method. In certain embodiments, the patient develops GM1 symptoms before 18 months of age. In certain embodiments, the patient develops GM1 symptoms at or below 6 months of age. In certain embodiments, the patient develops GM1 symptoms between 6 and 18 months of age. In certain embodiments, the patient has type 1 (infant) GM1. In other embodiments, the patient has type 2a (late infant) GM1. In certain embodiments, the subjects are at least 4 months of age, 4–36 months of age, 4–24 months of age, 6–36 months of age, 6–24 months of age, 12–36 months of age, or 12–24 months of age. In certain embodiments, the single dose is 3.3 × 10⁶ per gram of the patient's estimated brain mass. 10 It is GC. In a particular embodiment, the single dose is 2.1 × 10⁻⁶. 13 ~2.5×10 13 GC's rAAV or 2.6×10 13 ~3.1×10 13 This is GC's rAAV. In a particular embodiment, the single dose is 3.2 × 10⁻⁶. 13 ~4.5×10 13 This is GC's rAAV. In a particular embodiment, the single dose is 1.11 × 10⁶ per gram of the patient's estimated brain mass. 11 It is GC. In a particular embodiment, the single dose is 6.8 × 10 13 ~8.6×10 13 GC's rAAV, 8.7x10 13 ~0.9 × 10 14 GC's rAAV, or 1.0 × 10 14 ~1.5×10 14 This is GC's rAAV. In a specific embodiment, the patient is 4-8 months old, and the single dose is 2.1 × 10⁻⁶. 13This is GC's rAAV. In a specific embodiment, the patient is 4-8 months old, and the single dose is 6.8 × 10⁻⁶. 13 This is GC's rAAV. In a specific embodiment, the patient is 8-12 months old, and the single dose is 2.6 × 10⁻⁶. 13 This is GC's rAAV. In a specific embodiment, the patient is 8-12 months old, and the single dose is 8.7 × 10⁻⁶. 13 This is GC's rAAV. In a particular embodiment, the patient is at least 12 months old, and the single dose is 3.2 × 10⁻⁶. 13 This is GC's rAAV. In a particular embodiment, the patient is at least 12 months old, and the single dose is 1.0 × 10⁻⁶. 14 This is rAAV for GC. In some embodiments, the method further includes the step of hematopoietic stem cell transplantation. In certain embodiments, the method further includes the step of administering steroids to the patient. The steroids may be corticosteroids. In certain embodiments, the method includes daily administration of steroids for at least 21 days. In certain embodiments, the method includes daily administration of steroids for 30 days. In certain embodiments, the vector genome includes a sequence encoding human β-galactosidase, which includes the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 5, or a sequence encoding mature β-galactosidase from amino acids 24 to 677 of SEQ ID NO: 4, which is at least 95% identical to any one of SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 5. The human β-galactosidase has the amino acid sequence of SEQ ID NO: 4, or a functional fragment thereof. In certain embodiments, the vector genome has a sequence selected from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In certain embodiments, the vector genome has a sequence that is at least 95% identical to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In certain embodiments, the vector genome further comprises a 5' inverted terminal repeat (ITR) sequence, a regulatory element derived from the human ubiquitin C (UbC) promoter, a chimeric intron, a polyA signal, and / or a 3' ITR sequence.

[0010] In one embodiment, the herein provides a pharmaceutical composition in a unit dosage form, comprising 1 × 10⁶ units in a buffer. 13 GC~5×10 14 The pharmaceutical composition comprises a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV comprises an AAV capsid and a vector genome containing a sequence encoding human β-galactosidase under the control of a regulatory sequence that directs its expression in target cells. In certain embodiments, the composition is formulated for intracisor macromolar (ICM) injection. In certain embodiments, the buffer comprises sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and poloxamer 188. In further embodiments, the buffer comprises 1 mM sodium phosphate, 150 mM sodium chloride, 3 mM potassium chloride, 1.4 mM calcium chloride, 0.8 mM magnesium chloride, and 0.001% poloxamer 188. In certain embodiments, the composition comprises 2.1 × 10⁻¹⁶ 13 ~2.5×10 13 GC's rAAV, 2.6×10 13 ~3.1×10 13 GC's rAAV, 3.2x10 13 ~4.5×10 13 GC's rAAV, 6.8×10 13 ~8.6×10 13 GC's rAAV, 8.7x10 13 ~0.9 × 10 14 GC's rAAV, or 1.0 × 10 14 ~1.5×10 14The provided pharmaceutical composition includes an rAAV having a vector genome having a sequence encoding human β-galactosidase, which includes the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 5, or a sequence encoding mature β-galactosidase from amino acids 24 to 677 of SEQ ID NO: 4, and is at least 95% identical to any one of SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 5. In certain embodiments, the human β-galactosidase has the amino acid sequence of SEQ ID NO: 4, or a functional fragment thereof. In certain embodiments, the vector genome has a sequence selected from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In certain embodiments, the vector genome has a sequence at least 95% identical to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In certain embodiments, the vector genome includes a 5' inverted terminal repeat (ITR) sequence, a regulatory element derived from the human ubiquitin C (UbC) promoter, a chimeric intron, a poly(A) signal, and / or a 3' ITR sequence.

[0011] These and other aspects of the present invention will become apparent from the following detailed description of the invention. [Brief explanation of the drawing]

[0012] [Figure 1A] This provides a schematic diagram of the AAV vector genome (i.e., "AAVhu68.Ubc.hGLB1co.SV40") showing the 5'ITR, human ubiquitin C (UbC) promoter, chimeric intron, GLB1 gene encoding human β-galactosidase (β-gal), SV40 late polyA signaling, and 3'ITR. [Figure 1B] A schematic diagram of the cisplasmid pAAV.UbC.hGLB1co.SV40.KanR, which contains the AAV vector genome embodied by the cisplasmid, is provided. GLB1: β-galactosidase, ITR: inverted terminal repeat, KanR: kanamycin resistance, Ori: origin of replication, polyA: polyadenylation, and UbC: ubiquitin C. [Figure 1C]This diagram provides a schematic representation of a transplasmid containing the coding sequences for the full-length AAV2 replicase enzyme (AAV2 Rep) encoding four proteins and the AAVhu68 VP1 capsid gene (encoding VP1, VP2, and VP3 proteins). AAV2: adeno-associated virus serotype 2, AAVhu68: adeno-associated virus serotype hu68, Cap: capsid, KanR: kanamycin resistance, Ori: origin of replication, and Rep: replicase enzyme. [Figure 2A-2B] This study describes β-gal activity in the brain and cerebrospinal fluid (CSF) of wild-type mice treated with rAAVhu68.GLB1 expressing human β-gal using different promoters. Wild-type mice were treated with a single intraventricular (ICV) injection of rAAVhu68.GLB1 expressing human GLB1 from CB7, EF1a, or UbC promoters (n=10 per group). Untreated wild-type mice (n=5) served as controls. Brain (frontal cortex) and CSF were collected 14 days after rAAVhu68.GLB1 administration, and β-gal activity was measured using a fluorescent substrate. *p<0.05, **p<0.01, ***p<0.001, Kruskal-Wallis test, followed by Dunn test. [Figure 3A-3E]This study demonstrates serum and peripheral organ β-gal activity in GLB1 knockout mouse studies. Preclinical trials were conducted using GM1 GLB1 knockout mouse models (mice carrying homozygous mutations in the GLB1 gene, or GLB1- / - mice). These trials compared GLB1- / - mice treated with AAVhu68.UbC.hGLB1, GLB1- / - mice treated with a vehicle (phosphate-buffered saline, or PBS), and disease-free mice that were heterozygous GLB1 mutant carriers, or GLB1+ / - mice treated with a vehicle. All mice were treated at 1 month of age and observed until 4 months of age, typically when GM1 mice develop significant gait abnormalities associated with brain GM1 ganglioside levels similar to those seen in infant GM1 patients with progressive disease. All mice were treated with intraventricular (or ICV) injection of either one of the test vectors (shown as AAV in the figure below) or one of the vehicles. Ninety days after treatment, all animals were euthanized, and tissues were collected for histological and biochemical analysis (referred to as necropsy). Serum β-gal activity was measured at various time points before and after treatment (days 0, 10, 28, 60, and 90). At necropsy, β-gal activity in the brain, CSF, and peripheral organs was evaluated. β-gal activity was measured using a fluorescent substrate in serum (Figure 3A), as well as in lung (Figure 3B), liver (Figure 3C), heart (Figure 3D), and spleen (Figure 3E) samples, respectively. PBS: phosphate-buffered saline (vehicle), AAV: adeno-associated virus (AAVhu68.UbC.hGLB1). *p<0.05, **p<0.01, Kruskal-Wallis test, followed by Dunn test. NS: no significant difference. Figure 3A shows that GLB1- / - mice treated with AAVhu68.UbC.hGLB1 had substantially higher post-treatment serum β-gal activity than vehicle-treated GLB1- / - mice, and had β-gal activity similar to that of vehicle-treated heterozygous control mice.All AAVhu68.UbC.hGLB1-treated mice achieved elevated serum β-gal activity, measured in nanomoles / milliliters / hour or nmol / ml / hour, within a short period after treatment. This persisted throughout all studies except for two AAVhu68.UbC.hGLB1-treated mice, both of which exhibited antibodies against human β-gal. Figures 3B–3E show β-gal activity in the lungs, liver, heart, and spleen after necropsy. In each organ, β-gal activity in rAAV.hGLB1 GLB1- / - mice was higher than that in vehicle-treated GLB1- / - mice. This data supports the potential of hGLB1 to provide modified β-gal enzyme activity in peripheral organs, suggesting that treatment with the rAAV.hGLB1 vector could address both CNS and peripheral manifestations observed in GM1 patients. [Figure 4A-4B] This shows β-gal activity in the brain and post-necropsy CSF, measured in nanomoles / milligrams / hour or nmol / mg / hour. β-gal activity in AAVhu68.UbC.hGLB1-treated mice was higher than in vehicle-treated GLB1- / - mice in both brain and CSF. Brain (frontal cortex) and CSF were collected at necropsy, and β-gal activity was measured using a fluorescent substrate. PBS: phosphate-buffered saline (vehicle), AAV: adeno-associated virus (AAVhu68.UbC.hGLB1). *p<0.05, **p<0.01, Kruskal-Wallis test, then Dunn test. NS: no significant difference. Statistical significance is important and, where used herein, is indicated by the p-value. The p-value is the probability that the reported result was achieved purely by chance (e.g., p-value < 0.001 means there is less than a 0.1% chance that the observed change was purely due to chance). Generally, a p-value less than 0.05 is considered statistically significant. [Figure 5]This study shows a decrease in hexosaminidase (HEX) activity in the brains of GLB1- / - mice treated with rAAVhu68.GLB1. Brain (frontal cortex) samples were collected at necropsy, and HEX activity was measured using a fluorescent substrate. PBS: phosphate-buffered saline (vehicle), AAV: adeno-associated virus (AAVhu68.UbC.hGLB1). *p<0.05, **p<0.01, Kruskal-Wallis test followed by Dunn test. NS: no significant difference. Post-necropsy, biochemical and histological assays were used to assess correction of brain abnormalities. Lysosomal enzymes are frequently upregulated in lysosomal storage disorders, and this observation has been confirmed in GM1 patients. Therefore, the activity of the lysosomal enzyme HEX in brain lysates was measured. This figure shows that HEX activity in rAAV.hGLB1- / - mice treated with GLB1+ / - was normalized compared to GLB1+ / - control mice, while vehicle-treated GLB1- / - mice showed increased total HEX activity. [Figure 6] This shows the correlation between β-gal activity and anti-β-gal antibodies. β-gal activity and serum anti-β-gal antibodies were measured in serum samples collected from AAV-treated mice at necropsy. Each point represents an individual animal. [Figures 7A-7G]This shows the correction of gait abnormalities in AAV-treated GLB1- / - mice. Figures 7A and 7B show that untreated GLB1- / - mice (n=12) and GLB1+ / - controls (n=22), mean 5 months of age, were evaluated using CatWalk for two consecutive days. Average walking speed (Figure 7A) and hindlimb footprint length (Figure 7B) were quantified for each animal over at least three trials. **p<0.01 Mann-Whitney test. Figures 7C and 7D show that untreated GLB1+ / - mice (n=15) and vehicle-treated GLB1- / - mice (n=15), as well as AAV-treated GLB1- / - mice (n=14), were evaluated using CatWalk. Average walking speed (Figure 7C) and hindlimb footprint length (Figure 7D) were quantified for each animal on day 2 of the study over at least three trials. *p<0.05, **p<0.01, Kruskal-Wallis test, followed by Dunn test. NS: No significant difference. Figures 7E-7G show representative hindlimb footprints of AAV-treated GLB1- / - mice (Figure 7G) and vehicle-treated GLB1+ / - (Figure 7E) and GLB1- / - (Figure 7F) controls. [Figure 8A-8B] This study demonstrates the correlation between walking speed and gait parameters. GLB1+ / - controls (n=22) were evaluated for two consecutive days using the CatWalk system. Gait parameters measured in at least three trials on the second day of the test were recorded. Correlation analysis demonstrated a strong correlation between walking speed and gait parameters such as stride length (Spearman r=0.7432, p<0.001, Figure 8A). In contrast, hindlimb footprint length was not dependent on speed (Spearman r=-0.1239, p=0.423, Figure 8B). [Figures 9A-9G]ICV injection provides β-gal activity (Figure 9A), body weight (Figure 9B), neurological examination score (Neurological Examination Score, Figure 9C), hindlimb footprint length (Figure 9D), and swing time (Figure 9E) as well as hindlimb stride length (Figure 9F) of GLB1- / - mice that received one of four doses of rAAVhu68.UbC.GLB1 (1.3×10¹¹GC, 4.4×10¹⁰GC, 1.3×10¹⁰GC, or 4.4×10⁹GC) or a vehicle. GLB1+ / - mice administered a vehicle (Het+ vehicle) serve as controls. Further details are provided in Section A of Example 4. Figure 9G shows the mean serum β-gal activity in GLB1- / - mice administered the maximum dose of rAAV. GLB1 was approximately 10 times higher than that of normal vehicle-treated GLB1+ / - controls. At the second highest dose of rAAV.hGLB1, serum β-gal activity in GLB1- / - mice was similar to that of normal vehicle-treated GLB1+ / - controls. Serum β-gal activity at all other rAAV.hGLB1 doses in GLB1- / - mice was similar to that of vehicle-treated GLB1- / - controls. [Figure 10A-10B] This document provides the amino acid sequence of the vp1 capsid protein of AAVhu68 (SEQ ID NO: 2) (labeled as hu68.vp1 in the alignment) and its alignment with AAV9 (SEQ ID NO: 20), AAVhu31 (labeled as hu.31 in the alignment, SEQ ID NO: 21), and AAVhu32 (labeled as hu.32 in the alignment, SEQ ID NO: 22). Compared to AAV9, AAVhu31, and AAVhu32, two mutations (A67E and A157V) were found to be important in AAVhu68 and are circled in Figure 10A. [Figure 11A-11E] This provides alignments between the nucleic acid sequence encoding the vp1 capsid protein of AAVhu68 (SEQ ID NO: 1) and AAV9 (SEQ ID NO: 23), AAVhu31 (SEQ ID NO: 24), and AAVhu32 (SEQ ID NO: 25). [Figure 12A]rAAVhu68. Provides an exemplary flowchart of the manufacturing process for producing the GLB1 active pharmaceutical ingredient. AEX: Anion exchange, CRL: Charles River Laboratories, ddPCR: Droplet digital polymerase chain reaction, DMEM: Dulbecco's modified Eagle medium, DNA: Deoxyribonucleic acid, FFB: Final formulation buffer, GC: Genome copy, HEK293: Human embryonic kidney 293 cells, ITFFB: Intrathecal final formulation buffer, PEI: Polyethyleneimine, Ph.Eur.: European Pharmacopoeia, SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, TFF: Tangential flow filtration, USP: United States Pharmacopoeia, WCB: Working cell bank. [Figure 12B] rAAVhu68. Provides an exemplary flowchart of the manufacturing process for producing the GLB1 drug product. Ad5: Adenovirus serotype 5, AUC: Ultracentrifugation for analysis, BDS: Bulk drug substance, BSA: Bovine serum albumin, CZ: Crystal Zenith, ddPCR: Droplet digital polymerase chain reaction, E1A: Initial region 1A (gene), ELISA: Enzyme-linked immunosorbent assay, FDP: Final drug product, GC: Genome copy, HEK293: Human embryonic kidney 293 cells, ITFFB: Intrathecal final drug product buffer, KanR: Kanamycin resistance (gene), MS: Mass spectrometry, NGS: Next-generation sequencing, Ph.Eur.: European Pharmacopoeia, qPCR: Quantitative polymerase chain reaction, SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, TCID50: 50% tissue culture infectious dose, ULC: Ultrahigh performance liquid chromatography, USP: United States Pharmacopoeia. [Figure 13] The survival data for each cohort up to day 300 of the study are shown for doses of 1.3 × 10¹¹ GC, 4.4 × 10¹⁰ GC, 1.3 × 10¹⁰ GC, and 4.4 × 10⁹ GC, with vehicle controls for KO and vehicle controls for heterozygous mice. [Figure 14A-14C] Figure 14A shows the mean total severity score for each cohort at each neurological assessment period. Figure 14B shows the hindlimb footprint length (cm). Figure 14C shows the total score for the neurological examination. [Figure 15A-15C] Histological analyses comparing brain sections of rAAV.hGLB1-treated GLB1- / - mice, vehicle-treated GLB1- / - mice, and vehicle-treated GLB1+ / - control mice at baseline (Figure 15A, day 1, 1 month old), day 150 (Figure 15B), and day 300 (Figure 15C) are also performed and the results are provided. [Figure 16] Figure 16A provides serum β-gal activity (nmol / mL / hour), and Figure 16B shows that β-gal activity was detectable in the CSF of all mice evaluated. GLB1- / - mice administered the two maximum doses of rAAV.hGLB1 tested showed higher mean CSF β-gal activity levels than normal vehicle-treated GLB1+ / - controls. β-gal activity in CSF was generally dose-dependent, but β-gal activity appeared similar in the two minimum dose groups. [Figure 17A-17L] The results of the study evaluating β-galactosidase activity in the brain (Figure 17A, day 150 and Figure 17B, day 300), heart (Figure 17C, day 150 and Figure 17D, day 300), liver (Figure 17E, day 150 and Figure 17F, day 300), spleen (Figure 17G, day 150 and Figure 17H, day 300), lungs (Figure 17I, day 150 and Figure 17J, day 300), or kidneys (Figure 17K, day 150 and Figure 17L, day 300) of rAAV.hGLB1-treated GLB1- / - mice and vehicle-treated controls are shown. β-gal was detectable in the CSF of all mice evaluated. GLB1- / - mice administered the two maximum doses of rAAV.hGLB1 tested showed higher mean CSF β-gal activity levels than normal vehicle-treated GLB1+ / - controls. While β-gal activity in CSF was generally dose-dependent, β-gal activity appeared similar in the two minimum dose groups. [Figures 18A-18B]The severity of dorsal root ganglia (DRG) and spinal cord lesions at day 120 is shown, as measured by histological analysis and a severity scoring system from 0 (none) to 5 (severe). The arrows depict two animals that showed the most severe axonal loss and fibrosis, with reduced sensory nerve action potentials. [Figures 19A-19B] The images show median nerve axonal degeneration and median nerve periaxonal fibrosis at day 120, as measured by histological analysis and a severity scoring system of 0 (none) to 5 (severe) for lesions. The arrows indicate two animals that showed the most severe axonal loss and fibrosis, with reduced sensory nerve action potentials. [Figures 20A-20B] This shows changes in median sensory nerve conduction at each measurement point during the study up to day 120, as measured by median sensory action potentials in microvolts (MV). [Figures 21A-21B] The results for bilateral median nerve sensory action potential amplitude (SNAP) and conduction velocity are shown. Young NHPs received single ICM doses of either a vehicle (ITFFB, N=2 / group) or an rAAV.hGLB1 test vector at doses of 3.0 × 10¹² GC (low dose), 1.0 × 10¹³ GC (medium dose), or 3.0 × 10¹³ GC (high dose) (N=3 / group). Sensory nerve conduction studies were performed at BL and at 28±3, 60±3, 90±4, and 120±4 days. SNAP amplitude and conduction velocity for the left and right median nerves are presented. Abbreviations: BL: Baseline, GC: Genome copy, ICM: Intraciscomasal, ITFB: Intrathecal final preparation buffer, N: Number of animals, NHP: Non-human primate, SNAP: Sensory nerve action potential. [Figures 22A-22D]This figure shows the results for human β-galactosidase activity in CSF and serum of NHP treated with rAAV.hGLB1 test vector or vehicle. Young NHPs received a single ICM (Intracellular Carcinoma) dose of either vehicle (ITFFB, N=2 / group) or rAAV.GLB1 at doses of 3.0 × 10¹² GC (low dose), 1.0 × 10¹³ GC (medium dose), or 3.0 × 10¹³ GC (high dose) (N=3 / group). CSF and serum were collected on the indicated days and analyzed for human β-gal activity. The dashed line represents baseline endogenous β-gal activity levels. Figure 22A shows β-gal activity in CSF. Figure 22B shows β-gal activity in serum. Figures 22C and 22D show enlarged views of the results at day 14. White outlines indicate animals that were negative for serum circulating NAb against the vector capsid at the time of treatment. The black areas indicate animals that tested positive for serum circulating NAb against the vector capsid at the time of treatment. Abbreviations: β-gal: β-galactosidase, BL: baseline, GC: genome copy, ICM: cisterna magna, ITFFB: intrathecal final preparation buffer, N: number of animals, NAb: neutral antibody, NHP: non-human primate, SEM: standard error of mean. [Figure 23] This study provides the in vivo distribution of rAAV.hGLB1 vectors 60 days after ICM administration to NHPs. The tissues shown were collected at autopsy from young NHPs 60 days after a single ICM administration of rAAV.hGLB1 at doses of 3.0 × 10¹² GC (low dose), 1.0 × 10¹³ GC (medium dose), or 3.0 × 10¹³ GC (high dose) (N=3 / group). Tissue was also collected as a control from vehicle-treated (ITFFB) NHPs (N=2). Each bar graph represents the mean vector genome detected per 1 μg of DNA. Error bars represent SEM. The LOD was 50 GC / μg DNA. Abbreviations: DNA: deoxyribonucleic acid, GC: genome copy, ICM: intracisional, ITFFB: intrathecal final preparation buffer, LOD: limit of detection, N: number of animals, NHP: non-human primate, SEM: standard error of the mean. [Figure 24]This study provides the in vivo distribution of rAAV.hGLB1 vectors in NHPs 120 days after ICM administration. The tissues shown were collected at autopsy from young NHPs 120 days after a single ICM administration of rAAV.hGLB1 at doses of 3.0 × 10¹² GC (low dose), 1.0 × 10¹³ GC (medium dose), or 3.0 × 10¹³ GC (high dose) (N=3 / group). Tissue was also collected as a control from NHPs treated with vehicle-based (ITFFB) therapy (N=2). Each bar graph represents the mean vector genome detected per 1 μg of DNA. Error bars represent SEM. The LOD was 50 GC / μg DNA. Abbreviations: DNA: deoxyribonucleic acid, GC: genome copy, ICM: intracisional, ITFFB: intrathecal final preparation buffer, LOD: limit of detection, N: number of animals, NHP: non-human primate, SEM: standard error of the mean. [Modes for carrying out the invention]

[0013] Compositions and methods based on adeno-associated virus (AAV) for the treatment of GM1 gangliosidosis (GM1) are provided herein. An effective amount of genomic copy (GC) of recombinant AAV (rAAV) (rAAVhu68.GLB1), which has an AAVhu68 capsid and a vector genome containing a normal GLB1 gene encoding a human β-galactosidase enzyme, is delivered to the patient. Preferably, this rAAVhu68.GLB1 is formulated with an aqueous buffer. In certain embodiments, the suspension is suitable for intrathecal injection. In certain embodiments, rAAVhu68.GLB1 is AAVhu68.UbC.GLB1 (also referred to as AAVhu68.UbC.hGLB1), and the GLB1 gene (i.e., the β-galactosidase coding sequence (also referred to as GLB1 enzyme, β-gal, or galactosidase as used herein)) is under the control of a regulatory sequence containing a promoter derived from human ubiquitin C (UbC). In certain embodiments, the composition is delivered via intracisor macrocentromatic injection (ICM) injection.

[0014] The nucleic acid sequence encoding the capsid of lineage F adeno-associated virus, referred to herein as AAVhu68, is used for the production of recombinant AAV (rAAV) possessing the AAVhu68 capsid and vector genome. As used herein, the term “vector genome” refers to a nucleic acid molecule that is packaged in a viral capsid (e.g., an AAV capsid) and deliverable to a host cell or patient cell. In certain embodiments, the vector genome is an expression cassette having inverted end repeat (ITR) sequences at the 5' and 3' ends necessary to package the vector genome into the AAV capsid, and containing, between them, the GLB1 gene described herein, operably ligated to a sequence that directs its expression. Additional details relating to AAVhu68 are provided in WO2018 / 160582 (which is incorporated herein in its entirety by reference) and in this detailed description. The rAAVhu68.GLB1 described herein is highly suitable for delivering vector genomes containing the GLB1 gene to cells within the central nervous system (CNS), including the brain, hippocampus, motor cortex, cerebellum, and motor neurons. These rAAVhu68.GLB1 can be used to target other cells within the CNS, as well as certain other tissues and cells outside the CNS. Alternatively, the AAVhu68 capsid can be replaced with another capsid that is also suitable for delivering the vector genome to the CNS, such as AAVcy02, AAV8, AAVrh43, AAV9, AAVrh08, AAVrh10, AAVbb01, AAVhu37, AAVrh20, AAVrh39, AAV1, AAVhu48, AAVcy05, AAVhu11, AAVhu32, or AAVpi02.

[0015] I.GM1 and therapeutic GLB1 gene GM1 gangliosidosis (i.e., GM1) can be classified into three types based on its clinical phenotype: (1) Type 1 or infantile form, which develops between birth and 6 months of age, is rapidly progressive with hypotonia and severe central nervous system (CNS) degeneration, and results in death by 1-2 years of age; (2) Type 2 late infant or juvenile form, which develops between 7 months and 3 years of age, is characterized by delayed motor and cognitive development, and progresses more slowly; and (3) Type 3 adult or chronic variant, which develops later in life (3-30 years) and is accompanied by progressive extrapyramidal dysfunction due to local deposition of sphingoglycolipids in the caudate nucleus (Brunetti-Pierri and Scaglia, 2008. GM1 gangliosidosis: Review of clinical, molecular, and therapeutic aspects, Molecular Genetics and Metabolism, 94:391-96). Infant GM1 patients who develop symptoms before 6 months of age uniformly exhibit rapid and predictable progression of both motor and cognitive impairments. The majority of patients die within the first few years of life (median survival time is 46 months, Jarnes Utz et al., 2017). Despite the common underlying pathophysiology, the adult-onset (type 3) GM1 phenotype is variable, and the disease course is significantly milder. Most patients with type 3 GM1 first develop neurological symptoms in late childhood, with little subsequent progression in adulthood.

[0016] The severity of each type is inversely correlated with the residual activity of the β-gal enzyme encoded by the GLB1 gene (Brunetti-Pierri and Scaglia, 2008). In humans, more than 130 GLB1 mutations causing diseases have been identified (Hofer et al., 2010, Phenotype Determining). alleles in GM1 gangliosidosis patients bearing novel GLB1 mutations.Clinical Genetics.78(3):236-246, and Caciotti et al.,2011,M1 gangliosidosis and Morquio B disease:An update on genetic alterations and clinical findings.Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease.1812(7):782-790). While some GLB1 mutations have been genetically and biochemically analyzed and correlate with clinical phenotypes (Gururaj et al., 2005, Magnetic Resonance Imaging Findings and Novel Mutations in GM1 Gangliosidosis. Journal of Child Neurology. 20(1):57-60, Caciotti et al., 2011, and Sperb et al., 2013, Genotypic and phenotypic characterization of Brazilian patients with GM1 gangliosidosis. Gene. 512(1):113-116), many GLB1 mutations remain uncharacterized. In a broad sense, a patient's genotype results in varying levels of residual enzyme activity, but generally speaking, the higher the residual enzyme activity, the milder the phenotype (Ou et al., 2018, SAAMP 2.0: An algorithm to predict genotype-phenotype correlation of lysosomal storage diseases. Clinical Genetics. 93(5):1008-1014). The diagnosis of GM1 is, This is confirmed by biochemical assays of β-gal and neuraminidase, and / or by GLB1 molecular analysis. However, there are limitations to using genotype-phenotype correlations in predicting the clinical symptoms of affected individuals, as residual enzyme activity itself cannot predict the disease subtype caused by mutations in the GLB1 gene (Hofer et al., 2010, Caciotti et al., 2011, Ou et al., 2018). Predictions are generally best for individuals with two significant mutations (i.e., mutations that do not show GLB1 enzyme activity) that are present with a severe early-onset phenotype (Caciotti et al., 2011, Sperb et al., 2013). There is little data on sibling concordance, but the clinical course in siblings with infant GM1 is similar in terms of time to onset and general disease signs (Gururaj et al., 2005).

[0017] The gene therapy vectors provided herein, namely rAAV.GLB1 (e.g., rAAVhu68.GLB1, rAAVhu68.UbC.GLB1), or compositions containing them, are useful for treating conditions associated with deficiencies in normal levels of functional beta-galactosidase. Where used herein, gene therapy vectors refer to the rAAVs described herein and are suitable for use in treating patients. In certain embodiments, the gene therapy vectors or compositions provided herein are useful for treating GM1 type 1. In certain embodiments, the gene therapy vectors or compositions provided herein are useful for treating GM1 type 2. In certain embodiments, the gene therapy vectors or compositions provided herein are useful for treating GM1 type 3. In certain embodiments, the gene therapy vectors or compositions provided herein are useful for treating GM1 types 1 and 2. In certain embodiments, the gene therapy vectors or compositions provided herein are useful for treating GM1 patients 18 months of age or younger. In certain embodiments, the gene therapy vectors or compositions provided herein are useful for treating GM1 types 1 and 2. In certain embodiments, the gene therapy vectors or compositions provided herein are useful for treating GM1 patients who are 36 months of age or younger. In certain embodiments, the gene therapy vectors or compositions provided herein are for the treatment of GM1 other than type 3. In certain embodiments, the gene therapy vectors or compositions provided herein are useful for treating neurological conditions associated with deficiency of normal levels of functional β-galactosidase. In certain embodiments, the gene therapy vectors or compositions provided herein are useful for improving symptoms associated with GM1 gangliosidosis. In certain embodiments, the gene therapy vectors or compositions provided herein are useful for improving neurological symptoms associated with GM1 gangliosidosis.

[0018] In certain embodiments, the patient has infant gangliosidosis and is 18 months of age or younger. In certain embodiments, the patient receiving rAAV.GLB1 is between 1 month and 18 months of age. In certain embodiments, the patient receiving rAAV.GLB1 is between 4 months and 18 months of age. In certain embodiments, the infant is under 4 months of age. In certain embodiments, the patient receiving rAAV.GLB1 is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months of age. In certain embodiments, the patient is a toddler, for example, between 18 months and 3 years of age. In certain embodiments, the patient receiving rAAV.GLB1 is between 3 and 6 years of age, 3 and 12 years of age, 3 and 18 years of age, or 3 and 30 years of age. In certain embodiments, the patient is 18 years of age or older.

[0019] In certain embodiments, improvement in symptoms associated with GM1 gangliosidosis after treatment may include, for example, increased lifespan (survival), reduced need for feeding tubes, reduced incidence, frequency, and duration of seizures, and delayed seizure onset, as measured by, for example, PedsQL. Improvement in quality, reduced progression toward neurocognitive decline and / or improvement in neurocognitive development, e.g., improved development, or improvements in adaptive behavior, cognition, language (receptive and expressive communication), and motor function (gross and fine motor skills), as measured by the Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III) and the Vineland Adaptive Behavior Scales, Second Edition (Vineland-II), earlier age at achieving motor milestones and later age at losing them, delayed increase in brain tissue (cerebral cortex and other small structures) volume and ventricular volume, delayed decrease in size of hypobrain structures including the corpus callosum, caudate nucleus and putamen, and cerebellar cortex, as well as stabilization of brain atrophy and volume changes, delayed progression of abnormal T1 / T2 signal intensity in the thalamus and basal ganglia, increased β-gal enzyme activity in CSF and serum, CSF Observations include decreased GM1 ganglioside concentration, decreased serum and / or urinary keratan sulfate levels, decreased hexosaminidase activity, reduced inflammatory response in the brain, abnormally delayed liver and spleen volume, abnormally delayed EEG and visual evoked potentials (VEPs), and / or improvements in swallowing, gait function, motor skills, language, and / or respiratory function.

[0020] In certain embodiments, for patients who would not have been eligible without the AAV therapy described herein, the patient receives combination therapy after rAAV.GLB1 injection. Such combination therapy may include enzyme replacement therapy, substrate suppression therapy (e.g., with miglustat (OGT918, N-butyl-deoxynojirimycin), tanganyl (acetyl-DL-leucine) therapy, respiratory therapy, use of a feeding tube, antiepileptic drugs), or hematopoietic stem cell transplantation (HSCT) using bone marrow or umbilical cord blood.

[0021] Immunosuppressive combination therapy may be used selectively in the target population as needed. Immunosuppressants for such combination therapy include, but are not limited to, glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin or rapalog), and cell division inhibitors (including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents active against immunophilins). Immunosuppressants may include nitrogen mustard, nitrosourea, platinum compounds, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracyclines, mitomycin C, bleomycin, mitramycin, IL-2 receptor (CD25) specific antibodies or CD3 specific antibodies, anti-IL-2 antibodies, cyclosporine, tacrolimus, sirolimus, IFN-β, IFN-γ, opioids, or TNF-α (tumor necrosis factor-α) conjugates. In certain embodiments, immunosuppressive therapy may be initiated before or 0, 1, 2, 3, 4, 5, 6, 7, or more days after rAAV.GLB1 administration. Such immunosuppressive therapy may involve the administration of one, two, or more drugs (e.g., glucocorticoids, prednisolone, mycophenolate mofetil (MMF), and / or sirolimus (i.e., rapamycin)). Such immunosuppressants may be administered once, twice, or more times, in the same or adjusted doses, to patients / subjects who require them. Such therapy may involve the concomitant administration of two or more drugs (e.g., prednisolone, mycophenolate mofetil (MMF), and / or sirolimus (i.e., rapamycin)) on the same day. One or more of these drugs may be continued after rAAV.GLB1 administration in the same or adjusted doses. Such therapy may last for approximately one week (7 days), approximately 60 days, or longer, as needed. In certain embodiments, a regimen that does not include tacrolimus is selected.

[0022] In certain embodiments, the “effective dose” of rAAV.GLB1 (e.g., rAAV.GLB1, rAAV.UbC.GLB1) provided herein is the amount that achieves improvement of symptoms associated with GM1 gangliosidosis. An "effective dose" of rAAV.GLB1 is the amount that achieves one or more of the following endpoints: increased pharmacological and biological activity of β-gal in cerebrospinal fluid (CSF); increased pharmacological and biological activity of β-gal in serum; increased patient life expectancy (survival); delayed progression of GM1 gangliosidosis (assessed by one or more of the following: age at achievement, age at loss, and percentage of patients maintaining or gaining age-appropriate developmental and motor milestones); improved neurocognitive development based on one or more of the following changes in age-appropriate cognitive, gross motor, fine motor, receptive and expressive communication scores on the Bayley Scales of Infant and Toddler Development (BSID, e.g., BSID Third Edition (BSID-III)); or changes in standard scores for each domain of the Vineland Adaptive Behavior Scales. For older children and adults, the “effective dose” of rAAV.GLB1 provided herein may, in some embodiments, be an amount that improves swallowing difficulties, walking ability, motor skills, language, and / or respiratory function, changes in standard scores for each domain of the Vineland Adaptive Behavior Scales, Second Edition (Vineland-II), reduction in seizure frequency and age of seizure onset, and establishment of independence from a feeding tube at 24 months of age. Examples of age-appropriate developmental and motor milestones are provided by the World Health Organization (WHO). See, for example, Wijnhoven TM, et al. (2004). Assessment of gross motor development in the WHO Multicentre Growth Reference Study. Food Nutr Bull. 25(1 Suppl):S37-45, and the table below.In certain embodiments, the “effective amount” of rAAV.GLB1 provided herein (e.g., rAAVhu68,GLB1) is the amount of rAAV.GLB1 that achieves a pharmacodynamic effect on β-galactoside activity of CSF and serum, GM1 concentration of CSF, as well as keratan sulfate in serum and urine, changes in brain MRI, monitoring of liver and spleen volume, and monitoring of EEG and visual evoked potentials (VEPs). [Table 1]

[0023] The rAAV.GLB1 described herein, and compositions containing it, contain the GLB1 gene (i.e., the β-Gal coding sequence) that encodes and expresses human β-galactosidase (which may also be referred to as normal β-galactosidase) or a functional fragment thereof. The GLB1 enzyme catalyzes the hydrolysis of β-galactosides to monosaccharides. The amino acid sequence of human β-galactosidase (2034 bp, 677 aa, Genbank number AAA51819.1, EC3.2.1.23) is reproduced herein as Sequence ID No. 4 and is also recognized as isoform 1 of β-galactosidase. See, for example, UniProtKB-P16278(BGAL_HUMAN). In certain embodiments, the GLB1 enzyme may have the sequence of amino acids 24 to 677 of Sequence ID No. 4 (i.e., a mature GLB1 enzyme without the signal peptide). In certain embodiments, the GLB1 enzyme has sequence number The sequence of amino acids 31-677 of sequence number 4 (i.e., isoform 3 of β-galactosidase) may be present. In certain embodiments, the GLB1 enzyme is isoform 2 having the amino acid sequence of sequence number 26. Any fragment that retains the function of full-length β-galactosidase may be encoded by the GLB1 gene described herein and will be referred to as a “functional fragment”. For example, a functional fragment of β-galactosidase may have at least about 25%, 50%, 60%, 70%, 80%, 90%, 100%, or more of the activity of full-length β-galactosidase (i.e., a normal GLB1 enzyme which may be β-galactosidase having the sequence of amino acids 24-677 of sequence number 4, or any one of the three isoforms). Methods for evaluating the activity of β-galactosidase can be found in the examples and publications. For example, see Radoslaw Kwapiszewski, Determination of Acid β-Galactosidase Activity: Methodology and Perspectives. Indian J Clin Biochem. 2014 Jan;29(1):57-62. In certain embodiments, the functional fragment is a cleaved β-galactosidase lacking approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more amino acids at the N-terminus and / or C-terminus of the full-length β-galactosidase. In certain embodiments, the functional fragment contains about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more conserved amino acid substitutions compared to full-length β-galactosidase. As used herein, a conserved amino acid substitution is an amino acid substitution in a protein that changes a given amino acid to a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size).

[0024] In one embodiment, the GLB1 gene has the sequence of SEQ ID NO: 5. In a particular embodiment, the GLB1 gene is manipulated to have the sequence of SEQ ID NO: 6. In a particular embodiment, the GLB1 gene is manipulated to have the sequence of SEQ ID NO: 7. In a particular embodiment, the GLB1 gene is manipulated to have the sequence of SEQ ID NO: 8. In a particular embodiment, the GLB1 gene is manipulated to have a sequence that is at least 95% to 99.9% identical to SEQ ID NO: 6. In a particular embodiment, the GLB1 gene is manipulated to have a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.9% identical to SEQ ID NO: 6. In a particular embodiment, the GLB1 gene is manipulated to have a sequence that is at least 95% to 99.9% identical to SEQ ID NO: 7. In certain embodiments, the GLB1 gene is manipulated to have a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.9% identical to SEQ ID NO: 7. In certain embodiments, the GLB1 gene is manipulated to have a sequence that is at least 95% to 99.9% identical to SEQ ID NO: 8. In certain embodiments, the GLB1 gene is manipulated to have a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.9% identical to SEQ ID NO: 8. In further embodiments, the manipulated sequence encodes full-length β-galactosidase or a functional fragment thereof. In further embodiments, the manipulated sequence encodes amino acids 24 to 677 of SEQ ID NO: 4, or a functional fragment thereof. In another embodiment, the manipulated sequence encodes the amino acid sequence of SEQ ID NO: 4, or a functional fragment thereof.

[0025] In certain embodiments, the GLB1 gene encodes a β-galactoside enzyme comprising a signal (leader) peptide and a GLB1 mature protein, which is amino acids 24-677 of SEQ ID NO: 4. The leader sequence is preferably of human origin or a derivative of a human leader sequence, and comprises about 15-28 amino acids, preferably about 20-25 amino acids, or about 23 amino acids. This is the amino acid length. In certain embodiments, the signal peptide is a native signal peptide (amino acids 1-23 of SEQ ID NO: 4). In certain embodiments, the GLB1 enzyme contains an exogenous leader sequence instead of a native leader sequence (amino acids 1-23 of SEQ ID NO: 4). In other embodiments, the leader may be derived from human IL2 or a mutant leader. In other embodiments, the human serpin F1 secretion signal may be used as the leader peptide.

[0026] II.AAVhu68 AAVhu68 (formerly designated AAV3G2) differs from the virus AAV9 of another lineage F by two encoded amino acids at positions 67 and 157 of vp1, based on the numbering in Sequence ID No. 2. In contrast, AAVs of other lineage F (AAV9, hu31, hu31) have Ala at positions 67 and 157. Novel AAVhu68 capsids and / or manipulated AAV capsids are provided, having valine (Val or V) at position 157 based on the numbering in Sequence ID No. 2, and optionally having glutamic acid (Glu or E) at position 67 based on the numbering in Sequence ID No. 2.

[0027] 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, as determined using a neighbor-joining algorithm based on the alignment of the AAV vp1 amino acid sequence, with at least 75% bootstrap values ​​(out of at least 1000 replicas) and Poisson-corrected distance measurements of 0.05 or less. The neighbor-joining algorithm is described in the literature. For example, see M. Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York (2000)). Computer programs are available that can be used to implement this algorithm. For example, the MEGA v2.1 program implements the modified Nei-Gojobori method. Using these techniques and computer programs, as well as the sequences of the AAV vp1 capsid protein, those skilled in the art can easily determine whether a selected AAV belongs to one of the lineages identified herein, another lineage, or outside of these lineages. For example, see G Gao, et al, J Virol, 2004 Jun;78(10):6381-6388, which identifies lineages A, B, C, D, E, and F and provides the nucleic acid sequences of novel AAVs with GenBank accession numbers AY530553~AY530629. Also see WO2005 / 033321.

[0028] In certain embodiments, the AAVhu68 capsid is further characterized by one or more of the following: AAVhu68 capsid protein can be expressed as follows: AAVhu68 vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO: 1-736; vp1 protein produced from SEQ ID NO: 1; or vp1 protein produced from a nucleic acid sequence at least 70% identical to SEQ ID NO: 1 encoding the predicted amino acid sequence of SEQ ID NO: 2-736; AAVhu68 vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least approximately 138-736 amino acids of SEQ ID NO: 2; vp2 protein produced from a sequence containing at least nucleotides 412-2211 of SEQ ID NO: 1; or vp2 protein produced from a nucleic acid sequence at least 70% identical to at least nucleotides 412-2211 of SEQ ID NO: 1 encoding the predicted amino acid sequence of at least approximately 138-736 amino acids of SEQ ID NO: 2; and / or AAVhu68 produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least approximately 203-736 amino acids of SEQ ID NO: 2. vp3 protein, vp3 protein produced from a sequence containing at least nucleotides 607-2211 of SEQ ID NO: 1, or 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 It contains vp3 proteins produced from at least 70% identical nucleic acid sequences.

[0029] The vp1, vp2, and vp3 proteins of AAVhu68 are typically expressed as alternative splice variants encoded by the same nucleic acid sequence that encodes the full-length vp1 amino acid sequence (amino acids (aa)1-736). 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 encoding the AAVhu68 vp3 amino acid sequence (approximately aa203-736) that does not contain the vp1-specific region (approximately aa1-approximately aa137) and / or the vp2-specific region (approximately aa1-approximately aa202), or a complementary strand thereto, the corresponding mRNA or tRNA (e.g., mRNA transcribed from approximately nucleotide (nt)607-approximately 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 encoding the AAVhu68 vp2 amino acid sequence (approximately aa138-736) of SEQ ID NO: 2, which does not contain the vp1 specific region (approximately aa1-137) or a complementary strand, the corresponding mRNA or tRNA (e.g., mRNA transcribed from 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.

[0030] As described herein, rAAVhu68 is an rAAVhu68 capsid produced in a production system that expresses a capsid from 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., one encoding a 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. More specifically, the AAVhu68 capsid contains subpopulations within the vp1, vp2, and vp3 proteins that have modifications from the predicted amino acid residues of SEQ ID NO: 2. These subpopulations contain, at a minimum, deamidated asparagine (N or Asn) residues. For example, asparagine in an asparagine-glycine pair is highly deamidated.

[0031] 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 encoding the AAVhu68 vp3 amino acid sequence of SEQ ID NO: 2 (approximately aa203-736) are also provided, which do not include the vp1-specific regions (approximately aa1-137) and / or the vp2-specific regions (approximately aa1-1202), or complementary strands, the corresponding mRNA or tRNA (approximately nt607-1211 of SEQ ID NO: 1). In certain embodiments, nucleic acid sequences encoding the AAVhu68 vp2 amino acid sequence (approximately aa138-736) of SEQ ID NO: 2 are also provided, which do not include the vp1 specific region (approximately aa1-aa137) or a complementary strand, the corresponding mRNA or tRNA (nt412-2211 of SEQ ID NO: 1).

[0032] 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 may be the nucleic acid sequence of SEQ ID NO: 1, or at least 70% of the SEQ ID NO: 1 encoding SEQ ID NO: 2. The sequences are 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. In certain embodiments, the nucleic acid sequence 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 nucleic acid sequence of SEQ ID NO: 1, or at least nt412 to about nt2211 of SEQ ID NO: 1 encoding the vp2 capsid protein (about aa138 to 736) of SEQ ID NO: 2. In certain embodiments, the nucleic acid sequence has 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 sequences to the nucleic acid sequence of approximately nt607 to approximately nt2211 of SEQ ID NO: 1, which encodes the vp3 capsid protein (approximately aa203 to 736) of SEQ ID NO: 2.

[0033] Designing nucleic acid sequences encoding this AAVhu68 capsid, including DNA (genomic or cDNA) or RNA (e.g., mRNA), is within the scope of the art. In certain embodiments, the nucleic acid sequence encoding the AAVhu68 vp1 capsid protein is provided in SEQ ID NO: 1. See also Figures 11A–11E. In other embodiments, nucleic acid sequences 70%–99.9% identical to SEQ ID NO: 1 may be selected to express the AAVhu68 capsid protein. In certain other embodiments, the nucleic acid sequence is at least about 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%–99.9% identical to SEQ ID NO: 1. Such nucleic acid sequences may be codon-optimized for expression in a selected system (i.e., cell type) and can be designed in a variety of ways. This optimization can 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 2015 / 012924, which is incorporated in its entirety herein by reference. See also, for example, U.S. Patent Publication 2014 / 0032186 and U.S. Patent Publication 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 making actual changes to codons or for synthesizing codon-optimized coding regions designed as described herein. Such modifications or synthesis can be performed using standard and common 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 desired sequence length, are synthesized using standard methods.These oligonucleotide pairs are synthesized to form 80-90 base pair double-stranded fragments containing sticky ends when annealed, for example, each oligonucleotide in a pair is synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond the complementary region to the other oligonucleotide in the pair. The single-stranded end of each oligonucleotide pair is designed to anneal to the single-stranded end of another oligonucleotide pair. The oligonucleotide pairs are annealed, then approximately 5-6 of these double-stranded fragments are annealed together via sticky single-stranded ends, then ligated together, and cloned into a standard bacterial cloning vector, such as the TOPO® vector available from Invitrogen Corporation, Carlsbad, Calif. The construct is then sequenced by standard methods. Some of these constructs consist of 5-6 fragments from a combined ligated 80-90 base pair fragment, i.e., a fragment of approximately 500 base pairs is prepared, and as a result, the entire desired sequence is represented in a series of plasmid constructs. Then, inserts are placed into these plasmids. The molecules are then cleaved with appropriate restriction enzymes, ligated together, and the final construct is formed. The final construct is then cloned into a standard bacterial cloning vector and sequenced. Additional methods will readily become apparent to those skilled in the art. Furthermore, gene synthesis is readily available commercially.

[0034] In certain embodiments, the AAVhu68 capsid is produced using 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 nucleic acid sequence of SEQ ID NO: 1 or the vp1 amino acid sequence of SEQ ID NO: 2 having the modifications described herein (i.e., deamidated amino acids). In certain embodiments, the vp1 amino acid sequence is reproduced in SEQ ID NO: 2.

[0035] As used herein, when used to refer to vp capsid proteins, the term “heterogeneous” or any grammatical variation thereof refers to a population of non-identical elements having, for example, vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences. Sequence 2 provides the encoded amino acid sequence of the AAVhu68 vp1 protein. The term “heterogeneous” as used in relation to vp1, vp2, and vp3 proteins (alternatively referred to as isoforms) refers to the differences in the amino acid sequences of the vp1, vp2, and vp3 proteins within the capsid. The AAV capsid contains subpopulations within the vp1, vp2, and vp3 proteins that have the predicted amino acid residue modifications. These subpopulations contain, at a minimum, certain deamidated asparagine (N or Asn) residues. For example, certain subpopulations contain at least one, two, three, or four highly deamidated asparagine(N) positions in the asparagine-glycine pair, and optionally further contain other deamidated amino acids, where deamidation results in amino acid changes and other optional modifications.

[0036] As used herein, a “subpopulation” of vp proteins means, unless otherwise specified, a group of vp proteins that share at least one defined common feature and consist 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 vp1 protein and fewer than all vp1 proteins in an assembled AAV capsid. A “subpopulation” of vp3 proteins may be, unless otherwise specified, one vp3 protein and fewer than all vp3 proteins in an assembled AAV capsid. For example, in an assembled AAV capsid, vp1 proteins may be a subpopulation of vp proteins, vp2 proteins may be another subpopulation of vp proteins, and vp3 may be yet another subpopulation of vp proteins. 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.

[0037] Unless otherwise specified, high deamidation refers to deamidation 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%, or up to approximately 100% at the reference amino acid position, compared to the predicted amino acid sequence at the reference amino acid position (for example, based on the numbering of Sequence ID No. 2 (AAVhu68), at least 80% of asparagine at amino acid 57 may be deamidated based on the total vp1 protein, and may also be deamidated based on the total vp1, vp2, and vp3 proteins). Such percentages may be determined using 2D gel, mass spectrometry, or other preferred techniques.

[0038] While we do not wish to be bound by theory, the deamidation of at least highly deamidated residues in the vp protein within the AAV capsid is considered to be inherently non-enzymatic and is caused by functional groups in the capsid protein that deamidate selected asparagine, and to a lesser extent, glutamine residues. The efficient capsid assembly of most deamidated vp1 proteins indicates that these events occur after capsid assembly, or that deamidation in individual monomers (vp1, vp2, or vp3) is structurally well tolerated and mostly does not affect the assembly dynamics. Generally, extensive deamidation in the VP1-specific (VP1-u) region (approximately aa1-137), which is thought to be located internally before cell entry, suggests that VP deamidation may occur before capsid assembly. Deamidation of N may occur by nucleophilic attack on the side-chain amide group carbon atom of Asn via the skeletal nitrogen atom of its C-terminal residue. It is thought that an intermediate ring-closed succinimide residue is formed. Subsequently, the succinimide residue undergoes rapid hydrolysis to yield the final product, aspartic acid (Asp) or isoaspartic acid (IsoAsp). Thus, in certain embodiments, deamidation of asparagine (N or Asn) yields Asp or IsoAsp, which can be interconverted via succinimide intermediates, for example, as illustrated below. [ka]

[0039] As provided herein, each deamidated N in VP1, VP2, or VP3 may independently be aspartic acid (Asp), isoaspartic acid (isoAsp), aspartate, and / or an interconversion blend of Asp and isoAsp, or a combination thereof. Any preferred ratio of α-glutamic acid and isoaspartic acid may be present. For example, in certain embodiments, the ratio may be aspartic acid to isoaspartic acid at 10:1 to 1:10, aspartic acid to isoaspartic acid at about 50:50, or aspartic acid to isoaspartic acid at about 1:3, or another selected ratio.

[0040] In certain embodiments, one or more glutamines (Q) 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 can be interconverted via a common glutarimide intermediate. Any preferred ratio of α-glutamic acid and γ-glutamic acid may exist. For example, in certain embodiments, the ratio may be α:γ = 10:1 to 1:10, α:γ = about 50:50, or α:γ = about 1:3, or another selected ratio. [ka]

[0041] Therefore, rAAV contains subpopulations within the rAAV capsid of vp1, vp2, and / or vp3 proteins having deamidated amino acids, comprising at least one subpopulation containing at least one highly deamidated asparagine. Furthermore, other modifications may include isomerization at a selected aspartic acid (D or Asp) residue position. In yet another embodiment, the modification may include amidation at the Asp position.

[0042] In certain embodiments, the AAV capsid contains 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 encoded amino acid sequence of the vp protein. The majority of these may be N residues. However, Q residues may also be deamidated.

[0043] In certain embodiments, the rAAV has an AAV capsid having vp1, vp2, and vp3 proteins having subpopulations containing combinations of two, three, four or more deamidation residues at the positions shown in the table provided in Example 1 and incorporated herein by reference. Deamidation of the rAAV can be determined using 2D gel electrophoresis and / or mass spectrometry (MS) and / or protein modeling techniques. Online chromatography is performed using a Thermo UltiMate 3000 RSLC system (Thermo) coupled to a Q Exactive HF with an Acclaim PepMap column and a NanoFlex source (Thermo Fisher Scientific). This can be performed by Fisher Scientific. MS data are acquired using the Q Exactive HF's data-dependent top 20 method, which dynamically selects the most abundant yet unsequenced precursor ions from a survey scan (200–2000 m / z). Sequencing is performed by higher-energy collisional dissociation fragmentation with a target value of 1e5 ions determined by predictive autogain control, and precursor isolation was performed with a 4 m / z window. Survey scans were acquired at m / z 200, resolution 120,000. Resolution of HCD spectra may be set to 30,000 at m / z 200 with a maximum ion injection time of 50 ms and normalized collision energy of 30. S-lens RF level is set to the highest level in the m / z region occupied by peptides from digestion. To ensure proper transmission, it may be set to 50. A single unassigned charged state from fragmentation selection, or a precursor ion with more than 6 charged states, may be excluded. BioPharma Finder 1.0 software (Thermo Fischer Scientific) may be used for the analysis of acquired data. For peptide mapping, a search is performed using a single-entry protein FASTA database set to carbamide methylation as the fixed modification, oxidation, deamidation, and phosphorylation as variable modifications, 10 ppm mass precision, high protease specificity, and a confidence level of 0.8 in the MS / MS spectrum. Examples of suitable proteases may include, for example, trypsin or chymotrypsin. Identification of deamidated peptides by mass spectrometry is relatively straightforward, as deamidation adds +0.984 Da (mass difference between -OH and -NH2 groups) to the mass of the original molecule. The deamidation rate of a particular peptide is determined by dividing the mass area of ​​the deamidated peptide by the sum of the areas of the deamidated and native peptides. Given the number of potential deamidation sites, isobaric species deamidated at different sites may comigrate in a single peak. Consequently, multiple deamidation sites can be localized 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 isomeric species and independent of the deamidation site. It will be understood by those skilled in the art that many variations of these exemplary methods can be used. For example, suitable mass spectrometers include, for instance, quadrupole time-of-flight mass spectrometers (QTOF), e.g., Waters Xevo or Agilent 6530, or orbitrap instruments, e.g., Orbitrap Fusion or Orbitrap Velos (Thermo Fisher).Suitable liquid chromatography systems include, for example, the Waters Acquity UPLC system or the Agilent system (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 found, for example, in X. Jin et al, Hu Gene Therapy Methods, Vol. 28, No. 5, pp. 255-267, published online on June 16, 2017.

[0044] In addition to deamidation, other modifications may occur but one amino acid will not be converted to a different amino acid residue. Such modifications may include acetylation, isomerization, phosphorylation, or oxidation. Modification of deamidation: In certain embodiments, AAV 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, for example, 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 amine group). As used herein, amino acids lacking an amide or amine side group refer to, for example, glycine, alanine, valine, leucine, isoleucine, serine, threonine, cystine, 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 encoded AAV amino acid sequence. In certain embodiments, such modifications are not performed on all four asparagine-glycine pairs. Therefore, methods for reducing deamide of AAV and / or modified AAV variants have a lower deamide rate. Additionally, or alternatively, one or more other amide amino acids may be changed to non-amide amino acids. Deamidation of AAV can be reduced. In certain embodiments, the mutant AAV capsids described herein contain mutations in the asparagine-glycine pair such that glycine is changed to alanine or serine. The mutant AAV capsid may contain one, two, or three mutants at the position where the reference AAV naturally contains four NG pairs. In certain embodiments, the AAV capsid may contain one, two, three, or four such mutants at the position where the reference AAV naturally contains five NG pairs. In certain embodiments, the mutant AAV capsid contains only a single mutation in the NG pair. In certain embodiments, the mutant AAV capsid contains mutations in two different NG pairs. In certain embodiments, the mutant AAV capsid contains mutations in two different NG pairs located structurally distant from each other in the AAV capsid. In certain embodiments, the mutations are not in the VP1-specific region. In certain embodiments, one of the mutations is in the VP1-specific region. Selectively, mutant AAV capsids contain mutations to minimize or eliminate deamidation in one or more asparagine or glutamine located outside the NG pair, even if the mutations do not include modifications within the NG pair.

[0045] In certain embodiments, a method is provided for increasing the potency of rAAV, which involves manipulating an AAV capsid to eliminate one or more NGs in the wild-type AAV capsid. In certain embodiments, the coding sequence of "G" in "NG" is manipulated to code for a different amino acid. In the following specific examples, "S" or "A" is substituted. However, other suitable amino acid coding sequences may be selected. See the table in Example 1 (incorporated herein by reference).

[0046] In the AAVhu68 capsid protein, four residues (N57, N329, N452, N512) routinely exhibit deamidation levels exceeding 70%, and in most cases exceeding 90% across various lots. Additional 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] The AAVhu68 capsid contains subpopulations within the vp1, vp2, and vp3 proteins that have modifications from the predicted amino acid residues of SEQ ID NO: 2. These subpopulations contain, at a minimum, specific deamidated asparagine (N or Asn) residues. 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. SEQ ID NO: 3 provides the amino acid sequence of the modified AAVhu68 capsid, indicating positions that may have a certain percentage of deamidated amino acids or otherwise modified amino acids. Various combinations of these and other modifications are described herein.

[0048] In other embodiments, the method involves increasing the yield of rAAV, and therefore increasing the amount of rAAV present in the supernatant before or without requiring cell lysis. This method involves manipulating the AAV VP1 capsid gene based on an amino acid number sequence of the AAVhu68 vp1 capsid protein to express a capsid protein having Glu at position 67, Val at position 157, or both. In other embodiments, the method involves manipulating the VP2 capsid gene to express a capsid protein having Val at position 157. In yet another embodiment, rAAV includes both vp1 and vp2 capsid proteins having a modified capsid with Glu at position 67 and Val at position 157.

[0049] As used herein, "AAV9 capsid" refers to multiple AAV9 vp proteins. It is a self-assembling AAV capsid composed of quality. The AAV9 vp protein is typically expressed as an alternative splice variant encoded by 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 nucleic acid sequence of SEQ ID NO: 23, or the vp1 amino acid sequence of GenBank accession number AAS99264. In certain embodiments, the “AAV9 capsid” includes an AAV having an amino acid sequence that is 99% identical to AAS99264 or 99% identical to SEQ ID NO: 20. See US7906111 and WO2005 / 033321. As used herein, “AAV9 variant” includes, for example, those described in WO2016 / 049230, US8,927,514, US2015 / 0344911, and US8,734,809.

[0050] Methods for generating capsids, and therefore coding sequences, and methods for producing rAAV are described. See, for example, Gao, et al, Proc. Natl. Acad. Sci. USA100(10), 6081-6086 (2003) and US2013 / 0045186A1.

[0051] 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 is across the full-length sequence, its open reading frame, or another suitable fragment of at least 15 nucleotides in length. Examples of suitable fragments are described herein.

[0052] In the context of nucleic acid sequences, the terms “sequence identity,” “percent sequence identity,” or “percent identical” refer to residues in two sequences that are identical when aligned to the greatest extent possible. The length of the sequence identity comparison may be, and is preferable, over the entire genome, the entire gene coding sequence, or a fragment of at least about 500–5000 nucleotides. However, identity between smaller fragments, for example, at least about 9 nucleotides, typically at least about 20–24 nucleotides, at least about 28–32 nucleotides, or at least about 36 or more nucleotides, may also be desirable. Similarly, “percent sequence identity” can be readily determined for the entire amino acid sequence of a protein or a fragment thereof. Preferably, the fragment is at least about 8 amino acids long and may be up to about 700 amino acids long. Examples of preferred fragments are described herein.

[0053] 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 amino acid insertions or deletions, the aligned sequence has at least about 95–99% amino acid sequence identity. Preferably, the homology is across the full-length sequence, its open reading frame, or another suitable fragment that is at least 8 amino acids long, or more preferably at least 15 amino acids long. Examples of suitable fragments are described herein.

[0054] 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 by relying on algorithms and computer programs known to those skilled in the art.

[0055] Generally, the "identity," "homology," or "similarity" between two different adeno-associated viruses is not considered. When referring to "identity," "homology," or "similarity" is determined by referring to "aligned" sequences. An "aligned" sequence or "alignment" refers 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 an example, AAV alignment is performed using a publicly available AAV9 sequence as a reference point. Alignment is performed using one of the various publicly or commercially available multiple sequence alignment programs. 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. In addition, several algorithms known in the art exist and can be used to measure nucleotide sequence identity, including those included in the programs described above. As another example, polynucleotide sequences can be compared using Fasta®, a program from GCG version 6.1. Fasta® provides the best overlap region alignment and sequence identity percentage between query and search sequences. For example, percentage sequence identity between nucleic acid sequences can be determined using Fasta® with its default parameters (word size 6 and NOPAM factor for scoring matrix) provided in GCG Version 6.1, which is incorporated herein for reference. Several sequence alignment programs for amino acid sequences are also available, such as the “Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, one of these programs is used with its default settings, but those skilled in the art may change these settings as needed.Alternatively, a person skilled in the art may utilize another algorithm or computer program that provides at least the same level of identity or alignment as that provided by the algorithm and program of reference. See, for example, JDThomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

[0056] III. rAAV Recombinant adeno-associated viruses (rAAVs) have been described as suitable vehicles for gene delivery. Typically, an exogenous expression cassette containing a transgene (e.g., the GLB1 gene) for rAAV delivery replaces functional rep and cap genes from the natural AAV source, resulting in a non-replicable vector. The functions of these rep and cap genes are provided in trans during the vector production system but are not present in the final rAAV.

[0057] As shown above, an rAAV is provided, comprising an AAV capsid and a vector genome containing, at a minimum, an AAV inverted terminal repeat (ITR), the GLB1 gene, and a regulatory sequence that directs its expression, which are necessary to package the vector genome into the capsid. In certain embodiments, the AAV capsid is derived from AAVhu68. While the examples herein utilize a single-stranded AAV vector genome, in certain embodiments, an rAAV containing a self-complementary (sc)AAV vector genome may be utilized in the present invention.

[0058] The necessary regulatory elements are operably ligated to a gene (e.g., GLB1) in a manner that enables its transcription, translation, and / or expression in cells that take up rAAV. As used herein, “operably ligated” sequences include both expression regulatory sequences that are sequential to the gene of interest and expression regulatory sequences that act trans or asynchronously to control the gene of interest. Such regulatory sequences are typically, for example, promoters. This includes one or more of the following: enhancers, introns, poly(A), and self-cleaving linkers (e.g., furin, furin-F2A, IRES). The following examples utilize the CB7 promoter (e.g., SEQ ID NO: 10), the EF1a promoter (e.g., SEQ ID NO: 11), or the human ubiquitin C (UbC) promoter (e.g., SEQ ID NO: 9) for the expression of the GLB1 gene. However, in certain embodiments, other promoters or additional promoters may be selected.

[0059] In certain embodiments, in addition to the GLB1 gene, a non-AAV sequence encoding one or more other gene products may be included. Such gene products may be, for example, the peptide, polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor), or other gene products. A useful gene product may be miRNA. miRNAs and other small interfering nucleic acids regulate gene expression by cleaving / degrading target RNA transcripts or by repressing the translation of target messenger RNA (mRNA). miRNAs are typically expressed naturally as the final 19-25 untranslated RNA product. miRNAs exhibit their activity through sequence-specific interactions with the 3' untranslated region (UTR) of target mRNA. These endogenously expressed miRNAs form a hairpin precursor, which is then processed into a miRNA double-strand and further into a “mature” single-stranded miRNA molecule. This mature miRNA leads to the multiprotein complex miRISC, which identifies a target site on the target mRNA (e.g., in the 3'UTR region) based on its complementarity to the mature miRNA.

[0060] In certain embodiments, the vector genome may be engineered to include, in addition to the GBL1 coding sequence, one or more miRs useful for detargeting the dorsal root ganglia to improve safety and / or reduce side effects. Such drg detargeting sequences are operably ligated with the GLB1 coding sequence to minimize or prevent the expression of GLB1 products in the dorsal root ganglia. A preferred drg detargeting sequence is described in PCT / US19 / 67872, filed December 20, 2019, entitled "Compositions for DRG-specific reduction of transgene expression".

[0061] AAV vector genomes typically contain cis-acting 5' and 3' inverted terminal repeats (ITRs) (see, e.g., B.J. Carter, in “Handbook of Parvoviruses”, edited by P. Tijsser, CRC Press, pp. 155-168 (1990)). ITR sequences are approximately 145 base pairs (bp) in length. Preferably, substantially complete sequences encoding ITRs are used in the molecule, although some minimal modification of these sequences is acceptable. The ability to modify these ITR sequences is within the scope of those skilled in the art (see, e.g., Sambrook et al., “Molecular Cloning. A Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory, New York (1989), and K. Fisher et al., J. Virol., 70:520-532 (1996)). An example of such molecules used in the present invention is a “cis-acting” plasmid containing a transgene, where 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 a different AAV than the one supplying the capsid. In one embodiment, the ITR sequence is derived from AAV2. A shortened version of the 5' ITR, referred to as ΔITR, is described, with a deletion of the D sequence and terminal degradation sites (trs). In certain embodiments, the vector genome contains a 130-base pair shortened AAV2 ITR with a deletion of the outer A element. The shortened ITR is reverted to its wild-type length of 145 base pairs during vector DNA amplification using the inner A element as a template. In other embodiments, full-length AAV 5' and 3' ITRs are used. However, if other AAV sources are available... The originating ITR can be selected. If the ITR source is AAV2 and the AAV capsid is from another AAV source, the resulting rAAV may be described as pseudotype. However, other configurations of these elements may also be preferable.

[0062] In certain embodiments, the expression regulatory sequence (regulatory sequence) may include, for example, an additional or alternative promoter sequence located between the selected 5'ITR sequence and the coding sequence. Constitutive promoters, moduloable promoters (see, e.g., WO2011 / 126808 and WO2013 / 04943), tissue-specific promoters (e.g., neuron-specific promoters or glial cell-specific promoters, or CNS-specific promoters), or promoters that respond to physiological cues may be used in the rAAV described herein. The promoter can be selected from different sources, such as the human cytomegalovirus (CMV) pre-initial enhancer / promoter, SV40 initial enhancer / promoter, JC polymovirus 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 verta-actin promoter. Other suitable promoters may include the CB7 promoter. In addition to the promoter, the vector genome may contain one or more other suitable transcription start sequences, transcription termination sequences, 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, if necessary, 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 regulatory sequence comprises one or more expression enhancers. In one embodiment, the regulatory sequence comprises two or more expression enhancers. These enhancers may be the same or different from one another.For example, the enhancer may include a CMV pre-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 contains introns, such as chicken β-actin introns. In certain embodiments, the intron is a hybrid intron consisting of a chimeric intron (CI)-human β-globin splice donor and an immunoglobulin G (IgG) splice acceptor element. Other suitable introns include those known in the art, such as those described in WO2011 / 126808. Examples of suitable polyA sequences include, for example, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyA. Optionally, one or more sequences may be selected to stabilize the mRNA. An example of such a sequence is a modified WPRE sequence, which can be manipulated upstream of the poly-A sequence and downstream of the coding sequence (see, for example, MA Zanta-Boussif, et al, Gene Therapy (2009) 16:605-619). In certain embodiments, the WPRE sequence is absent.

[0063] In certain embodiments, a vector genome is constructed containing a 5'AAV ITR-promoter-optional enhancer-optional intron-GLB1 gene-poly-A-3'ITR. In certain embodiments, the ITR is not derived from AAV2. In certain embodiments, two or more promoters are present. In certain embodiments, the enhancer is present in the vector genome. In certain embodiments, two or more enhancers are present. In certain embodiments, the intron is present in the vector genome. In certain embodiments, the Hansers and introns are present. In certain embodiments, the intron is a chimeric intron (CI), which is a hybrid intron consisting of a human beta-globin splice donor and an immunoglobulin G (IgG) splice acceptor element. In certain embodiments, polyA is SV40 polyA (i.e., a polyadenylation (polyA) signal derived from the late gene of monkey virus 40 (SV40)). In certain embodiments, polyA is rabbit beta-globin (RBG) polyA. In certain embodiments, the vector genome includes a 5'AAV ITR-CB7 promoter-GLB1 gene-RBG polyA-3'ITR. In certain embodiments, the vector genome includes a 5'AAV ITR-EF1a promoter-GLB1 gene-SV40 polyA-3'ITR. In certain embodiments, the vector genome includes a 5'AAV ITR-UbC promoter-GLB1 gene-SV40 polyA-3'ITR. In certain embodiments, the GLB1 gene has sequence number 5. In certain embodiments, the GLB1 gene has sequence number 6. In certain embodiments, the GLB1 gene has sequence number 7. In certain embodiments, the GLB1 gene has sequence number 8. In certain embodiments, the vector genome has the sequence of sequence number 12, or a sequence that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to about 99.9% identical thereto. In certain embodiments, the vector genome has the sequence of sequence number 13, or a sequence that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to about 99.9% identical thereto. In certain embodiments, the vector genome has the sequence of SEQ ID NO: 14, or a sequence that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to about 99.9% identical thereto. In certain embodiments, the vector genome has the sequence of SEQ ID NO: 15, or a sequence that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to about 99.9% identical thereto.In certain embodiments, the vector genome has the sequence of sequence number 16, or a sequence that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to about 99.9% identical thereto.

[0064] IV.rAAV production For use in the production of AAV virus vectors (e.g., recombinant(r)AAV), the vector genome may be encapsulated on any suitable vector, e.g., a plasmid, to be delivered to a packaging host cell. Plasmids useful for the present invention may be engineered to be particularly suitable for replication and packaging in vitro into prokaryotic cells, insect cells, and mammalian cells. Suitable transfection techniques and packaging host cells are known and / or can be readily designed by those skilled in the art. Exemplary production processes are provided in Figures 12A-12B.

[0065] Methods for generating and isolating AAVs suitable for use as vectors are known in the art. See, for example, Grieger & Samulski, 2005, Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications, Adv. Biochem.Engin / Biotechnol.99:119-145, Buning et al., 2008, Recent developments in adeno-associated virus vector technology, J.Gene Med.10:717-733, and the references cited below (each of these is incorporated herein by reference in its entirety). For packaging 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 gene. The cap and rep genes may be supplied trans.

[0066] In one embodiment, selected gene elements may be delivered to AAV-packaging cells by any preferred method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Suitable AAV-packaging cells may also be constructed. Methods used to construct such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Molecular Cloning: A Laboratory Manual, edited by Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0067] The terms “AAV intermediate” or “AAV vector intermediate” refer to assembled rAAV capsids that have a deletion of the desired genomic sequence packaged therein. These may also be referred to as “empty” capsids. Such capsids may contain no detectable genomic sequence of the expression cassette at all, or only a partially packaged genomic sequence that is insufficient to achieve the expression of a gene product (e.g., β-gal). These empty capsids are nonfunctional for introducing the gene of interest into a host cell. In certain embodiments, rAAV.GLB1 or the compositions described herein may not contain at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% of AAV intermediates, i.e., they may contain less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0.1% of AAV intermediates.

[0068] The recombinant adeno-associated viruses (AAVs) described herein can be produced using known techniques. See, for example, WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, and US7588772B2. Such methods involve culturing host cells containing a nucleic acid sequence encoding an AAV capsid protein, a functional rep gene, an expression cassette consisting of at least an AAV inverted terminal repeat (ITR) and a transgene, and sufficient helper function to enable packaging the expression cassette into an AAV capsid protein. Methods for producing the capsid, the coding sequence therefor, and 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.

[0069] In one embodiment, a production cell culture useful for producing recombinant AAV (e.g., rAAVhu68) is provided. Such a cell culture contains nucleic acids expressing the AAV capsid protein in host cells, nucleic acid molecules suitable for packaging in the AAV capsid, such as a vector genome containing AAV ITR, and a GLB1 gene operably linked to a regulatory sequence that directs gene expression in cells (e.g., cells in a patient requiring it), as well as sufficient AAV rep and adenovirus helper functions to enable packaging the vector genome in the recombinant AAV capsid. In one embodiment, the cell culture consists of mammalian cells (e.g., particularly human embryonic kidney 293 cells) or insect cells (e.g., Spodoptera frugiperda (Sf9) cells). In certain embodiments, a baculovirus provides the helper functions necessary for packaging the vector genome in the recombinant AAVhu68 capsid.

[0070] Optionally, the rep function is provided by an AAV other than AAVhu68, which is the capsid-source AAV. In certain embodiments, at least part of the rep function is derived from AAVhu68. In other embodiments, the rep protein is a heterologous rep protein other than AAVhu68, for example, but not limited to, AAV1 rep protein, AAV2 It may be the 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 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 host cells.

[0071] In one embodiment, cells are prepared in a suitable cell culture (e.g., HEK293 or Sf9) or suspension. Methods for preparing gene therapy vectors described herein include methods well known in the art, such as generating plasmid DNA used for gene therapy vector production, vector production, and vector purification. In some embodiments, the gene therapy vector is rAAV, and the produced plasmids are AAV cis-plasmids encoding the AAV vector genome containing the gene of interest, AAV trans-plasmids containing AAV rep and cap genes, and adenovirus helper plasmids. 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 vector-containing cells and culture medium. 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 an overview of these production systems, please refer to, 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, the contents of which are incorporated herein by reference in their entirety. Methods for preparing and using these and other AAV production systems are also described in U.S. Patents No. 5,139,941, 5,741,683, 6,057,152, 6,204,059, 6,268,213, 6,491,907, 6,660,514, 6,951,753, 7,094,604, 7,172,893, 7,201,898, 7,229,823, and 7,439,065, the contents of each of these, are incorporated herein by reference in their entirety.

[0072] The crude cell recovery can then be subjected to method steps such as concentration of the rAAV recovery, dialysfiltration of the rAAV recovery, microsolution of the rAAV recovery, nuclease digestion of the rAAV recovery, filtration of the microsolution intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration for preparing bulk rAAV.

[0073] The rAAV drug product is purified using a two-step high-salt affinity chromatography purification followed by anion exchange resin chromatography to remove empty capsids. These methods are described in more detail in WO2017 / 160360, International Patent Application PCT / US2016 / 065970, filed 9 December 2016, and its priority documents, U.S. Patent Application 62 / 322,071, filed 13 April 2016, and U.S. Patent Application 62 / 226,357, “Scalable Purification Method for AAV9,” filed 11 December 2015, which are incorporated herein by reference.

[0074] To calculate the content of empty and complete particles, the selected sample (e.g., in the examples herein, a preparation purified by an iodixanol gradient, genome copy (GC)) The VP3 band volume for each loaded GC particle (where pt = 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 material peak. Then, the number of particles per 20 μL loaded (pt) is multiplied by 50 to obtain particles (pt) / mL. Pt / mL is divided by GC / mL to obtain the ratio of particles to genome copies (pt / GC). Pt / mL - GC / mL gives empty pt / mL. The percentage of empty particles is obtained by dividing empty pt / mL by pt / mL and multiplying by 100.

[0075] Generally, assay methods for rAAV particles containing empty capsids and packaged vector genomes are known in the 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, this method comprises subjecting a treated AAV stock to SDS-polyacrylamide gel electrophoresis (e.g., a gradient gel containing 3-8% tris acetate in buffer) of any gel capable of separating three capsid proteins, then trialing the gel until the sample material is separated, and blotting the gel on a nylon or nitrocellulose membrane, preferably nylon. Then, an anti-AAV capsid antibody, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody, is used as the primary antibody to bind to the denatured capsid protein (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, for SDS-PAGE, a sample may be taken from a column fragment and heated in an SDS-PAGE loading buffer containing a reducing agent (e.g., DTT) to resolve the capsid protein on a precast gradient polyacrylamide gel (e.g., Novex). Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions, or using other suitable staining methods, namely SYPRO ruby ​​or Coomassie dye. 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 digested with DNase 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 primer and the DNA sequence between 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 rAAV. The vector genome titer is 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 are also available.

[0076] In one embodiment, an optimized q-PCR method is used that utilizes a broad-spectrum serine protease, such as protease 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 proteinase K buffer, treated with proteinase K, and then thermally inactivated. Preferably, the sample is diluted with an amount of proteinase K buffer equal to the sample size. The proteinase K buffer is twice as thick. The concentration may be higher than the above. Typically, the proteinase K treatment is about 0.2 mg / mL, but can vary between 0.1 mg / mL and about 1 mg / mL. The treatment step is generally carried out at about 55°C for about 15 minutes, but may be carried out at a lower temperature (e.g., about 37°C to about 50°C) for a longer time (e.g., about 20 minutes to about 30 minutes), or at a higher temperature (e.g., up to about 60°C) for a shorter time (e.g., about 5 to 10 minutes). Similarly, thermal inactivation is generally at about 95°C for about 15 minutes, but the temperature may be lowered (e.g., about 70 to about 90°C) and the time may be extended (e.g., about 20 minutes to about 30 minutes). The sample is then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in the standard assay.

[0077] Additionally, or alternatively, droplet digital PCR (ddPCR) may be used. For example, a method for determining single-stranded and self-complementary AAV vector genome titers by ddPCR has been described. See, for example, M. Lock et al, Hum Gene Ther Methods. 2014 Apr;25(2):115-25. doi:10.1089 / hgtb.2013.131.Epub 2014 Feb 14.

[0078] In short, a method for separating rAAVhu68 particles with a packaged genome sequence from genome-deficient AAVhu68 intermediates involves subjecting a suspension containing recombinant AAVhu68 virus particles and AAVhu68 capsid intermediates to high-performance liquid chromatography (HFL). The AAVhu68 virus particles and intermediates are bound to a strong anion exchange resin equilibrated at approximately 10.2 pH and subjected to a salt gradient while monitoring the eluate for UV absorbance at approximately 260 nanometers (nm) and approximately 280 nm. While not ideal for rAAVhu68, the pH can be in the range of approximately 10.0–10.4. In this method, the complete AAVhu68 capsid is collected from the fragment and elutes when the A260 / A280 ratio reaches an inflection point. For example, for the affinity chromatography step, the dialyzed 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 proteins flows through the column, while AAV particles are efficiently captured.

[0079] Also provided herein are production vectors (such as plasmids), or host cells for producing the vector genome and / or rAAV.GLB1 described herein. A production vector, when used herein, for carrying the vector genome into host cells to generate and / or package the gene therapy vector described herein.

[0080] rAAV.GLB1 (e.g., rAAVhu68.GLB1) is suspended in a suitable physiologically compatible composition (e.g., buffered saline). This composition may be frozen for storage, subsequently thawed, and optionally diluted with a suitable diluent. Alternatively, rAAV.GLB1 may be prepared as a composition suitable for delivery to the patient without going through the freezing and thawing steps.

[0081] V. Composition and Use Provided herein are compositions comprising at least one rAAV stock (e.g., rAAVhu68 stock or mutant rAAVhu68 stock) and an optional carrier, excipient, and / or preservative.

[0082] As used herein, “stock” of rAAV refers to a population of rAAVs. Despite heterogeneity of capsid proteins resulting from deamidation, rAAVs within stock The stocks are expected to share the same vector genome. The stock may include, for example, a selected AAV capsid protein and an rAAV having a capsid with a heterogeneous deamidation pattern characteristic of the selected production system. The 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.

[0083] In particular, the composition is for the treatment of GM1 gangliosidosis. In one embodiment, the composition is suitable for administration to patients with GM1 gangliosidosis or to infants under 18 months of age with gangliosidosis. In one embodiment, the composition is suitable for administration to patients with GM1 gangliosidosis or to infants under 36 months of age with gangliosidosis. In one embodiment, the composition is suitable for administration to patients who require it to improve the symptoms of GM1 gangliosidosis or to improve the neurological symptoms of GM1 gangliosidosis. In some embodiments, the composition is for use in the manufacture of pharmaceuticals for the treatment of GM1 gangliosidosis.

[0084] 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.

[0085] In certain embodiments, provided herein are compositions comprising rAAV.GLB1 as described herein and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar adverse reaction when administered to a host.

[0086] In certain embodiments, provided herein are compositions comprising rAAV.GLB1 as described herein and a delivery vehicle. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc., may be used to introduce the compositions of the present invention into suitable host cells. In particular, the rAAV delivery vector genome may be formulated for delivery encapsulated in any of the following: lipid particles, liposomes, vesicles, nanospheres, or nanoparticles.

[0087] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject / patient, 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 may be transported as a concentrate that is diluted for administration to a subject. In yet another embodiment, the composition may be lyophilized and reconstituted at the time of administration.

[0088] Suitable surfactants, or combinations of surfactants, may be selected from non-toxic nonionic surfactants. In one embodiment, for example, a difunctional block copolymer surfactant with primary hydroxyl groups at the terminal end is selected, such as Pluronic® F68 [BASF] (also known as poloxamer 188), which has a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers include nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) adjacent to two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS15 (macrogol-15 hydroxysteart), LABRASOL (polyoxycapric 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 poloxamer. These copolymers are generally named with the letter "P" (in the case of poloxamer) followed by a three-digit number, where the first two digits × 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit × 10 gives the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. In one embodiment, the surfactant may be present in an amount of up to about 0.0005% to about 0.001% (w / w%, based on weight ratio) of the suspension. In another embodiment, the surfactant may be present in an amount of up to about 0.0005% to about 0.001% (v / v%, based on volume ratio) of the suspension. In yet another embodiment, the surfactant is present in an amount of up to about 0.0005% to about 0.001% of the suspension, where n% represents n grams per 100 mL of suspension.

[0089] rAAV.GLB1 is administered in an amount sufficient to transfect cells, providing sufficient levels of gene transfer and expression to deliver a therapeutic effect without excessive adverse effects or with medically acceptable physiological effects, which can be determined by those skilled in the art of medicine. Conventional pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the desired organ (e.g., brain, CSF, liver (optionally, via hepatic artery), lung, heart, eye, kidney), oral, inhalation, intranasal, intrathecal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, intraparenchymal, intraventricular, intrathecal, ICM, lumbar puncture, and other parenteral routes of administration. Routes of administration may be combined if desired.

[0090] The dosage of rAAV.GLB1 depends primarily on factors such as the condition being treated, the patient's age, weight, and health status, and therefore can vary among patients. For example, the effective human dose of rAAV.GLB1 for treatment is generally about 1 × 10⁶ per mL. 9 ~1 × 10 16The volume ranges from approximately 25 to approximately 1000 microliters for a region of approximately 100 mL containing 1 vector genome copy. In certain embodiments, a suspension volume of approximately 1 mL to approximately 15 mL, or approximately 2.5 mL to approximately 10 mL, or approximately 5 mL is delivered. In certain embodiments, a suspension volume of approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, or approximately 15 mL is delivered.

[0091] In some embodiments, the composition is intended for single-dose administration. In some embodiments, the composition is intended for multiple-dose administration.

[0092] In a particular embodiment, approximately 8 × 10 per patient 12 rAAV.GLB1 genome copy (GC) is approximately 3 × 10 per patient. 14 The dose of GC rAAV.GLB1 is administered in the volume described herein. In certain embodiments, approximately 2 × 10⁶ doses are administered per patient. 12 GC rAAV.GLB1~ Approximately 3 x 10 per patient 14 GC rAAV.GLB1, or approximately 2 × 10⁶ per patient 13 GC rAAV.GLB1~ Approximately 3 x 10 per patient 14 GC rAAV.GLB1, or approximately 8 × 10 per patient 13 GC rAAV.GLB1~ Approximately 3 x 10 per patient 14 GC rAAV.GLB1, or approximately 9 x 10 per patient 13 GC's rAAV.GLB1, or approximately 8.9 × 10 in total 12 ~2.7×10 14 The GC dose is administered in the volume described above.

[0093] In a particular embodiment, 1 × 10⁶ per gram of brain mass 10 rAAV.GLB1~3.4×10⁻¹⁰ GC (GC / g brain mass) 11 The dose of GC / g brain mass is administered in the volume described herein. In certain embodiments, 3.4 × 10⁻⁶ 10 GC / g brain mass ~3.4×10 11GC / g brain mass, or 1.0×10 11 GC / g brain mass to 3.4×10 11 GC / g brain mass, or approximately 1.1×10 11 GC / g brain mass, or approximately 1.1×10 10 GC / g brain mass to approximately 3.3×10 11 The dose of GC / g brain mass is administered in the volume described above. In certain embodiments, approximately 3.0×10 9 , approximately 4.0×10 9 , approximately 5.0×10 9 , approximately 6.0×10 9 , approximately 7.0×10<00000​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​, about 5.5×10 11 , about 6.0×10 11 , about 6.5×10 11 , about 7.0×10 11 , about 7.5×10 11 , about 8.0×10 11 , about 8.5×10 11 , about 9.0×10 11 The dose of GC is administered in the volume described above. In certain embodiments, the dose was adjusted for use in human patients based on the minimum effective dose shown in the GM1 animal model and on genome copies per gram of brain mass. In one embodiment, the dose for use in human patients is calculated using the assumed brain mass listed in the table below. [Table 2] [Table 3]

[0094] To balance the therapeutic effect with any side effects, the dosage may be adjusted, and such dosages may vary depending on the therapeutic use for which rAAV.GLB1 is utilized. Monitoring the expression level of the transgene product (e.g., β-gal) can determine the dosage frequency that yields rAAV containing rAAV.GLB1, preferably a minigene (e.g., the GLB1 gene). Optionally, dosages similar to those described for therapeutic purposes may be used. The regimen can be used for immunization using the composition of the present invention.

[0095] The replication-deficient virus composition is formulated in dose units, and for human patients, approximately 1.0 × 10⁶ units are used (to treat the target). 9 GC~approx. 1.0×10 16 It contains an amount of replication-deficient virus (e.g., rAAV.GLB1, rAAVhu68.GLB1, or rAAVhu68.UbC.GLB1) within the GC range, and all integers or small quantities within that range, preferably 1.0 × 10⁻¹⁶. 12 GC~1.0×10 14It may contain GC. In one embodiment, the composition contains at least 1 × 10 per dose, including all integers or decimals within the range. 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 composition is formulated to contain GC. In another embodiment, the composition contains at least 1 × 10 per dose, including all integers or decimals 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 decimals 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 decimals 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 12 The formulation is to include GC. In another embodiment, the composition contains at least 1 × 10 per dose, including all integers or decimals within the range. 13 , 2×10 13 , 3 x 10 13 , 4×1013 , 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 decimals 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 decimals 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 It is formulated to contain GC. In one embodiment, for human application, the dose is 1 × 10 per dose, including all integers or decimals within the range. 10 ~Approx. 1×10 12 It could be within the scope of garbage collection.

[0096] These aforementioned doses may be administered in carrier, excipient, or buffer formulations of varying volumes, ranging from about 25 to about 1000 microliters, or larger volumes, or any number within that range, depending on the size of the area being treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume of the carrier, excipient, or buffer is at least about 25 μL. In another embodiment, the volume is about 50 μL. In yet another embodiment, the volume is about 75 μL. In yet another embodiment, the volume is about 100 μL. In yet another embodiment, the volume is about 125 μL. In yet another embodiment, the volume is about 150 μL. In yet another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In yet 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 another embodiment, the volume is approximately 350 μL. In another embodiment, the volume is approximately 375 μL. In another embodiment, the volume is approximately 400 μL. In another embodiment, the volume is approximately 450 μL. In another embodiment, the volume is approximately 500 μL. In another embodiment, the volume is approximately 550 μL. In another embodiment, the volume is approximately 600 μL. In another embodiment, the volume is approximately 650 μL. In another embodiment, the volume is approximately 700 μL. In another embodiment, the volume is approximately 700 to 1000 μL. In some embodiments, the volume is approximately 1 mL to 10 mL, and in some embodiments, the volume is less than 15 mL.

[0097] In certain embodiments, the dose is approximately 1 × 10 9 Approximately 1 × 10⁻⁶ brain mass from GC / g brain mass. 12 The GC / g brain mass may be in the range of GC / g brain mass. In certain embodiments, the dose is approximately 3 × 10⁻⁶ 10 GC / g brain mass ~ approx. 3×10 11 The GC / g brain mass may be in the range of GC / g brain mass. In certain embodiments, the dose is approximately 5 × 10⁻⁶ 10 GC / g brain mass ~ approx. 1.85×10 11It could be within the range of GC / g brain mass.

[0098] 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 13 It can be delivered in GC doses. Suitable volumes for the delivery of 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, with higher volumes selected for adults. Typically, for neonates, suitable volumes may be about 0.5 mL to 10 mL, and for older infants, about 0.5 mL to 15 mL. For toddlers, volumes of about 0.5 mL to 20 mL may be selected. For children, volumes up to about 30 mL may be selected. For early adolescents and teenagers, volumes up to about 50 mL may be selected. In yet another embodiment, the patient may receive intrathecal administration in volumes of about 5 mL to 15 mL, which may be selected, or about 7.5 mL to 10 mL. Other suitable volumes and dosages can be determined. Doses may be adjusted to balance the therapeutic effect with respect to any side effects, and such dosages may vary depending on the therapeutic use for which rAAV.GLB1 is utilized.

[0099] The rAAV.GLB1 described above can be delivered to host cells according to the published methods. Preferably, rAAV suspended in a physiologically compatible carrier can be administered to human or non-human mammalian patients. In certain embodiments, for administration to human patients, rAAV is preferably suspended in an aqueous solution containing physiological saline, a surfactant, and a physiologically compatible salt or mixture of salts. Preferably, the formulation is adjusted to a physiologically acceptable pH range, for example, pH 6 to 9, or pH 6.0 to 7.5, or pH 6.2 to 7.7, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8, or about pH 7.0. In certain embodiments, the formulation is adjusted to a pH of approximately 6.0, approximately 6.1, approximately 6.2, approximately 6.3, approximately 6.4, approximately 6.5, approximately 6.6, approximately 6.7, approximately 6.8, approximately 6.9, approximately 7.0, approximately 7.1, approximately 7.2, approximately 7.3, approximately 7.4, approximately 7.5, approximately 7.6, approximately 7.7, or approximately 7.8. In certain embodiments, for intrathecal delivery, pH values ​​of approximately 7.28–7.32, 6.0–7.5, 6.2–7.7, 7.5–7.8, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8 are desirable, while for intravenous delivery, pH values ​​of approximately 6.8–7.2 may be desirable. However, other pH ranges, and sub-ranges of these, are available and can be selected for other delivery routes.

[0100] 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 in terms of the indications targeted by the introduced virus. For example, one suitable carrier comprises saline and can be formulated using various buffering 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. A suitable surfactant, or combination of surfactants, may be selected from non-toxic nonionic surfactants. In one embodiment, for example, poloxamer 188 (also known as Pluronic® F68 [BASF], Lutrol® F68, Synperonic® F68, and Kolliphor® P188) has a neutral pH and an average molecular weight of 8400. A bifunctional block copolymer surfactant with a primary hydroxyl group at the end, such as ), is selected. Other surfactants and other poloxamers, namely nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (macrogol-15 hydroxysteart), LABRASOL (polyoxycapric acid glyceride), polyoxy-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 with the letter "P" (in the case of a poloxamer) followed by a three-digit number, where the first two digits × 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit × 10 gives the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount of approximately 0.0005% to 0.001% of the suspension.

[0101] For example, the formulation may contain a buffered saline solution containing, for instance, 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 milliosmoles / liter (mOsm / L) to about 290 mOsm / L). 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 other optional excipients. See, for example, Elliotts B® solution [Lukare Medical]. Each 10 mL of Elliotts B solution contains: sodium chloride, USP-73 mg; sodium bicarbonate, USP-19 mg; dextrose, USP-8 mg; magnesium sulfate, 7H2O, USP-3 mg; potassium chloride, USP-3 mg; calcium chloride, 2H2O, USP-2 mg; dibasic sodium phosphate, 7H2O, USP-2 mg; water for injection, USP-10 mL. Electrolyte concentrations: sodium (149 mEq / liter); bicarbonate (22.6 mEq / liter); potassium (4.0 mEq / liter); chloride (132 mEq / liter); calcium (2.7 mEq / liter); sulfate (2.4 mEq / liter); magnesium (2.4 mEq / liter); phosphate (1.5 mEq / liter).

[0102] The formulas and molecular weights of the components are as follows: [Table 4]

[0103] The pH of Elliotts B solution is 6 - 7.5, and the molar osmotic concentration is 288 mOsmol per liter (calculated value). In certain embodiments, the intrathecal final formulation buffer (ITFFB) formulation buffer comprises buffered saline, and artificial cerebrospinal fluid containing one or more of sodium, calcium, magnesium, potassium, or mixtures thereof, and a surfactant. In certain embodiments, the surfactant comprises from about 0.0005% to about 0.001% of the suspension. In further embodiments, the percentage (%) is calculated based on the weight (w) ratio (i.e., w / w).

[0104] In certain embodiments, the composition containing rAAVhu68.GLB1 (e.g., ITFFB formulation) has a pH in the range of 6.0 - 7.5, or 6.2 - 7.7, or 6.8 - 8, or 7.2 - 7.8, or 7.5 - 8. In certain embodiments, the final formulation has a pH of about 7, or 7 - 7.4, or 7.2. In certain embodiments, for intrathecal delivery, a pH above 7.5, e.g., 7.5 - 8, or 7.8 may be desirable.

[0105] In certain embodiments, for intrathecal delivery as well as other delivery routes, a pH of about 7 is desirable.

[0106] In certain embodiments, the formulation may contain a buffered saline aqueous solution free of sodium bicarbonate. Such a formulation may contain a buffered saline aqueous solution containing one or more of sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and combinations thereof in water, e.g., Harvard buffer. The aqueous solution may further contain Kolliphor® P188, a poloxamer commercially available from BASF, which was formerly sold under the trade name Lutrol® F68. In certain embodiments, the aqueous solution may have a pH of 7.2. In certain embodiments, the aqueous solution may have a pH of about 7.

[0107] In another embodiment, the formulation may contain a buffered saline solution containing 1 mM sodium phosphate (Na3PO4), 150 mM sodium chloride (NaCl), 3 mM potassium chloride (KCl), 1.4 mM calcium chloride (CaCl2), 0.8 mM magnesium chloride (MgCl2), and 0.001% poloxamer (e.g., Kolliphor®) 188. In certain embodiments, the formulation has a pH of about 7.2. In certain embodiments, the formulation has a pH of about 7. See, for example, harvardapparatus.com / harvard-apparatus-perfusion-fluid.html. In certain embodiments, Harvard buffer is preferred because better pH stability is observed with Harvard buffer. The following table provides a comparison of Harvard buffer and Elliot's B buffer.

[0108] Cerebrospinal fluid (CSF) composition [Table 5]

[0109] In certain embodiments, the formulation buffer is an artificial CSF containing Pluronic F68. In other embodiments, the formulation may contain one or more osmotic enhancers. Examples of suitable osmotic enhancers may include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, or EDTA.

[0110] 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.

[0111] The compositions according to the present invention may comprise a pharmaceutically acceptable carrier as defined above. Preferably, the compositions described herein comprise an effective amount of one or more AAVs suspended in a pharmaceutically suitable carrier and / or mixed with a suitable excipient designed for delivery to a target via injection, infiltration pump, intrathecal catheter, or by another device or route. In one example, the composition is formulated for intrathecal delivery. In one embodiment, the composition is formulated for administration via intracisional injection (ICM). In one embodiment, the composition is formulated for administration via CT-guided suboccipital injection into the cisterna magna.

[0112] As used herein, the terms “intrathecal delivery” or “intrathecal administration” refer to the route of drug administration via injection into the spinal canal, more specifically, via injection into the subarachnoid space to reach the cerebrospinal fluid (CSF). Intrathecal delivery may include lumbar puncture, intraventricular (including lateral ventricles (ICV)), suboccipital / intracisional, and / or C1-2 puncture. For example, material may be introduced by means of lumbar puncture to diffuse throughout the subarachnoid space. In this example, injection into the cisterna magna may also be used.

[0113] 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 magnum of the cerebellum, more specifically, a route of drug administration by suboccipital puncture, direct injection into the cisterna magnum, or a permanently placed tube.

[0114] In certain embodiments, an aqueous composition comprising a formulation buffer and rAAV.GLB1 (e.g., rAAVhu68.GLB1) provided herein is delivered to a patient in need. In certain embodiments, rAAV.GLB1 has a vector genome comprising an AAV capsid (e.g., AAVhu68 capsid) and a 5'AAV ITR-promoter-optionally selected enhancer-optionally selected intron-GLB1 gene-polyA-3'ITR. In certain embodiments, the ITR is not derived from AAV2. In certain embodiments, two or more promoters are present. In certain embodiments, the enhancer is present in the vector genome. In certain embodiments, two or more enhancers are present. In certain embodiments, the intron is present in the vector genome. In certain embodiments, both enhancers and introns are present. In certain embodiments, polyA is SV40 polyA. In certain embodiments, polyA is rabbit beta-globin (RBG) polyA. In certain embodiments, the vector genome includes a 5'AAV ITR-CB7 promoter-GLB1 gene-RBG poly-A-3'ITR. In certain embodiments, the vector genome includes a 5'AAV ITR-EF1a promoter-GLB1 gene-SV40 poly-A-3'ITR. In certain embodiments, the vector genome includes a 5'AAV ITR-UbC promoter-GLB1 gene-SV40 poly-A-3'ITR. In certain embodiments, the GLB1 gene has sequence number 5. In certain embodiments, the GLB1 gene has sequence number 6. In certain embodiments, the GLB1 gene has sequence number 7. In certain embodiments, the GLB1 gene has sequence number 8. In certain embodiments, the vector genome has the sequence of sequence number 12. In certain embodiments, the vector genome has the sequence of sequence number 13. In certain embodiments, the vector genome has the sequence of sequence number 14. In certain embodiments, the vector genome has the sequence of sequence number 15. In certain embodiments, the vector genome has the sequence of sequence number 16.

[0115] In certain embodiments, the final formulation buffer comprises buffered saline, artificial cerebrospinal fluid containing one or more of sodium, calcium, magnesium, potassium, or a mixture thereof, and a surfactant. In certain embodiments, the surfactant is present in an amount of about 0.0005% to about 0.001% of the suspension. In certain embodiments, the surfactant is Pluronic F68. In certain embodiments, Pluronic F68 is present in an amount of about 0.0001% of the suspension. In certain embodiments, the composition has a pH in the range of 7.5 to 7.8 for intrathecal delivery. In certain embodiments, the composition has a pH in the range of 6.2 to 7.7, or 6.9 to 7.5, or about 7 for intrathecal delivery. In one embodiment, the percentage (%) is calculated based on weight ratio or volume ratio. In another embodiment, the percentage represents "grams per 100 ml of final volume".

[0116] In certain embodiments, the treatment of the compositions described herein has minimal to mild asymptomatic degeneration of DRG sensory neurons in animals and / or human patients, and is well tolerable with respect to sensory neurotoxicity and asymptomatic sensory neuronal lesions.

[0117] In certain embodiments, the compositions described herein are useful in improving the functional and clinical outcomes of the subject / patient being treated. Such outcomes are observed approximately 30 days, 60 days, 90 days, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, and approximately 16 months after administration of the composition. Measurements may be taken at 17 months, approximately 18 months, approximately 19 months, approximately 20 months, approximately 21 months, approximately 22 months, approximately 23 months, approximately 24 months, approximately 2.5 years, approximately 3 years, approximately 3.5 years, approximately 4 years, and approximately 4.5 years, and then annually for up to approximately 5 years. The measurement frequency may be approximately every month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, or every twelve months.

[0118] In certain embodiments, the compositions described herein exhibit pharmacokinetics and clinical efficacy measured in a treated subject compared to an untreated control.

[0119] In certain embodiments, pharmacokinetic efficacy, clinical efficacy, functional outcome, or clinical outcome may be measured by one or more of the following: (1) survival, (2) independence from feeding tubes, (3) seizure diaries, e.g., seizure incidence, onset, frequency, duration, and type, (4) quality of life, e.g., as measured by PedsQL, (5) neurocognitive and behavioral development, (6) e.g., β-gal enzyme expression or activity in serum or CSF, and (7) other parameters described herein. The effects of the composition on development and / or change in adaptive behavior, cognition, language, motor function, and health-related quality of life may be quantified using the Bailey Infant Development and Vineland Scales.

[0120] In certain embodiments, neurocognitive development is based on one or more of the following: changes in age-appropriate cognition, gross motor, fine motor, receptive and expressive communication scores on the Bailey Infant Development Scale; changes in standard scores for each domain of the Vineland Adaptive Behavior Scale; and changes in total scores on the Quality of Child Life Survey and the Infant Scale of the Quality of Child Life Survey (PedsQL and PedsQL-IS).

[0121] The Bayley Scales of Infant and Toddler Development (BSID) is primarily used to assess the development of infants and toddlers aged 1–42 months (Albers and Grieve, 2007; Test Review: Bayley, N. (2006). Bayley Scales of Infant and Toddler Development - Third Edition. San Antonio, TX: Harcourt Assessment. Journal of Psychoeducational Assessment. 25(2):180–190). It consists of a standardized set of developmental play tasks, and the developmental index is derived by converting the raw scores of well-completed items into scores and composite scores, and comparing the scores to standards typically obtained from developing children of the same age. The Bayley-III has the following three main subtests: The cognitive scale includes items such as attention to familiar and unfamiliar objects, searching for dropped objects, and pretending to play; a language scale assesses language comprehension and expression (e.g., ability to follow instructions and ability to name objects); and a motor scale measures gross and fine motor skills (e.g., grasping, sitting, stacking blocks, climbing stairs). The latest version is BSID-III.

[0122] Vineland assesses adaptive behavior from birth to adulthood (0-90 years) across five domains: communication, daily living skills, social skills, motor skills, and maladaptive behaviors. The latest version is Vineland III. Improvements from Vineland-II to Vineland-III include questions that help to better understand developmental disorders.

[0123] BSID and Vineland are selected based solely on data from prospective studies of infant GM1 gangliosidosis patients (Brunetti-Pierri and Scaglia, 2008, GM1 gangliosidosis: Review of clinical, molecular, and therapeutic asp). (ects. Molecular Genetics and Metabolism. 94(4):391-396). Age-equivalent scores for BSID-III showed a floor decline in test scales by 28 months of age in both cognitive and gross motor domains, while Vineland-II Adaptive Behavior Scale scores remained measurable by 28 months of age, albeit far below normal. Although these tools exhibit a floor effect, they have been shown to be appropriate measures for measuring developmental changes in this severely disabled population, and their cross-cultural validity makes them suitable for international research.

[0124] PedsQL and PedsQL-IS: As with severe childhood illnesses, the burden of illness on families is significant. The Pediatric Quality of Life Inventory (trademark) is a validated tool for assessing the quality of life of children and their parents (based on parental proxy reports). This has been validated in healthy children and adolescents and has been used in a variety of childhood illnesses (Iannaccone et al., 2009, The PedsQL in pediatric patients). With Spinal Muscular Atrophy: feasibility, reliability, and validity of the Pediatric Quality of Life Inventory Generic Core Scales and Neuromuscular Module. Neuromuscular disorders: NMD. 19(12):805-812, Absoud et al., 2011, Paediatric UK demyelinating disease longitudinal study (PUDDLS).” BMC Pediatrics. 11(1):68, and Consolaro and Ravelli, 2016, Chapter 5 - Assessment Tools in Juvenile Idiopathic Arthritis. Handbook of Systemic Autoimmune Diseases. R. Cimaz and T. Lehman, Elsevier. 11:107-127). Therefore, PedsQL is included to evaluate the impact of rAAV.GLB1 on the quality of life of patients and their families. This can be applied to parents of children aged 2 years and older and can thus be useful as the age of the child over a 5-year follow-up period. The Pediatric Quality of Life Inventory (trademark) Infant Scale (Varni et al., 2011, “The PedsQL (trademark) Infant Scales: feasibility, internal consistency reliability, and validity in healthy and ill infants.” Quality of Life Research. 20(1):45-55) is a validated modular instrument completed by parents and is designed to measure health-related quality of life measures specifically for healthy and ill infants aged 1 to 24 months.

[0125] Given the severity of the disease in the target population, subjects may have achieved motor skills by the time of 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. Motor milestone achievement is defined into six gross milestones based on WHO criteria outlined in the table provided herein under GM1 and therapeutic GLB1 genes in Section I. Given that subjects with infant GM1 gangliosidosis may develop symptoms within the first few months of life, and that the acquisition of the first WHO motor milestone (sitting without support) typically does not occur before 4 months of age (median: 5.9 months), this endpoint may lack sensitivity to assess the degree of therapeutic benefit, particularly in subjects with more apparent symptoms at the time of treatment. (Scharf et al., 2016, Developmental) Milestones.Pediatr Rev.37(1):25-37;quiz 38,47). One drawback is that the published tools are intended for use by clinicians and parents and organize skills around typical ages of milestone acquisition without referring to normal ranges. However, the data may be useful for summarizing retention, acquisition, or loss of developmental milestones over time compared to typical acquisition times in untreated children with infant GM1 disorder or typically developing children.

[0126] As the disease progresses, children may experience seizures. The onset of seizure activity allows for the determination of whether treatment with rAAV.GLB1 will prevent or delay the onset of seizures, or reduce the frequency of seizure events in this population. Parents are asked to keep a seizure diary, tracking the onset, frequency, duration, and type of seizures.

[0127] Furthermore, in certain embodiments, pharmacokinetic efficacy, clinical efficacy, functional outcomes, or clinical outcomes may include CNS symptoms of the disease, such as volume changes measured over time by MRI. The phenotype of all gangliosidosis infants has been shown to have a consistent pattern with macrocephaly and rapid increases in intracranial MRI volume, both in brain tissue volume (cerebral cortex and other smaller structures) and ventricular volume. In addition, various small subbrain structures, including the corpus callosum, caudate nucleus, and putamen, as well as the cerebellar cortex, generally decrease in size with disease progression (Regier et al., 2016s, and Nestrasil et al., 2018, cited herein). Treatment with rAAV.GLB1 can slow or halt the progression of CNS disease symptoms, with evidence of stabilization in atrophy and volume changes. Changes in T1 / T2 signal intensity (normal / abnormal) in the thalamus and basal ganglia may also be included, based on reported evidence regarding changes in thalamic structure in patients with GM1 and GM2 gangliosidosis (Kobayashi and Takashima, 1994, "Thalamic hyperdensity on CT in infantile GM1-gangliosidosis." Brain and Development. 16(6):472-474). In certain embodiments, pharmacokinetic efficacy, clinical efficacy, functional outcome, or clinical outcome may include changes in total brain volume, hypobrain volume, and lateral ventricular volume as measured by MRI, as well as / or changes in T1 / T2 signal intensity in thalamic and basal ganglia activity.

[0128] Alternatively or additionally, pharmacodynamic efficacy, clinical efficacy, functional outcomes, or clinical outcomes may include biomarkers, e.g., pharmacodynamic and biological activity of rAAV.GLB1, β-gal enzyme activity that can be measured in CSF and serum, CSF GM1 concentration, serum and urine keratan sulfate levels, decreased hexosaminidase activity, and brain MRI showing consistent rapid atrophy in infantile GM1 gangliosidosis (Regier et al., 2016b, cited herein).

[0129] In certain embodiments, the compositions described herein are useful in slowing disease progression and are evaluated, for example, by age at achievement, age at loss, and the percentage of children who maintain or achieve age-appropriate developmental and motor milestones (as defined by World Health Organization (WHO) criteria).

[0130] In certain embodiments, pharmacokinetic efficacy, clinical efficacy, functional outcome, or clinical outcome may include liver and spleen volume, as well as / or EEG and visual evoked potentials (VEP).

[0131] VI. Apparatus and method for delivering pharmaceutical compositions to cerebrospinal fluid In one embodiment, the rAAV or composition provided herein is provided in this section and is described in WO2018 / 160582 (which is incorporated herein by reference). It may be administered intrathecally via a method and / or device. Another suitable device is the "Microcatheter for Therapeutic and / or Diagnostic Interventions in" filed on January 31, 2020. This is described in PCT / US20 / 14402, titled "the Subarachnoid Space," which is incorporated herein by reference. Alternatively, other devices and methods may be selected.

[0132] In certain embodiments, the method includes the step of a CT-guided suboccipital injection into the patient's cisterna magna via a spinal needle. 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.

[0133] On the day of treatment, prepare rAAV.GLB1 at the appropriate concentration. Send a syringe containing the appropriate volume (e.g., 3.6 mL, 4.6 mL, or 5.6 mL) of rAAV.GLB1 at the appropriate concentration to the treatment room. The following persons will be present during the administration of the investigational drug: the interventional physician performing the procedure, the anesthesiologist and respiratory technician, the nurse and medical assistant, the CT (or operating room) technician, and the site research coordinator. Prior to drug administration, perform a lumbar puncture to remove a predetermined volume of CSF (e.g., approximately 5 mL), and then inject an iodine contrast agent intrathecally (IT) to aid in visualization of the relevant anatomical structures of the cisterna magna. Intravenous (IV) contrast agents may be administered as an additive to the intrathecal contrast agent before or during needle puncture. The subject is anesthetized, intubated, and placed on the treatment table. Prepare and drape the injection site using sterile techniques. Under fluoroscopy guidance, advance the spinal needle (e.g., a 2-inch or 3-inch 25G spinal needle for subjects aged 3 months to 18 years) into the cisterna magna. A larger introducer needle may be used to assist in needle placement. After confirming needle placement, attach the extension set to the spinal needle and fill it with CSF. At the discretion of the intervening physician, a syringe containing contrast material may be connected to the extension set and a small amount injected to confirm needle placement in the cisterna magna. After confirming needle placement with CT guidance + / - contrast injection, connect a syringe containing an appropriate volume of rAAV.GLB1 to the extension set. Inject the contents of the syringe slowly (e.g., over approximately 1-2 minutes) without applying excessive force to the syringe plunger during injection. Inject a total of 3 mL, 4 mL, or 5 mL of rAAV.GLB1, leaving 0.6 mL of rAAV.GLB1 in the device. The needle, extension tube, and syringe are slowly removed from the subject and placed on a surgical tray for disposal in an appropriate biohazard waste container. The needle insertion site is examined for signs of bleeding or CSF leakage and treated as indicated by the surgeon. The site is treated with gauze, surgical tape, and / or a clear wound dressing (e.g., Tegaderm) as indicated. After bandaging, the subject remains prone for at least 20 minutes. The subject is removed from the CT scanner and placed supine on a stretcher.During transport and positioning, a sufficient number of staff must be present to ensure the patient's safety. Anesthesia is discontinued, and the patient recovers according to facility guidelines for post-anesthetic care. Neurophysiological devices are removed, if applicable. The stretcher head is lowered to approximately 20-30 degrees for approximately one hour of recovery. The patient is transported to a suitable post-anesthetic care unit according to facility guidelines.

[0134] Additional or alternative routes of administration to the intrathecal methods described herein include, for example, systemic, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration.

[0135] In one embodiment, the dose is scaled by brain mass to provide an approximation of the size of the CSF compartment. In further embodiments, the dose conversion is based on ventricular volume of 0.4 g in adult mice, 90 g in juvenile rhesus monkeys, and 800 g in children aged 4–18 months. The following table provides exemplary doses of mouse MED studies, NHP toxicity studies, and equivalent human doses. . [Table 6]

[0136] In certain embodiments, rAAV.GLB1 is administered to the subject in a single dose. In certain embodiments, multiple doses (e.g., two doses) may be desired. For example, in infants under 6 months of age, multiple doses at intervals of several days, weeks, or months may be desirable.

[0137] In certain embodiments, the single dose of rAAV.GLB1 is approximately 1 × 10⁻⁶ 9 GC / g brain mass ~ approx. 5×10 11 This is GC / g brain mass. In certain embodiments, a single dose of rAAV.GLB1 is approximately 1 × 10⁻⁶ 9 GC / g brain mass ~ approx. 3×10 11 It is GC. In certain embodiments, the single dose of rAAV.GLB1 is approximately 1 × 10⁻⁶. 10 GC / g brain mass ~ approx. 3×10 11This is GC / g brain mass. In a particular embodiment, the dose of rAAV.GLB1 is 1 × 10⁻⁶ 10 GC / brain mass ~ approx. 3.33×10 11 This is GC / brain mass. In certain embodiments, the dose of rAAV.GLB1 is 1 × 10⁻⁶. 11 GC / brain mass ~ approx. 3.33×10 11 This is GC / brain mass. In a particular embodiment, the single dose of rAAV.GLB1 is 1.11 × 10⁻⁶. 10 GC / g brain mass ~3.33×10 11 This is GC / g brain mass.

[0138] In a particular embodiment, the single dose of rAAV.GLB1 is 1 × 10⁻⁶ 10 GC / g brain mass ~3.4×10 11 This is GC / g brain mass. In a particular embodiment, the single dose of rAAV.GLB1 is 3.4 × 10⁻⁶. 10 GC / g brain mass ~3.4×10 11 This is GC / g brain mass. In a particular embodiment, the single dose of rAAV.GLB1 is 1.0 × 10⁻⁶. 11 GC / g brain mass ~3.4×10 11 This is GC / g brain mass. In certain embodiments, a single dose of rAAV.GLB1 is approximately 1.1 × 10⁻⁶. 11 This is GC / g brain mass. In certain embodiments, the single dose of rAAV.GLB1 is at least 1.11 × 10⁻¹⁶. 10 This is GC / g brain mass. In other embodiments, different doses may be selected.

[0139] In a preferred embodiment, the subject is a human patient. In this case, the single dose of rAAV.GLB1 is approximately 1 × 10⁻⁶. 12 GC ~ approx. 3×10 14 It is GC. In a particular embodiment, the single dose of rAAV.GLB1 is 9 × 10 12 GC~3×10 14 It is GC. In a particular embodiment, the dose of rAAV.GLB1 is 5 × 10 13 GC~3×10 14 It is GC. In a particular embodiment, the single dose of rAAV.GLB1 is 8.90 × 10⁻⁶. 13 GC~2.70×1014 It is GC. In a particular embodiment, the single dose of rAAV.GLB1 is 8 × 10 per patient. 12 Genome copy (GC) ~ 3 x 10⁶ per patient 14 It is GC. In a particular embodiment, the single dose of rAAV.GLB1 is 2 × 10⁶ per patient. 13 GC ~ 3 x 10 per patient 14 It is GC. In a particular embodiment, the single dose of rAAV.GLB1 is 8 × 10 per patient. 13 GC ~ 3 x 10 per patient 14 It is GC. In certain embodiments, a single dose of rAAV.GLB1 is approximately 9 × 10⁶ per patient. 13 It is GC. In certain embodiments, the single dose of rAAV.GLB1 is at least 8.90 × 10⁻¹⁰ 13 It is GC. In other embodiments, different doses may be selected.

[0140] The composition is approximately 1 x 10 (to treat an average subject weighing 70 kg). 9 Genome copy (GC) ~ approximately 5 × 10⁻⁶ 14 It can be formulated in dosage units containing an amount of AAV within the range of GC. In some embodiments, the composition is 1 × 10 9 Genome copy (GC) ~ 5 × 10⁻⁶ 13 GC, 1×10 10 Genome copy (GC) ~ 5 × 10⁻⁶ 14 GC, 1×10 11 GC~5×10 14 GC, 1×10 12 GC~5×10 14 GC, 1×10 13 GC~5×10 14 GC, 8.9×10 13 GC~5×10 14 GC, or 8.9 × 10 13 GC~2.7×10 14 The formulation is prepared in dosage units that contain an amount of AAV within the range of GC. In certain embodiments, the composition contains at least 1 × 10⁻⁶ 13 GC, 2.7×10 13 GC, or 8.9 × 10 13It is formulated in dosage units that contain the same amount of AAV as GC.

[0141] In one embodiment, a spinal puncture is performed, approximately 15 mL (or less) to 40 mL of CSF is removed, rAAV.GLB1 is mixed with the CSF and / or suspended in a suitable carrier and delivered to the subject. In one example, the rAAV.GLB1 concentration is 1 × 10⁻⁶. 10 Genome copy (GC) ~ 5 × 10⁻⁶ 14 GC, 1×10 11 GC~5×10 14 GC, 1×10 12 GC~5×10 14 GC, 1×10 13 GC~5×10 14 GC, 8.9×10 13 GC~5×10 14 GC, or 8.9 × 10 13 GC~2.7×10 14 GC is approximately 1 × 10 9 GC, approx. 5×10 9 GC, approx. 1×10 10 GC, approx. 5×10 10 GC, approx. 1×10 11 GC, approx. 5×10 11 GC, approx. 1×10 12 GC, approx. 5×10 12 GC, approx. 1.0×10 13 GC, approx. 5×10 13 GC, approx. 1.0×10 14 GC, or approximately 5 × 10 14 Other quantities such as GC. In certain embodiments, the concentration in GC is expressed as GC per spinal puncture. In certain embodiments, the concentration in CG is expressed as GC per mL.

[0142] Combination therapies may be delivered together with the rAAV.GLB1 compositions provided herein. Combination therapies as described earlier in this application are incorporated herein by reference.

[0143] One such combination therapy may involve immunomodulatory agents. Immunosuppressants for such combination therapies include, but are not limited to, glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin or rapalog), and cell division inhibitors (including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents active against immunophilins). Immunosuppressants may include nitrogen mustard, nitrosourea, platinum compounds, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracyclines, mitomycin C, bleomycin, mitramycin, IL-2 receptor (CD25) specific antibodies or CD3 specific antibodies, anti-IL-2 antibodies, cyclosporine, tacrolimus, sirolimus, IFN-β, IFN-γ, opioids, or TNF-α (tumor necrosis factor-α) conjugates. In certain embodiments, immunosuppressive therapy may be initiated before gene therapy administration. Such therapy may involve the combined administration of two or more drugs on the same day (e.g., prednisolone, mycophenolate mofetil (MMF), and / or sirolimus (i.e., rapamycin)). One or more of these drugs may be continued at the same or adjusted dose after gene therapy administration. Such therapy may last, as needed, about one week, about 15 days, about 30 days, about 45 days, 60 days, or longer.

[0144] For example, if nutrition is a concern in GM1, a gastrostomy tube may be appropriate. If respiratory function deteriorates, a tracheostomy or non-invasive respiratory support may be provided. Power chairs and other devices can improve quality of life.

[0145] The words “comprise,” “comprises,” and “comprising” should be interpreted comprehensively, not exclusively. The words “consist,” “consisting,” and their variations should be interpreted exclusively, not comprehensively. Various embodiments are presented using the language "comprising," but in other circumstances, the relevant embodiments are also intended to be interpreted and described using the language "consisting of" or "essentially consisting of."

[0146] 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” are used particularly in relation to the production of peptides or proteins. Expression may be transient or stable.

[0147] As used herein, the term “NAb titer” is a measure of how much neutralizing antibody (e.g., anti-AAV Nab) is produced that neutralizes the physiological effects of its targeted epitope (e.g., AAV). Anti-AAV NAb titer may be measured as described, for example, in Calcedo, R., et al., *Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses*, *Journal of Infectious Diseases*, 2009. 199(3): pp. 381-390, which is incorporated herein by reference.

[0148] In some embodiments, administration of AAV or the composition improves the symptoms of GM1 gangliosidosis or the neurological symptoms of GM1 gangliosidosis. In some embodiments, after treatment, the patient has one or more of the following: increased life expectancy, reduced need for a feeding tube, reduced incidence and frequency of seizures, reduced progression to neurocognitive decline, and / or improved neurocognitive development.

[0149] 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.

[0150] When used in reference to a protein or nucleic acid, the term "heterogeneous" indicates that the protein or nucleic acid contains two or more sequences or subsequences that are not found in the same relationships with each other as they are 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 a coding sequence derived from a different gene. Thus, with reference to the coding sequence, the promoter is heterogeneous.

[0151] A “replication-deficient virus” or “viral vector” refers to a synthetic or artificial viral particle in which a vector genome containing an expression cassette with a gene of interest (e.g., GLB1) is packaged in a viral capsid (e.g., AAV or bocavirus) or envelope, and any viral genome sequence packaged within the viral capsid or envelope is replication-deficient, i.e., they 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 gene of interest adjacent to the signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. Therefore, Because replication and infection by progeny virions cannot occur without the presence of the viral enzymes necessary for replication, they are considered safe for use in gene therapy.

[0152] As used herein, “effective dose” refers to the amount of rAAV composition that delivers and expresses a quantity of genomic products from the vector genome in target cells. The effective dose may be determined based on animal models rather than human patients. Examples of suitable mouse or NHP models are described herein.

[0153] The terms "a" or "an" refer to one or more, for example, "enhancer (an It should be noted that "enhancer)" refers to one or more enhancers. Therefore, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably in this specification.

[0154] As mentioned above, unless otherwise specified, the term "approximately" means a variation of ±10% when used to adjust a number.

[0155] As stated above, “increase,” “decrease,” “decrease,” “improve,” “enhance,” “delay,” “early,” “slow down,” “stop,” or any grammatical variation thereof, or any similar term, indicates a change and, unless otherwise specified, means a change of approximately 5 times, approximately 2 times, approximately 1 time, approximately 90%, approximately 80%, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 10%, and approximately 5% compared to the corresponding reference (e.g., an untreated control, a GM1 patient at the corresponding level or a GM1 patient at a particular stage, or a healthy subject or healthy human without GM1).

[0156] As used herein, “patient” or “subject” refers to a mammal and includes humans, veterinary or agricultural animals, domestic or companion animals, and animals typically used in clinical research. In one embodiment, the subject of these methods and compositions is a human. In a particular embodiment, the patient has GM1.

[0157] 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]

[0158] The following examples are for illustrative purposes only and are not intended to limit the present invention.

[0159] Example 1: Deamidation of AAVhu68+ AAVhu68 was analyzed for modifications. In short, AAVhu68 was produced using a vector genome unrelated to this study, each produced using a conventional triple transfection method in 293 cells. For a general explanation of these techniques, see, for example, Bell CL, et al., The AAV9 receptor and its modification to improve in vivo lung gene transfer in mice. J Clin Invest. 2011;121:2427-2435. In short, for example, a plasmid encoding a packaging sequence adjacent to an AAV2 inverted terminal repeat (a transgene expressed from the chicken β-actin promoter, an intron, and poly(A) derived from the late gene of monkey virus 40 (SV40)) was transfected into the AAV2 rep gene. The AAV virus particles were packaged by triple transfection of HEK293 cells using a plasmid encoding the AAVhu68 cap gene and an adenovirus helper plasmid (pAdΔF6). The resulting AAV virus particles can be purified and concentrated using CsCl gradient centrifugation and frozen for later use.

[0160] Denaturation and Alkylation: Add 2 μl of 1 M dithiothreitol (DTT) and 2 μl of 8 M guanidine hydrochloride (GndHCl) to 100 μg of thawed virus preparation (protein solution) and incubate at 90°C for 10 minutes. Cool the solution to room temperature, then add 5 μl of freshly prepared 1 M iodoacetamide (IAM) and incubate in the dark at room temperature for 30 minutes. After 30 minutes, quench the alkylation reaction by adding 1 μl of 1 M DTT.

[0161] Digestion: Add 20 mM ammonium bicarbonate at pH 7.5-8 to the denatured protein solution by volume to dilute the final GndHCl concentration to 800 mM. Add trypsin solution to achieve a trypsin-to-protein ratio of 1:20 and incubate overnight at 37°C. After digestion, add TFA to a final concentration of 0.5% to quench the digestion reaction.

[0162] Mass Spectrometry: A combined digest mixture of approximately 1 microgram is analyzed by UHPLC-MS / Ms. LC is performed using an UltiMate 3000 RSLCnano System (Thermo Scientific). Mobile phase A is MilliQ water containing 0.1% formic acid. Mobile phase B is acetonitrile containing 0.1% formic acid. An LC gradient is performed from 4%B to 6%B over 15 minutes, then to 10%B over 25 minutes (total 40 minutes), and then to 30%B over 46 minutes (total 86 minutes). The sample is loaded directly into the column. The column size is 75 cm × 15 μm inner diameter and packed with 2 microns of C18 medium (Acclaim PepMap). LC is interfaced to a quadrupole-orbitrap mass spectrometer (Q-Exactive HF, Thermo Scientific) via Nanoflex electrospray ionization using a source. The column is heated to 35°C, and an electrospray voltage of 2.2kV is applied. The mass spectrometer is programmed to acquire tandem mass spectra from the top 20 ions. The maximum MS resolution is 120,000, and the MS / MS resolution is 30,000. The normalized collision energy is set to 30, the automatic gain control to 1e5, the maximum packed MS to 100ms, and the maximum packed MS / MS to 50ms.

[0163] Data Processing: Raw data files from the mass spectrometer were analyzed using BioPharma Finder 1.0 (Thermo Scientific). Briefly, all searches required a precursor mass tolerance of 10 ppm, a fragment mass tolerance of 5 ppm, tryptic cleavage, up to one missed cleavage, fixed modification of cysteine ​​alkylation, oxidation of methionine / tryptophan, deamidation of asparagine / glutamine, phosphorylation, methylation, and variable modification of amidation.

[0164] In the table below, T refers to trypsin and C refers to chymotrypsin. [Table 7-1] [Table 7-2] [Table 7-3]

[0165] In the AAVhu68 capsid protein, four residues (N57, N329, N452, N512) routinely exhibit deamidation levels exceeding 70%, and in most cases exceeding 90% across various lots. Additional 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.

[0166] Therefore, AAVs containing the AAVhu68 capsid protein may contain a heterogeneous population of capsid proteins, as the AAV may contain AAVhu68 capsid proteins exhibiting different levels of deamidation. A heterogeneous population of AAVhu68 vp1 proteins with varying levels of deamidation may include vp1 proteins produced from expression of nucleic acid sequences encoding the predicted amino acid sequence of SEQ ID NO: 1-736, vp1 proteins produced from SEQ ID NO: 1, or vp1 proteins produced from nucleic acid sequences at least 70% identical to SEQ ID NO: 1, which encodes the predicted amino acid sequence of SEQ ID NO: 2-736. AAVhu68 vp2 proteins with varying levels of deamidation may also exist. The heterogeneous population of the protein may be a vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least approximately 138-736 amino acids of SEQ ID NO: 2, 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, which encodes the predicted amino acid sequence of at least approximately 138-736 amino acids of SEQ ID NO: 2. The heterogeneous population of AAVhu68 vp3 proteins with varying levels of deamidation may be a vp3 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least approximately 203-736 amino acids of SEQ ID NO: 2, 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, which encodes the predicted amino acid sequence of at least approximately 203-736 amino acids of SEQ ID NO: 2.

[0167] Adult rhesus macaques were given AAVhu68.CB7.CI.eGFP.WPRE.rBG(3.00×10 13 GC) was administered via ICM, and autopsy was performed 28 days later to evaluate vector transduction. Transduction of AAVhu68 was observed in a wide range of brain regions (data not shown). Therefore, AAVhu68 capsid offers the potential for cross-correction in the CNS.

[0168] Example 2: Manufacturing - Components and Materials The vector is constructed from a cis-plasmid containing the coding sequence of human GLB1 expressed from a chicken beta-actin promoter with a cytomegalovirus enhancer (CB7) [SEQ ID NO: 10], a human elongation initiation factor 1 alpha promoter (EF1a) [SEQ ID NO: 11], or a human ubiquitin C promoter (UbC) [SEQ ID NO: 9] (1229 bp, GenBank#D63791.1) adjacent to an AAV2 inverted terminal repeat. Various coding sequences of human GLB1 [the aa sequence in SEQ ID NO: 4] are constructed. The wild-type sequence is reproduced in SEQ ID NO: 5. Various manipulated GLB1 coding sequences are generated and provided in SEQ ID NOs: 6, 7, or 8.

[0169] The vector was packaged into an AAV serotype hu68 capsid by triple transfection of adherent HEK293 cells. (Lock, M., et al. Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors) As already described in Scale. Human Gene Therapy 21, 1259-1271 (2010), it is purified by iodixanol gradient centrifugation. The AAV serotype Hu68 capsid is described in WO2018 / 160582 (the entire text is incorporated herein by reference).

[0170] More specifically, AAVhu68.GLB1 is produced by triple plasmid transfection of human HEK293WCB cells with 1) an AAV cis-vector genome plasmid, 2) an AAV transplasmid called pAAV2 / hu68.KanR that encodes AAV2 replication enzyme (rep) and AAVhu68 capsid (cap), and 3) a helper adenovirus plasmid called pAdΔF6.KanR.

[0171] Description of sequence elements in AAV cis vector genome plasmids: • Inverted Terminal Repeats (ITRs): ITRs are identical reverse complementary sequences derived from AAV2 (130 bp, GenBank#NC001401) that are adjacent to all components of the vector genome. When AAV and adenovirus helper functions are provided in trans, the ITR sequence serves as both the origin of vector DNA replication and the packaging signal for the vector genome. It functions as a vector. Therefore, the ITR sequence represents the only cis sequence necessary for vector genome replication and packaging.

[0172] • Promoter: Regulatory element derived from the human ubiquitin C (UbC) promoter: This ubiquitous promoter (1229 bp, GenBank#D63791.1) was selected to drive transgene expression in any CNS cell type.

[0173] • Coding sequence: The GLB1 gene encodes beta-galactosidase and is based on maximized human codon use. The GLB1 enzyme catalyzes the hydrolysis of β-linked galactose from gangliosides (2034 bp polynucleotide and stop codon for 677 aa, Genbank #AAA51819.1, EC3.2.1.23).

[0174] • A hybrid intron consisting of a chimeric intron (CI) - human β-globin splice donor and immunoglobulin G (IgG) splice acceptor element.

[0175] • SV40 polyadenylation signal (232 bp): The SV40 polyadenylation signal promotes efficient polyadenylation of gene mRNA in cis. This element functions as a signal for transcription termination, specific cleavage events at the 3' end of nascent transcripts, and the addition of long polyadeny tails.

[0176] AAVhu68 transplasmid: pAAV2 / hu68.KanR The AAV2 / hu68 transplasmid pAAV2 / hu68.KanR was constructed in the laboratory of Dr. James M. Wilson at the University of Pennsylvania. The AAV2 / hu68 transplasmid encodes four wild-type (WT) AAV2 replicase (Rep) proteins required for the replication and packaging of the AAV vector genome. The AAV2 / hu68 transplasmid also encodes three WT AAVhu68 virion protein capsid (Cap) proteins, which are assembled into a virion shell of AAV serotype hu68 to accommodate the AAV vector genome. The AAVhu68 sequence was obtained from human cardiac tissue DNA.

[0177] To construct the AAV2 / hu68 transplasmid, the AAV9 cap gene from the plasmid pAAV2 / 9n, 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 also replaced with the kanamycin resistance (KanR) gene to obtain pAAV2 / hu68.KanR. The AAV p5 promoter, which normally drives rep expression, was moved from the 5' end of the rep to the 3' end of the cap, leaving the cleaved p5 promoter upstream of the rep. This cleaved promoter downregulates rep expression, thereby maximizing vector production (Figure 1C). All component parts of the plasmid were confirmed by direct sequencing.

[0178] pAdDeltaF6(KanR) adenovirus helper plasmid: Plasmid pAdDeltaF6(KanR) is 15,774 bp in size. This plasmid contains regions of the adenovirus genome important for AAV replication, namely E2A, E4, and VA RNA (adenovirus E1 function is provided by HEK293 cells), but does not contain any other adenovirus replication or structural genes. The plasmid does not contain critical cis elements for replication, such as adenovirus inverted terminal repeats, and therefore is not expected to produce infectious adenovirus. The plasmid was derived from an Ad5 E1, E3 deletion molecular clone (pBHG10, pBR322-based plasmid). Deletions were introduced into the Ad5 DNA to remove unwanted A Denovirus gene expression was removed, reducing the amount of adenovirus DNA from 32kb to 12kb. Finally, the ampicillin resistance gene was replaced with a kanamycin resistance gene to generate pAdeltaF6(KanR). The adenovirus genes E2, E4, and VAI remaining in this plasmid, as well as E1 present in HEK293 cells, are necessary for AAV vector production.

[0179] AAVhu68.GM1 is produced by transient transfection of HEK293 cells, followed by downstream purification. Figures 12A-12B show a flowchart of the production process. The main reagents used in product preparation are shown on the left side of the figure, and the quality assessments within the process are shown on the right side. Descriptions of each production and purification step are also provided.

[0180] Cell culture and recovery: The cell culture and recovery process involves four main manufacturing steps: cell seeding and proliferation, transient transfection, vector recovery, and vector clarification (Figure 12A).

[0181] Cell seeding and proliferation: A fully characterized HEK293 cell line is used in the production process.

[0182] Transient transfection: After approximately 4 days of growth (DMEM medium + 10% FBS), the cell culture medium is replaced with fresh serum-free DMEM medium, and the cells are transfected with three production plasmids using a polyethyleneimine (PEI)-based transfection method. First, a DNA / PEI mixture containing the cis (vector genome) plasmid, trans (rep and cap gene) plasmids, and helper plasmids is prepared in a ratio with GMP-grade PEI (PEIPro HQ, PolyPlus Transfection SA). This plasmid ratio was determined to be optimal for AAV production in small-scale optimization studies. After thorough mixing, the solution is allowed to stand at room temperature for up to 25 minutes, then added to serum-free medium to quench the reaction, and finally added to the iCELLis bioreactor. The reactor is temperature and dissolved oxygen (DO) controlled, and the cells are incubated for 5 days.

[0183] Vector Recovery: Transformed cells and culture media are recovered from the PALL iCELLis bioreactor using disposable bioprocess bags by aseptically pumping the culture media from the bioreactor. After recovery, detergent, endonuclease, and MgCl2 (a cofactor for endonuclease) are added to release the vector and digest the unpackaged DNA. The product (in the disposable bioprocess bag) is incubated at 37°C for 2 hours in a temperature-controlled, single-use mixer to provide sufficient time for enzymatic digestion of any residual cell and plasmid DNA present in the recovered product resulting from the transfection procedure. This step is performed to minimize the amount of residual DNA in the final vector formulation (drug product, DP). After incubation, NaCl is added to a final concentration of 500 mM to aid in the recovery of the product during filtration and downstream tangential flow filtration (TFF).

[0184] Vector clarification: Cells and cellular debris are removed from the product using a pre-filter and depth filter capsule (1.2 / 0.22 μm) connected in series as a sterile, sealed tube and bag set driven by a peristaltic pump. Clarification ensures that downstream filters and chromatography columns are protected from contamination, and filtration reduces bioburden. At the end of the filter train, it is ensured that any bioburden that may have been introduced during the upstream production process is removed before downstream purification.

[0185] Purification Process: The purification process includes four main manufacturing steps: concentration and buffer exchange with TFF, affinity chromatography, anion exchange chromatography, and concentration and buffer exchange with TFF. These process steps are shown in the overview process diagram (Figure 12B). A general description of each of these processes is provided below.

[0186] Large-scale tangential flow filtration (TFF) is achieved by TFF using custom sterile closed bioprocess tubing, bags, and membrane sets, achieving a 20-fold volume reduction of the clarified product. The principle of TFF is to flow a solution through a membrane of suitable porosity (100 kDa) under parallel pressure. The pressure difference effectively drives smaller molecules through the membrane to the waste channel, while retaining larger molecules. By recirculating the solution, the parallel flow pushes the membrane surface, preventing membrane pore fouling and product loss due to binding to the membrane. By selecting appropriate membrane pore diameter and surface area, liquid samples can be rapidly reduced in volume while retaining and concentrating the desired molecules. Dialysis filtration in TFF applications involves adding fresh buffer to the recirculating sample at the same rate that the liquid passes through the membrane to the waste channel. The increase in the amount of small molecules due to the volume increase of dialysis filtration is removed from the recirculating sample. This diafiltration provides moderate purification of the clarified product while also achieving buffer exchange compatible with the subsequent affinity column chromatography step. Therefore, the inventors utilize a 100 kDa PES membrane for concentration and perform diafiltration with a buffer consisting of 20 mM Tris (pH 7.5) and 400 mM NaCl in a minimum of 4 diafiltration volumes. The diafiltration product is then further clarified using a 1.2 / 0.22 μm depth filter capsule to remove all precipitated material.

[0187] Affinity chromatography: The dialyzed product is applied to Poros® Capture-Select® AAV affinity resin (Life Technologies) which efficiently captures AAVhu68 serotypes. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, and AAV particles are efficiently captured. After application, the column is treated with 5 volumes of low-salt endonuclease solution (250 U / mL endonuclease, 20 mM Tris (pH 7.5), and 40 mM NaCl, 1.5 mM MgCl2) to remove any remaining host cell and plasmid nucleic acids. The column is washed to remove additional feed impurities, followed by washing with a low-pH step elution (400 mM NaCl, 20 mM sodium citrate, pH 2.5), which is immediately recovered in 1 / 10 volume neutralizing buffer (200 mM Bis Tris Propane, pH 10.2) for neutralization.

[0188] Anion exchange chromatography: To achieve further reduction of in-process impurities, including empty AAV particles, the Poros AAV elution pool is diluted 50-fold (20 mM bistrispropane, 0.001% Pluronic F68, pH 10.2) to reduce ionic strength and enable binding to the CIMultus® QA monolithic matrix (BIA Separations). After low-salt washing, the vector product is eluted using a 60 column volume (CV) NaCl linear salt gradient (10–180 mM NaCl). This shallow salt gradient effectively separates capsid particles (empty particles) that do not contain the vector genome from particles containing the vector genome (complete particles), resulting in a preparation concentrated for complete particles. The peak eluate of complete particles is collected, neutralized, diluted 20-fold with 20 mM bistrispropane and 0.001% Pluronic F68 (pH 10.2), and reapplied to the same column washed in situ. A 10–180 mM NaCl salt gradient is reapplied to collect the appropriate complete particle peak. The peak area is evaluated and compared to previous data to determine the approximate vector yield.

[0189] Concentration and buffer exchange by hollow fiber tangential flow filtration: pooled anion exchange intermediate The product is concentrated, and the buffer is replaced using a TFF. A 100 kDa hollow fiber TFF membrane is used in this step. During this step, the product is brought to the target concentration, and then the buffer is replaced with intrathecal final formulation buffer (ITFFB, i.e., artificial CSF containing 0.001% Pluronic® F68). The product is sterile filtered (0.22 μm), stored in a sterile container, and kept frozen at -60°C or below in an isolated location until released for final filling.

[0190] Final Filling: The frozen product is thawed, pooled, and adjusted to the target concentration using final formulation buffer (dilution or concentration step via TFF). This product is finally filtered through a 0.22 μm filter and filled to the determined volume into sterile West Pharmaceutical's Crystal Zenith (cyclic olefin polymer) vials and stoppers with crimp seals. The vials are individually labeled. Labelled vials are stored below 60°C.

[0191] Example 3 We developed an AAV vector expressing human β-gal and evaluated the effects of administering the vector to CSF ​​on brain enzyme activity, lysosomal accumulation lesions, and neurological signs using a mouse disease model. Neurological assessments were adapted from previous studies of the GM1 mouse model [Ichinomya, S., et al., Brain Dev 2007;29:210-216]. These assessments were selected to reflect neurological signs characteristic of this model. Blinded participants evaluated nine different parameters: gait, forelimb position, hindlimb position, trunk position, tail position, avoidance reaction, rollover, vertical righting reflex, and parachute reflex. Each test item was assigned one of four scores: 0 (normal), 1 (slightly abnormal), 2 (moderately abnormal), and 3 (very abnormal). The scores for each parameter were added together to calculate the total score.

[0192] A. Materials and Methods Animal Procedures: All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. GLB1 knockout mice were obtained from the RIKEN BioResource Research Center. Mice were maintained as heterozygous carriers on a C57BL / 6J background. For ICV injection, the vector was diluted to 5 μL by volume with sterile phosphate-buffered saline (Gibco) and injected freehand into isoflurane-anesthetized mice using a custom-made airtight syringe (Hamilton) and a cemented 10 mm 27 gauge needle, with a plastic tube attached to the base of the needle to limit penetration to a depth of 3 mm. Submandibular blood collection was performed on isoflurane-anesthetized mice. Blood was collected in a serum separatory tube, allowed to coagulate, separated by centrifugation, divided into aliquots, and frozen at -60°C or below. At necropsy, mice were sedated with ketamine and xylazine, and CSF was collected by suboccipital puncture using a 32 gauge needle connected to a polyethylene tube. Euthanasia was performed by cervical dislocation. The CSF, heart, lungs, liver, and spleen were immediately frozen on dry ice and stored at -60°C or below. The brain was removed, and a coronal section of the frontal lobe was recovered and frozen for biochemical testing. The remaining brain was used for histological analysis.

[0193] Vectors were generated as described in Examples 1 and 2.

[0194] Empty: Perfect particle ratio: Load the vector sample into a cell with a 2-channel charcoal-epon centerpiece having a 12 mm optical path length. Load the supplied dilution buffer into the reference channel of each cell. Then, place the loaded cells in an AN-60Ti analytical rotor and load them into a Beckman-Coulter ProteomeLab XL-I analytical ultracentrifuge equipped with both absorbance and RI detectors. Allow to fully temperature-level at 20°C. After balancing, the rotor is set to a final running speed of 12,000 rpm. Absorbance in the 280 nm scan is recorded approximately every 3 minutes for about 5.5 hours (110 total scans for each sample). The raw data is analyzed using the c(s) method and implemented in the analysis program SEDFIT. The resulting size distribution is graphed and the peaks are merged. The percentage value associated with each peak represents the peak area fraction of the total area under all peaks, based on the raw data generated at 280 nm. Many laboratories use these values ​​to calculate the empty:perfect particle ratio. However, empty and perfect particles have different absorption coefficients at this wavelength, so the raw data can be adjusted accordingly. The empty:perfect particle ratio is determined using the ratio of empty particle and perfect monomer peak values ​​both before and after absorption coefficient adjustment.

[0195] Assay for the replication-capable AAV: Samples are analyzed for the presence of replication-capable AAV2 / hu68 (rcAAV) that may occur during the production process. The cell-based component consists of inoculating a monolayer of HEK293 cells (P1) with the test sample and a dilution of wild-type (WT) human adenovirus type 5 (Ad5). The maximum amount of product tested is 1.0 × 10⁶. 10 This is a GC vector product. The presence of adenovirus amplifies rcAAV in cell cultures. After 2 days, cell lysates are prepared and Ad5 is thermally inactivated. The clarified lysates are then passaged through a second batch of cells (P2) to enhance sensitivity (again, in the presence of Ad5). After 2 days, cell lysates are prepared and Ad5 is thermally inactivated. The clarified lysates are then passaged through a third batch of cells (P3) to enhance sensitivity (again, in the presence of Ad5). After 2 days, cells are lysed to release DNA, which is then subjected to qPCR to detect the AAVhu68 cap sequence. The presence of rcAAV is indicated by the amplification of the AAVhu68 cap sequence in an Ad5-dependent manner. Using AAV2 / hu68 surrogate positive controls containing AAV2 rep and the AAVhu68 cap gene allows for the determination of the assay's detection limits (0.1, 1, 10, and 100 IU). rAAV(1.0 × 10⁻⁶) 10, 1.0 × 10 9 , 1.0 × 10 8 , and 1.0 × 10 7 The approximate amount of rcAAV present in the test sample can be quantified using serial dilutions (GC).

[0196] In vitro efficacy: To correlate ddPCR GC titers with gene expression, an in vitro relative efficacy bioassay is performed. Briefly, cells are seeded in a 96-well plate and incubated overnight at 37°C / 5%CO2. The following day, cells are infected with serially diluted AAV vector and incubated at 37°C / 5%CO2 for up to 3 days. Cell supernatant is collected and analyzed for β-gal activity based on cleavage of the fluorescence-generating substrate.

[0197] Total protein, capsid protein, protein purity, and capsid protein ratio: Using a bicinchoninic acid (BCA) assay, vector samples are first quantified for total protein against a standard curve of bovine serum albumin (BSA) protein. The determination is made by mixing equal volumes of the sample with the Micro-BCA reagent provided in the kit. The same procedure is applied to the dilution of the BSA standard. The mixture is incubated at 60°C and the absorbance is measured at 562 nm. A standard curve is generated from the standard absorbances of known concentrations using a 4-parameter fit. Unknown samples are quantified according to a 4-parameter regression. To provide a semi-quantitative determination of rAAV purity, the samples are normalized for genomic titer and measured at 5.0 × 10⁻⁶. 9 The GC samples are separated under reducing conditions by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE gel is then stained with SYPRO Ruby dye. All impurity bands are quantified by concentration measurement. Stained bands appearing in addition to the three AAV-specific proteins (VP1, VP2, and VP3) are considered protein impurities. The impurity mass percentage and approximate molecular weight of the impurity bands are reported. The SDS-PAGE gel is also used to quantify the VP1, VP2, and VP3 proteins and determine their proportions.

[0198] Enzyme activity assay: Tissues were homogenized in 0.9% NaCl (pH 4.0) using a steel bead homogenizer (TissueLyzer, Qiagen). After three freeze-thaw cycles, samples were clarified by centrifugation, and protein content was quantified by bicinchoninic acid assay (BCA). Serum samples were used directly in the enzyme assay. For the β-gal activity assay, 1 μL of sample was combined with 99 μL of 0.5 mM 4-methylumbelliferyl β-D-galactopyranoside (Sigma M1633) in 0.15 M NaCl, 0.05% Triton-X100, and 0.1 M sodium acetate (pH 3.58). The reaction was incubated at 37°C for 30 minutes, then stopped by adding 150 μL of 290 mM glycine and 180 mM sodium citrate (pH 10.9). Fluorescence was compared to a 4 MU standard dilution. β-gal activity is expressed as nmol of 4 MU freed per hour per 1 mg of protein (tissue) or per 1 ml of serum or CSF. Using 1 mM 4-methylumbelliferyl N-acetyl-β-D-glucosaminide (Sigma M2133) as the substrate, a HEX assay was performed in the same manner as the β-gal activity assay, using a sample volume of 1 μL for tissue lysates and a sample volume of 2 μL for serum.

[0199] Histology: In addition to the knockout mouse model, histological analyses were also performed comparing post-necrop rAAV.hGLB1-treated GLB1- / - mice with both vehicle-treated GLB1- / - mice and GLB1+ / - control mice. Lysosomal storage disorders were evaluated by staining brain sections with Philippine, a fluorescent molecule that binds to GM1 gangliosides, as well as by immunostaining for lysosomal-associated membrane protein 1. Philippine staining revealed significant accumulation of GM1 gangliosides in cortical, hippocampal, and thalamic neurons in vehicle-treated GLB1- / - mice, which normalized in rAAV.hGLB1-treated GLB1- / - mice. Immunohistochemistry showed increased lysosomal membrane staining in the cortex and thalamus of vehicle-treated GLB1- / - mice, which decreased in rAAV.hGLB1-treated GLB1- / - mice, similar to GLB1+ / - control mice. Brain tissue was fixed overnight in 4% paraformaldehyde, equilibrated in 15% and 30% sucrose, and then frozen in OCT-embedded cultures. Frozen sections were stained with Philippine (Sigma, 10 μg / mL) or antibodies against GFAP or LAMP1.

[0200] Anti-β-gal antibody ELISA: High-binding polystyrene ELISA plates were coated overnight with 100 μL of recombinant human β-gal per well at a concentration of 1 μg / mL in PBS (R&D Systems). The plates were washed and blocked with 2% bovine serum albumin in PBS at room temperature for 2 hours. Duplicate wells were incubated with serum samples diluted 1:1,000 in PBS at room temperature for 1 hour. The plates were washed and incubated with horseradish peroxidase conjugate anti-mouse IgG polyclonal antibody diluted 1:5,000 in blocking solution for 1 hour, and then developed using TMB substrate.

[0201] Evaluation of therapeutic effects on neurological function To evaluate neurological function in rAAV.hGLB1- / - mice treated with GLB1, gait analysis was performed for two consecutive days at 4 months of age (3 months after rAAV.hGLB1 or vehicle administration) using the CatWalk XT gait analysis system (Noldus), a commonly used assessment of motor performance in mice, according to the manufacturer's instructions. Mice were tested for two consecutive days. At least three complete trials were performed for each animal on each test day. Trials exceeding 5 seconds, or trials in which the animal stopped or reversed without crossing the entire length of the apparatus, were excluded from the analysis. Over at least three evaluations, on the second day of the test, the average walking speed and hindlimb footprint length were quantified for each animal. Slower speeds and elongated footprint lengths indicate impaired motor performance. As shown in the figure below, in rAAV.hGLB1- / - mice treated with vehicle-treated GLB1, - / - Compared to a mouse Walking speed and footprint length were significantly improved, and GLB1 + / - The results were similar to those of the control mice. See, for example, Figures 7C and 7D.

[0202] Lifetime assessment included survival monitoring, neurological examination, gait analysis, and evaluation of serum transgene expression (β-gal activity). Untreated GLB1 - / - Mouse and normal GLB1 + / - For mice, necropsy was performed on the day of drug administration (day 1) to assess the severity of baseline brain accumulation lesions. Vehicle-treated mice and vector-treated mice were necropsyed on days 150 and 300.

[0203] In the 150-day cohort, all mice received one vehicle-treated GLB1. - / - With the exception of the mouse, the animals survived until the scheduled autopsy (Figure 13). This animal died two days after vector administration due to intracranial hemorrhage, which was likely caused by the ICV injection procedure.

[0204] In the 300-day cohort, all 12 animals received vehicle-based GLB1 treatment before the planned trial endpoint. - / -Mice were euthanized according to the euthanasia criteria defined in the study. The mice exhibited neurological signs (i.e., ataxia, tremor, and / or limb weakness) as a result of disease progression. Vehicle treatment GLB1 - / - The median survival time for mice was 268 days (ranging from 185 to 283 days). (Minimum dose group: 4.4 × 10⁻⁶) 9 In the GC study, 5 out of 12 animals (41.7%) were euthanized due to disease progression during a survival period ranging from 268 to 297 days. 1.3 × 10 10 One animal (1 / 12 [8.3%]) in the GC dose cohort was euthanized due to disease progression 290 days after treatment. 4.4 × 10 10 GC or 1.3 × 10 11 All animals that received the GC vector dose survived until the study endpoint.

[0205] Gait analysis was performed at baseline (-7 days to 0 days) and every 60 days until day 240, evaluating stride length and hindlimb footprint length in vehicle-treated and vector-treated mice. - / - The study revealed progressive abnormalities in mice, while also identifying two optimal vector doses (1.3 × 10⁻¹⁰). 11 GC and 4.4×10 10 GLB1 treated with GC - / - The mice showed consistent improvement in both walking parameters.

[0206] At baseline, the mean stride length of vehicle-treated GLB1- / - mice was significantly shorter than that of normal GLB1+ / - controls, and this abnormality persisted until day 240. The stride length abnormality was partially relieved in rAAV.hGLB1-treated GLB1- / - mice, and by day 60, all doses showed a statistically significant increase in mean stride length compared to vehicle-treated GLB1- / - mice. However, in the two maximum dose groups (1.3 × 10⁻⁶), 11 GC and 4.4×10 10 Only the GC mice maintained a significantly longer average stride length up to day 240 compared to vehicle-treated GLB1- / - mice.

[0207] On day 60, the mean hindlimb footprint length of vehicle-treated GLB1- / - mice was significantly shorter than that of normal GLB1+ / - controls, and this abnormality persisted until day 240. The hindlimb footprint length abnormality was observed at three maximum doses (1.3 × 10⁻⁶). 11 GC, 4.4×10 10 GC, 1.3 × 10 10 In GC, rAAV.hGLB1 administration partially rescued GLB1- / - mice, resulting in a statistically significant reduction in mean hindlimb footprint length up to day 240 compared to vehicle-treated GLB1- / - mice.

[0208] Pharmacological examination of dosage range Pharmacological studies were conducted to evaluate the minimum effective dose (or MED) and β-gal expression levels of GM1 in a GLB1 knockout mouse model after ICV administration of rAAV.hGLB1. In this study, GLB1- / - mice were given rAAV.hGLB1 The vehicle was administered via ICV at four separate dose levels. GLB1- / - mice and heterozygous GLB1 mice, or HET mice, were administered the vehicle via ICV. In this study, ICV administration of rAAV.hGLB1 resulted in a stable dose-dependent increase in transgene product expression in the brain and peripheral organs, degradation of brain lysosomal accumulation lesions, improvement of the neuronal phenotype, and increased survival rate in GLB1- / - mice. The minimum dose evaluated was considered the MED based on statistically significant improvements in survival rate, neurological examination scores, and brain accumulation lesions.

[0209] B. Results Transgene cassettes were designed consisting of human GLB1 cDNA driven by a chicken beta-actin promoter along with a cytomegalovirus enhancer (CB7) and either a human elongation initiation factor 1 alpha promoter (EF1a) or a human ubiquitin C promoter (UbC). Each cassette was packaged in an AAVhu68 capsid, and 10 11A single dose of one genome copy (GC) was administered to wild-type mice by intraventricular (ICV) injection. Two weeks after injection, β-gal activity was measured in the brain and CSF (Figure 2A-2B). The vector with the UbC promoter showed a statistically significant increase in β-gal activity in both the brain and CSF, with enzyme activity being nearly twice as high in the brain and tenfold higher in the CSF compared to untreated wild-type mice. Therefore, the AAVhu68.UbC.hGLB1 vector was selected for further study.

[0210] The effectiveness of the optimization vector is measured in GLB1. - / - The condition was evaluated using a mouse model. A mouse model of GM1 gangliosidosis has been developed by targeting and inserting a neomycin-resistant cassette into the 6th and / or 15th exon of the GLB1 gene. Hahn, CN, et al. Generalized CNS disease and massive GM1-ganglioside accumulation in Mice defective in lysosomal acid beta-galactosidase. Human Molecular Genetics 6, 205-211 (1997), and Matsuda, J., et al. Beta-galactosidase-deficient mouse as an animal model for GM1-gangliosidosis. Glycoconjugate Journal 14, 729-736 (1997). Similar to infant GM1 gangliosidosis patients, these mice do not express functional β-gal and show rapid accumulation of GM1 gangliosides in the brain. Brain GM1 accumulation is already evident by the first week of life, and by 3 months of age, GLB1 - / - Mice have brain levels of GM1 comparable to those of 8-month-old infant GM1 patients (Hahn 1997, cited above). GLB1 - / -The clinical phenotype of mice most closely models the clinical phenotype of infantile GM1 gangliosidosis, with motor abnormalities appearing by 4 months of age and severe neurological symptoms (e.g., ataxia or paralysis) requiring euthanasia by 10 months of age (Hahn 1997, Matsuda 1997, cited above). GLB1 - / - The mouse model does not show any involvement of peripheral organs and differs from infant GM1 patients who often develop bone deformities and hepatosplenomegaly (Hahn 1997, Matsuda 1997, cited above). Therefore, GLB1 - / - Mice are a representative model of the neurological features of infantile GM1 gangliosidosis, but they are not a model of the signs of the systemic disease.

[0211] GLB1 - / - Mice are treated at 1 month of age and observed until 4 months of age, at which point they will typically develop marked gait abnormalities associated with brain GM1 levels similar to those seen in infants with progressive GM1 gangliosidosis (Matsuda 1997, cited above). GLB1 - / - The mouse, 1.0 x 10 11 Treatment involved a single ICV injection of either AAVhu68.UbC.hGLB1 (n=15) or vehicle (n=15) from genome copies (GC). Heterozygous (GLB1) patients treated with vehicle (n=15) + / - The group of mice was It served as a normal control. Serum was collected on the day of injection (day 0), as well as on days 10, 28, 60, and 90. Ninety days after treatment, motor function was assessed using the CatWalk XT gait analysis system (Noldus Information Technology, Wageningen, The Netherlands), after which the animals were euthanized and tissues were collected for histological and biochemical analysis. The CatWalk XT tracks the footprints of mice walking across a glass plate. The system quantifies the dimensions of each footprint and statistically analyzes the animal's speed and other gait characteristics. To perform this assessment, the CatWalk XT was calibrated with a set of walkways of appropriate width before the start of the experiment. The animals were brought indoors and adapted in darkness for at least 30 minutes before running the CatWalk XT. Once adaptation was complete, animals were selected and placed at the entrance of the walkway. The researchers started the acquisition software and allowed the animals to walk the walkway. The animals' home cages were placed at the end of the walkway to encourage them. The run was considered complete when the animal successfully walked to the end of the catwalk within the allotted time limit. Otherwise, the run was repeated. The animals performed three trials with a minimum duration of 0.50 seconds and a maximum duration of 5.00 seconds. Three successful runs were required for a trial to be considered complete. If an animal was unable to complete three runs after a 10-minute trial, only the completed runs were used for analysis. This analysis was performed by blinded evaluators for each animal ID and treatment group. Catwalk XT software was used to automatically classify runs, and then the accuracy and appropriate labeling of the footprints were verified. Data other than footprints were manually removed. Average speed, stride length, and hind limb footprint length were automatically measured by the program. The average of the left and right hind limb footprint lengths was calculated and analyzed for each group. The average of the stride length measured from each foot was calculated and analyzed for each group. Analysis was performed using Prism 7.0 (GraphPad Software). Neurological examination scores and gait analysis parameters (walking speed and hindlimb footprint length) were compared between groups at each time point using two-way analysis of variance (ANOVA).Survival curves were compared between groups using the log-rank (Mantel-Cox) test. Brain LAMP1 data were compared using one-way ANOVA, followed by Dunnett's test, after log-transformation.

[0212] During the ICV injection procedure, one mouse treated with AAV died. All other mice survived to the 90-day study endpoint. AAV delivery to CSF ​​has been shown to result in vector distribution in peripheral blood and significant hepatic transduction (Hinderer, C., et al. Intrathecal gene therapy corrects CNS pathology in a feline model of mucopolysaccharidosis I. Molecular therapy: the journal of the American Society of Gene Therapy 22, 2018-2027 (2014), Gray, SJ, Nagabhushan Kalburgi, S., McCown, TJ & Jude Samulski, R. Global CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathecal AAV administration in non-human primates. Gene therapy 20, 450-459 (2013), Haurigot, V., et al. Whole body correction of mucopolysaccharidosis IIIA by intracerebrospinal fluid gene therapy.The Journal of clinical investigation(2013), Hinderer, C., et al.Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cisterna magna.Molecular therapy.Methods & clinical deve lopment 1,14051 (2014), Hordeaux, J., et al.Toxicology Study of Intra-Cisterna Magna Adeno-Associated Virus 9 Expressing Human Alpha-L-Iduronidase in Rhesus Macaques.Molecular therapy.Methods & clinical development 10,79-88(2018)). GLB1 treated with AAVhu68.UbC.hGLB1 - / - Mice were heterozygous (GLB1) 10 days after vector administration. + / - The mice showed higher serum β-gal activity than the control group (Figure 3A). Serum antibodies against human β-gal were detectable in 5 / 15 mice treated with AAVhu68.UbC.hGLB1 by day 90. In all but two mice, elevated serum β-gal activity persisted throughout the study, and both expressed antibodies against human β-gal (Figure 6). Peripheral organs, including the heart, lungs, liver, and spleen, also showed elevated β-gal activity (Figures 3B-3E). Some animals that expressed antibodies against the human transgene product had lower β-gal activity in their peripheral organs.

[0213] CSF collected at autopsy was treated with GLB1 from AAVhu68.UbC.hGLB1. - / - The vector-treated mice exhibited higher β-gal activity than heterozygous controls (Figure 4B). β-gal activity in the brains of vector-treated mice was similar to that of heterozygous controls (Figure 4A). The anti-β-gal antibody does not appear to affect β-gal levels in the brain or CSF.

[0214] Correction of brain abnormalities was evaluated using biochemical and histological assays. Lysosomal enzymes were frequently upregulated in the context of lysosomal accumulation, and this observation has been confirmed in patients with GM1 gangliosidosis (Van Hoof, F. & Hers, HG: The abnormalities of lysosomal enzymes). in mucopolysaccharidoses. European Journal of Biochemistry 7, 34-44 (1968). Therefore, the activity of hexosaminidase (HEX), a lysosomal enzyme, was measured in brain lysates. Vehicle therapy GLB1 - / - In mouse-derived brain samples, HEX activity was elevated, and in vector-treated animals, it was normalized (Figure 5).

[0215] To assess the progression of lysosomal accumulation lesions, brain sections were stained for the lysosomal membrane protein LAMP1 with Philippine, a fluorescent molecule that binds to GM1 ganglioside, as well as immunostaining for lysosomal-associated membrane 1 (protein LAMP1). Philippine also binds to non-esterified cholesterol, although previous studies have shown that Philippine staining is primarily for GLB1. - / - This study demonstrates that it reflects GM1 accumulation in mice (Arthur, JR, Heinecke, KA & Seyfried, TN: Filipin recognizes both GM1 and cholesterol in GM1 gangliosidosis mouse brain. Journal). of lipid research 52,1345-1351(2011). Philippine staining is vehicle treatment GLB1 - / - We revealed significant GM1 accumulation in mice cortical, hippocampal, and thalamic neurons, which normalized in mice treated with AAVhu68.UbC.hGLB1 (data not shown). LAMP1 immunohistochemistry showed that GLB1 - / -Increased lysosomal membrane staining was observed in the cortex and thalamus of mice, and decreased in vector-treated mice (data not shown). Gliosis was assessed by staining for the astrocyte marker, glial fibrillary acidic protein (GFAP). Vector-treated GLB1 - / - Mice showed a significant reduction in thalamic astrogliosis compared to vehicle-treated controls (data not shown).

[0216] Vector therapy GLB1 - / - To evaluate neurological function in mice, 4-month-old mice Gait analysis was performed (3 months after vector or vehicle administration). Untreated GLB1 - / - It has been previously noted that mice exhibit clinically significant gait abnormalities by 3-4 months of age. Untreated GLB1 - / - Quantitative gait assessments performed using the CatWalk system in a cohort of mice and normal controls revealed various abnormalities, including slower spontaneous walking speed, differences in stride length, and duration of several stages in the step cycle (Figures 7C and 7D). GLB1 - / - Due to the significantly slower walking speed of the mice, many interpretations of these apparent differences were complicated by the speed dependence of most walking parameters (Figures 8A and 8B) (Batka, RJ, et al. The need for speed in rodent locomotion). analyzes.Anatomical record(Hoboken, NJ:2007)297,1839-1864(2014)). GLB1 - / - The mice also showed consistent abnormalities in hindlimb positioning, which could be measured as an increase in hindlimb footprint length (Figure 7D). This abnormality was found to be unrelated to walking speed, consistent with previous reports (Batka, et al., cited above), and this suggests that GLB1 - / -This gait signature proved useful for evaluating speed-independent gait impairment in mice (Figures 8A and 8B). In a study conducted on two consecutive days using mice from the same cohort, slower spontaneous walking speed and increased hindlimb footprint length were associated with untreated GLB1. - / - We demonstrated that the observational results were reproducible in mice (Figures 7A and 7B). Vehicle-based treatment GLB1 - / - Mice exhibited gait abnormalities similar to those previously identified in untreated animals (Figures 7A-7G). Walking speed and footprint length were observed in mice treated with vector-treated GLB1. - / - The condition was normalized in mice (Figures 7A-7G).

[0217] Survival Data: Figure 13 shows survival data for each cohort in the trial up to day 300. All 12 vehicle-treated GLB1- / - mice were euthanized before the planned trial endpoint according to the trial-defined euthanasia criteria due to disease progression accompanied by neurological signs characterized by ataxia, tremor, and limb weakness. The median survival time for this group was 268 days. In the lowest dose group, 5 out of 12 animals were euthanized due to disease progression. In the second lowest dose cohort, 1 out of 12 animals were euthanized due to disease progression. All animals in the two highest dose cohorts survived to the trial endpoint.

[0218] Neurological examination: Standardized neurological examinations were performed every 60 days for up to 240 days in a blinded format, and the mean total severity score was obtained. Figure 14C shows the mean total severity score for each cohort at each evaluation period. Starting with evaluation at day 120, vehicle or minimum dose vector (4.4 × 10) 9 Glb1 administered with either GC - / - The mice progressively showed higher total severity scores, indicating an increase in the severity of neurological signs. However, Glb1 administered at the minimum dose... - / - The total severity score in mice was determined by vehicle treatment Glb1. - / - This is significantly lower than in mice, which suggests that this dose (4.4 × 10) 9This suggests that GC partially relieved the neurological phenotype. The next maximum dose (1.3 × 10⁻¹⁰) 10 In GC, minimal abnormalities were detectable in 7 out of 12 animals (58.3%) at 240 days of evaluation, suggesting substantial relief of neurological phenotypes. Two highest vector doses (1.3 × 10⁻¹⁰) 11 GC and 4.4×10 10 In GC, no neurological abnormalities were evident, and the total severity score in these groups was Glb1 with normal vehicle treatment at each time point. + / - This is similar to the control group, suggesting complete relief of the neurological phenotype.

[0219] Vehicle treatment GLB1 - / - The results in mice novelly showed a higher total severity score, indicating progressive neurological signs starting from the 120-day assessment. With the lowest dose of rAAV.hGLB1, the total severity score was higher than that of vehicle-treated GLB1 at the same point in time. - / - Although significantly lower than in mice, a gradual increase in the total severity score was also observed by the 120-day evaluation. At the minimum rAAV.hGLB1 dose, minimal abnormalities were detectable in 7 out of 12 animals at day 240. At the two maximum doses of rAAV.hGLB1, no neurological abnormalities were evident, and the overall severity score in these groups was normal for vehicle-treated GLB1. + / - It was the same as the one at that time.

[0220] Histological analysis: Brain sections of rAAV.hGLB1-treated GLB1- / - mice, vehicle-treated GLB1- / - mice, and vehicle-treated GLB1+ / - control mice were also compared at baseline, day 150, and day 300. Brain frozen sections were stained overnight at 4°C with an antibody against lysosomal-associated membrane protein (LAMP1) (Abcam, catalog no. Ab4170). The following day, slides were washed and incubated with an anti-rabbit IgG TritC conjugate secondary antibody for 1 hour at room temperature. Slides were washed and coverslips were applied. LAMP1 staining was quantified as positive cells per field of view of the entire cerebral cortex from a single coronary brain section using VisioPharm image analysis software. Cortical cells positive for LAMP1 (i.e., cells showing lysosomal expansion) were quantified in sections scanned using an automated program. For animals that did not survive until the scheduled 300-day necropsy due to disease progression, the brain was collected at euthanasia, and the data are presented as part of the 300-day cohort. Untreated GLB1- / - baseline mice necropsed on day 1 showed a higher percentage of LAMP1-positive cells in the brain compared to normal untreated GLB1+ / - baseline controls. At both days 150 and 300, rAAV.hGLB1-treated mice showed a dose-dependent reduction in the percentage of LAMP1-positive cells compared to vehicle-treated GLB1- / - controls. At the two maximum doses of rAAV.hGLB1, the percentage of LAMP1-positive cells decreased to levels similar to those of normal vehicle-treated GLB1+ / - controls.

[0221] β-gal activity: β-gal activity was measured in serum on the day of administration and then every 60 days until day 240. At autopsy, β-gal activity was measured in the brain and peripheral organs (heart, liver, spleen, lungs, and kidneys). As shown in Figure 9C, the maximum dose test hAAV.hGLB1(1.3 × 10⁶) 11 GLB1 administered to GC - / - The mean β-gal activity in mouse serum was approximately 10 times greater than that of normal vehicle-treated GLB1+ / - controls. The second highest dose trial hAAV.hGLB1(4.4 × 10⁻¹⁰)10 GC) GLB1 - / - Serum β-gal activity in mice was similar to that of normal vehicle-treated GLB1+ / - controls. All other rAAV.hGLB1 doses of GLB1 - / - Serum β-gal activity in mice is related to vehicle-based GLB1 - / - It was similar to the control.

[0222] For each tissue type examined, the mean β-gal activity levels within each group were similar at both time points (days 150 and 300) (Figures 17A-L). In the brain, vector therapy Glb1 - / - In mice, β-gal activity increased in a dose-dependent manner. The mean β-gal activity across all dose groups was greater than that of vehicle-treated Glb1. - / - It was higher than the control group. However, the two maximum dose groups (1.3 × 10) 11 GC and 4.4×10 10 GC) Only normal vehicle treatment Glb1 at both time points + / - The average β-gal activity was higher than that of the control group. Several peripheral organs (e.g., liver and spleen), though not all organs (e.g., lungs and kidneys), showed an increase in β-gal activity after vector administration (Figures 17A-L). It should be noted that the heart showed a dose-dependent increase in β-gal activity, and at all doses, vehicle treatment Glb1 - / - It yielded higher average levels than mice. However, the two maximum doses (1.3 × 10⁻⁶) 11 GC and 4.4×10 10 GC) alone shows β-gal activity at both time points, normal vehicle treatment Glb1 + / - The levels were restored to the same or higher level as the control group.

[0223] β-gal activity was measured in all animals in the 300-day cohort that survived until the scheduled autopsy. Measured in the CSF of the substance. Vehicle treatment Glb1 - / -None of the animals survived until day 300 due to disease progression; therefore, the β-gal activity levels of vector-treated mice were normal vehicle-treated Glb1. + / - Compared to the control group (Figure 16C). β-gal activity was measured in the CSF of all animals in the cohort at day 300, surviving until scheduled necropsy. Since none of the vehicle-treated Glb1- / - animals survived to day 300 due to disease progression, the β-gal activity levels of vector-treated mice were compared to normal vehicle-treated Glb1 mice. + / - It was compared with the control (Figure 16C). As shown in Figure 16C, β-gal activity was detectable in the CSF of all mice evaluated. Two maximum dose tests were conducted: rAAV.hGLB1 (1.3 × 10⁶). 11 GC and 4.4×10 10 GLB1- / - mice administered GC showed higher mean CSF β-gal activity levels than normal vehicle-treated GLB1+ / - controls. β-gal activity in CSF was generally dose-dependent, but two minimum dose groups (1.3 × 10⁻⁶) 10 GC and 4.4×10 9 β-gal activity in GC is related to vehicle treatment Glb1 + / - It appeared to be similar to that of the other. The reason why the β-gal activity levels were similar at the two minimum doses is that the minimum vector dose (4.4 × 10) 9 This may be related to the number of CSF samples from animals treated with GC, and was limited by the high mortality rate in this group. Animals that survived in this group may have had higher β-gal expression than other animals that did not survive. In all groups, β-gal activity levels were related to prior treatment with the control vehicle Glb1. - / - The observed range of CSF from mice was exceeded.

[0224] Figures 17A-L show β-gal activity in the brain, heart, and liver after autopsy. In the brain, β-gal activity increased in a dose-dependent manner in experimental rAAV.hGLB1- / - mice treated with GLB1. The mean β-gal activity in all dose groups was higher than that of vehicle-treated GLB1- / - controls. However, only the two maximum dose groups showed higher mean β-gal activity than normal vehicle-treated GLB1+ / - controls at both time points. Several peripheral organs also showed a dose-dependent increase in β-gal activity after administration of experimental rAAV.hGLB1. The heart showed a dose-dependent increase in β-gal activity, resulting in higher mean levels than vehicle-treated GLB1- / - mice at all doses. However, only the two maximum doses restored β-gal activity to levels equivalent to or higher than that of normal vehicle-treated GLB1+ / - controls at both time points. The liver showed a dose-dependent increase in β-gal activity after administration of experimental rAAV.hGLB1. At all doses except the minimum dose, the mean β-gal activity levels at both time points were higher than those of vehicle-treated GLB1- / - mice and similar to, or higher than, those of normal vehicle-treated GLB1+ / - controls.

[0225] C. Consideration These results suggest that administration of rAAVhu68.hGLB1 to CSF ​​increases brain β-gal activity, reduces neuronal lysosomal accumulation lesions, prevents neurological decline, and gene transfer can prevent and reverse GM1 accumulation in the brain.

[0226] This study demonstrated the absence of neuronal accumulation lesions in Glb1- / - mice treated with AAV vectors at 4 weeks of age, even when significant brain accumulation lesions were already present in this model. These results suggest that gene transfer can prevent and reverse GM1 accumulation in the brain. Patients with infantile GM1 gangliosidosis are a suitable population for AAV gene therapy because they are frequently diagnosed based on subtle neurological findings that appear at 6 months of age, before the onset of rapid developmental regression that inevitably follows within 1-2 years.

[0227] Example 4: Animal Model A.GLB1 - / - Identification of the minimum effective dose (MED) of AAVhu68.UbC.GLB1 in a mouse model. The effects of different doses of rAAVhu68.UbC.GLB1 - / - CNS lesions and neurological signs were evaluated in a mouse model. Efficacy was assessed by serum enzyme activity, reduction of brain lesions, neurological signs measured by automated walking analysis (e.g., via the CatWalk system), and standardized neurological examinations performed by blinded re-evaluators (e.g., a 9-point scale for posture, motor function, sensation, and reflexes), as well as survival rate. Safety analyses (including blood sampling and analysis) were also performed. 4-week-old GLB1 mice - / - Mice were given four doses of rAAVhu68.UbC.GLB1 (1.3 × 10) by ICV injection. 11 GC, 4.4×10 10 GC, 1.3 × 10 10 GC or 4.4×10 9 They received either GC (Gross Classification) or a vehicle (n=24 per group). Heterozygous littermates treated with a vehicle (n=24) served as normal controls.

[0228] Serum β-gal enzyme activity, gait analysis, and neurological examinations were performed on half of the animals in each group every 60 days, while body weight was measured at least every 30 days during the 120-day observation period. The results are plotted in Figures 9A-9F and briefly explained below.

[0229] All treated mice appeared healthy and showed normal weight gain. No significant differences in body weight were detected between the groups during the observation period (Figure 9B).

[0230] Serum enzyme expression was consistent with the test discussed in Example 3. As shown in Figure 9A, vehicle-based GLB1 - / - The β-gal enzyme activity in mice (which function as negative controls) remained at approximately 10 nmol / mL / hour, while the positive control group (vehicle-treated GLB1) + / -The mice showed an enzyme activity of approximately 100 nmol / mL / hour. (4.4 × 10⁶ mice) 10 Treatment with rAAVhu68.UbC.GLB1 at GC doses significantly increased β-gal enzyme activity compared to negative controls at both day 60 and day 120. (1.3 × 10⁶ per mouse) 11 Higher doses of GC-mediated rAAVhu68.UbC.GLB1 resulted in higher β-gal enzyme activity than the positive control at day 60, and further increases at day 120.

[0231] The gait phenotype of GM1 mice was also consistent with the previous results shown in Example 3. Neurological examination scores, hindlimb footprint length, hindlimb swing time, and hindlimb stride length were obtained, and the results were plotted in Figures 9C-9F. For all four plotted parameters, there were significant statistical differences between negative and positive controls, indicating that these parameters can serve as good indicators for evaluating efficacy. Vehicle-based treatment GLB1 - / - Compared to a mouse, 4.4 x 10 10 Mice treated with GC's rAAVhu68.UbC.GLB1 showed significant improvements in hindlimb footprint length, hindlimb swing time, and hindlimb stride length. 1.3 × 10 11 Higher doses of GC provided increased hindlimb swing time and longer stride length, indicating successful modification. Neurological examination was more sensitive than gait analysis. As shown in Figure 9C, dose-dependent improvement was observed, indicated by a decrease in neurological scores with increasing dose, but only by 1.3 × 10⁻⁶. 10 Treatment with GC's rAAVhu68.UbC.GLB1 showed statistical significance in total score compared to the negative control group. Evidence of phenotypic modification was only 1.3 × 10⁶ per mouse. 10 Observed at GC doses.

[0232] If all untreated animals are expected to remain alive, the same set of parameters will be collected within this animal cohort for at least another 150 days. Untreated GLB1- / - We will evaluate the change in survival rate compared to mice.

[0233] Half of the animals discussed above will be euthanized 270 days after treatment. The other half will be euthanized 150 days after treatment. Another 24 mice will be provided as baseline necropsy controls. Histological and biochemical comparisons will be performed on all euthanized animals. This is performed between treated and untreated animals. After necropsy, the brain is sectioned and stained for LAMP1 to assess lysosomal accumulation lesions, which are quantified using an automated imaging system. β-gal activity is measured in the brain, serum, and peripheral organs. For safety analysis, blood is collected at necropsy for whole blood count and serum chemistry panels, and the brain, spinal cord, heart, lungs, liver, spleen, kidneys, and gonads are collected for histopathological evaluation by a committee-certified veterinary pathologist. Vehicle-based therapeutic GLB1 - / - The minimum dose of rAAVhu68.UbC.GLB1 that achieves a significant reduction in brain accumulation lesions compared to mice is selected as the minimum effective dose (MED).

[0234] Results: 4.40 × 10⁶ in 4-week-old GLB1- / - mice 9 Genome copy (GC) ~ 1.30 × 10⁻⁶ 11 Single intraventricular (ICV) administration of rAAVhu68.UbC.GLB1 at dose levels within the GC range resulted in increased β-galactosidase activity measured in the brain and supported by degradation of cerebral accumulation lesions. Survival, degradation of cerebral accumulation lesions, and neurological function, as measured by autogastric analysis and standardized neurological examinations, were improved in a dose-dependent manner. Hepatic transduction and serum β-galactosidase activity in rAAVhu68.UbC.GLB1-treated mice were significantly higher than that of heterozygous controls (Glb+ / - mice). Biochemical modifications in peripheral organs were observed with rAAVhu68.UbC.GLB1 treatment, suggesting the potential to treat both central and peripheral diseases with a single ICV administration. Minimum dose evaluated (4.4 × 10⁻⁶) 9GC was considered MED based on statistically significant improvements in survival rate, neurological examination scores, and brain accumulation lesions.

[0235] B. Toxicity testing in non-human primates (NHPs) Rhesus monkeys were selected for toxicity studies because they best replicate the size and CNS anatomical structure of the patient population (infants aged 4–18 months) and can be treated using the clinical route of administration (ROA). Juvenile animals were selected to represent the pediatric study population. In one embodiment, juvenile rhesus monkeys were 15–20 months old. The similarity in size, anatomical structure, and ROA results in representative vector distribution and transduction profiles, enabling accurate assessment of toxicity. In addition, more rigorous neurological assessment is performed in NHP than in rodent models, allowing for more sensitive detection of CNS toxicity.

[0236] A 120-day GLP-compliant safety study was conducted in juvenile rhesus monkeys to investigate the toxicity of AAVhu68.UbC.GLB1 after ICM administration. The 120-day evaluation period was chosen to allow sufficient time for the secreted transgene product to reach stable steady-state levels after AAV administration of ICM. The study design is outlined in the table below. Rhesus monkeys received one of the following three dose levels: a total of 3.0 × 10⁶ 12 GC, total 1.0 × 10 13 GC, or 3.0 × 10 in total 13 GC (n=6 / dose) or vehicle (n=4). Dose levels were selected to be equivalent to those assessed in the minimum effective dose (MED) study when scaled by brain mass (assuming 0.4 g for mice and 90 g for rhesus monkeys). Baseline neurological examination, clinicopathology (cell count with differential diagnosis, clinical chemistry, and coagulation panel), CSF chemistry, and CSF cytology were performed. Animals were monitored daily for signs of distress and abnormal behavior after administration of AAVhu68.UbC.GLB1 or vehicle.

[0237] Hematological and CSF clinicopathological evaluations, as well as neurological examinations, were performed weekly for 30 days after administration of rAAVhu68.UbC.GLB1 or vehicle, and every 30 days thereafter. Neutralizing antibodies against AAVhu68, as well as cytotoxic T lymphocyte (CTL) responses to AAVhu68 and AAVhu68.UbC.GLB1 transgene products, were assessed at baseline and at each subsequent 30-day time point by interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assay.

[0238] Good Laboratory Practice (GLP) toxicity testing of rhesus monkeys [Table 8]

[0239] After administration of either rAAVhu68.UbC.GLB1 or the vehicle, half of the animals were euthanized on day 60 and the other half on day 120. Tissues were collected for comprehensive microscopic histopathological examination. Histopathological examination focused on central nervous system tissues (brain, spinal cord, and dorsal root ganglia) and the liver because these were the tissues most severely transduced after ICM administration of the AAVhu68 vector. In addition, lymphocytes were collected from the spleen and bone marrow at autopsy to assess the presence of T cells that respond to both the capsid and the transgene product in these organs.

[0240] The in vivo distribution of the vector was evaluated by quantitative PCR in tissue samples. The vector genome was quantified in serum and CSF samples.

[0241] result: To evaluate the safety, tolerability, and in vivo distribution and excretion (shedding) profiles of the vector after ICM administration, a 120-day toxicity study in good laboratory practice (Good Laboratory Practice) or GLP-compliant manner was performed at the NHP. Juvenile male and female rhesus monkeys received either a single ICM dose of the vehicle or one of three dose levels of rAAV.hGLB1. Animals from each cohort were euthanized either 60 or 120 days after administration.

[0242] Lifetime assessments included daily clinical observations, multiple scheduled physical examinations, standardized neurological monitoring, sensory nerve conduction studies, or clinicopathological assessments of NCS, body weight, blood, and CSF, evaluation of serum circulating neutralizing antibodies, and evaluation of vector pharmacokinetics and vector excretion.

[0243] Animals were autopsied, tissues were collected, and comprehensive histopathological examinations, T-cell response measurements, and in vivo distribution analyses were performed.

[0244] C. Sensory neurotoxicity in nonclinical AAV trials Non-clinical studies evaluating systemic and intrathecal (IT) administration of AAV have consistently demonstrated evidence of efficient transduction of sensory neurons within the dorsal root ganglia (DRG) and, in some cases, toxicity associated with these cells. Intrathecal administration may enable sensory neuron transduction because their central axons are exposed to the CSF, or rAAV may reach the cell body directly because the DRG is exposed to the spinal cord CSF. Non-clinical results suggest that ICM administration of rAAV.hGLB in 1-24 month-old subjects with GM1 gangliosidosis increases central beta-galactosidase levels and prevents disease progression. Non-clinical toxicity data indicate that clinical safety monitoring should consist of assessments typically used for other AAV gene therapies, in addition to peripheral neuropathy safety monitoring.

[0245] Sensory nerve conduction studies were performed on all animals prior to rAAV.hGLB administration and monthly thereafter, measuring bilateral median sensory action potential amplitude and conduction velocity. Animals were sedated with a ketamine / dexmedetomidine combination. To maintain body temperature, sedated animals were placed lateral or supine on a treatment table equipped with a heat pack. Electronic heating devices were not used due to the potential interference with electrical signal acquisition.

[0246] Sensory nerve conduction studies (NCS) were performed using the Nicolet EDX® system (Natus Neurology) and Viking® analysis software. In short, the stimulation probe was positioned over the median nerve with the cathode closest to the recording site. Two needle electrodes were inserted subcutaneously into the second finger at the level of the distal phalanx (reference electrode) and proximal phalanx (recording electrode), while a ground electrode was positioned proximal to the stimulation probe (cathode). (WR50Comfort Plus Probe pediatric stimulator (Natus)) Neurology was used. The excited response was differentially amplified and displayed on a monitor. The initial acquired stimulation intensity was set to 0.0 mA to ensure there was no background electrical signal. To find the optimal stimulation position, the stimulation intensity was increased up to 10.0 mA, and the probe was moved along the median nerve while generating repeated stimuli until the maximum position, determined by the largest clear waveform, was found. While keeping the probe in the optimal position, the stimulation intensity was gradually increased up to 10.0 mA until the peak amplitude response no longer increased. Each stimulation response was recorded and saved in the software. Up to 10 maximum stimulation responses were averaged and reported for the median nerve. The distance (cm) from the recording site to the stimulation cathode was measured and entered into the software. Conduction velocity was calculated using the response onset latency and distance (cm). The average conduction velocity and sensory nerve action potential (SNAP) amplitude were reported. The median nerve was tested bilaterally. All raw data generated by this device was retained as part of the test file.

[0247] While inter-animal and intra-animal variability was evident in SNAP amplitude, the values ​​typically remained within the baseline range (Figures 21A-21B). One animal administered a moderate dose (animal 17-226 [1.0 × 10⁻¹⁶]). 13 GC, group 7) and one animal (animal 17-205 [3.0 × 10) administered a high dose 13 Animals [GC, group 8] showed a significant decrease in bilateral median nerve sensory amplitude 28 days after rAAV.GLB administration, which persisted until autopsy (Figures 21A-21B). These animals did not exhibit any abnormal clinical findings, but they correlated with histopathological findings of the peripheral nerves. In animals exhibiting a significant decrease in SNAP (animals 17-226 [1.0 × 10¹³ GC, group 7] and animal 17-205 [3.0 × 10¹³ GC, group 8]), the onset latency could not be determined, thus hindering the measurement of conduction velocity. In all other animals, no significant changes in median nerve conduction velocity were observed throughout the study (Figures 21A-21B).

[0248] Histopathological first look A. Histopathology is performed by hematoxylin and eosin staining or trichrome staining. That was my assessment. Hematoxylin and Eosin Staining: All tissues and any generalized lesions were collected and labeled according to SOP4019. Samples in pre-labeled cassettes were fixed in 10% neutral buffer formalin, modified Davidson solution (eye), or Davidson solution (testis) according to SOP4003. All wet tissues were sent to Histo-Scientific Research Laboratories for tissue processing, embedding, sectioning, and hematoxylin and eosin (H&E) staining. For histopathological evaluation, histopathology slides were first evaluated by a primary examination pathologist, and a preliminary pathology report was prepared based on the histological evaluation, autopsy findings, relevant clinicopathological results, and any supporting data to aid in the interpretation of the histopathological findings. Upon completion of the primary review, the draft pathology report, slides, and any supporting materials used in preparing the draft report were submitted to a peer review pathologist for peer review. The pathology peer review memo was prepared, signed, and dated by the peer review pathologist. The memo contained documentation regarding the materials, methods, and implementation of the peer review process, as well as the peer review pathologist's general agreement on the primary examination pathologist's pathology report. Any discrepancies between the primary examination pathologist and the peer review pathologist were reconciled, and the final report was prepared upon completion of the peer review. The final examination report incorporated input from the peer review report and was reviewed for quality assurance by the GTP Quality Assurance Unit (QAU).

[0249] For trichrome staining: At the discretion of the study pathologist, Masson's trichrome histochemical staining was used to further evaluate the target findings identified by H&E staining (i.e., periaxonal fibrosis). Slides of the left and right proximal median nerves were stained using Masson's Trichrome Stain Kit (Polysciences, Inc., catalog no. 25088-1). For histopathological evaluation, the slides were examined by light microscopy and scored in a blinded manner by the primary study pathologist using the same semi-quantitative scoring system used for H&E stained slides. The slides were also digitally scanned using the Aperio VERSA Scanning System (Leica Biosystems) and quantified using VIS image analysis software (Visiopharm, Hoersholm, Denmark, Version 2019.07.0.6328).

[0250] B. Histopathological findings Findings related to the test substance were observed primarily in the DRG, trigeminal ganglion (TRG), posterior column of spinal white matter, and peripheral nerves. These findings consisted of neuronal degeneration within the DRG / TRG, as well as axonal degeneration (i.e., axonal degeneration) within the posterior column of spinal white matter and peripheral nerves. Overall, these findings were observed across all GTP-203 treatment groups; however, the incidence and severity were lower at both time points for the medium dose (1.0 × 10⁻⁶). 13 GC) group and high dose (3.0 × 10 13 There was a tendency for elevated levels in individual animals from the GC group. Findings associated with other test items generally included small foci of gliosis in various nuclei and white matter tracts of the brain, in addition to mononuclear cell infiltration in skeletal muscle and adipose tissue at the injection site.

[0251] Histopathological findings associated with the test material observed across all dose groups at days 60 and 120 consisted of degeneration of neuronal cell bodies with mononuclear cell infiltration in the DRG, with axons projecting centrally into the posterior column of the spinal white matter and peripherally into the peripheral nerves. Similar findings were observed in the TRG. At day 60, the incidence and severity of DRG / TRG degeneration were (none to minimum [3.0 × 10⁻⁶]) in the low-dose group. 12 In the GC, group 2, 1 / 3 animal group, the medium-dose group (1.0 × 10) 13 GC, group 3, 3 / 3 animals) and high-dose group (3.0 × 10) 13 GC, Group 4, 2 / 3 Animal) (None to Moderate) This was slightly lower compared to both groups, suggesting a dose-dependent response. At day 120, the severity of DRG / TRG degeneration was (none to minimum [3.0 × 10)) in the low-dose group. 12 The lowest levels were observed in the GC, group 6, 2 / 3 animals, and in the medium-dose group (none to mild [1.0 × 10⁻¹⁰]). 13 GC, group 7, 3 / 3 animals) to high-dose group (none to moderate [3.0 × 10 13 The incidence and severity of DRG / TRG neuronal degeneration increased to GC (group 8, 3 / 3 animals), which also showed a dose-dependent response. When compared between time points, the incidence and severity of DRG / TRG neuronal degeneration were relatively similar among the rAAV.GLB1 treatment groups, suggesting the absence of a time-dependent response. The absence of a time-dependent response suggests that further progression of DRG / TRG neuronal degeneration does not occur between days 60 and 120.

[0252] DRG degeneration caused axonal degeneration of the posterior column of the spinal white matter and peripheral nerves, which was microscopically consistent with axonal degeneration. At day 60, the overall incidence and severity (from dose-dependent response to mild) were similar across all rAAV.hGLB1 treatment groups, so a dose-dependent response was not observed for posterior column axonal degeneration. At day 120, the low-dose group (minimum [3.0 × 10⁻¹⁰) 12 In the GC, group 6, 2 / 3 animals, the medium-dose group (1.0 × 10) 13 GC, group 7, 3 / 3 animals) and high-dose group (3.0 × 10)13 A dose-dependent response was observed, as the incidence and severity of posterior column axonal degeneration were lowest in the GC (group 8, 3 / 3 animals) group compared to both groups (minimum to moderate). Comparing across time points, the moderate dose (1.0 × 10⁻¹⁰) was most effective between days 60 and 120. 13 GC) and high dose (3.0 × 10 13 In both the GC group and the GC group, the severity and incidence of posterior column axonal degeneration of white matter increased, and to a lesser extent, indicating a time-dependent response and progression of findings. However, an important caveat to this conclusion is that at day 120, one-third of the animals in the medium-dose group (animal 17-226 [1.0 × 10 13 GC, group 7) and 1 / 3 of the animals in the high-dose group (animals 17-205 [3.0 × 10 13 The GC group (group 8) had significantly higher severity than the other animals in both groups, which influences the interpretation of these results. In contrast, the incidence and severity were higher in the low-dose group (3.0 × 10) from day 60 to day 120. 12 The GC showed a decrease, indicating that posterior column axonal degeneration of white matter did not progress at this dose. Regarding peripheral nerve axonal degeneration at day 60, the severity observed in the high-dose group was (minimum to moderate [3.0 × 10⁻¹⁰). 13 Compared to GC, Group 4, 20 / 24 neurological, 3 / 3 animal, the low dose (3.0 × 10) 12 GC, Group 2, 20 / 24 neurological, 3 / 3 animal) and medium dose (1.0 × 10) 13 A dose-dependent response was observed, as the severity was lowest in the GC, Group 3, 22 / 24 neurological, 3 / 3 animal groups (minimum to mild). At day 120, the severity of peripheral nerve axonal degeneration was lower in the moderate dose group (1.0 × 10⁻⁶). 13 GC, Group 7, 30 / 30 neurological, 3 / 3 animal) and high dose (3.0 × 10) 13 In the GC, Group 8, 30 / 30 neurological, 3 / 3 animal groups (minimum to significant), low dose (3.0 × 10) 12The severity observed was higher in the GC (Group 6, 29 / 30 nerves, 3 / 3 animals) and vehicle-treated (ITFFB, Group 5, 30 / 30 nerves, 2 / 2 animals) groups (minimum), showing a dose-dependent response. At day 120, the vehicle-treated animals (ITFFB, Group 5, 30 / 30 nerves, 3 / 3 animals) showed minimal axonal degeneration, which was observed in peripheral nerves and DRG axons. The degree of axonal degeneration observed in these vehicle-treated animals at day 120 was lower in the low-dose group (3.0 × 10⁻⁶). 12 GC, group 6) 3 / 3 animals and medium-dose group (1.0 × 10 13 The degree of peripheral nerve and DRG axons was comparable to that of one-third of the animals in group GC (7). Comparing peripheral nerve axonal degeneration across multiple time points, the incidence and severity increased from day 60 to day 120 in all groups, indicating a time-dependent response, although the difference was less pronounced with moderate dose (1.0 × 10⁻⁶). 13 It was the most dramatic in GC.

[0253] The significant difference in peripheral nerve findings at day 120 compared to day 60 was in the medium-dose group (1.0 × 10⁻⁶). 13 GC, group 7, 2 / 3 animals) and high-dose group (3.0 × 10) 13 Perisonal fibrosis (minimal to prominent) was observed only in GC, group 8, 3 / 3 animals. In periaxonal fibrosis, a dose-dependent response was observed, but in the medium-dose group (1.0 × 10⁻⁶), 13 The GC group (group 7) showed the highest severity. Considering the absence of periaxonal fibrosis at day 60, a time-dependent response was observed.

[0254] To further evaluate periaxonal fibrosis observed by H&E staining, Masson trichrome staining was performed. This stain highlights fibrous connective tissue from surrounding muscles and other tissues. The proximal portions of the left and right median nerves were selected for trichrome staining due to their large circumference, allowing for additional re-transection. Since periaxonal fibrosis was absent in all animals at day 60, trichrome staining was performed on all animals at day 120. Semi-quantitative scoring of trichrome staining by blinded evaluators was as follows: moderate dose group (1.0 × 10⁶). 13 GC, group 7, 2 / 3 animals) and high-dose group (3.0 × 10) 13 The presence of dose-dependent periaxonal fibrosis was confirmed in the GC group (8 animals, 3 / 3 of which were 3 animals). Severity was 1.0 × 10⁻⁶ in the moderate-dose group. 13 In the GC (Group 7, 3 / 3 animals) group, the effects ranged from none to significant, while in the high-dose group (3.0 × 10⁻⁶ 13 In GC (group 8, 3 / 3 animals), the range was minimal to significant. Consistent with findings based on H&E, the most severe periaxonal fibrosis (moderate to significant) occurred in 1 / 3 animals (animals 17-226, 1.0 × 10⁻⁶) at moderate doses. 13 GC, Group 7), at high doses 1 / 3 animal (animal 17-205, 3.0 × 10) 13 This occurred in GC (group 8), and this correlated with a significant decrease in SNAP amplitude observed in these animals from day 28 to day 120. Furthermore, quantification of trichrome staining using VIS image analysis software indicated that low doses (3.0 × 10⁻⁶) were associated with this condition. 12 Compared to the GC (group 6) and vehicle therapy group (ITFFB, group 5), the moderate dose (1.0 × 10) 13 GC, group 7) and high-dose group (3.0 × 10) 13 In the GC group (8), a dose-dependent decrease in nerve tissue volume and a dose-dependent increase in white space within tissue sections were observed. These findings were observed in the medium-dose group (1.0 × 10⁻⁶). 13 GC, group 7) and high-dose group (3.0 × 10) 13 This showed axonal loss in GC, group 8).

[0255] Other findings related to test items in CNS include one animal (animal 17-216 [3.0 × 10)) administered a high dose. 13 The GC group (group 4) at day 60 included mild gliosis and satellite lesions in the ventral angle of the lumbar spinal cord. Minimal gliosis, with or without satellite lesions, was sporadically observed in the brains of animals across all rAAV.hGLB1 treatment groups at both time points. At day 60, particularly in animals 17-213, the high-dose group (3.0 × 10⁻⁶) 13 In the GC (Group 4, 2 / 3 animals) group, the low-dose group (3.0 × 10⁻⁶) 12 GC, group 2, 1 / 3 animal) and medium-dose group (1.0 × 10) 13 Compared to GC (group 3, 1 / 3 animals), the incidence of gliosis was slightly higher, regardless of the presence or absence of satellite lesions. At day 120, sporadic minimal perivascular infiltration and small gliotic lesions were observed across all rAAV.hGLB1 treatment groups, but the incidence of these findings decreased at day 120 compared to day 60, suggesting degradation.

[0256] Local injection site findings in skeletal muscle and adipose tissue across the ICM / CSF harvesting site were observed in all groups, including vehicle-treated animals (ITFFB, group 1), at day 60. However, at day 60, the composition of the infiltrates changed, with increased severity in rAAV.hGLB1-treated animals. At day 60, the infiltrates in the vehicle-treated group (ITFFB, group 1, 1 / 2 animals) consisted mostly of histiocytes (minimal), while GTP-203-treated animals had minimal to mild muscle fiber changes, or no changes at all, and mainly consisted of lymphocytes and plasma cells (minimal to moderate). Muscle fiber changes, including degeneration and atrophy, were observed only at day 60 in the high-dose group (3.0 × 10⁻⁶). 13 GC, group 4, 1 / 3 animals). At day 120, all rAAV.hGLB1-treated animals were receiving a low dose (3.0 × 10⁻⁶). 12 Minimum to medium dose (1.0 × 10) in GC, group 6, 3 / 3 animals. 13 GC, group 7, 3 / 3 animals) and high dose (3.0 × 10) 13Minimum in GC, group 8, 3 / 3 animals. Mononuclear cell infiltration was observed in skeletal muscle and / or adipose tissue ranging from minimal to mild, which likely suggests a dose-dependent response. The severity of injection site findings at day 120 (minimal to mild) decreased compared to the severity observed at day 60 (minimal to moderate, with muscle fiber changes), indicating degradation and suggesting a time-dependent response. These findings are likely attributable to the initial injection, possibly due to contributions from repeated CSF harvesting, but components attributable to a local response to the test material may also have been present.

[0257] Vector pharmacokinetics and excretion After ICM administration, rAAV.hGLB1 vector DNA was detectable in CSF and peripheral blood, and the peak concentration in CSF correlated with the dose. The concentration of rAAV.hGLB1 in CSF decreased rapidly after the first time point evaluated (day 7), and in one animal (animal 17-212 [3.0 × 10⁻¹⁰]) in the high-dose group. 13 With the exception of GC (group 8), it was not detected in most animals by day 60. At necropsy on day 60, the concentration of rAAV.hGLB1 vector DNA in CSF showed a decreasing trend. The concentration of rAAV.hGLB1 vector DNA in the blood decreased more slowly, which may be due to transduction of peripheral blood cells.

[0258] On day 0, two animals in the high-dose group (animals 17-197 and 17-205 [3.0 × 10 13 rAAV.hGLB1 vector DNA was detected in CSF from group 8 (GC), but not in the blood. On day 0, the rAAV.hGLB1-positive CSF samples were retested, and the results were confirmed. The detection of rAAV.hGLB1 vector DNA in CSF on day 0 is highly likely to be due to contamination of the CSF sample during the ICM administration procedure.

[0259] rAAV.GLB1 vector DNA was detectable in urine and feces on day 5 after vector administration. Peak levels were generally proportional to the administered dose. rAAV.hGLB1 vector DNA was not detectable in the urine and feces of any animals by day 60 after vector administration.

[0260] Evaluation of transgene expression Human β-gal activity was measured in CSF and serum. Briefly, 1–10 μL of either CSF or serum was mixed in a 96-well black plastic assay plate with 99 μL of reaction mixture (0.5 mM 4-methylumbelliferyl β-D-galactopyranoside [Sigma M1633], 0.15 M NaCl, 0.05% Triton-X100, and 0.1 M sodium acetate, pH 3.58). The plate was sealed and incubated at 37°C for 30 minutes, and the reaction was stopped by adding 150 μL of stop solution (290 mM glycine and 180 mM sodium citrate, pH 10.9). Fluorescence from the reaction product was measured at an emission wavelength of 450 nm when excited at 365 nm.

[0261] Transgene product expression (β-gal activity) in major organs could not be evaluated due to the high levels of endogenous rhesus macaque β-gal enzyme activity in normal NHP. Lower levels of endogenous rhesus macaque β-gal enzyme are present in CSF and serum, enabling transgene expression analysis of CSF at days 0, 7, 14, 28, 60, 90, and 120, as well as baseline and serum at days 14, 28, 60, 90, and 120. However, it should be noted that the analysis of transgene product activity in CSF and serum of NHP was limited by the nature of the assay and could not distinguish between human β-gal enzyme and endogenous rhesus macaque β-gal enzyme. This limited sensitivity and the need to use baseline endogenous β-gal activity levels for each animal for analysis (shown by dashed lines in Figures 22A-22D). The rapid loss of transgene product activity after day 14 is related to human transgene products. The analysis was complex because it was highly likely to be caused by an antibody response (Figure 22A).

[0262] Despite these precautions, β-gal activity in CSF and serum was detected above baseline levels in animals from all dose groups 14 days after rAAV.hGLB1 administration (Figure 22B). In CSF, two higher doses (1.0 × 10⁶) were detected. 13 GC[1.1×10 11 GC / g brain] or 3.0 × 10 13 GC[3.3×10 11 Animals receiving GC / g brain showed β-gal activity levels approximately 2-fold and 4-fold higher, respectively, than those of the vehicle treatment control. Furthermore, expression in the CSF was unaffected by the presence of pre-existing NAbs against the vector capsid, which supports the possibility of achieving therapeutic activity in the target organ system (CNS) in infant / late infant GM1 patients regardless of NAb status.

[0263] In serum, animals lacking pre-existing NAbs against the vector capsid (indicated by white outlines in Figure 22B) tended to have higher β-gal enzyme activity compared to either vehicle-treated controls or animals positive for pre-existing NAbs against the vector capsid (indicated by black outlines in Figure 22B). This result suggests potential therapeutic activity in peripheral organs of NAb-negative infants / late infant GM1 patients.

[0264] In vivo distribution: At autopsy, tissue was collected for in vivo distribution, placed on dry ice in labeled vials, and stored at -60°C or below before analysis. DNA was extracted from tissue by trained practitioners and performed TaqMan qPCR according to SOP3001. Briefly, tissue was mechanically homogenized and digested with proteinase K. Samples were treated with RNAse A, and cells were lysed by incubation in buffer AL (catalog no. #19075, QIAGEN) at 70°C for 1 hour. DNA was extracted and purified on a QIAGEN spin column. After dilution to concentrations of ≥90 ng / μl and ≥110 ng / μl, qPCR was performed in duplex using vector-specific primers and / or transgene-specific primers. Signals were compared to standard curves of washed plasmid DNA against a background of known concentrations of DNA from naive or negative control animals from the same test. Genomic copies per microgram of DNA were calculated. Additional controls were used to exclude cross-contamination and sample interference in the PCR reaction. Raw data were analyzed based on predefined acceptance criteria for Ct values, and the limit of quantification was determined for each run. All data were included in and / or attached to batch record forms.

[0265] The vector genome was detected at high levels in the brain, spinal cord, DRG, liver, and spleen at day 60 (Figure 23) and day 120 (Figure 24), which is consistent with previous studies of ICM AAV administration. The amount of vector genome detected in CNS tissue was generally observed to be dose-dependent. The vector genome in CNS tissue appeared to be stable between 60 and 120 days post-administration. At day 120, the medium-dose group (1.0 × 10⁶) 13 All three animals registered in GC (group 7) had baseline NAb for AAVhu68, which correlated with very low vector distribution to the liver. Vector genomes were detected in several samples from two vehicle-treated control animals (animals 17-199 [group 1] and 17-204 [group 5]). These samples were tested twice to confirm the presence of vector genomes.

[0266] Conclusion: ICM administration of rAAV.hGLB was well tolerable at all evaluated doses. rAAV.hGLB did not cause adverse effects on clinical and behavioral signs, body weight, or neurological and physical examination. In some animals, mild transient CSF leukocytes were observed. Aside from an increase, there were no other clinicopathological abnormalities in blood and CSF associated with rAAV.hGLB administration.

[0267] rAAV.hGLB administration induced asymptomatic degeneration of TRG and DRG sensory neurons, as well as their associated central and peripheral axons. The severity of these lesions was typically minimal to mild. These findings were observed in the medium-dose group (1.0 × 10⁻⁶). 13 GC) and high-dose group (3.0 × 10) 13 In GC (Generalized Clinical Practice), there was a tendency towards more severe lesions, and this was dose-dependent.

[0268] Degeneration of sensory neuron cell bodies was less severe at day 120 than at day 60. While this result indicated that these lesions were not progressive, subsequent axonal degeneration and fibrosis may continue to progress over several months. Consistent with these findings, the two animals (animals 17-226 and 17-205) that showed the most severe axonal loss and median nerve fibrosis at autopsy at day 120 showed decreased median nerve sensory action potential amplitude by day 28, but did not progress further. NOAEL could not be defined as asymptomatic sensory neuron lesions were present in all treatment groups. The maximum dose evaluated was 3.0 × 10⁻⁶. 13 GC) was considered MTD. Two animals showing the most severe axonal loss and fibrosis, with reduced sensory nerve action potentials, are indicated by arrows. (Figures 18A-18B, 19A-19B. Figures 20A-20B show changes in median sensory nerve conduction at each measurement point during the test, as measured by median sensory action potentials in microvolts.)

[0269] Transgene expression (i.e., β-gal enzyme activity) in CSF and serum was detectable above baseline levels in animals from all dose groups 14 days after ICM administration of rAAV.hGLB. In CSF, animals receiving the two higher doses (1.0 × 10¹³ GC or 3.0 × 10¹³ GC) showed β-gal activity levels approximately 2-fold and 4-fold higher, respectively, than those of the vehicle treatment control. Expression in CSF was unaffected by the presence of pre-existing NAbs against the vector capsid, which supports the potential to achieve therapeutic activity in the target organ system (CNS) in infant / late infant GM1 patients regardless of NAb status.

[0270] ICM administration of rAAV.hGLB induced vector distribution in the CSF, as well as high levels of gene transfer into the brain, spinal cord, and DRG. rAAV.hGLB also reached significant concentrations in peripheral blood and the liver.

[0271] Evaluation of rAAV.hGLB DNA excretion showed detectable vector DNA in urine and feces 5 days after administration, and it reached undetectable levels within 60 days.

[0272] T cell responses to vector capsids and / or human transgene products were detectable in PBMCs and / or tissue lymphocytes (liver, spleen, bone marrow) in the majority of rAAV.hGLB-treated animals. The T cell responses were generally not associated with any abnormal clinical or histological findings.

[0273] Existing NAbs against vector capsids were detectable in several animals and did not appear to affect gene delivery to the brain and spinal cord; however, the presence of existing NAbs correlated with a significant reduction in hepatic gene delivery.

[0274] Example 5: Phase 1 / 2 open-label multicenter dose-escalation study to evaluate the safety and tolerability of a single dose of rAAVhu68.GLB1 delivered to the cisterna magna (ICM) in a child with infantile GM1 gangliosidosis. Enroll GM1 subjects up to 24 months of age who developed symptoms in the first 18 months. This includes, The study includes subjects with type 1 (infant) GM1 and type 2a (late infant) GM1. Subjects with type 1 (infant) GM1 may present with symptoms at birth. Therefore, treatment should be initiated as early as possible to maximize potential benefits, and this trial includes subjects at least one month of age. Another consideration in selecting the lower age limit is to ensure that the ICM procedure can be performed safely. The proposed ICM procedure includes preoperative brain MRI and MR angiography and CT / CTA-guided ICM injection. There are no age-specific safety concerns in administering ICM to infants older than one month of age.

[0275] ICM vector administration results in immediate vector distribution within the CNS compartment. Therefore, the clinical dose is determined by scaling based on brain mass, providing an approximation of the CNS compartment size. Both efficacy and toxicity are expected to be related to CNS vector exposure. Dose conversion is based on brain mass ranges of 0.4 g in juvenile adult mice, 90 g in juvenile and adult rhesus monkeys (Herndon 1998), and 370 g to 1080 g in human infants aged 0–30 months (Dekaban, 1978). Nonclinical and corresponding human doses are shown in the table below.

[0276] [Table 9]

[0277] Considering the differences in brain weight (for example, there is approximately a threefold difference between newborns and 2-year-old subjects), a slide scale is used to determine the amount of drug product administered to individual subjects in FIH studies (in units of gene copies [GC]) based on the published mean brain weight of infants and children up to 24 months of age. In this way, subjects are administered the volume of drug product closest to the intended dose in terms of estimated grams of gene copies / brain weight. [Table 10] [Table 11] [Table 12]

[0278] This study is a Phase 1 / 2 open-label dose-escalation study of AAVhu68.GLB1 to evaluate safety, tolerability, and exploratory efficacy endpoints after a single dose of AAVhu68.GLB1 delivered via the incisor macromolar (ICM) in children with infantile GM1 (type 1) or late infantile (type 2a) dysplasia. Up to 24 children will be enrolled in this study to receive a single dose of AAVhu68.GLB1 administered via the ICM.

[0279] Type 1 (infant) GM1 • GM1 subjects before symptomatic onset (under 6 months of age, with confirmed mutation and decreased serum β-gal activity) were identified through prenatal screening or by a family history of older siblings with the same genotype and a confirmed diagnosis of GM1 gangliosidosis. Siblings must have developed symptoms by 6 months of age or younger.

[0280] Symptomatic GM1 subjects (those with confirmed mutations and decreased serum β-gal activity) must have hypotonia or any documented symptoms consistent with GM1 gangliosidosis, and at least 70% age-adjusted expected motor development (BSID-III) at the time of medication, and have medical record documentation of onset at 6 months of age or younger.

[0281] Type 2 (Late-stage infant) GM1 Symptomatic subjects with onset between 6 months and 18 months of age, exhibiting any documented symptoms consistent with GM1 gangliosidosis, including hypotonia or plateauing or delay in achieving further developmental milestones, and having at least 70% age-corrected expected motor development (BSID-III).

[0282] Two doses of rAAVhu68.GLB1 will be evaluated in alternating sequential administration to the subjects. The rAAVhu68.GLB1 dose levels will be determined based on data from mouse MED and GLP NHP toxicity studies, consisting of a low dose (administered to cohort 1) and a high dose (administered to cohort 2). The high dose is based on the maximum tolerable dose (MTD) in the NHP toxicity study scaled to equivalent human doses. A safety margin will be applied so that the high dose selected for human subjects is one-third to one-half of the equivalent human dose. The low dose is typically two to three times lower than the selected high dose, as long as it exceeds the MED scaled to equivalent in the animal study. This ensures that both dose levels have the potential to provide therapeutic benefits, and that the higher dose is advantageous if tolerated. It is expected that this will be the case. Sequential evaluation of low dose followed by high dose will allow for the identification of the maximum tolerated dose (MTD) of the two doses tested. Finally, the expanded cohort (Cohort 3) will accept the MTD of rAAVhu68.GLB1. Six subjects in Cohort 3 (MTD) will be enrolled simultaneously without staggered dosing. Cohort 3 may receive combination therapy with hematopoietic stem cell transplantation (HSCT) and rAAVhu68.GLB1. If tolerated, higher doses are expected to be advantageous.

[0283] The primary focus of this trial is to evaluate the safety and tolerability of rAAVhu68.GLB1. Since NHP studies of ICM AAVhu68 delivery have demonstrated minimal to mild asymptomatic degeneration of DRG sensory neurons in some animals, detailed examinations will be conducted to assess sensory neurotoxicity, and sensory nerve conduction studies will be used in this trial to monitor asymptomatic sensory neuronal lesions. Notably, sensory neuronal loss (attributable to potential dorsal root ganglion toxicity) will be assessed by sensory nerve conduction studies performed at 30 days, 3 months, 6 months, 12 months, 18 months, and 24 months, and thereafter at annual intervals. Given that sensory neuronal lesions appeared within 2–4 weeks post-AAV administration in nonclinical NHP studies, more frequent assessments throughout the 3 months post-treatment will allow for the evaluation of similar events in humans and enable assessment of potential variability in toxicity kinetics. By conducting follow-up observations throughout this trial, we will be able to evaluate the delayed effects when the time course differs in humans or when clinical complications are observed, and assess how long they last, improve over time, remain stable, or worsen.

[0284] In this trial, pharmacokinetic and efficacy endpoints were also evaluated and selected for their potential to demonstrate meaningful functional and clinical outcomes in this population. Endpoints were measured at 30, 90, 6, 12, 18, and 24 months, except for those requiring sedation and / or LP, and thereafter annually for a maximum follow-up period of 5 years. In the long-term follow-up phase, the measurement frequency was reduced to once every 12 months. These time points were selected to facilitate a complete assessment of the safety and tolerability of rAAVhu68.GLB1. Early time points and 6-month intervals were also selected considering the rapid rate of disease progression in untreated infant GM1 patients. This approach allows for a complete assessment of pharmacokinetic and clinical efficacy measures in the treated population over a follow-up period where untreated comparative data are available and a significant reduction in untreated patients is expected.

[0285] Secondary and exploratory efficacy endpoints include survival, independence from feeding tubes, seizure incidence and frequency, quality of life as measured by PedsQL, and neurocognitive and behavioral development. The Bailey Infant Development and Vineland Scales will be used to quantify the impact of rAAVhu68.GLB1 on development and / or change in adaptive behavior, cognition, language, motor function, and health-related quality of life. Each measure will be used in either the GM1 disease population or an associated population, further refined based on parental and family input, and the most meaningful and influential measure for them will be selected. To standardize assessments, participants in the trial will be trained on administering various measures by experienced neuropsychologists.

[0286] Considering the severity of the disease in the target population, participants may have achieved motor skills by the time of enrollment, developed other motor milestones and subsequently lost them, or have not yet shown signs of development of a motor milestone. Assessment tracks age at achievement and age at loss for all milestones. Motor milestone achievement is defined by six gross milestones based on WHO criteria.

[0287] Subjects with infant GM1 gangliosidosis may develop symptoms within the first few months of life. Given the possibility that the first WHO motor milestone (sitting without support) is not typically achieved before 4 months of age (median: 5.9 months), this endpoint may lack sensitivity to assess the degree of therapeutic benefit, particularly in subjects with more pronounced symptoms at the time of treatment. Therefore, assessment of age-appropriate developmental milestones applicable to infants is also necessary (Scharf et al., 2016, Developmental Milestones. Pediatr Rev. 37(1):25-37; quiz 38, 47). These data may be useful for summarizing the retention, acquisition, or loss of developmental milestones over time compared to typical acquisition times in untreated or typically developing children with infant GM1 disorder.

[0288] As the disease progresses, children may experience seizures. The onset of seizure activity allows for the determination of whether treatment with rAAVhu68.GLB1 will prevent or delay the onset of seizures, or reduce the frequency of seizure events in this population. Parents are asked to keep a seizure diary, tracking the onset, frequency, duration, and type of seizures. These entries will be reviewed and interpreted by the clinician at each visit.

[0289] To evaluate the effect of rAAVhu68.GLB1 on disease-related volume changes in CNS signs, measurements were performed over time by MRI. The phenotype of all gangliosidosis infants has been shown to have a consistent pattern with macrocephaly and rapid increases in intracranial MRI volume, both in brain tissue volume (cerebral cortex and other smaller structures) and ventricular volume. In addition, various small brain substructures, including the corpus callosum, caudate nucleus, and putamen, as well as the cerebellar cortex, generally decrease in size with disease progression (Regier et al., 2016, and Nestrasil et al., 2018, cited herein). Treatment with rAAVhu68.GLB1 is expected to slow or halt the progression of symptoms of CNS disease, with evidence of stabilization in atrophy and volume changes. Exploratory endpoints evaluating changes in T1 / T2 signal intensity (normal / abnormal) in the thalamus and basal ganglia are based on reported evidence regarding changes in thalamic structure in patients with GM1 and GM2 gangliosidosis (Kobayashi and Takashima, 1994, Thalamic hyperdensity on CT in infantile). GM1-gangliosidosis.Brain and Development.16(6):472-474).

[0290] Biomarkers for clinical trials include β-gal enzyme (GLB1) activity, which can be measured in CSF and serum, as well as brain MRI showing consistent and rapid atrophy in infantile GM1 gangliosidosis (Regier et al., 2016b, cited herein). Additional biomarkers will be investigated in CSF and serum derived from recovered samples.

[0291] A. Main purpose: To evaluate the safety and tolerability of rAAVhu68.GLB1 over two years following a single dose administration to the incisor meristem (ICM), adverse events, neurological examinations, sensory nerve conduction studies, total Neuropathy Score-Nurse, hematology, serological chemistry, liver function tests, coagulation (PT, aPTT, INR), troponin-If, CSF anti-AAVhu68 nAb, vector loss, urinalysis, seizure diaries, physical examination, vital signs, ECG, brain MRI, and CSF cytology and chemistry (cell count, protein, glucose) will be assessed over five years.

[0292] • Evaluate the efficacy of rAAVhu68.GLB1 after a single dose administration to the cisterna magna. The primary secondary endpoints* will be evaluated at 2 and 5 years. ● Vineland Adaptive Behavior Scale, 2nd Edition ●Other secondary endpoints will be evaluated over the second and fifth years. ● Bailey's Infant Development Scale, 3rd Edition ● WHO Multicenter Growth Reference Study Performance Criteria ● Developmental milestone assessment ● Hammersmith Infant Neurological Examination ●Global Impressions of Severity and Changes Among Clinicians and Caregivers ●Interview * No clinical outcome assessment suitable for this purpose exists for GM1 gangliosidosis. Therefore, in parallel with the conduct of this trial, the sponsor is working with experts in the subject to collect data from clinical experts and parents / caregivers to identify the primary efficacy endpoint for Cohort 3 and, where necessary, develop outcome measurement strategies, including designing composite endpoints derived from the above scales, modifying existing COAs, or developing patient-centered GM1-specific supplemental items or scales. See the Statistical Analysis section for further details.

[0293] B. Secondary objectives: • Evaluate the pharmacokinetics and biological activity of rAAVhu68.GLB1 over 24 months following a single dose to the cisterna magna. Evaluation: CSF biomarkers: β-galactosidase activity, hexosaminidase activity, GM1 ganglioside levels; serum biomarkers: β-galactosidase activity, hexosaminidase activity; urinary biomarkers: keratan sulfate levels; all evaluated at day 30 and over 5 years.

[0294] • Evaluate the effect of rAAVhu68.GLB1 on disease progression after a single dose administration to the cisterna magna. Evaluation: Total brain volume, volume of hypocerebral structures, and ventricular volume as well as T1 / T2 signal intensity measured by MRI; skeletal abnormalities measured by lateral spinal X-ray; cardiomyopathy measured by echocardiography; hepatosplenomegaly measured by abdominal ultrasound; brain function and diffuse delayed changes measured by continuous electroencephalography; assessment of survival without mechanical ventilation; and assessment of nutritional status based on the need for placement and use of feeding tubes. All of these will be evaluated over a 5-year period.

[0295] To evaluate the effects of a single dose of rAAVhu68.GLB1 administered to the cisterna magna on quality of life and resource utilization. Assessment: Quality of life: Pediatric Quality of Life Survey / Infant Scale of the Pediatric Quality of Life Survey; Resource utilization: Chart review of clinic days, ER visits, ICU admissions, surgeries, interviews, and need for visual aids, all assessed over a 5-year period.

[0296] C. Test Design: A multicenter, open-label, single-arm, dose-escalation study of rAAVhu68.GLB1 (see table below). A total of 12 pediatric subjects with infantile GM1 gangliosidosis were enrolled in two dose cohorts and received a single dose of rAAVhu68.GLB1 administered by ICM injection. Safety and tolerability were evaluated over 2 years, and all subjects were followed up for 5 years after administration of rAAVhu68.GLB1 to long-term evaluate safety and tolerability, pharmacokinetics (durability of transgene expression), and durability of clinical outcomes. [Table 13]

[0297] AAVhu68.UbC.GLB1 is supplied frozen (-60°C or below) as a sterile solution in ITFFB (Intrathecal Final Preparation Buffer). Depending on the target dose level and age group, dilution of AAVhu68.UbC.GLB1 DP with ITFFBD01 (test drug diluent) may be required before administration. The AAVhu68.UbC.GLB1 DP and ITFFBD01 preparations consist of 1 mM sodium phosphate, 150 mM sodium chloride, 3 mM potassium chloride, 1.4 mM calcium chloride, 0.8 mM magnesium chloride, 0.001% poloxamer 188, and pH 7.2.

[0298] Potential participants will be screened between 35 days prior to administration and 1 day prior to administration to determine their eligibility for the study. Up to 24 pediatric participants with type 1 (infant) and type 2a (late infancy) GM1 gangliosidosis will be enrolled in this study. Participants meeting the inclusion / exclusion criteria will be admitted to the hospital on the morning of day 1 or according to the facility's medical procedures. Participants will receive a single ICM dose of rAAVhu68.GLB1 on day 1 and remain in the hospital for at least 24 hours post-administration for observation. Subsequent evaluations will be conducted 7, 14, and 30 days after administration, then every 60 days in the first year and every 90 days in the second year. The safety and tolerability of rAAVhu68.GLB1 are monitored through assessment of adverse events (AEs) and serious adverse events (SAEs), vital signs, physical examination, sensory nerve conduction studies, and clinical laboratory tests (chemistry, hematology, coagulation tests, CSF analysis). The immunogenicity of AAV and the transgene product is also evaluated. Efficacy evaluation includes survival rates, measurements of cognitive, motor, and social development, changes in visual function and electroencephalography (EEG), changes in liver and spleen volume, and biomarkers in CSF, serum, and urine.

[0299] This study consists of the following three cohorts, in which rAAVhu68.GLB1 was administered as a single ICM injection. • Cohort 1 (low dose): Three eligible subjects (subjects #1-#3) will be enrolled and administered a low dose of rAAVhu68.GLB1 with a 4-week safety observation period between subjects #1 and #2. If no safety review trigger (SRT) is observed, all available safety data will be evaluated by an independent safety board 4 weeks after subject #3 of Cohort 1 receives rAAVhu68.GLB1.

[0300] • Cohort 2 (high dose): If progression is determined, three eligible subjects (subjects #4-#6) will be enrolled and administered a high dose of rAAVhu68.GLB1 with a 4-week safety observation period between subjects 4 and 5. If no SRT is observed, an independent safety committee will evaluate all available safety data, including safety data from subjects in Cohort 1, 4 weeks after subject 3 of Cohort 2 receives rAAVhu68.GLB1.

[0301] • Cohort 3 (MTD): If the Safety Committee has not yet made a positive recommendation, up to 6 additional subjects will be enrolled and administered a single ICM dose of rAAVhu68.GLB1 in the MTD. Administration to subjects in this cohort must be conducted without delay within the 4-week safety observation period between subjects, and the Safety Committee must review the administration after the first 3 subjects in this cohort have received the drug.

[0302] D. Inclusion Criteria: 1. At the time of registration, the animal is between 1 month and 24 months old, and has type 1 (onset at 6 months or earlier) or type 2a (onset between 6 months and 18 months).

[0303] a. Type 1 infant GM1 i. Subjects who were symptomatic (under 6 months of age, with confirmed mutation and decreased serum β-gal activity) were identified by prenatal screening or by a family history of older siblings with the same genotype and a confirmed diagnosis of GM1 gangliosidosis. Siblings must have developed symptoms by 6 months of age or younger.

[0304] or ii. Symptomatic subjects (with confirmed mutations and decreased serum β-gal activity) must have hypotonia or any documented symptoms consistent with GM1 gangliosidosis, and at least 70% age-adjusted expected motor development (BSID-III) at the time of medication, and have medical record documentation of onset at 6 months of age or younger.

[0305] b.2a Late-stage infant GM1: i. Symptomatic subjects with onset between 6 months and 18 months of age, exhibiting any documented symptoms consistent with GM1 gangliosidosis, including hypotonia or plateauing or delay in achieving further developmental milestones, and having at least 70% age-corrected expected motor development (BSID-III).

[0306] 2. The subjects have been documented to be homozygous or compound heterozygous due to GLB1 gene deletion or mutation, and reduced β-gal activity (less than 20% of the lower normal value in leukocytes).

[0307] E. Exclusion Criteria: 1. Any clinically significant neurocognitive deficit not attributable to GM1 gangliosidosis, or any other condition that, in the opinion of the principal investigator, could cause confusion in interpreting the study results.

[0308] 2. If any participant has an acute illness requiring hospitalization within 30 days of registration, the medical history of that participant must be discussed with the clinical trial sponsor's medical monitor before permitting their registration.

[0309] 3. History of ventilatory support or respiratory assistance, or need for tracheostomy.

[0310] 4. Intractable seizures or uncontrolled epilepsy, defined as having had an epileptic episode or a seizure requiring hospitalization within 30 days prior to administration of the investigational drug.

[0311] 5. Any contraindications to the ICM administration procedure, including contraindications to fluoroscopic imaging and anesthesia.

[0312] 6. Any contraindications to MRI or LP.

[0313] 7. Previous gene therapies.

[0314] 8. Use of miglustat within 48 hours prior to administration of the investigational drug.

[0315] 9. Use of enzyme replacement therapy or other investigational drug therapy within 5 half-lives prior to administration of the investigational drug.

[0316] 10. Any condition that, in the opinion of the principal investigator, could unduely expose the subject to risk during the procedure, interfere with the evaluation of the investigational drug, or interfere with the interpretation of the subject's safety or the results of the study (e.g., medical history, evidence of current disease, findings on physical examination, or any laboratory anomaly). This includes: a. Abnormal laboratory values ​​that the principal investigator determined to be clinically significant b. A decrease in the 20th percentile (20 / 100) of body weight at 3 months prior to screening / baseline, as defined below. c. Fundamental defects in immune function d. A history of multiple severe, life-threatening infections.

[0317] F. Route of administration and procedure rAAVhu68.GLB1 is administered as a single dose to subjects on day 1 via CT-guided suboccipital injection into the cisterna magna.

[0318] On day 1, rAAVhu68.GLB1 at the appropriate concentration will be prepared by the investigational pharmacy department associated with the study. A syringe containing 5.6 mL of rAAVhu68.GLB1 at the appropriate concentration will be delivered to the treatment room. The following persons will be present during the administration of the investigational drug: the intervening physician, anesthesiologist and respiratory technician, nurse and medical assistant, CT (or operating room) technician, and site research coordinator.

[0319] Prior to administering the test drug, a lumbar puncture is performed to remove a predetermined volume of CSF, and then an iodine contrast agent is injected intrathecally (IT) to aid in visualization of the relevant anatomical structures of the cisterna magna. Intravenous (IV) contrast agents may be administered before or during needle puncture as an alternative to intrathecal contrast agents. The decision of whether to use IV or IT contrast agents is left to the discretion of the intervening physician. The subject is anesthetized, intubated, and placed on a treatment table. The injection site is prepared and covered using sterile techniques. Under fluoroscopy guidance, a spinal needle (22-25G) is advanced into the cisterna magna. A larger introducer needle may be used to assist in needle placement. After confirming needle placement, an extension set is connected to the spinal needle and filled with CSF. At the discretion of the intervening physician, a syringe containing contrast material may be connected to the extension set and a small amount injected to confirm needle placement in the cisterna magna. After confirming needle placement with CT guidance + / - contrast agent injection, connect a syringe containing 5.6 mL of rAAVhu68.GLB1 to the extension set. Slowly inject the contents of the syringe over 1-2 minutes to deliver a volume of 5.0 mL. Slowly withdraw the injection needle from the subject.

[0320] A single dose of GLB1 administered into the intracisor major cell (ICM) is safe and tolerable throughout the 5-year period following administration.

[0321] A single dose of rAAVhu68.GLB1 into the cisterna magna (ICM) improves survival rates, reduces the probability of tube feeding at 24 months of age, and / or reduces disease progression as assessed by age at achievement, age at loss, and the percentage of children who maintain or achieve age-appropriate developmental and motor milestones.

[0322] Treatment slows the loss of neurocognitive function.

[0323] To prevent potential immune-mediated injuries such as hepatotoxicity, patients will receive systemic corticosteroids. Systemic corticosteroids equivalent to oral prednisolone will be administered at a dose of 1 mg / kg body weight per day for approximately 30 days (or until the scheduled 1-month follow-up visit, whichever comes first), starting one day prior to rAAVhu68.GLB1 administration. During this visit, clinical and laboratory tests should be performed according to the evaluation schedule. For patients with insignificant findings, the principal investigator should reduce the corticosteroid dose over the next 21 days, based on clinical judgment. The daily dose should begin at 0.75 mg / kg during week 5, then 0.5 mg / kg during week 6, and subsequently 0.25 mg / kg during week 7. If a patient does not adequately respond to the 1 mg / kg / day regimen, consult a specialist. In the opinion of the principal investigator, if a subject develops clinical symptoms or clinical / laboratory signs of potential immunomediated toxicity, the dosage, type, and schedule of immunosuppression must be changed and the principal investigator notified. Regular vaccination schedules and local guidelines must be followed, including recommendations for adjusting the timing of vaccinations while the subject is receiving steroid treatment.

[0324] Example 6: Phase 1 / 2 open-label multicenter dose-escalation study to evaluate the safety and tolerability of a single dose of rAAVhu68.GLB1 delivered to the cisterna magna (ICM) in a child with infantile GM1 gangliosidosis. Enroll GM1 subjects up to 24 months of age who developed symptoms in the first 18 months. This includes subjects with type 1 (infant) GM1 and type 2a (late infant) GM1. Subjects with type 1 (infant) GM1 may show symptoms at birth. Therefore, treatment should be initiated as early as possible to maximize potential benefits, and this trial will include subjects at least 1 month of age. Another consideration in selecting the lower age limit is to ensure that the ICM procedure can be performed safely. The proposed ICM procedure includes preoperative brain MRI and MR angiography and CT / CTA-guided ICM injection. There are no age-specific safety concerns in administering ICM to infants older than 1 month of age.

[0325] ICM vector administration is CNS This results in immediate vector distribution within the compartment. Therefore, the clinical dose is determined by scaling according to brain mass. Provides an approximate value for the size of the CNS compartment. Effectiveness and Both toxicity is expected to be related to CNS vector exposure. Dose conversion was performed at 0.4g in juvenile adult mice and juvenile and adult rhesus monkeys (Herndon). The 1998 study was based on a brain mass of 90g, and the 1978 study was based on brain mass in human infants aged 0-30 months with a range of 370g-1080g. Non-clinical and equivalent human doses are shown in the table below. [Table 14]

[0326] Considering the differences in brain weight (for example, there is approximately a threefold difference between newborns and 2-year-old subjects), a slide scale is used to determine the amount of drug product administered to individual subjects in FIH studies (in units of gene copies [GC]) based on the published mean brain weight of infants and children up to 24 months of age. In this way, subjects are administered the volume of drug product closest to the intended dose in terms of estimated grams of gene copies / brain weight. [Table 15] [Table 16] [Table 17]

[0327] This study is a Phase 1 / 2 open-label dose-escalation study of AAVhu68.GLB1 to evaluate safety, tolerability, and exploratory efficacy endpoints after a single dose of AAVhu68.GLB1 delivered via the incisor macromolar (ICM) in children with infantile GM1 (type 1) or late infantile (type 2a) dysplasia. Up to 28 children will be enrolled in this study and will receive a single dose of AAVhu68.GLB1 administered via the ICM.

[0328] Inclusion Criteria: This study may include infants who have either type 1 (infant) GM1, characterized by a confirmed GLB1 mutation (homozygous or compound heterozygous due to GLB1 gene deletion or mutation), decreased β-gal activity (less than 20% of the lower normal value in leukocytes), age between 4 months and 24 months at registration, early onset (6 months or less), and rapid progression, or type 2a (late infant) GM1, characterized by late onset presentation (more than 6 months but 18 months or less) and slower progression.

[0329] Type 1 (infant) GM1 ●(a) Prenatal screening or family history of an older sibling with the same genotype and a history of onset before 6 months of age, with a confirmed diagnosis of GM1, or (b) a pre-symptomatic subject identified by signs of prenatal GM1 disorder, such as intrauterine growth restriction, hydrops fetalis, or placental vacuolation.

[0330] Symptomatic subjects must have a medical record documenting the onset of symptoms at 6 months of age or younger, have hypotonia and / or developmental delay and / or other signs consistent with GM1 (e.g., hepatosplenomegaly, skeletal dysplasia, cherry blossom erythema, cardiomyopathy, and rough facial features), and have at least one of the following remaining developmental skills within the past week as confirmed / observed by an inspector at the facility.

[0331] - Demonstrates the ability to intentionally move the arms and legs.

[0332] - Look at the target object continuously for at least 3 seconds.

[0333] -When held securely against the caregiver's chest, the head can be rolled from one side to the other (for example, if a child is lying on their side with their left ear against the caregiver's shoulder, they can be switched to lying on their side with their right ear against the caregiver's shoulder without assistance or repositioning).

[0334] - To voice specific feelings.

[0335] - Communication accom...

Claims

1. A therapeutic regimen useful for treating GM1 gangliosidosis in human patients, comprising the administration of a recombinant adeno-associated virus (rAAV) vector having an AAV capsid and a vector genome containing a sequence encoding human β-galactosidase under the control of a regulatory sequence that directs its expression in target cells, wherein the administration is (i) The patient is approximately 1 month to 4 months old, approximately 1.6 × 10 １３ ~Approx. 1.6×10 １４ GC, (ii) The patient is at least about 4 months old and less than 8 months old, about 2.1 × 10 １３ ~Approx. 2.1×10 １４ GC, (iii) The patient is at least 8 months old and at most 12 months old, approximately 2.6 × 10 １３ ~Approx. 2.6×10 １４ GC, or (iv) The patient is at least 12 months old, approximately 3.2 × 10 １３ ~Approx. 3.2×10 １４ A treatment regimen including a single dose intracisional cisterna macrocephala (ICM) injection containing GC.

2. The regimen according to claim 1, wherein the human β-galactosidase coding sequence includes a sequence that is at least 95% identical to the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 5, or to any one of SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 5, which codes for mature β-galactosidase from amino acids 24 to 677 of SEQ ID NO:

4.

3. The encoded human β-galactosidase, (a) Approximately amino acids 1 to 677 of SEQ ID NO: 4, and (b) The regimen according to claim 1 or 2, comprising a sequence selected from a synthetic human enzyme containing a heterologous leader sequence fused to approximately amino acids 24-677 of SEQ ID NO:

4.

4. The regimen according to any one of claims 1 to 3, wherein the vector genome further comprises a 5' inverted terminal repeat (ITR) sequence, a regulatory element derived from a human ubiquitin C (UbC) promoter, a chimeric intron, a polyA signal, and / or a 3' ITR sequence.

5. The regimen according to any one of claims 1 to 4, wherein the patient is identified as having type 1 (infant) GM1 or type 2a (late infant) GM1.

6. The regimen according to any one of claims 1 to 5, further comprising administering at least one immunosuppressive combination therapy to the patient at least one day prior to or on the day of delivery of the rAAV.

7. The regimen according to claim 6, wherein the immunosuppressive combination therapy comprises one or more corticosteroids.

8. The regimen according to claim 6 or 7, wherein the immunosuppressive combination therapy comprises oral prednisolone.

9. The regimen according to claim 8, wherein oral prednisolone is administered at a dose of approximately 1 mg / kg body weight.

10. The regimen according to any one of claims 5 to 9, wherein the administration of at least one immunosuppressive combination therapy is continued for at least 3 to 4 weeks after the administration of rAAV.

11. The effectiveness of the aforementioned treatment is due to a delay in the onset of seizures, a reduction in the frequency of seizures, and the effects on serum and / or cerebral spine. A regimen according to any one of claims 1 to 10, which is evaluated by one or more of the following: β-galactosidase in cerebrospinal fluid and volume changes of brain tissue measured by magnetic resonance imaging (MRI).

12. A composition comprising a recombinant AAV (rAAV) vector comprising an AAV capsid, a vector genome containing a human β-galactosidase coding sequence, and an expression regulatory sequence that directs its expression in target cells, wherein the rAAV vector is used in human subjects that require it. (i) The patient is from about 1 month old to about 4 months old, about 1.6×10 １３ to about 1.6×10 １４ GC, (ii) The patient is at least about 4 months old and less than 8 months old, about 2.1 × 10 １３ ~Approx. 2.1×10 １４ GC, (iii) The patient is at least 8 months old and at most 12 months old, approximately 2.6 × 10 １３ ~Approx. 2.6×10 １４ GC, or (iv) The patient is at least 12 months old, approximately 3.2 × 10 １３ ~Approx. 3.2×10 １４ A composition formulated for intracisor macromolar (ICM) injection to administer a dose of GC.

13. The composition according to claim 12, wherein the human β-galactosidase coding sequence includes a sequence that is at least 95% identical to the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 5, or to any one of SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 5, which codes for mature β-galactosidase from amino acids 24 to 677 of SEQ ID NO:

4.

14. The composition according to claim 12 or 13, wherein the vector genome further comprises a 5' inverted terminal repeat (ITR) sequence, a regulatory element derived from a human ubiquitin C (UbC) promoter, a chimeric intron, a polyA signal, and / or a 3' ITR sequence.

15. The aforementioned rAAV is 3.33 × 10⁻¹⁶ per gram of brain mass. １０ GC - 3.33 x 10⁻¹⁰ per gram of brain mass １１ The composition according to any one of claims 12 to 14, formulated in suspension form to deliver GC, and optionally having a dose volume of about 3.0 mL to about 5.0 mL.

16. The composition according to any one of claims 12 to 15, wherein the rAAV is in a formulation buffer having a pH of 6 to 9, and optionally the pH is about 7.

2.

17. The composition according to any one of claims 12 to 16, for use in a combination therapy comprising administering at least one immunosuppressant to the patient at least one day before or on the day of delivery of the rAAV.

18. The composition according to claim 17, wherein the immunosuppressant is a corticosteroid or prednisolone delivered orally as optionally.

19. A method for treating a patient having GM1 gangliosidosis, the method comprising administering a single dose of recombinant adeno-associated virus (rAAV) to the patient by intracisor macrocystic (ICM) injection, The rAAV comprises an AAV capsid and a vector genome containing a sequence encoding human β-galactosidase under the control of a regulatory sequence that directs its expression in target cells. The aforementioned single dose is 1 × 10 per gram of the patient's estimated brain mass. １０ GC ~ 3.4 x 10 １１ The method of GC.

20. The method according to claim 19, wherein the patient develops GM1 symptoms before 18 months of age.

21. The method according to claim 20, wherein the patient develops GM1 symptoms at six months of age or younger.

22. The method according to claim 20, wherein the patient develops GM1 symptoms at 6 to 18 months of age.

23. The method according to any one of claims 19 to 21, wherein the patient has type 1 (infant) GM1.

24. The method according to any one of claims 19, 20, or 22, wherein the patient has type 2a (late infant) GM1.

25. The method according to any one of claims 19 to 24, wherein the patient has been diagnosed with having type 1 GM1 or type 2a GM1.

26. The method according to any one of claims 19 to 25, wherein the subject is at least 4 months old.

27. The method according to claim 26, wherein the subject is 4 to 36 months old.

28. The method according to claim 26, wherein the subject is a human patient aged 4 to 24 months.

29. The method according to claim 26, wherein the patient is a human patient aged 6 to 36 months.

30. The method according to claim 26, wherein the patient is a human patient aged 6 to 24 months.

31. The method according to claim 26, wherein the patient is a human patient aged 12 to 36 months.

32. The method according to claim 26, wherein the patient is a human patient aged 12 to 24 months.

33. The aforementioned single dose is 3.3 × 10⁶ per gram of the patient's estimated brain mass. １０ The method according to any one of claims 19 to 32, wherein GC.

34. The aforementioned single dose is 2.1 × 10 １３ ~2.5 x 10 １３ The method according to claim 33, wherein the rAAV of GC.

35. The aforementioned single dose is 2.6 × 10 １３ ~3.1 x 10 １３ The method according to claim 33, wherein the rAAV of GC.

36. The aforementioned single dose is 3.2 × 10 １３ ~4.5 x 10 １３ The method according to claim 33, wherein the rAAV of GC.

37. The aforementioned single dose is 1.11 × 10⁻¹⁰ per gram of the patient's estimated brain mass. １１ The method according to any one of claims 19 to 32, wherein GC.

38. The aforementioned single dose is 6.8 × 10 １３ ~8.6 x 10 １３ The method according to claim 37, wherein the rAAV of GC.

39. The aforementioned single dose is 8.7 × 10 １３ ~0.9 x 10 １４ The rAAV of GC, The method described in item 37.

40. The aforementioned single dose is 1.0 × 10 １４ ~1.5 x 10 １４ The method according to claim 37, wherein the rAAV of GC.

41. The patient is 4 to 8 months old, and the single dose is 2.1 × 10 １３ The method according to any one of claims 19 to 25, wherein the rAAV of GC.

42. The patient is 4 to 8 months old, and the single dose is 6.8 × 10 １３ The method according to any one of claims 19 to 25, wherein the rAAV of GC.

43. The patient is 8 to 12 months old, and the single dose is 2.6 × 10 １３ The method according to any one of claims 19 to 25, wherein the rAAV of GC.

44. The patient is 8 to 12 months old, and the single dose is 8.7 × 10 １３ The method according to any one of claims 19 to 25, wherein the rAAV of GC.

45. The patient is at least 12 months old, and the single dose is 3.2 × 10 １３ The method according to any one of claims 19 to 25, wherein the rAAV of GC.

46. The patient is at least 12 months old, and the single dose is 1.0 × 10 １４ The method according to any one of claims 19 to 25, wherein the rAAV of GC.

47. The method according to any one of claims 19 to 46, further comprising the step of hematopoietic stem cell transplantation.

48. The method according to any one of claims 19 to 47, further comprising the step of administering a steroid to the patient.

49. The method according to claim 48, wherein the steroid is a corticosteroid.

50. The method according to claim 48 or 49, wherein the steroid is administered systemically on a daily basis for at least 21 days.

51. The method according to claim 48 or 49, wherein the steroid is administered systemically on a daily basis for 30 days.

52. The method according to any one of claims 19 to 51, wherein the sequence encoding human β-galactosidase includes a nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 5, or a sequence that is at least 95% identical to one of SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 5, which encodes mature β-galactosidase of amino acids 24 to 677 of SEQ ID NO:

4.

53. The method according to any one of claims 19 to 51, wherein the human β-galactosidase has the amino acid sequence of SEQ ID NO: 4 or a functional fragment thereof.

54. The method according to any one of claims 19 to 51, wherein the vector genome has a sequence selected from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO:

15.

55. The vector genome is sequence number 12, sequence number 13, sequence number 14, or sequence number The method according to any one of claims 19 to 51, having an arrangement that is at least 95% identical to that of 15.

56. The method according to any one of claims 19 to 51, wherein the vector genome further comprises a 5' inverted terminal repeat (ITR) sequence, a regulatory element derived from a human ubiquitin C (UbC) promoter, a chimeric intron, a polyA signal, and / or a 3' ITR sequence.

57. A pharmaceutical composition in unit dosage form, 1 × 10⁻⁶ in buffer solution １３ GC ~ 5 x 10 １４ Contains recombinant adeno-associated virus (rAAV) vector, The rAAV comprises an AAV capsid and a vector genome containing a sequence encoding human β-galactosidase under the control of a regulatory sequence that directs its expression in target cells, as a pharmaceutical composition.

58. The pharmaceutical composition according to claim 57, formulated for intracisor macromolar (ICM) injection.

59. The pharmaceutical composition according to claim 58, wherein the buffer solution comprises sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and poloxamer 188.

60. The pharmaceutical composition according to any one of claims 57 to 59, wherein the buffer solution comprises 1 mM sodium phosphate, 150 mM sodium chloride, 3 mM potassium chloride, 1.4 mM calcium chloride, 0.8 mM magnesium chloride, and 0.001% poloxamer 188.

61. A pharmaceutical composition according to any one of claims 57 to 60, which is in liquid form.

62. The pharmaceutical composition according to claim 61, having a volume of 3.0 mL, 4.0 mL, or 5.0 mL.

63. 2.1 × 10 １３ ~2.5 x 10 １３ A pharmaceutical composition according to any one of claims 57 to 62, comprising the rAAV of GC.

64. 2.6 x 10 １３ ~3.1 x 10 １３ A pharmaceutical composition according to any one of claims 57 to 62, comprising the rAAV of GC.

65. 3.2 x 10 １３ ~4.5 x 10 １３ A pharmaceutical composition according to any one of claims 57 to 62, comprising the rAAV of GC.

66. 6.8 x 10 １３ ~8.6 x 10 １３ A pharmaceutical composition according to any one of claims 57 to 62, comprising the rAAV of GC.

67. 8.7 x 10 １３ ~0.9 x 10 １４ A pharmaceutical composition according to any one of claims 57 to 62, comprising the rAAV of GC.

68. 1.0 x 10 １４ ~1.5 x 10 １４ A pharmaceutical composition according to any one of claims 57 to 62, comprising the rAAV of GC.

69. The sequence encoding the human β-galactosidase is the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 5, or the amino acid 2 of SEQ ID NO:

4. A pharmaceutical composition according to any one of claims 57 to 68, comprising a sequence that is at least 95% identical to one of SEQ ID NOs. 8, SEQ ID NOs. 7, SEQ ID NOs. 6, or SEQ ID NOs.

70. The pharmaceutical composition according to any one of claims 57 to 68, wherein the human β-galactosidase has the amino acid sequence of SEQ ID NO: 4 or a functional fragment thereof.

71. The pharmaceutical composition according to any one of claims 57 to 68, wherein the vector genome has a sequence selected from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO:

15.

72. The pharmaceutical composition according to any one of claims 57 to 68, wherein the vector genome has a sequence that is at least 95% identical to sequence number 12, sequence number 13, sequence number 14, or sequence number 15.

73. The pharmaceutical composition according to any one of claims 57 to 68, wherein the vector genome further comprises a 5' inverted terminal repeat (ITR) sequence, a regulatory element derived from a human ubiquitin C (UbC) promoter, a chimeric intron, a polyA signal, and / or a 3' ITR sequence.

Images