Methods and compositions for treating leukodystrophies

Abstract

Recombinant adeno-associated virus vectors expressing aspartoacylase (ASPA) protein and related uses for treating leukodystrophies are disclosed herein. The present disclosure relates to recombinant adeno-associated virus (AAV) vectors engineered to express aspartoacylase (ASPA) in subjects in need of such expression. Aspects of the present disclosure relate to methods of treating neurodegenerative diseases (e.g., leukodystrophies such as Canavan disease) in subjects in need of such treatment. In some embodiments, the methods provided herein include regulating N-acetylaspartate (NAA) levels in a subject. NAA is metabolized by aspartoacylase (ASPA) to acetate and L-aspartate.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 254,885, filed October 12, 2021, and U.S. Provisional Application No. 63 / 352,049, filed June 14, 2022, the contents of each of which are incorporated herein by reference in their entirety for all purposes.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (ASPA_004_02WO_SeqList_ST26.xml; size: 32,450 bytes; and creation date: October 11, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0003] Leukodystrophies are a group of rare, mostly inherited neurological disorders that result from abnormal production, processing, or development of myelin and other components of central nervous system (CNS) white matter, such as cells called oligodendrocytes and astrocytes. Deterioration of myelin function leads to progressive loss of neuronal function.

[0004] More than 50 different leukodystrophies have been identified, including: Alexander disease autosomal dominant leukodystrophy with autonomic diseases (ADLD), Canavan disease, cerebrotendinous xanthomatosis (CTX), metachromatic leukodystrophy (MLD), Pelizaeus-Merzbacher disease, and Refsum disease. The specific symptoms of leukodystrophies vary widely depending on the type of disease.

[0005] There are no cures for leukodystrophies that significantly alter the course of the disease. Instead, treatments aim to alleviate symptoms and preserve some neurological function. Thus, there is an unmet need to develop new therapeutic agents for leukodystrophic disease types such as Canavan disease. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides a method for treating a subject with leukodystrophy by gene therapy. Gene therapy can include administration of a recombinant adeno-associated virus vector comprising a nucleic acid molecule encoding aspartoacylase (ASPA).

[0007] In one aspect, the disclosure provides a method comprising administering to a subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, the rAAV vector comprising: (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); and (ii) a non-AAV nucleotide sequence encoding aspartoacylase (ASPA), the non-AAV nucleotide sequence being operably linked to a promoter.

[0008] In a related aspect, the disclosure provides a method for expressing aspartoacylase (ASPA) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, the rAAV vector comprising (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and (ii) a non-AAV nucleotide sequence encoding ASPA, wherein the non-AAV nucleotide sequence is operably linked to a promoter.

[0009] In another aspect, the disclosure provides a method for increasing the level of aspartoacylase (ASPA) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, the rAAV vector comprising (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and (ii) a non-AAV nucleotide sequence encoding ASPA, wherein the non-AAV nucleotide sequence is operably linked to a promoter.

[0010] In a further aspect, the disclosure provides a method of treating a leukodystrophy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV vector comprises (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); and (ii) a non-AAV nucleotide sequence encoding ASPA, wherein the non-AAV nucleotide sequence is operably linked to a promoter.

[0011] In another aspect, the disclosure provides a method of treating Canavan disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV vector comprises (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); and (ii) a non-AAV nucleotide sequence encoding ASPA, wherein the non-AAV nucleotide sequence is operably linked to a promoter.

[0012] In another aspect, the disclosure provides for the use of a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector in the treatment of a leukodystrophy, wherein the rAAV vector comprises (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); and (ii) a non-AAV nucleotide sequence encoding ASPA, wherein the non-AAV nucleotide sequence is operably linked to a promoter.

[0013] In another aspect, the disclosure provides for the use of a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector in the treatment of Canavan disease, wherein the rAAV vector comprises (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and (ii) a non-AAV nucleotide sequence encoding ASPA, wherein the non-AAV nucleotide sequence is operably linked to a promoter.

[0014] In a further aspect, the disclosure provides a composition for the treatment of leukodystrophy, comprising a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector in the treatment of Canavan disease, the rAAV vector comprising (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR), and (ii) a non-AAV nucleotide sequence encoding ASPA, wherein the non-AAV nucleotide sequence is operably linked to a promoter.

[0015] In a further aspect, the disclosure provides a composition for the treatment of Canavan disease, comprising a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector in the treatment of Canavan disease, the rAAV vector comprising (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and (ii) a non-AAV nucleotide sequence encoding ASPA, wherein the non-AAV nucleotide sequence is operably linked to a promoter.

[0016] In another aspect, the disclosure provides a method of use of a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector in the manufacture of a medicament for the production of a substance for treating a leukodystrophy, wherein the rAAV vector comprises (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and (ii) a non-AAV nucleotide sequence encoding ASPA, wherein the non-AAV nucleotide sequence is operably linked to a promoter.

[0017] In another aspect, the disclosure provides a method of use of a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector in the manufacture of a medicament for the production of a substance for treating Canavan disease, wherein the rAAV vector comprises (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and (ii) a non-AAV nucleotide sequence encoding ASPA, wherein the non-AAV nucleotide sequence is operably linked to a promoter.

[0018] In some embodiments of any of the foregoing aspects, the leukodystrophy is associated with a condition selected from the group consisting of Canavan disease, adrenomyeloneuropathy, Alexander disease, cerebrotendinous xanthomatosis, Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, and Refsum disease, hi some embodiments, the leukodystrophy is associated with Canavan disease.

[0019] In some embodiments of any of the aforementioned aspects, the therapeutically effective amount is about 10 13 Vector genomes / kilogram (vg / kg) ~ approx. 10 15 In some embodiments, the therapeutically effective amount is in the range of about 10 14 vg / kg ~ approx. 5×10 14 In some embodiments, the therapeutically effective amount is in the range of at least about 1.32×10 14 In some embodiments, the therapeutically effective amount is about 3×10 14 vg / kg.

[0020] In some embodiments of any of the aforementioned aspects, the rAAV is administered by intravenous infusion.

[0021] In some embodiments of any of the aforementioned aspects, the subject is 30 months of age or younger. [Brief description of the drawings]

[0022] [Figure 1]1 shows the design of the self-complementary AAV (scAAV) vector used in the study of Example 1, designated "scAAV9-CB6-hASPAopt," which contains the CB6 promoter and a codon-optimized transgene encoding the human ASPA protein. Abbreviations: CMV: cytomegalovirus; IE: immediate early; ITR: inverted terminal repeat.

[0023] [Diagram 2] Body weights of animals treated with the medium and high dose regimens of BBP-812 are shown. Body weights were measured every other day for the first 32 days and then every other week (n=10 each).

[0024] [Diagram 3] Brain magnetic resonance spectroscopy analysis showing total N-acetylaspartate (tNAA) levels normalized to total creatine (tCr) (n=3).

[0025] [Figure 4] Motor behavior testing times at postnatal days (PND) 27, 90, 180, and 364 for wild-type (WT), untreated, and BBP-812-treated mice are shown.

[0026] [Figure 5A] Brain histology in wild-type and Aspa- / - (knockout, KO) mice treated on PND1 with BBP-812 at 2.6x1013, 8.8x1013, and 2.6x1014 vector genomes / kilogram (vg / kg) (CA3: cornu ammonis 3; Ce: cerebellum; Cx: cortex; DG: dentate gyrus). [Figure 5B] Brain histology in wild-type and Aspa- / - (knockout, KO) mice treated on PND1 with BBP-812 at 2.6x1013, 8.8x1013, and 2.6x1014 vector genomes / kilogram (vg / kg) (CA3: cornu ammonis 3; Ce: cerebellum; Cx: cortex; DG: dentate gyrus).

[0027] [Figure 6]Eleven different regions of the CNS analyzed for vector genome copy number per cell using ddPCR at 364 days after vector administration are shown (BS: brainstem; CBL: cerebellum; CSC: cervical spinal cord; CNS: central nervous system; Cx: cortex; ddPCR: droplet digital polymerase chain reaction; HPC: hippocampus; LMN: tectal plate; LSC: lumbar spinal cord; MB: midbrain).

[0028] [Figure 7A] Brain histology in wild-type Aspa− / − mice treated with BBP-812 on PND1 (postnatal day 1). [Figure 7B] Brain histology in wild-type Aspa− / − mice treated with BBP-812 on PND1 (postnatal day 1).

[0029] [Figure 8] The locations of samples taken and used to determine the biodistribution of BBP-812 and the expression of the hASPAOpt transgene are indicated.

[0030] [Figure 9] Assessment of BBP-812 in the brain (Figure 9A), spinal cord (Figure 9B), liver (Figure 9C), heart (Figure 9D), and kidney (Figure 9E) is shown. For the brain and spinal cord, each individual tissue region is plotted as an individual data point.

[0031] [Figure 10A] Measurements of creatine kinase (FIG. 10A) and lactate dehydrogenase (FIG. 10B) are shown. [Figure 10B] Measurements of creatine kinase (FIG. 10A) and lactate dehydrogenase (FIG. 10B) are shown.

[0032] [Figure 11]ELISA results showing the reactivity of spleen cells from vehicle (V), 1×10 vg / kg treated animals (L), or 3×10 vg / kg treated mice (H) with medium (negative control), ConA (positive control), and peptide pools to AAV9 and ASPA (SFU: spot forming units).

[0033] [Figure 12] Vector genome (upper panel) and transgene RNA (lower panel) detected in the 3x1014 vg / kg dose group from 4-, 12-, and 24-week biodistribution samples are shown.

[0034] [Figure 13] FIG. 1 shows the timeline and overall design of a Phase 1 / 2 study of gene therapy to treat Canavan disease. (d: days; DSMC: Data and Safety Monitoring Committee; kg: kilograms; mo: months; N: number of participants; vg: vector genome).

[0035] [Figure 14] The study dose setting, observation, and enrollment expansion sequence is shown.

[0036] [Figure 15] Glucocorticoid prophylaxis and tapering regimens are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present disclosure relates to recombinant adeno-associated virus (AAV) vectors engineered to express aspartoacylase (ASPA) in a subject in need thereof. Aspects of the present disclosure relate to methods of treating neurodegenerative diseases (e.g., leukodystrophies such as Canavan disease) in a subject in need thereof. In some embodiments, the methods provided herein include regulating N-acetylaspartate (NAA) levels in a subject. NAA is metabolized by aspartoacylase (ASPA) to acetate and L-aspartate.

[0038] The headings used herein are merely for organizational purposes and should not be construed as limiting the subject matter described. All documents or portions of documents cited herein, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference in their entirety for all purposes. In the event that one or more of the incorporated documents or portions of documents defines a term and it conflicts with the definition of that term in this application, the definition set forth in this application shall prevail. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be taken as, any form of acknowledgment or suggestion that they constitute valid prior art or form part of the general knowledge in any country in the world.

[0039] Any concentration range, percentage range, ratio range, or integer range herein should be understood to include any integer value within the stated range, and, where appropriate, fractions thereof (e.g., tenths and hundredths of integers), unless otherwise specified. The term "about," when immediately preceding a number or numeral, means that the number or numeral is within a range of plus or minus 10%. As used herein, the terms "a" and "an" should be understood to refer to "one or more" of the indicated components, unless otherwise specified. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. The term "and / or" should be understood to mean either one or both of the alternatives following. As used herein, the terms "include" and "comprise" are used interchangeably.

[0040] Recombinant AAV vectors and particles In one aspect, the present disclosure provides a viral vector for delivering a nucleic acid sequence encoding ASPA to a cell, such as a cell in need of treatment. Thus, in some embodiments, the present disclosure relates to a recombinant adeno-associated viral (rAAV) vector comprising a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleotide sequence (also referred to as a heterologous polynucleotide) encoding ASPA, wherein the non-AAV nucleotide sequence is operably linked to a promoter.

[0041] As used herein, the term "operably linked" or "operably linked" refers to the physical or functional juxtaposition of the described components that allows them to function in their intended manner. In the example of an expression control element (e.g., a promoter or enhancer) operably linked to a polynucleotide, the relationship is such that the control element regulates expression of the nucleic acid. More specifically, for example, two deoxyribonucleic acid (DNA) sequences are operably linked means that the two DNAs are positioned in a relationship (in cis or trans) such that at least one of the DNA sequences can exert a physiological effect on the other sequence. "Operably linked" can mean that the nucleic acid sequences being linked are contiguous or substantially contiguous, and, where necessary to link two protein coding regions, contiguous and in reading frame.

[0042] In some embodiments, the rAAV vector expresses an ASPA protein that is a human ASPA protein. In some cases, the ASPA protein expressed by the rAAV vector described herein is a natural (e.g., wild-type) ASPA protein. The ASPA protein or polypeptide encoded by the nucleotide sequence includes the full-length natural sequence, as well as functional subsequences, modified forms, or sequence variants, as in the naturally occurring ASPA protein, provided that the subsequences, modified forms, or variants retain some degree of function of the natural full-length ASPA protein. In the methods and uses of the present disclosure, the ASPA protein and polypeptide encoded by the nucleotide sequence in the rAAV vector can be, but is not required to be, identical to the endogenous ASPA protein that is missing or insufficiently expressed or deficient in the treated subject. In some embodiments, ASPA comprises the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identity to SEQ ID NO:6.

[0043] In some embodiments, the non-AAV nucleotide sequence (e.g., heterologous sequence) encoding the ASPA protein is a wild-type ASPA gene sequence. In some embodiments, the non-AAV nucleotide sequence (e.g., heterologous sequence) encoding the ASPA protein is codon-optimized with respect to the wild-type ASPA gene sequence. In some embodiments, the nucleotide sequence encoding the ASPA of the present disclosure is a codon-optimized sequence and comprises or consists of SEQ ID NO:1. In other embodiments, the non-AAV nucleotide sequence encoding the ASPA protein comprises a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identity to SEQ ID NO:1. In some embodiments, the non-AAV nucleotide sequence (e.g., heterologous sequence) encoding the ASPA protein is a human ASPA complementary DNA (cDNA), optionally linked to a nucleotide sequence encoding a hemagglutinin (HA) tag. In some embodiments, the non-AAV nucleotide sequence (e.g., a heterologous sequence) encoding the ASPA protein is linked to a nucleotide sequence encoding a tag, e.g., hemagglutinin (HA), UA, cMyc, or any suitable tag.

[0044] Codon optimization exploits the redundancy of the genetic code, allowing nucleotide sequences to be altered while maintaining the same amino acid sequence of the encoded protein. In some embodiments, codon optimization is performed to facilitate increased or decreased expression of the encoded protein. This is accomplished by matching the codon usage in the nucleotide sequence to that of a particular cell type, thus exploiting cellular codon biases that correspond to biases in the relative abundance of particular transfer ribonucleic acids (tRNAs) in the cell type. Expression can be increased by altering the codons in the nucleotide sequence to match the relative abundance of the corresponding tRNA. Conversely, expression can be decreased by selecting codons whose corresponding tRNAs are known to be rare in a particular cell type.

[0045] In some embodiments, the codon-optimized nucleotide sequence encoding the ASPA protein is more stable than the wild-type cDNA sequence, thereby avoiding the generation of alternatively spliced ​​variants or truncated proteins when non-AAV nucleotide sequences are introduced into the transcriptional machinery via gene therapy. [Table 1-1] [Table 1-2]

[0046] The term "homologous" or "homology" means that two or more referenced entities are the same over a defined region (e.g., region, domain, or portion) (e.g., when the entities are aligned). An "aligned" sequence refers to a sequence of multiple polynucleotides or proteins (amino acids), often containing corrections of missing or additional bases or amino acids (gaps) compared to a reference sequence. If two sequences are at least partially homologous, they share at least partial identity. An "area", "region" or "domain" of homology or identity means that parts of two or more referenced entities are identical such that they share homology or identity. Thus, if two sequences are identical over one or more sequence regions, they share identity in these regions. By way of example, if two polypeptide sequences are identical, they have the same amino acid sequence, at least within the referenced region or portion. Similarly, if two polynucleotide sequences are identical, they have the same polynucleotide sequence, at least within the referenced region or portion. "Substantial homology" means that a molecule is structurally or functionally preserved such that it has, or is predicted to have, one or more of the structures or functions (e.g., biological functions or activities) of the reference molecule, or at least a partial structure or function of a related / corresponding region or portion of the reference molecule with which it shares homology.

[0047] The identity or homology between two sequences can extend over the entire length of the sequence or over a portion of the sequence.In some embodiments, the length of the sequence that shares percent identity is 2, 3, 4, 5 or more consecutive polynucleotides or amino acids, for example, at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive amino acids.In some embodiments, the length of the sequence that shares identity is 20 or more consecutive polynucleotides or amino acids, for example, at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more consecutive amino acids. In some embodiments, the length of the sequences that share identity is 35 or more consecutive polynucleotides or amino acids, e.g., at least 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more consecutive amino acids. In some embodiments, the length of the sequences that share identity is 50 or more consecutive polynucleotides or amino acids, e.g., at least 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-110, or more consecutive polynucleotides or amino acids.

[0048] The degree of identity (homology) between two sequences can be ascertained using computer programs and mathematical algorithms. Percent identity can be calculated using the alignment program Clustal Omega, available at www.ebi.ac.uk / Tools / msa / clustalo using default parameters. See, for example, Sievers et al., "Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega." (2011 October 11) Molecular systems biology 7:539. Extensions such as tags are not included in the calculation of identity for sequences.

[0049] A vector genome sequence, including the rAAV vector genome sequence described herein, can include one or more "expression control elements." Typically, an expression control element is a nucleic acid sequence that affects the expression of an operably linked polynucleotide. Control elements, including the expression control elements described herein, such as promoters and enhancers present in the vector, are included to facilitate transcription and / or translation (e.g., promoters, enhancers, splicing signals for introns, maintaining the correct reading frame of the gene to allow in-frame translation of the mRNA, etc.) of the appropriate heterologous polynucleotide (e.g., ASPA gene). Expression control elements include appropriate transcription initiation, termination, promoter, and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals, sequences that stabilize cytoplasmic mRNA, sequences that increase translation efficiency (i.e., Kozak consensus sequences), sequences that increase protein stability, and, in some cases, sequences that increase secretion of the encoded product (e.g., ASPA). In some embodiments, the rAAV vector genome sequence of the disclosure comprises a consensus sequence such as a Kozak sequence (e.g., a DNA sequence that is transcribed into an RNA Kozak sequence). In some embodiments, the rAAV vector genome sequence of the disclosure comprises a Kozak sequence upstream of a nucleotide sequence encoding an ASPA protein. In some embodiments, the RNA Kozak sequence comprises or consists of ACCAUGG (SEQ ID NO:44), GCCGCCACCAUGG (SEQ ID NO:45), CCACCAUG (SEQ ID NO:46), or CCACCAUGG (SEQ ID NO:47).

[0050] Expression control can occur at the levels of transcription, translation, splicing, message stability, etc. Typically, expression control elements that regulate transcription are juxtaposed near the 5' end (i.e., "upstream") of the transcribed polynucleotide. Expression control elements can also be located at the 3' end (i.e., "downstream") of the transcribed sequence or within the transcript (e.g., within an intron). Expression control elements can be located at a significant distance, but far from the transcribed sequence (e.g., 100-500, 500-1000, 2000-5000, 5000-10,000, or more nucleotides from the nucleotide sequence expressing ASPA). Nevertheless, due to polynucleotide length limitations, for AAV vectors, such expression control elements will typically be within 1-1000 nucleotides from the nucleotide sequence encoding ASPA.

[0051] Functionally, the expression of the operably linked nucleotide sequence encoding ASPA can be at least partially controlled by an element (e.g., a promoter) that regulates the transcription of the nucleotide sequence and, if necessary, the translation of the transcript. A specific example of an expression control element is a promoter, which is usually located 5' of the transcribed sequence. Another example of an expression control element is an enhancer, which can be located 5' of the transcribed sequence, 3' of the transcribed sequence, or within the transcribed sequence.

[0052] As used herein, a "promoter" can refer to a nucleic acid sequence that is located adjacent to a nucleic acid sequence (e.g., a heterologous polynucleotide) that encodes a recombinant product (e.g., ASPA). A promoter is typically operably linked to an adjacent sequence, e.g., a heterologous polynucleotide. A promoter usually increases the amount of expression from a heterologous polynucleotide compared to the amount expressed in the absence of the promoter.

[0053] "Enhancer" as used herein can refer to a sequence located adjacent to the nucleotide sequence encoding ASPA. Enhancer elements are typically located upstream of promoter elements, but can also be functional and located downstream or within a DNA sequence (e.g., the nucleotide sequence encoding ASPA). Thus, enhancer elements can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream or downstream of a heterologous polynucleotide. Enhancer elements usually increase the expression of a heterologous polynucleotide beyond the level of increased expression provided by promoter elements.

[0054] In some embodiments, expression control elements include ubiquitous, constitutive or promiscuous promoters and / or enhancers capable of driving expression of the polynucleotide in many different cell types. Such elements include the cytomegalovirus / β-actin hybrid (e.g., CAG, CB6, or CBA) promoter, the phosphoglycerol kinase (PGK) promoter, the cytomegalovirus (CMV) immediate early promoter and / or enhancer sequence, the Rous sarcoma virus (RSV) promoter and / or enhancer sequence, as well as other viral promoters and / or enhancers active in a variety of mammalian cell types, or non-naturally occurring synthetic elements (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the avian β-actin (CBA) promoter, the EF1 promoter (Invitrogen), the immediate early CMV enhancer combined with the CBA promoter (Beltran et al., Gene Therapy, 17(9):1162-1174 (2010)), and the CBh promoter (Gray et al., Hum Gene Ther, 22(9):1143-1153(2011)). In some embodiments, the rAAV of the present disclosure comprises a synthetic CASI promoter that contains a portion of the CMV enhancer, a portion of the avian beta actin promoter, and a portion of the UBC enhancer. (See, e.g., International Patent Publication No. WO2012115980). In some embodiments, the promoter is an astrocyte-specific promoter, a glial fibrillary acidic protein (GFAP) promoter, or an enhanced avian beta-actin promoter. In some embodiments, the promoter is a cytomegalovirus / beta-actin hybrid promoter, or a PGK promoter. In some embodiments, the cytomegalovirus / beta-actin hybrid promoter is a CAG promoter, a CB6 promoter, or a CBA promoter.

[0055] In some embodiments, the rAAV vector comprises a CAG promoter sequence comprising SEQ ID NO:2, or a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% identity to SEQ ID NO:2. In some embodiments, the rAAV vector comprises a PGK promoter sequence comprising SEQ ID NO:3, or a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% identity to SEQ ID NO:3. In some embodiments, the rAAV vector comprises a CB6 promoter sequence comprising SEQ ID NO: 4, or a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% identity to SEQ ID NO: 4. In some embodiments, the rAAV vector comprises a CBA promoter sequence comprising SEQ ID NO: 5, or a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% identity to SEQ ID NO: 5. Table 1 includes non-limiting examples of promoter sequences.

[0056] Inducible promoters allow the control of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a particular physiological state, acute phase, a particular differentiation state of cells, or only in replicating cells. Inducible promoters and induction systems are available from a variety of commercial sources, including but not limited to Invitrogen and Clontech. Many other systems have been described and can be used. Examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, the ecdysone insect promoter, the tetracycline repression system, the tetracycline induction system, the RU486 induction system, and the rapamycin induction system. Any type of inducible promoter that is tightly regulated and specific to the particular target cell type in which ASPA expression is intended may be used.

[0057] Expression control elements (e.g., promoters) include those active in a specific tissue or cell type, referred to herein as "tissue-specific expression control elements / promoters". Tissue-specific expression control elements are typically active in a specific cell or tissue (e.g., brain, central nervous system, spinal cord, etc.). Expression control elements are typically active in a specific cell, tissue, or organ type because they are recognized by transcriptional activator proteins or other transcriptional regulators that are specific to these cells, tissues, or organs. Thus, in some cases, the rAAV vectors of the present disclosure include a promoter that directs the expression of a nucleotide sequence encoding an ASPA protein in a host cell (e.g., an oligodendrocyte, a Schwann cell, a microglial cell, a neuronal cell such as an astrocyte or a neuron, or a non-neuronal cell such as a hepatocyte).

[0058] In some embodiments, the regulatory sequence useful in the rAAV vector of the present disclosure also contains an intron, which is optionally located between the promoter / enhancer sequence and the ASPA gene. In some embodiments, the intron sequence is a 100 bp mini-intron splice donor / splice acceptor derived from SV-40, called SD-SA. In some embodiments, the rAAV vector comprises a post-transcriptional regulatory element. One example of a post-transcriptional regulatory element is the woodchuck hepatitis virus post-transcriptional element (WPRE). (See, e.g., Wang and Verma, Proc. Natl. Acad. Sci., USA, 96:3906-3910 (1999)). In certain embodiments, the post-transcriptional regulatory element is the hepatitis B virus post-transcriptional regulatory element (HBVPRE) or the RNA transport element (RTE). In some embodiments, the WPRE or HBVPRE sequence is any of the WPRE or HBVPRE sequences disclosed in U.S. Patent No. 6,136,597 or U.S. Patent No. 6,287,814. In some embodiments, the WPRE sequence comprises or consists of: [ka]

[0059] In some embodiments, the rAAV vector comprises a poly A signal. The poly A signal may be derived from a number of suitable species, including, but not limited to, rabbit, SV-40, human, and bovine. In some embodiments, the rAAV vector comprises a rabbit globin poly A signal, such as a rabbit β-globin poly A signal. In some embodiments, the rabbit β-globin poly A signal has a nucleic acid sequence of SEQ ID NO: 101 or has a nucleotide sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% identical to SEQ ID NO: 101 (SEQ ID NO: 101-aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctca).

[0060] Another useful regulatory component that may be included in a rAAV vector is an internal ribosome entry site (IRES). An IRES sequence or other suitable system may be used to generate more than one polypeptide from a single gene transcript. An IRES (or other suitable sequence) is used to generate a protein containing more than one polypeptide chain, or to express two different proteins from or within the same cell. An exemplary IRES is the poliovirus internal ribosome entry sequence. The IRES may be located 5' or 3' to the ASPA transgene in the rAAV vector. In other embodiments, the rAAV vector may include a nucleotide sequence encoding a 2A peptide, which allows for the expression of multiple polypeptides from a single promoter.

[0061] As used herein, a recombinant "vector" or "rAAV vector" refers to a vector derived from the wild-type genome of a virus, such as AAV, by using molecular methods to remove the wild-type genome from the virus and replace it with a non-native nucleic acid, such as a heterologous polynucleotide sequence (e.g., a therapeutic gene expression cassette expressing ASPA). Typically, for AAV, one or both inverted terminal repeat (ITR) sequences of the wild-type AAV genome are retained in the AAV vector. A rAAV vector can be distinguished from a viral genome because all (or a portion) of the viral genome has been replaced with a non-native sequence with respect to the viral genome nucleic acid. Thus, the incorporation of a non-native sequence, such as a heterologous polynucleotide, defines the viral vector as a "recombinant" vector, and in the case of AAV, can be referred to as a "rAAV vector." A rAAV vector containing a nucleic acid molecule encoding ASPA may also be referred to as "scAAV9-CB6-hASPAopt" or "ASPA vector." As the context makes clear, a "vector" may refer to an isolated recombinant nucleotide sequence, or an AAV particle or virion containing a recombinant nucleotide sequence.

[0062] In some embodiments, rAAV vector does not contain any binding site for miRNA (microRNA). In some embodiments, rAAV vector contains one, two, three, four, five or more binding sites for miRNA expressed in cells where ASPA protein expression is not desired (i.e., non-targeted). In some embodiments, rAAV vector contains one or more binding sites for miR-122. Binding of miR-122 to ASPA coding sequence can reduce the expression of this sequence in liver cells where miR-122 is very prevalent (Thakral and Ghoshal, Curr Gene Ther. 2015; 15(2): 142-150).

[0063] The rAAV nucleic acid sequences can be packaged into viruses (also referred to herein as "particles" or "virions") for subsequent infection (transformation) of cells ex vivo, in vitro, or in vivo. When recombinant vector sequences are enclosed or packaged in an AAV particle, the particle can be referred to as "rAAV". Such particles or virions typically include proteins that encapsidate or package the vector genome. Particular examples include viral envelope proteins and, in the case of AAV, capsid proteins.

[0064] The AAV components of the rAAV vectors and rAAV particles described herein may be selected from various AAV serotypes. In some embodiments, the rAAV vector comprises AAV nucleic acid sequences from rh10, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or rh74 serotypes. In some embodiments, the rAAV vector comprises AAV nucleic acid sequences from the AAV9 serotype. These AAV components can be easily isolated from AAV serotypes using a variety of techniques. Such AAV may be isolated or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, VA). Alternatively, the AAV sequences may be obtained by synthesis or other suitable means, by reference to published sequences such as those available in the literature or in databases such as GenBank™, PubMed, etc.

[0065] In certain embodiments, the rAAV vector or particle comprises an AAV nucleic acid sequence or an AAV protein, for example, as disclosed in U.S. Patent No. 7,906,111 or U.S. Patent No. 7,629,322, which are incorporated herein by reference in their entirety. In some embodiments, the rAAV vector or particle comprises an AAV nucleic acid sequence or an AAV protein from AAV serotype AAV8 or a variant thereof, for example, as disclosed in U.S. Patent No. 7,282,199, U.S. Patent No. 9,587,250 or U.S. Patent No. 9,677,089, which are incorporated herein by reference in their entirety. In some embodiments, the rAAV vector or particle comprises an AAV nucleic acid sequence or an AAV protein from AAV serotype AAV9 or a variant thereof, for example, as disclosed in U.S. Patent No. 7,198,951, which is incorporated herein by reference in its entirety. In some embodiments, the rAAV vector or particle comprises an AAV nucleic acid sequence or an AAV protein from AAV serotype rh74 or a variant thereof.

[0066] In some embodiments, the rAAV vector of the present disclosure comprises a nucleic acid molecule comprising at least one AAV ITR sequence. In certain embodiments, the rAAV vector comprises two ITR sequences, which may be of the same or different AAV serotypes. The AAV ITRs may be selected from any useful AAV serotype, including, but not limited to, rh10, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh74, and other AAV serotypes. In some embodiments, the rAAV vector described herein comprises a genome comprising one or two AAV2 ITR sequences.

[0067] The present disclosure further provides an rAAV particle comprising the rAAV vector described herein.Thus, in some aspects, the present disclosure relates to an rAAV particle comprising a nucleic acid molecule comprising at least one AAV ITR and a non-AAV nucleotide sequence (also referred to as a heterologous polynucleotide) encoding an ASPA protein, wherein the non-AAV nucleotide sequence is operably linked to a promoter.In some embodiments, the rAAV particle comprises at least one capsid protein selected from the group consisting of AAV serotypes rh10, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh74, and other AAV serotypes.In some embodiments, the rAAV particle comprises at least one AAV9 capsid protein.

[0068] In some embodiments, the rAAV vector or particle comprises a nucleic acid molecule comprising an AAV9 capsid protein, a human ASPA cDNA, a CAG, PGK, CBA or CB6 promoter, and optionally one or two AAV2 ITR sequences. In some embodiments, the rAAV vector or particle is an AAV9-CAG-hASPAopt, AAV9-PGK-hASPAopt, AAV9-CBA-hASPAopt or AAV9-CB6-hASPAopt vector. In some embodiments, the promoter comprises or consists of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In some embodiments, the rAAV vector comprises a Kozak sequence. In some embodiments, the Kozak sequence can comprise or be transcribed to SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47. In some embodiments, the rAAV vector further comprises a HBVPRE or WPRE sequence (e.g., SEQ ID NO: 51).

[0069] In some embodiments, an rAAV vector or particle comprises a capsid ("Cap") protein (e.g., including vp1, vp2, vp3 and hypervariable regions), a viral replication ("Rep") protein (e.g., rep78, rep68, rep52, or rep40), and / or a sequence encoding one or more such proteins. These AAV components can be readily utilized in a variety of vector systems and host cells. Such components may be used alone, in combination with other AAV serotype sequences or components, or in combination with elements derived from non-AAV viral sequences. As used herein, an artificial AAV serotype includes, but is not limited to, an AAV with a non-naturally occurring capsid protein. Such artificial capsids may be generated by any suitable technique using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with a heterologous sequence that may be obtained from a different selected AAV serotype, from non-contiguous portions of the same AAV serotype, from a non-AAV viral source, or from a non-viral source. The artificial AAV serotype may be, but is not limited to, a pseudo AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. A pseudo vector in which the capsid of one AAV is replaced with a heterologous capsid protein is useful in the present disclosure. In some embodiments, the AAV is AAV2 / 5. In some embodiments, the AAV is AAV2 / 8. See, for example, Mussolino et al., Gene Therapy, 18(7):637-645 (2011); Rabinowitz et al., J Virol, 76(2):791-801 (2002).

[0070] In some embodiments, vectors useful in the compositions and methods described herein contain at least sequences encoding a selected AAV serotype capsid or fragments thereof. In some embodiments, useful vectors contain at least sequences encoding a selected AAV serotype rep protein or fragments thereof. Optionally, such vectors may contain both AAV cap and rep proteins. In vectors where both AAV rep and cap are provided, the AAV rep and AAV cap sequences can both be of one serotype. Alternatively, vectors may be used in which the rep sequence is from one AAV serotype and the cap sequence is from a different AAV serotype. In some embodiments, the rep and cap sequences are expressed from separate sources (e.g., separate vectors, or host cells and vectors). In some embodiments, these rep sequences are fused in frame to cap sequences of a different AAV serotype to form chimeric AAV vectors, such as AAV2 / 8, described in U.S. Pat. No. 7,282,199, which is incorporated herein by reference in its entirety.

[0071] A suitable rAAV can be produced by culturing a host cell that contains a nucleic acid molecule consisting of a nucleic acid sequence encoding an AAV serotype capsid protein, or a fragment thereof, as defined herein, a functional rep gene, a nucleic acid sequence encoding at least an AAV inverted terminal repeat (ITR) and ASPA, and sufficient helper functions to allow packaging of the nucleic acid molecule into an AAV capsid protein. In some aspects, the disclosure provides a host cell that contains an rAAV vector or rAAV particle disclosed herein. The components required to be present in the host cell to package the rAAV vector into an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., vector, rep sequence, cap sequence, and / or helper functions) may be provided by a stable host cell that has been engineered to contain one or more of the required components using various methods. Most preferably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. In the above discussion of regulatory elements suitable for use with non-AAV nucleotide sequences, i.e., ASPA, examples of suitable inducible and constitutive promoters are provided herein. In yet another alternative, the selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated that is derived from 293 cells (containing E1 helper functions under the control of a constitutive promoter) but contains rep and / or cap proteins under the control of an inducible promoter. Still other stable host cells may be generated.

[0072] The rAAV vectors, rep sequences, cap sequences, and helper functions used to produce the rAAV of the present disclosure can be delivered to the packaging host cell in the form of any genetic element that transfers the sequences carried thereon. The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of the present disclosure, including the generation of rAAV particles, are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY; Fisher et al, J. Virol., 70:520-532 (1993); and US 5,478,745, each of which is incorporated herein by reference in its entirety.

[0073] In one aspect, the disclosure provides a method for producing rAAV particles, the method comprising culturing a host cell that contains (a) a nucleic acid molecule comprising or consisting of a rAAV vector genome expressing an ASPA described herein, (b) a nucleic acid molecule encoding AAV rep, (c) a nucleic acid molecule encoding at least one AAV capsid protein, and (d) sufficient helper functions for packaging the rAAV vector genome into an rAAV particle.

[0074] The rAAV particles of the present disclosure may be purified by various methods. In some embodiments, the rAAV virus may be purified by anion exchange chromatography. For example, see US Patent Publication No. 2018 / 0163183A1. Further details regarding the construction and characterization of the AAV vectors and particles disclosed herein are described in International Patent Publication No. WO2019 / 143803, the entire contents of which are incorporated herein by reference. Further details regarding rAAV particles encoding ASPA can be found in US Patent No. 9,102,949, US Patent Publication No. 2019 / 0125899A1, and US Patent Publication No. 2018 / 0311323A1, the contents of each of which are incorporated herein by reference in their entirety.

[0075] In some embodiments, a composition comprising a non-replicating recombinant AAV serotype 9 (AAV9) vector containing an expression cassette of the human ASPA transgene disclosed herein has the AAV-hASPAopt-Opt vector design shown in Figure 2. In some embodiments, the expression cassette of the rAAV vector comprises a 5'ITR comprising the nucleic acid sequence of SEQ ID NO: 102 (SEQ ID NO: 102-ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct). In some embodiments, the expression cassette of the rAAV vector comprises a codon-optimized nucleic acid sequence encoding hASPA of SEQ ID NO: 1. In some embodiments, the expression cassette of the rAAV vector comprises a rabbit β-globin poly A comprising the nucleic acid sequence of SEQ ID NO: 101. In some embodiments, the expression cassette of the rAAV vector comprises a 3' ITR comprising the nucleic acid sequence of SEQ ID NO: 103 (SEQ ID NO:103 - aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag).

[0076] Pharmaceutical Compositions The rAAV vector or rAAV particle of the present disclosure can be incorporated into a pharmaceutical composition suitable for administration. In one aspect, the present disclosure provides a pharmaceutical composition comprising a rAAV vector or rAAV particle disclosed herein (e.g., a rAAV particle comprising a nucleic acid sequence encoding ASPA) and a pharma- ceutically acceptable carrier, diluent, or excipient. As used herein, the term "pharma- ceutically acceptable" refers to molecular entities and compositions that do not generally produce allergic or other serious adverse reactions when administered using established routes. Molecular entities and compositions approved by a regulatory agency of the U.S. Federal or U.S. State government or listed in the U.S. Pharmacopeia or other pharmacopoeias generally recognized for use in animals, more specifically in humans, are considered "pharma- ceutically acceptable". As used herein, the term "pharma-ceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference text in this field, and is incorporated herein by reference.Some examples of such carriers or diluents include, but are not limited to, water, saline, buffered saline, Ringer's solution, dextrose solution, 5% human serum albumin, and other buffers for maintaining pH at an appropriate physiological level, such as HEPES.Except insofar as any medium or agent is incompatible with the active ingredient, its use in the composition is contemplated.Auxiliary active compounds can also be incorporated into the composition.

[0077] Throughout this description, "vg" can refer to the "viral genome" or the "vector genome."

[0078] Examples of pharmaceutical compositions and delivery systems that can be used to administer the rAAV disclosed herein are described in Remington: The Science and Practice of Pharmacy (2003) 20 thed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18 th ed.,Mack Publishing Co.,Easton,Pa.;The Merck Index(1996)12 th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technomic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11 th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, NY, pp. 253-315.

[0079] The pharmaceutical composition of the present disclosure may be formulated to suit its intended route of administration. Examples of routes of administration include parenteral administration, e.g., intravenous administration, or injection. Solutions or suspensions used for parenteral (e.g., intravenous or injection) applications may contain the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium hydrogen sulfate; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates, and agents for adjusting osmolality such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0080] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents such as sugars, polyols such as mannitol and sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable composition can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0081] Sterile injectable solution can be prepared by incorporating the required amount of active compound into suitable solvent with one or combination of the above-listed ingredients as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into sterile vehicle that contains basic dispersion medium and other ingredients required from above-listed.In the case of sterile powder for preparing sterile injectable solution, the preparation method is vacuum drying and freeze-drying, which obtains powder of active ingredient plus any additional ingredient from its solution that has been previously sterile-filtered.

[0082] For injection, the pharma- ceutically acceptable carrier can be a liquid. Exemplary physiologically acceptable carriers include sterile pyrogen-free water and sterile pyrogen-free phosphate-buffered saline. A variety of such known carriers are provided in U.S. Patent No. 7,629,322, which is incorporated by reference in its entirety. In some embodiments, the carrier is an isotonic sodium chloride solution. In some embodiments, the carrier is a balanced salt solution. In some embodiments, the carrier comprises TWEEN® (polysorbate). For long-term storage of rAAV, it may be frozen in the presence of glycerol or TWEEN® (polysorbate) 20.

[0083] It is particularly advantageous to formulate parenteral compositions in dosage unit forms for ease of administration and uniformity of dosage. As used herein, dosage unit forms refer to physically discrete units suitable as unitary dosages for the subject to be treated, each unit containing a predetermined amount of active agent (e.g., rAAV) calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of dosage unit forms of the present disclosure are necessitated by and directly depend on the characteristics of the active agent (e.g., rAAV) and the particular therapeutic effect to be achieved, as well as the limitations inherent in the art of compounding such active agents (e.g., rAAV) for the treatment of individuals. The unit forms can be, for example, in ampoules and vials that can contain liquid compositions, or compositions in lyophilized or lyophilized form, to which, for example, a sterile liquid carrier can be added prior to administration or delivery in vivo. Individual unit forms can be included in multi-dose kits or containers. Recombinant vector (e.g., rAAV) sequences, plasmids, vector genomes, recombinant viral particles (e.g., rAAV), and pharmaceutical compositions thereof can be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0084] Pharmaceutical compositions containing a rAAV vector or rAAV particles containing a nucleic acid sequence encoding ASPA may be included in a container, pack, or dispenser together with instructions for administration.

[0085] In some embodiments, the composition includes a surfactant. In some embodiments, the composition includes a poloxamer, such as poloxamer 188. In some embodiments, the composition includes a thickener, a plasticizer, and / or a cryoprotectant. In some embodiments, the composition includes a sugar alcohol, such as sorbitol. In some embodiments, the composition includes a buffer. In some embodiments, the composition is formulated as a sterile solution in phosphate buffered saline, pH 7.0, 5 weight / volume (w / v) sorbitol, 0.001% w / v poloxamer 188. It may be supplied in a 5 milliliter (mL) pyrogen-free vial with a fill volume of 2.5 mL and sealed with a latex-free rubber stopper and an aluminum flip-off seal. In some embodiments, the composition is formulated as a sterile solution containing 137 millimolar (mM) sodium chloride, 8.1 mM dibasic sodium phosphate, 1.47 mM potassium dihydrogen phosphate, 2.7 mM potassium chloride, 5% w / v sorbitol, 0.001% w / v poloxamer 188, and water for injection. The vector concentration of the formulated product is not limited and can be about 10 12 vg / mL ~ approx. 10 15 vg / mL, e.g., about 5 × 10 12 vg / mL, approximately 1×10 13 vg / mL, approximately 5×10 13 vg / mL, approximately 1×10 14 vg / mL, approximately 5×10 14 In some embodiments, the vector concentration of the formulated product can range from about 1.8×10 vg / mL, including all values ​​and subranges therebetween. In some embodiments, the vector concentration of the formulated product can range from about 1.8×10 13 vg / mL ~ approx. 5.0×10 13 In some embodiments, the vector concentration of the formulated product is in the range of about 1.8×10 13 vg / mL ~ approx. 4.2×10 13The vials can be stored frozen at -60°C or colder until ready for use.

[0086] In one aspect, the present disclosure provides a kit comprising a rAAV vector, or a particle comprising the same, wherein the rAAV vector comprises a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleotide sequence encoding an ASPA protein, the non-AAV nucleotide sequence being operably linked to a promoter. In some embodiments, the kit further comprises instructions for administering the rAAV vector to a subject. In some embodiments, the kit further comprises instructions for administering the rAAV vector by intravenous infusion.

[0087] In another aspect, the disclosure provides a unit dose comprising a rAAV vector, or a particle comprising the same, wherein the rAAV vector comprises a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleotide sequence encoding an ASPA protein, the non-AAV nucleotide sequence being operably linked to a promoter, and the therapeutically effective amount is about 10 13 vg / kg~about 10 15 In some embodiments, the therapeutically effective amount is in the range of about 1.32×10 14 In some embodiments, the therapeutically effective amount is about 3×10 14 vg / kg. In some embodiments, the unit dose comprises a liquid formulation. In some embodiments, the unit dose is configured to be administered to a subject by intravenous infusion.

[0088] In some embodiments of the above aspects, the ASPA protein is a human ASPA protein. In some embodiments of the above aspects, the non-AAV nucleotide sequence encoding the ASPA protein comprises or consists of a human ASPA cDNA. In some embodiments of the above aspects, the non-AAV nucleotide sequence encoding the ASPA protein comprises or consists of a codon-optimized nucleotide sequence. In some embodiments of the above aspects, the non-AAV nucleotide sequence encoding the ASPA protein comprises or consists of SEQ ID NO:1. In some embodiments of the above aspects, the non-AAV nucleotide sequence encoding the ASPA protein encodes the amino acid sequence of SEQ ID NO:1, or an amino acid sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:1. In some embodiments of the above aspects, the promoter directs expression of the ASPA protein in a host cell, such as a neuronal cell. In some embodiments of the above aspects, the promoter is a cytomegalovirus / β-actin hybrid promoter, or a PGK promoter. In some embodiments of the above aspects, the cytomegalovirus / β-actin hybrid promoter is a CAG, CB6, or CBA promoter. In some embodiments of the above aspects, the promoter comprises or consists of the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In some embodiments of the above aspects, the ITR is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, or rh74 serotype ITR. In some embodiments of the above aspects, the rAAV is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, or rh74 serotype. In some embodiments of the above aspects, the rAAV is an AAV9 serotype. In some embodiments of the above aspects, the nucleic acid molecule further comprises a Kozak sequence.In some embodiments of the above aspects, the nucleic acid molecule further comprises a miR-122 binding site.

[0089] Methods of Use of Recombinant AAV Vectors and Particles The present disclosure provides a method involving the use of an AAV (e.g., recombinant AAV) vector, such as in the treatment of a subject in need thereof. The method provided herein includes, for example, administering to a subject a therapeutically effective amount of any one of rAAV or compositions disclosed herein. In some embodiments, administration of the rAAV vector results in the expression of ASPA in the tissue of the subject. In some embodiments, the tissue is a peripheral tissue or a central nervous system (CNS) tissue. In some embodiments, administration of the rAAV vector results in the expression of ASPA in the cells of the subject. In some embodiments, the cell is a neuronal cell, such as an oligodendrocyte.

[0090] In some embodiments, the subject is a human, a non-human primate, a pig, a horse, a cow, a dog, a cat, a rabbit, a guinea pig, a hamster, a mouse, or a rat. In certain embodiments, the subject is a human. The human subject may be a human female or a human male. In some embodiments, the subject is a human who has previously undergone a hormone therapy program. In some embodiments, the subject is undergoing a hormone therapy program. In some embodiments, the subject is undergoing or has undergone physical therapy or other physical interventions, such as supportive care or the use of a feeding tube. In some embodiments, the subject is undergoing or has undergone treatment for one or more symptoms of Canavan disease, for example, with anti-seizure or anti-convulsant medications. In some embodiments, the subject is undergoing or has undergone investigational treatment for Canavan disease, such as lithium gluconate, glyceryl triacetate (GTA), cord blood cell therapy, or ASPA gene therapy. In some embodiments, the subject is a human infant. In certain cases, the subject is a human infant about 1 month old, about 2 months old, about 3 months old, about 4 months old, about 5 months old, about 6 months old, about 7 months old, about 8 months old, about 9 months old, about 10 months old, about 11 months old, or about 1 year old. In some embodiments, the subject is a human infant less than 3 months old, less than 6 months old, less than 9 months old, less than 1 year old, or less than 18 months old. In some embodiments, the subject is a human infant less than 30 months old. In some embodiments, the subject is a human child (e.g., under 18 years old), such as a child between 13 and 18 years old, between 12 and 18 years old, between 10 and 18 years old, between 8 and 18 years old, between 6 and 18 years old, between 2 and 18 years old, between 10 and 13 years old, between 8 and 13 years old, between 6 and 13 years old, between 2 and 13 years old, between 10 and 12 years old, between 8 and 12 years old, between 6 and 12 years old, between 2 and 12 years old, between 2 and 10 years old, between 2 and 8 years old, or between 2 and 6 years old, or any range therein. In some embodiments, the subject is a human adult (e.g., 18 years old or older).

[0091] As used herein, the term "patient in need" or "subject in need" refers to a patient or subject at risk for or suffering from a disease, disorder, or condition that can be treated or ameliorated with a rAAV comprising a nucleic acid sequence encoding ASPA or a composition comprising such a rAAV provided herein, such as leukodystrophy. In some embodiments, a "patient or subject in need" is a patient or subject at risk for developing or suffering from a disease associated with dysfunction of ASPA, such as Canavan disease. In some embodiments, a "patient in need" or "subject in need" has one or more amino acid mutations in the ASPA gene that result in altered ASPA function. "Subject" and "patient" are used interchangeably herein.

[0092] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of an agent, e.g., rAAV, sufficient to reduce or ameliorate the severity and / or duration of a disorder, e.g., a leukodystrophy such as Canavan disease, or one or more symptoms thereof, prevent progression of the disorder, cause regression of the disorder, prevent the recurrence, onset, onset or progression of one or more symptoms associated with the disorder, detect the disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., a prophylactic or therapeutic agent). In some embodiments, an effective amount of rAAV may, for example, increase expression of ASPA and / or alleviate to some extent one or more symptoms associated with a disease associated with a lack of adequate ASPA levels and / or sufficient ASPA function.

[0093] The present disclosure provides methods and uses of rAAV, including nucleic acid molecules encoding ASPA as described herein, for providing therapeutic benefit to a subject having a disorder or disease characterized by ASPA deficiency or dysfunction. In some embodiments, the methods include treating a disorder or disease characterized by ASPA deficiency or dysfunction in a subject by administering a therapeutically effective amount of rAAV or a composition described herein to a subject in need thereof.

[0094] In some cases, the present disclosure provides a method for the expression of ASPA in a subject that requires the expression of ASPA, comprising administering to the subject a therapeutically effective amount of rAAV particles described herein or a pharmaceutical composition described herein, thereby expressing ASPA in the subject.In certain embodiments, the present disclosure provides a method for the expression of ASPA in a subject that requires the expression of ASPA, comprising administering to the subject a therapeutically effective amount of rAAV particles described herein or a pharmaceutical composition comprising particles, thereby increasing the expression of ASPA in the subject.In some embodiments, the method disclosed herein causes the expression of ASPA or increases the expression of ASPA in the neural cells of the subject.

[0095] In some embodiments, the subject requires expression of ASPA. In some embodiments, the subject has ASPA deficiency. The present disclosure provides a method for treating a subject with ASPA deficiency, comprising administering to the subject a therapeutically effective amount of a rAAV particle or a pharmaceutical composition comprising the particle as described herein, thereby treating the ASPA deficiency in the subject. The present disclosure provides a method for treating a subject with leukodystrophy, such as Canavan disease, comprising administering to the subject a therapeutically effective amount of a rAAV particle or a pharmaceutical composition comprising the particle as described herein, thereby treating the leukodystrophy in the subject.

[0096] The leukodystrophy may be any leukodystrophy identified as such by a physician, including more than 50 types of leukodystrophies that affect myelin function resulting in progressive loss of neurological function. In some embodiments, the leukodystrophies are selected from the group consisting of adult-onset autosomal dominant leukodystrophy (ADLD), Aicardi-Goutières syndrome, Alexander disease, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), Canavan disease, cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), cerebrotendinous xanthomatosis, childhood ataxia, cerebral hypomyelination (CACH) / vanishing white matter disease (VWMD), Fabry disease, fucosidosis, GM1 gangliosidosis, Krabbe disease, L-2 hydroxyglutaric aciduria, macrocephalic leukoencephalopathy with subcortical cysts, metachromatic leukodystrophy, multiple sulfatase deficiency, Pelizaeus-Merzbacher disease, Pol III Related leukodystrophies include Refsum disease, Salla disease, (free salaic acid storage disease), Sjögren-Larsson syndrome, X-linked adrenoleukodystrophy or Zellweger syndrome spectrum disorders.

[0097] The present disclosure further provides a method for treating, reducing, ameliorating, slowing the progression, or preventing symptoms of leukodystrophy (e.g., Canavan disease) in a subject having leukodystrophy, comprising administering to the subject a therapeutically effective amount of an rAAV particle or a pharmaceutical composition comprising the particle described herein, thereby treating, reducing, ameliorating, slowing the progression, or preventing symptoms of leukodystrophy in the subject. Non-limiting examples of symptoms of leukodystrophy include problems with one or more of balance, breathing, cognition (learning, thinking, memory), eating and swallowing, hearing, movement, balance and coordination, speech, and vision.

[0098] In some embodiments, the leukodystrophy is Canavan disease. The present disclosure provides a method of treating a subject with Canavan disease, comprising administering to the subject a therapeutically effective amount of a rAAV particle or a pharmaceutical composition comprising the particle as described herein, thereby treating the subject's Canavan disease. In some embodiments, the method further comprises selecting a subject with Canavan disease before administering the rAAV vector. The present disclosure further provides a method of treating, alleviating, ameliorating, delaying progression, or preventing symptoms of Canavan disease in a subject with Canavan disease, comprising administering to the subject a therapeutically effective amount of a rAAV particle or a pharmaceutical composition comprising the particle as described herein, thereby treating, alleviating, ameliorating, delaying progression, or preventing symptoms of Canavan disease in the subject. Non-limiting examples of symptoms of Canavan disease may include one or more of the following: lack of motor development; difficulty eating; abnormal muscle tone (weakness or stiffness); abnormally large and poorly controlled head; paralysis, blindness; or hearing loss. In some embodiments, symptoms of Canavan disease are poor head control, macrocephaly (abnormally large head), hypotonia (poor muscle tone), apathy (unresponsiveness), lethargy, irritability, dysphagia (difficulty swallowing), feeding disorders, delay in reaching developmental milestones, failure to walk independently, psychomotor regression (progressive loss of skill and coordination), mental retardation, intellectual disability, seizures, sleep disorders, nasal regurgitation, regurgitation that may be associated with vomiting, optic nerve deterioration, optic nerve atrophy, decreased visual responsiveness, hearing loss, seizures, decerebrate posturing, paralysis, or increased NAA in the urine.

[0099] In some embodiments, the method further comprises selecting a subject with leukodystrophy prior to administering the rAAV vector. In some embodiments, the subject may be screened and identified or diagnosed (e.g., by genetic or physiological testing) as having leukodystrophy, even though the subject does not have one or more symptoms of the disease. In other embodiments, the subject has one or more symptoms of leukodystrophy. In certain embodiments, the subject has a mutation in a gene associated with leukodystrophy, such as Canavan disease. In some embodiments, the subject has a loss-of-function mutation in a gene that controls the expression and / or function of aspartoacylase (ASPA), such as the gene encoding ASPA. In some embodiments, the subject is diagnosed with Canavan disease based on a prenatal blood test that screens for ASPA levels and / or mutations in the gene encoding ASPA.

[0100] In some embodiments, the method includes reducing the level of N-acetylaspartic acid (NAA) in the subject's biological sample compared to (a) the level of NAA in the subject's biological sample prior to administration of an rAAV particle or a pharmaceutical composition comprising the particle described herein, and / or (b) the level of NAA in the biological sample of a control subject, where the control subject has a leukodystrophy (such as Canavan disease) and is not administered an rAAV particle or a pharmaceutical composition comprising the particle described herein. In some embodiments, the method involves reducing the level of N-acetylaspartic acid (NAA) in a biological sample obtained from the subject by at least about 2% (e.g., about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and subranges therebetween), compared to (a) the level of NAA in the biological sample of the subject prior to administration of a rAAV particle or a pharmaceutical composition comprising a particle as described herein, and / or (b) the level of NAA in a biological sample of a control subject, where the control subject has a leukodystrophy (such as Canavan disease) and is not administered a rAAV particle or a pharmaceutical composition comprising a particle as described herein. In some embodiments, the biological sample is blood, urine, peripheral tissue, CNS fluid, or CNS tissue.

[0101] In some embodiments, it has been determined that the subject has a metabolic imbalance comprising a transition from glycolysis to beta-oxidation. In some embodiments, the method further comprises detecting the metabolic imbalance by assessing the level of one or more glycolytic and / or beta-oxidation factors. In some embodiments, the level of one or more glycolytic and / or beta-oxidation factors is assessed using central nervous system (CNS) fluid obtained from the subject. In some embodiments, the method further comprises (a) obtaining CNS fluid from the subject, (b) detecting an increase in beta-oxidation in the CNS fluid, and (c) administering rAAV to the subject based on the detection in (b). In some embodiments, the method further comprises (a) measuring a metabolic profile of a biological sample obtained from the subject, and (b) identifying a metabolic imbalance comprising a transition from glycolysis to beta-oxidation based on the metabolic profile. In some embodiments, measuring the metabolic profile comprises assaying the biological sample using liquid chromatography (LC), mass spectrometry (MS), liquid chromatography / mass spectrometry (LC / MS), or ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). In some embodiments, the biological sample comprises CNS tissue or cerebrospinal fluid (CSF). In some embodiments, the CNS tissue is brain tissue. In some embodiments, the metabolic profile comprises a level of a first biomarker selected from the group consisting of glucose, glucose-6-phosphate, 3-phosphoglycerate, pyruvate, lactate, and phosphoenolpyruvate. In some embodiments, the metabolic profile comprises a level of a second biomarker selected from the group consisting of carnitine, malonylcarnitine, myristoylcarnitine, palmitoylcarnitine, malonylcarnitine, and beta-hydroxybutyrate.

[0102] In some embodiments, the rAAV particle or pharmaceutical composition comprising the particle that comprises the nucleic acid molecule encoding ASPA is administered to the subject systemically.In some embodiments, the rAAV particle or pharmaceutical composition comprising the particle that comprises the nucleic acid molecule encoding ASPA is administered to the subject intravenously by direct injection via laparotomy or laparoscopy, or by injection into artery or vein via catheter placement.In any of the treatment methods described herein, the rAAV particle or pharmaceutical composition comprising the particle that comprises the nucleic acid molecule encoding ASPA can be administered to the subject using intravenous infusion.

[0103] The present disclosure further contemplates the use of the agent described herein (e.g., rAAV or a pharmaceutical composition comprising rAAV) in the manufacture of a medicament for treating a disorder or disease characterized by ASPA dysfunction or deficiency in a subject.The present disclosure also includes the use of the agent described herein (e.g., rAAV or a pharmaceutical composition comprising rAAV) for treating a disorder or disease characterized by ASPA dysfunction or deficiency in a subject.

[0104] The composition may be delivered in a volume of about 50 microliters (μL) to about 1 mL, including all values ​​within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In some embodiments, the volume is about 50 μL. In another embodiment, the volume is about 70 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 250 μL. In another embodiment, the volume is about 300 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 750 μL. In another embodiment, the volume is about 850 μL. In another embodiment, the volume is about 1000 μL.

[0105] In some embodiments, the lowest effective concentration of virus is utilized to reduce the risk of undesirable effects such as toxicity or adverse immune response.Other dosages within these ranges can be selected by the attending physician, taking into account the physical condition of the subject (e.g., human) being treated, the subject's age, the specific ASPA deficiency disorder, and the degree of development if the disorder is progressive.

[0106] In some embodiments, an rAAV vector or rAAV particle comprising a nucleic acid sequence encoding ASPA is administered intravenously to about 10 subjects. 12 ~about 10 16 vg / kg body weight of a subject, e.g., about 10 13 vg / kg~about 10 15 In some embodiments, the rAAV vector or particle comprising a nucleic acid sequence encoding ASPA is administered to the subject at a dose ranging from about 10 vg / kg body weight. 14 vg / kg ~ approx. 5×10 14 In some embodiments, the rAAV vector or particle comprising a nucleic acid sequence encoding ASPA is administered to a subject at a dose ranging from about 1.32×10 14 In some embodiments, the rAAV vector or particle comprising a nucleic acid sequence encoding ASPA is administered to a subject at a dose of about 3×10 14 The subject is administered a dose of 1000 mg / kg.

[0107] In some embodiments, the therapeutically effective amount is about 10 14 vg / kg ~ approx. 5x10 14 vg / kg range, e.g., about 1.1x10 14 vg / kg, approx. 1.2x10 14 vg / kg, approx. 1.3x10 14 vg / kg, approx. 1.4x10 14 vg / kg, approx. 1.5x10 14 vg / kg, approx. 2x10 14 vg / kg, approx. 2.5x10 14 vg / kg, approx. 3x10 14 vg / kg, approx. 3.5x10 14 vg / kg, approx. 4x1014 vg / kg, approx. 4.5x10 14 vg / kg, or approximately 5x10 14 vg / kg, including all values ​​and subranges therebetween. In some embodiments, the therapeutically effective amount is about 1.32×10 14 In some embodiments, the therapeutically effective amount is about 3×10 14 In some embodiments, the therapeutically effective amount is about 1.32×10 14 vg / kg ~ approx. 3×10 14 vg / kg range.

[0108] In some embodiments, the subject is administered a single dose of rAAV vector, rAAV particle, or composition disclosed herein.In some embodiments, the subject is administered multiple doses of rAAV vector, rAAV particle, or composition disclosed herein, for example, 2, 3, 4, or 5 doses.In some embodiments, the subject is administered a single dose of rAAV vector, rAAV particle, or composition disclosed herein by intravenous infusion.

[0109] The compositions disclosed herein may be administered to a subject at any frequency. In some embodiments, the compositions may be administered to a subject once a day or more than once a day. In some embodiments, the compositions may be administered to a subject two, three, four, five, six, seven, eight, nine, or ten times a day. In some embodiments, the compositions may be administered to a subject every day, every other day, every third day, every fourth day, every fifth day, or every sixth day. In some embodiments, the compositions may be administered to a subject every week, every other week, or every three weeks. In some embodiments, the compositions may be administered to a subject every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months. In some embodiments, the compositions may be administered to a subject every year. In some embodiments, the compositions may be administered to a subject every 1, 2, 3, 4, 5, 10, 15, or 20 years. In certain embodiments, the compositions may be administered to a subject only once during the subject's lifetime.

[0110] In some embodiments, any one of the methods disclosed herein further comprises administering a therapeutically effective amount of an immunosuppressant. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a rAAV or composition disclosed herein and a therapeutically effective amount of an immunosuppressant. The present disclosure provides a method of treatment in which a therapeutically effective amount of a rAAV or composition disclosed herein and a therapeutically effective amount of an immunosuppressant are administered to the subject.

[0111] In some embodiments, the immunosuppressant is administered prior to, concurrently with, and / or after administration of the rAAV vector. In some embodiments, the immunosuppressant is administered prior to administration of the rAAV vector. In some embodiments, the immunosuppressant is administered at least 12 hours prior to administration of the rAAV vector. In some embodiments, the immunosuppressant is administered about 2 days prior to administration of the rAAV vector.

[0112] In some embodiments, the immunosuppressant is a non-glucocorticoid immunosuppressant. In some embodiments, the immunosuppressant is an inhibitor of calcineurin. In some embodiments, the immunosuppressant is cyclosporine, tacrolimus, sirolimus, everolimus, zotarolimus, or any combination thereof. In some embodiments, the immunosuppressant is tacrolimus. Other non-limiting examples of immunosuppressants include alkylating agents such as nitrogen mustard (cyclophosphamide), nitrosoureas, platinum compounds, folic acid analogs such as methotrexate, purine analogs such as azathioprine and mercaptopurine, pyrimidine analogs such as fluorouracil, protein synthesis inhibitors, cytotoxic antibodies such as dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, polyclonal antibodies that inhibit T lymphocytes, IL-2 receptor-directed monoclonal antibodies such as basiliximab or daclizumab, anti-CD3 monoclonal antibodies such as muromonab, opioids, TNF-alpha binding proteins such as infliximab, etanercept, or adalimumab, mycophenolate, fingolimod, and myriocin. Other examples of biological immunosuppressants include, but are not limited to, monoclonal antibodies, such as monoclonal antibodies that block costimulatory pathways (e.g., antibodies against CTLA4, ICOS, CD80, OX40, or other appropriate targets), interfering RNA (e.g., siRNA, dsRNA, shRNA, miRNA, etc.) that target immune stimulatory molecules (e.g., cytokines), and proteins (e.g., proteasome inhibitors).

[0113] In some embodiments, the immunosuppressant is administered orally. In some embodiments, the therapeutically effective amount of the immunosuppressant is in the range of about 0.005 milligrams per kilogram (mg / kg) to about 0.1 mg / kg, e.g., about 0.01 mg / kg, about 0.015 mg / kg, about 0.02 mg / kg, about 0.025 mg / kg, about 0.03 mg / kg, about 0.035 mg / kg, about 0.04 mg / kg, about 0.045 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, or about 0.1 mg / kg, including all values ​​and subranges therebetween. In some embodiments, the therapeutically effective amount of the immunosuppressant is in the range of about 0.01 mg / kg to about 0.05 mg / kg. In some embodiments, the therapeutically effective amount of the immunosuppressant is about 0.025 mg / kg. In some embodiments, a therapeutically effective amount of the immunosuppressant is administered twice daily.

[0114] In some embodiments, the method includes administering a therapeutically effective amount of a steroid to the subject. In some embodiments, the steroid is a mineralocorticoid. In some embodiments, the steroid is a glucocorticoid. In some embodiments, the glucocorticoid is prednisolone, methylprednisolone, cortisol, cortisone, prednisone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone, deoxycorticosterone (DOCA), aldosterone, or any combination thereof. In some embodiments, the steroid is hydrocortisone. In some embodiments, the steroid is administered prior to, concurrently with, and / or after administration of the rAAV vector. In some embodiments, a therapeutically effective amount of the steroid administered to the subject prior to administration of the rAAV vector is greater than a therapeutically effective amount of the steroid administered to the subject after rAAV administration.

[0115] In some embodiments, any one of the methods disclosed herein further comprises administering a therapeutically effective amount of an antihistamine. In some embodiments, the antihistamine is administered prior to administration of the rAAV vector, concurrently with administration of the rAAV vector, and / or after administration of the rAAV vector. In some embodiments, the antihistamine is diphenhydramine, hydroxyzine, chlorpheniramine, or any combination thereof.

[0116] In some embodiments, the method further comprises (a) administering to the subject a small molecule metabolic regulator; (b) prescribing a dietary intervention to the subject, where the dietary intervention promotes glycolysis and / or reduces beta-oxidation in the subject; and / or (c) administering to the subject an immunosuppressant.

[0117] In some embodiments, after administration of a vector described herein, N-acetylaspartic acid (NAA) levels are reduced in urine, cerebrospinal fluid (CSF), and / or brain tissue. In some embodiments, after administration, NAA levels in urine are reduced. In some embodiments, NAA levels in urine are reduced by at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, NAA levels in urine remain reduced for at least 6 months, 9 months, 12 months, 18 months, 24 months, or more compared to pre-treatment levels. In some embodiments, NAA levels in CSF are reduced after administration. In some embodiments, NAA levels in CSF are reduced by at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, NAA levels in CSF remain reduced for at least 6 months, 9 months, 12 months, 18 months, 24 months, or more compared to pre-treatment levels. In some embodiments, after administration, NAA levels in brain tissue are reduced. In some embodiments, NAA levels in brain tissue are reduced by at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, NAA levels in brain tissue remain reduced for at least 6 months, 9 months, 12 months, 18 months, 24 months, or more compared to pre-treatment levels.

[0118] In some embodiments, after administration of a vector described herein, neomyelination is observable using magnetic resonance imaging (MRI). EXAMPLES

[0119] Example 1: BBP-812 Gene Therapy Vector for Canavan Disease The BBP-812 active agent is a non-replicating rAAV serotype 9 (AAV9) vector containing a self-complementary expression cassette of the human ASPA transgene. The scAAV9-CB6-hASPAopt vector design is shown in Figure 1. It contains a codon-optimized human aspartoacylase transgene under the control of the CB6 promoter containing the cytomegalovirus immediate early enhancer flanked by 5' and 3' terminal repeats. The site of Rep nicking is removed in the 5' inverted terminal repeat, thereby creating a self-complementary AAV genome.

[0120] BBP-812 is formulated as a sterile solution for injection in phosphate buffered saline, pH 7.0, 5 weight / volume (w / v) sorbitol, 0.001% w / v poloxamer 188. It is supplied in 5 milliliter (mL) pyrogen-free vials with a fill volume of 2.5 mL, sealed with a latex-free rubber stopper and aluminum flip-off seal. The vector concentration of the formulated product is 1.8 x 10 13 ~5.0×10 13 The vials are stored frozen at or below -60°C until ready for use.

[0121] BBP-812 formulation (DP) was 1.8 × 10 13 ~4.2×10 13 A single dose, preservative-free, sterile solution, intravenous (IV) injection of a non-replicating recombinant AAV9 vector at a concentration of 10 ...

[0122] This study will evaluate the safety, tolerability, pharmacodynamic (PD) activity, and clinical activity of two dose levels of AAV9 vectors (shown in Figure 1) encoding ASPA in pediatric patients with Canavan disease aged 30 months or younger. Dosing will occur by the date before participants reach 31 months of age. Evaluation of safety, tolerability, PD activity, and preliminary clinical activity of BBP-812 in the first six participants will inform dose selection and enrollment expansion to a total of 15 participants at the selected dose. All participants will be followed for at least 5 years from the date of treatment with BBP-812.

[0123] BBP-812 at 1.32 x 10 14 in cohort 1 at a dose of vector genome (vg) / kilogram (kg) body weight, 3.0 × 10 14 Cohort 2 will be administered a dose of 100 mg / kg body weight. No placebo will be administered in this study. A maximum of 18 participants will be enrolled in the study, with a sample size of 15 participants treated with the selected dose.

[0124] For pharmacodynamic analysis, changes in N-acetylaspartate (NAA) levels following administration of BBP-812 are measured in urine and CNS tissues. For clinical changes, for example, brain imaging is used to assess motor, cognitive and language development and function following administration of BBP-812.

[0125] Example 2: Non-clinical trials of BBP-812 Demonstration of the therapeutic potential and safety of BBP-812 in the treatment of Canavan disease is provided by nonclinical studies examining efficacy in the Aspa- / - mouse model of Canavan disease, as well as safety evaluation in wild-type mice and nonhuman primates. For the efficacy studies described below, heterozygous Aspa+ / - mice were crossed to generate homozygous pups. Animals were genotyped immediately after birth (PND0 / 1) using a polymerase chain reaction (PCR)-based assay utilizing genomic deoxyribonucleic acid (gDNA) templates prepared from tail tissue samples. For efficacy studies, all mice were treated via IV injection into the facial vein on PND1. Nonclinical safety and pharmacology studies performed with BBP-812 are summarized in Table 2. The main results of the studies are discussed in the following sections. [Table 2]

[0126] Canavan disease mouse model The safety and efficacy of BBP-812 was investigated using the Aspa- / - mouse model of Canavan disease. Aspa- / - mice were the first developed animal model of Canavan disease and mimic the clinical phenotype of Canavan disease patients, exhibiting ataxia, instability, muscle weakness, failure to thrive, craniofacial abnormalities, and cognitive impairment within the first month of life. Without intervention, Aspa- / - mice die around PND28 (Ahmed 2013).

[0127] Several other mouse models of Canavan disease have been reported, but they display less severe phenotypes (Carpinelli 2014, Mersmann 2011, Traka 2008). Notably, these other models all display normal life spans and do not develop measurable motor deficits until a few weeks after birth. This phenotype is reminiscent of milder forms of Canavan disease (Janson 2006a, Mendes 2017, Yalcinkaya 2005).

[0128] For the PoC study, Aspa- / - mice were injected between PND0 and 20 for initial experiments and at PND1 for determination of the minimum effective dose (MED). From the literature, neuronal development in mice ranging from PND0 to PND20 is equivalent to human neurodevelopment from 35 weeks gestation to approximately 2.5 years of age (Dutta 2016). Therefore, the timing of treatment in the PoC study supports the use of BBP-812 in pediatric patients aged 30 months or younger in a Phase 1 / 2 pediatric clinical trial (CVN102).

[0129] Dose-ranging efficacy study in postnatal day 1 Aspa- / - mice A dose-ranging study (Gessler 2017) was initiated to establish the MED in animal disease models and to determine the starting dose of CVN102 for the proposed Phase 1 / 2 pediatric clinical trial. Key parameters for determining the MED included survival and weight gain, reduction in CNS NAA levels, improvement in motor function, and improvement in brain histopathology. Biodistribution in the brain was also assessed.

[0130] Study design: Randomization and dose justification The dose evaluated in this study was 2.6 × 10 13 , 8.8×10 13 , and 2.6 × 10 14 The doses were 100-250 vg / kg. The highest dose was based on previous studies with earlier generation vectors (Ahmed 2013). The medium and low doses represent a 3- and 10-fold reduction in vector. Mice received a single IV injection on PND1 into the facial vein.

[0131] For these studies, animals were born between March 2014 and January 2016. Randomization was performed by dosing all animals in the fetus in the same manner. Only 25% of pregnant fetuses are Aspa- / -. Therefore, it was not always possible to test multiple doses in the same fetus. Initial dosing included the highest dose, vehicle (0.9% NaCl) treated, and untreated animals. As efficacy data were generated, lower doses were tested as additional fetuses were born to establish the minimum effective dose. However, throughout the study period, random fetuses received the highest dose or vehicle, or remained untreated, to demonstrate consistency of results over time and across vector lots. A complete list of animals used in the development and establishment of the MED for BBP-812 is listed in Table 3. [Table 3]

[0132] Study Design: Efficacy parameters measured in the study Survival and weight gain: Survival was monitored daily and body weights were measured every other day from PND1 to PND32, then every other week from PND42 (6 weeks) to PND364 (1 year).

[0133] Reduction in CNS and peripheral NAA levels: Reduction in CNS and peripheral NAA levels was measured by magnetic resonance spectroscopy (MRS) and mass spectrometry.

[0134] Motor function and spatial memory tests: The motor performance of mice was assessed using an accelerating rotarod for motor function and endurance, a balance beam for vestibular function and ataxia, and an inverted screen for grip strength. With the exception of the rotarod (rotarod series 8, IITC Life Sciences, Woodland Hills, CA), the equipment was manufactured by the University of Massachusetts Medical School Machine Shop. For each motor function test, mice were injected and tested independently. Controls for these experiments included untreated Aspa- / - mice, Aspa- / - mice treated with 0.9% saline, and wild-type (WT) mice. The treatments that animals received were blinded to the assessors, and animals were tested over a 2-year period for all evaluations in the study.

[0135] Accelerating rotarod: Mice were trained 2 days before the test day and ran three times each. On the test day, mice were placed on the rotarod and allowed to acclimate for 1 min. Each mouse was tested three times and the best value was used for analysis. Acceleration and timing were set at 4-40 rpm for 5 min.

[0136] Balance beam: Animals were placed in the middle of a horizontal wooden balance beam (1.5 × 100 cm) with a pad placed underneath to protect the mouse from any possible fall from the beam. The latency to fall was recorded and a time limit of 5 min was imposed on each trial. To increase the stringency of this test, the cutoff time was extended to 5 min from the previously used 3 min. Animals were tested three times and the best value was counted.

[0137] Inverted screen: Place the mouse in a horizontal position on the grid (25 square millimeters (mm 2 30 square centimeters (cm) with a hole of 2)) and allowed to acclimate for 1 min. The grid was slowly rotated to 125° within 15 s so that the mouse was hanging upside down. The latency to fall was measured. The cutoff for PND27 testing was 3 min as previously published. For all other time points, the cutoff was 5 min to increase the stringency of the testing. Each animal was tested three times and the best value was counted.

[0138] Brain histopathology was evaluated by hematoxylin and eosin (H&E) staining.

[0139] Biodistribution was determined by droplet digital polymerase chain reaction (ddPCR).

[0140] Test results: survival rate and weight gain BBP-812 treatment increased survival and promoted weight gain compared to untreated Aspa- / - animals (Figure 2). Untreated animals (red line) began to lose weight around 2 weeks of age and died by 4 weeks of age. Treated animals gained weight comparable to WT animals and survived to the end of the experiment.

[0141] No unscheduled deaths due to treatment were reported during the study, as outlined in Table 4. Causes of death included malocclusion (n=2) and infanticide (n=5). Given the nature of these events, no further pathology or histopathology evaluation was performed. [Table 4]

[0142] Test Results: Decreased CNS NAA levels Using MRS analysis, animals administered BBP-812 were monitored for CNS levels of NAA (Figure 3 and Table 5). 13 vg / kg and 2.6 × 10 14 Animals receiving BBP-812 at a dose of 2.6×10 14 vg / kg treatment group was 8.8 × 1013 vg / kg group. Compared to untreated Aspa− / − animals, the lower 13 No changes in NAA were observed at vg / kg. [Table 5]

[0143] Functional and spatial memory tests Mice were tested for improvement in three motor function assessments. At PND27, 2.6×10 13 Animals receiving 8.8×10 vg / kg showed improvement compared to untreated Aspa− / − mice, but did not behave similarly to WT animals. 13 vg / kg or 2.6 × 10 14 Motor function tests (rotarod, balance beam, and inversion screen) in mice treated with 1.5 vg / kg were comparable to WT mice on PND27 and PND90 in all three assessments. Results on PND180 and PND360 were 2.6 × 10 14 The results showed that BBP-812 at a dose of 8.8×10 vg / kg rescued motor function. 13 Mice receiving 100 vg / kg showed limited improvement in motor function at PND180 and no benefit at the final time point (Figure 4).

[0144] Test results: Brain histopathology Brain histopathology in animals receiving BBP-812 was evaluated by H&E staining and compared with WT and untreated Aspa− / − mice (FIGS. 5A and 5B). 14 Pathology from animals receiving the 8.8×10 vg / kg dose was indistinguishable from WT animals. 13 Animals receiving the 2.6×10 vg / kg dose showed significant improvement with reduced vacuolation, although some disease was still present (note the pons (Po), cerebellum (Ce), and striatum (St) as examples). 13 Mice treated with 1000 mg / kg showed little, if any, improvement compared to untreated Aspa− / − mice.

[0145] Test results: Biodistribution Vector genomes per diploid cell (vg / cell) was determined by ddPCR in samples from different regions of the brain (Figure 6). One year after dosing, 0.5-1.5 vg / cell was detected, demonstrating that IV administration of BBP-812 is able to access and persist in all regions of the brain tested.

[0146] As a final determination of efficacy, brain histopathology in Aspa − / − mice receiving BBP-812 was evaluated by H&E staining and compared to wild-type and untreated Aspa − / − mice (FIGS. 7A and 7B). 14 Histopathology from Aspa− / − animals receiving a dose of 8.8×10 vg / kg was indistinguishable from wild-type animals. 13 Animals receiving the 2.6×10 vg / kg dose showed marked improvement compared to untreated controls, with reduced vacuolization, although some disease features were still present (note the pons [Po], cerebellum [Ce], and striatum [St] as examples). 13 Aspa− / − mice treated with 1000 vg / kg showed little, if any, improvement compared to untreated mice.

[0147] The results of these analyses are 2.6x10 14 showed that a complete reversal of the disease phenotype could be achieved at a dose of 10 × 10 vg / kg, whereas a 10-fold lower dose provided minimal benefits on motor function but no benefits on NAA metabolism or histopathology. 13 vg / kg was sufficient to significantly improve all efficacy endpoints, but not as well as the highest dose. Based on these findings, the MED of BBP-812 is approximately 8.8 × 10 13 vg / kg. Safety and biodistribution in non-human primates The objective of this study was to evaluate the safety (clinical observations, clinical chemistry [serum, urine, and CSF], hematology, immune responses, and histopathology) and biodistribution of BBP-812 in the CNS and peripheral tissues. Three routes of administration were compared in this study: IV into the saphenous vein, intrathecal (IT) into the lumbar subarachnoid space, and intracerebroventricular (ICV) into the left lateral ventricle. Animals were sacrificed at 3 and 8 weeks post-dose. The overall study design is outlined in Table 6. [Table 6]

[0148] Study Design: Dose Justification Dose was selected based on the minimal effective dose identified in a PoC study in the Aspa- / - mouse model of Canavan disease. The IV administration route was directly compared to IT and ICV delivery to determine which route promoted superior general CNS biodistribution in a large animal model.

[0149] Study design: Outcome measures Animals were monitored regularly throughout the study by daily clinical observations. Neurological evaluations were performed on days 22 and 57. Blood was collected for clinical pathology and hematology on days -1, 3, 8, 22, 36, and 57 post-dose. Antibody responses to AAV9 capsid and ASPA protein were determined in serum and CSF pre-dose and on days 22 and 57 post-dose. Biodistribution was assessed as vector genomes per diploid cell measured by droplet digital polymerase chain reaction (ddPCR) using a transgene-specific TaqMan assay. Histopathology included H&E staining of all tissues, and the CNS underwent additional immunostaining for ionized calcium-binding adaptor molecule 1 (IBA-1) and glial fibrillary acidic protein (GFAP). At least eight dorsal root ganglia and associated spinal nerve roots were examined per animal. Test results: Biodistribution Biodistribution of BBP-812 and expression of the hASPAOpt transgene were evaluated in samples from animals in groups 1, 2, 3, 7, and 9. Expression in the brain was evaluated from 3 mm x 3 mm tissue punches. A list of all punches collected and analyzed (bold) from the brain can be seen in Figure 8. A complete list of tissues analyzed, including spinal cord (thoracic, lumbar, and cervical) and peripheral organs, is shown in Table 7. [Table 7]

[0150] Intravenously treated animals exhibit a dose-dependent increase in vector genomes across the CNS and in peripheral tissues. In the high dose group, vector genomes in brain punches at necropsy on day 57 ranged from 0.26 to 2.16. Animals receiving IT or ICV administration of BBP-812 had significantly lower levels of transduction in the CNS compared to all three IV doses. In the highest dose IV animals, vector was detectable in all brain regions tested in each animal. In the IT and ICV dosing groups, vector was undetectable in 5 / 30 and 13 / 30 brain punches, respectively. Detection of vector genomes in peripheral tissues was also dose-dependent for the IV group, with the highest levels detected in the liver and heart. Animals receiving IT and ICV administration of BBP-812 had detectable genomes in peripheral tissues, but at much lower levels than animals administered by the IV route (Figures 9A-9E). Intravenous dosing was observed to penetrate far superior to either IT or ICV into the deep cortical white matter and all other brain regions thought to be most relevant in Canavan disease.

[0151] Test results: Detection of anti-AAV9 and anti-ASPA antibodies All animals were screened negative for neutralizing antibodies to AAV9 prior to randomization and dosing. Total antibodies to AAV9 and ASPA were measured pre- and post-dose in animals in groups 1, 2, 3, 7, and 9 using a qualified enzyme-linked immunosorbent assay (ELISA). Serum analysis included samples collected pre-dose and on days 22 and 57 post-dose (Table 8). No animals tested positive for antibodies to AAV9 prior to dosing. After dosing, 11 / 12 IV-treated animals, 5 / 6 IT-treated animals, and 5 / 6 ICV-treated animals developed antibodies to AAV9. Before dosing, one animal in the high-dose IV group had detectable antibodies to ASPA. After dosing, 10 / 12 IV-treated animals, 1 / 6 IT-treated animals, and 1 / 6 ICV-treated animals had antibodies to ASPA. [Table 8]

[0152] Test Results: Neurological Examination Animals underwent neurological examinations on days 22 and 57. This included assessment of general sensory and motor function, brain reflexes, and spinal reflexes. No BBP-812-related changes were noted during these neurological examinations.

[0153] Test Results: Clinical Chemistry and Hematology and Coagulation Evaluation There were no conclusive BBP-812-related changes in hematological parameters, and coagulation values ​​were unremarkable in all groups. Changes in serum chemistry were limited to transient elevations in liver enzymes in the high-dose IV group. These values ​​returned to normal without intervention. There were no significant changes in urinalysis parameters for any of the animals. Changes in CSF chemistry following administration of BBP-812 included decreases in albumin on days 22 and 57 in animals administered via the IV route.

[0154] Study results: Autopsy and histopathological evaluation and findings A thorough histopathological examination was performed. At necropsy, the brain was sectioned in a brain matrix with a coronal section thickness of 3 mm. The first slice and all other slices thereafter were fixed in 10% neutral buffered formalin for histopathological analysis. The spinal cord (cervical, thoracic, and lumbar) was cut into 1 cm sections, with the catheter tip defined as the zero point for IT animals and the thoracic-lumbar junction for IV animals. The first slice and all subsequent second slices (odd numbers) were fixed in 10% neutral buffered formalin for histopathological evaluation. A complete list of tissues analyzed, and specific information regarding sampling and analysis, is provided in Table 9. [Table 9]

[0155] Test results: Histopathological findings 3.14×10 14 There were no adverse test article-related microscopic or functional changes in NHPs following IV administration of BBP-812 at doses up to 1.8 × 10 vg / kg. Test article-related changes were observed at necropsy on days 22 and 57, with a mean of 1.8 × 10 14 This was only observed in the liver (portal vein infiltration and / or increased cellularity [Kupffer cells]) in two of three animals that received BBP-812 IV at 1000 vg / kg. These changes are commonly observed in studies involving IV administration of AAV products and were not considered adverse.

[0156] Findings of cellular infiltration in the dorsal root ganglia (DRG) were reported in three of six BBP-812-treated IV high-dose animals, affecting a total of four of 48 DRGs. The infiltration was not associated with evidence of neurodegeneration and / or necrosis and was indistinguishable from the same findings routinely observed in control NHPs. The DRG findings in animals receiving BBP-812 by IV administration were not adverse, did not affect functional activity, and had no toxicological consequences. From this study, 3.14 × 10 14 The no observed adverse effect level (NOAEL) in vg / kg was determined.

[0157] 5.0×1012 There were no test sample-related adverse microscopic changes at IT doses containing 5.0×10 total vg BBP-812. 12 Two equivocal test sample-related changes were observed at the total vgBBP-812 dose level. First, there was a mild increase in cellularity in a single dorsal root ganglion in two of three animals (sacrificed on day 22). This was not associated with any neuronal changes and was not interpreted as adverse. Similar changes were not observed in animals necropsied on day 57. Second, at sacrifice on day 22, bilateral microgliosis (mild) in the ventral gray matter was present in one of three animals. This change was not associated with obvious neuronal loss or degeneration, was absent in any other animals, including the day 57 animal, and may have been strictly attributable to enhanced staining of resident microglial cells. This level of microgliosis was not considered adverse.

[0158] Non-clinical toxicity: study design A GLP-compliant safety and biodistribution study was conducted in wild-type C57BL / 6 mice. The experimental design for this study is shown in Table 10. Three groups of C57BL / 6 mice were administered a low dose (1 × 10 14 vg / kg) or high dose (3 × 10 14 Animals received BBP-812 at 100 mg / kg (600 mg / kg) or vehicle and were monitored for 24 weeks. These BBP-812 dose levels are considered clinically relevant. Clinical observations, body weight, and food intake were recorded for the duration of the study. Animals were euthanized at 4, 12, and 24 weeks post-dosing. Tissues were collected, weighed, and preserved for histopathology, in vivo analysis, and immunogenicity. Blood (terminal collection) was collected for hematology, clinical chemistry, biodistribution, and immunogenicity. [Table 10]

[0159] Dosage Justification Doses were selected based on dose range-finding studies performed in the Aspa- / - mouse model of Canavan disease. The IV administration route was selected due to efficacy data generated in Aspa- / - mice and improved distribution to the CNS when compared to IT and ICV dosing observed in NHPs. Based on the concentration of vector used in the PoC study, the retro-orbital route was implemented to allow for administration of larger volumes necessary to achieve the desired higher dose in mice. Doses administered in GLP toxicity studies support the proposed clinical dosing regimen. Age of intervention is also an important factor for successful long-term outcomes after correcting the underlying disease pathology.

[0160] Outcome measures up to 24 weeks Animals underwent daily and then weekly clinical observations. Animals were sacrificed at 4, 12, and 24 weeks post-dosing where peripheral blood samples were collected for clinical pathology and hematology. Immunoassays evaluating antibodies to AAV9 and ASPA at each necropsy and T cell reactivity were determined at all time points by enzyme-linked immunosorbent spot (ELISpot) against peptide pools for full-length AAV9 and human ASPA. Biodistribution was assessed by quantitative polymerase chain reaction (qPCR) for vector genomes and reverse transcription quantitative polymerase chain reaction (RT-qPCR) for transgene RNA. A comprehensive panel of tissues was collected for histological examination with H&E staining.

[0161] Study results: Clinical observation up to 24 weeks Detailed clinical observations were performed at randomization, the day before dosing, and on days 0, 1, 2, and 3 after dosing, weekly for the first 12 weeks, and every other week for the remainder of the study. Similarly, body weights were measured at randomization, the day before dosing, and on days 0, 1, 2, and 3 after dosing, weekly for the first 12 weeks, and every other week for the remainder of the study. Final body weights were collected at necropsy. Finally, food intake per cage and per week was monitored weekly for the first 12 weeks starting on the day of dosing, and then every other week for the remainder of the study. No test sample-related findings were observed. Study results: Hematology and clinical chemistry through 24 weeks Hematology and clinical chemistry panels were evaluated in all groups at weeks 4, 12, and 24. There were no clinically significant test sample-related changes. There was a dose-dependent decrease in creatine kinase and lactate dehydrogenase that was not associated with any microscopic changes (Figures 10A and 10B).

[0162] Histopathology up to 24 weeks There were no test sample related observations in the tissues examined.

[0163] Anti-drug antibody response up to 24 weeks Blood samples were collected for analysis of circulating antibodies against AAV9 and human ASPA at necropsy at 4, 12, and 24 weeks. Blood was processed to serum and samples were stored at <-60°C until analysis. Antibody titers were measured by qualified ELISA.

[0164] At 4 weeks, no animals had an antibody response to the transgene, and all animals that received BBP-812 developed antibodies to AAV9.

[0165] At weeks 12 and 24, one vehicle-treated animal was screened positive for antibodies to AAV9 at each time point, and only at week 12, one animal receiving BBP-812 developed a mild antibody response to the transgene. All animals except one that received BBP-812 developed antibodies to AAV9 (at week 12). A summary of the data is shown in Table 11 and is presented as number of positive animals / number of animals tested. [Table 11]

[0166] To evaluate the cellular immune response to AAV9 and ASPA, splenocytes were isolated from a cohort of animals in each dose group at necropsy at 12 and 24 weeks. Cells were stimulated with 15-mer peptide pools spanning the full length of AAV9 and ASPA, resulting in an overlap of 2 amino acids per peptide. Reactivity to peptide pools was measured by interferon gamma secretion as determined by ELISpot assay. Concanavalin A (ConA) was used as a positive control. All samples tested showed a strong response to ConA, which was absent in vehicle-treated cells, indicating the health and responsiveness of the cells. There was no response to the ASPA peptide. Two of the AAV9 peptide pools showed responses in treated mice that were not observed in the control group. The values ​​observed were just above the detection limit of the assay and >20-fold lower than those observed with ConA (Figure 11).

[0167] Biodistribution up to 24 weeks Tissues collected are listed in Table 12. Tissues were collected at each scheduled necropsy and flash frozen in liquid nitrogen before storage at <-60°C. Blood was washed with K 2 The tissue was collected in EDTA tubes and flash frozen in liquid nitrogen. [Table 12]

[0168] There was no detectable vector in tissues from vehicle-treated animals. All tissues from treated animals were positive for vector genome and transgene RNA (Figure 12). The highest levels of vector were detected in the liver. The highest levels of transgene RNA were in the heart, skeletal muscle, and liver.

[0169] Due to the lack of adverse findings in any of the parameters evaluated, the NOAEL for BBP-812 under the conditions of this study is 3.0 × 10 14 vg / kg.

[0170] Example 3: A Phase 1 / 2 Open Label Study of the Safety and Clinical Activity of Gene Therapy for Canavan Disease by Administration of Adeno-Associated Virus (AAV) A Phase 1 / 2 open-label study of the safety and clinical activity of gene therapy for Canavan disease by administration of an adeno-associated virus serotype 9 (AAV9)-based recombinant vector encoding the human ASPA gene (also referred to herein as "BBP-812") is being conducted. The study timeline and overall study design are shown in FIG. 13.

[0171] The open-label, controlled study is designed to evaluate the safety, tolerability, pharmacodynamic (PD) activity, and clinical activity of BBP-812 at two planned dose levels administered to pediatric participants aged 30 months or younger with Canavan disease. Evaluation of safety, tolerability, PD activity, and preliminary clinical activity of BBP-812 in the first six participants will inform dose selection and enrollment expansion to a total of 15 participants at the selected dose. All participants will be followed for at least five years from the date of treatment with BBP-812.

[0172] Screening Period After the patient's parent(s) and / or legal guardian(s) provide written consent, the patient will be considered a study participant and enrolled in the study. Study participants will be assessed for treatment eligibility during a screening period. For all participants, the standard screening period will begin at the time of the first screening assessment. The screening period will be up to 42 days prior to treatment with BBP-812 (Day 0). If necessary, the screening period may be extended due to transient illness, unavoidable logistical challenges, or other factors as determined by the investigator. If >90 days pass between key screening assessments, those assessments will be repeated (i.e., rescreening).

[0173] Baseline, treatment, and acute follow-up periods The combined duration of the baseline, treatment, and acute follow-up periods will be 52 weeks. The long-term follow-up period will last for at least four years after the end of the acute follow-up period. In total, all participants will be followed for at least five years from the date of treatment with BBP-812.

[0174] After completion of screening procedures, participants' eligibility for treatment will be confirmed during a baseline period. Participants eligible for treatment with BBP-812 will begin glucocorticoid prophylaxis the day before BBP-812 dosing to prevent or suppress potential immune responses to BBP-812, and on the day of BBP-812 dosing, will receive antihistamine prophylaxis to prevent infusion reactions. Participants will receive an intravenous (IV) infusion of BBP-812 on day 0, administered during hospitalization at a sponsor-designated treatment center. Participants will receive only a single dose of BBP-812. After receiving BBP-812, participants will remain at the sponsor-designated treatment center for safety observations for at least 72 hours after dosing completion, or longer, as medically indicated at the investigator's discretion. Upon discharge, participants will continue glucocorticoid prophylaxis for the first month after BBP-812 dosing, followed by a protocol-defined tapering regimen.

[0175] The study uses a conservative approach to the selection of viral vector dose in this pediatric / infant patient population with an extremely rare disease. In total, at least six participants will be treated during the dose-finding phase, with at least three participants planned at each dose level. After the first participant receives BBP-812 at the starting dose, treatment of subsequent participants will occur only after review of the first participant's safety data through at least the 28-day post-dose visit. This review will be conducted by the sponsor and the Data and Safety Monitoring Committee (DSMC) to evaluate the safety and tolerability of BBP-812. In addition to the ongoing sponsor safety data review after each participant is dosed, the DSMC and sponsor require a review of all participants' cumulative post-dose safety data for at least 4 weeks prior to dose escalation, dosing of a second participant in a dose cohort, and cohort expansion at a given dose level. Additionally, MRI and CSF results at month 3 from at least one participant in cohort 1 will be evaluated before dose escalation proceeds to cohort 2. For cohort 2, dose expansion may be considered after at least three participants have been dosed and at least 4 weeks of safety data have been reviewed. Before proceeding with dose expansion, 3-month MRI and CSF results from at least three participants in cohort 1 and at least one participant in cohort 2 will be evaluated. After 12-week post-treatment data are available from the third participant treated in cohort 2, all available data from study participants, including safety and NAA levels, will be evaluated to inform dose selection and support enrollment expansion to a total of 15 participants at the selected dose level.

[0176] During the first 4 weeks after treatment, all evaluations will take place at the treatment center. After this period, subsequent evaluations will be allowed at follow-up centers or, depending on the type of evaluation, in the participant's home.

[0177] Adverse events (AEs) and use of concomitant medications will be monitored on an ongoing basis. Standard safety assessments, laboratory measurements, physical examination, standard 12-lead electrocardiogram (ECG), and baseline electroencephalogram will be performed. Cerebrospinal fluid (CSF) samples will be obtained via lumbar puncture while participants are under sedation / anesthesia at time points consistent with MRI and MRS. Immunogenicity will be assessed by evaluating the development of antibody and T cell responses to the capsid and transgene product. Biodistribution of AAV9 vector in blood, and vector shedding in saliva, urine, and feces will be monitored. During the first 12 weeks following treatment with BBP-812, participants' caregivers will be contacted weekly for safety assessments if such contact does not coincide with a scheduled study visit.

[0178] The PD activity of BBP-812 treatment is evaluated by measuring NAA levels in urine and CNS. Brain NAA levels are measured non-invasively by MRS and also directly in CSF via samples obtained by lumbar puncture coincident with the MRS time point. Concurrently, the effect of BBP-812 treatment on brain anatomy and tissue composition characteristics is evaluated by MRI.

[0179] Functional measures will be assessed through measures and tests of fine and gross motor development and cognition and language development. Participant adaptive behavior and family / caregiver(s) quality of life (QOL) will include measures validated in pediatric populations. The Canavan Disease Rating Scale will be used specifically to assess the neurological domain of participants with Canavan Disease.

[0180] A standard neurological examination will be performed to assess for changes related to Canavan disease. In addition, an ophthalmologic evaluation will be performed, including visual evoked potential (VEP) testing.

[0181] Long-term follow-up period After the treatment and acute follow-up period, the long-term follow-up period will last for at least 4 years. In addition to safety, motor development, cognitive and language development, assessment of family / caregiver(s)'s QOL, neurological status, and NAA levels will be tracked to evaluate the durability of the effects of treatment with BBP-812. In total, all participants will be followed for at least 5 years from the date of treatment with BBP-812. The long-term follow-up monitoring plan describes the process of ongoing safety monitoring to be conducted by the Sponsor in collaboration with DSMC.

[0182] Safety Monitoring The DSMC will monitor and evaluate AEs, serious adverse events (SAEs), and post-dose toxicities occurring during the study in accordance with the Study Safety Management Plan and the DSMC Charter. The DSMC will review cumulative safety data and evaluate the safety and tolerability of BBP-812 in relation to continued treatment and enrollment in Cohort 1 and Cohort 2 during the dose-finding phase, dose selection, and enrollment and treatment during the enrollment expansion phase of the study.

[0183] Stopping rules, dose-limiting toxicities, and dosage adjustments This trial may be suspended, temporarily discontinued, or terminated early by the Sponsor.

[0184] Toxicity will be determined by Common Terminology Criteria for Adverse Events (CTCAE) v5.0. Suspected dose-limiting toxicities include, but are not limited to, SAEs, grade ≥ 3 adverse events, or grade ≥ 3 clinical AEs assessed by the investigator related to BBP-812 and not attributable to Canavan disease or other causes. BBP-812 doses will not be adjusted per participant.

[0185] Prohibited drugs The use of any gene therapy other than BBP-812 is prohibited before and during the study. The use of other investigational drugs for the treatment of Canavan disease or other diseases is not permitted within 30 days (or 5 half-lives, whichever is longer) before BBP-812 administration. Any hepatotoxic agents are prohibited for at least 4 weeks before and 12 weeks after BBP-812 is administered. Participants' parents / caregivers should be advised not to administer acetaminophen to participants during the same period. If medically necessary, the use of potentially hepatotoxic drugs will be determined on an individual participant basis after discussion between the investigator and sponsor. Investigators will make every effort to minimize hepatotoxicity risk by selecting appropriate antiepileptic drugs and sedative or anesthetic agents as necessary for protocol-specified evaluations. Immunosuppressants cannot be used during the study unless otherwise defined in the protocol.

[0186] Number of participants (estimated) Planned Enrollment: Up to 18 participants receiving doses (total): Dose-finding phase: 6 or more participants ● Registration expansion phase: up to 12 participants

[0187] Inclusion and Exclusion Criteria Inclusion Criteria:

[0188] To be eligible for this clinical trial, participants must meet all of the following criteria: - Be male or female and aged 30 months or younger on the anticipated date of BBP-812 infusion (i.e., dosing could occur up to the day before the participant turns 31 months of age). - In the opinion of the investigator, the patient is in stable health and has no acute or chronic hematologic, renal, hepatic, immune, or neurologic disease (other than Canavan disease) as determined by medical history and clinical examination. Those who have received a biochemical and genetic diagnosis of Canavan disease: • Elevated urinary NAA and biallelic variants in the ASPA gene determined at screening or recorded in the participant's medical history. • Have a clinical diagnosis and active signs of Canavan disease (including but not limited to hypotonia, developmental delay, and macrocephaly). ● Are up to date on all immunizations according to geographically applicable guidelines (e.g., Centers for Disease Control and Prevention, World Health Organization) and the participant's parent(s) and / or legal guardian(s) have consented to the administration of standard and study-specific immunizations required during the course of the clinical trial. • Have parent(s) and / or legal guardian(s) who are willing and able to read / understand and provide written, signed informed consent after the nature of the study has been explained to them and before any study-related procedures are performed. Have a parent(s) and / or legal guardian(s) who are willing and able to allow the participant to participate in the clinical trial and comply with all clinical trial requirements, including concomitant medication and other treatment restrictions.

[0189] Exclusion criteria:

[0190] Participants who meet any of the following criteria will be excluded from the study: • A positive total anti-AAV9 antibody (>1:50) test as determined by enzyme-linked immunosorbent assay. Have received previous gene therapy or other therapy (including vaccines) involving AAV. - Receiving high-dose immunosuppressant therapy. Significant progression of Canavan disease, characterized by: the presence of continuous / constant decerebrate or decorticate posturing; Recurrent status epilepticus, or Refractory seizures that do not respond while taking three or more types of antiepileptic drugs Have the ASPA genotype, which is known to be associated with a milder Canavan disease phenotype, as determined by the investigator. • Have a history of liver disease (e.g., cirrhosis or current active liver insufficiency) evidenced by medical history or clinical and / or laboratory findings during screening. Have abnormal laboratory values ​​deemed clinically significant by the investigator: Alanine aminotransferase or aspartate aminotransferase > upper limit of normal (ULN) ●Conjugated bilirubin>ULN Gamma glutamyltransferase > ULN -Estimated glomerular filtration rate (eGFR) is below the lower limit of normal for your age Hemoglobin < 8g / dL ● White blood cell (WBC) count outside the normal range for age ●Platelet count<150,000 / μL - Partial thromboplastin time outside the normal range ● International normalized ratio outside the reference range Have clinical and / or laboratory evidence of active or latent infection, including a positive screening test for severe acute respiratory syndrome coronavirus 2 (SARS CoV2; i.e., coronavirus disease-2019 [COVID19]), human immunodeficiency virus type 1 or 2 (HIV1 or HIV2), hepatitis B or C, or tuberculosis. • The diagnosis of hereditary fructose intolerance has been confirmed based on the results of ALDOB genetic testing during screening. ●Currently participating in another interventional clinical trial or have completed another clinical trial with an investigational drug or device within 30 days or 5 half-lives (at the investigator's discretion) prior to receiving BBP-812. ●Has a past or present medical or behavioral condition, findings on a physical exam or other medical evaluation, or other circumstances that may be relevant, as determined by the investigator. adversely affecting the safety and health of participants in the clinical trial; interferes with the completion of study procedures or follow-up; or • Confusing interpretations of study findings. - History of hypersensitivity to any of the excipients in the investigational drug.

[0191] Investigational drug and placebo, dosage, and administration method Prophylaxis: On day -1, at least 24 hours prior to BBP-812 dosing, participants will begin glucocorticoid prophylaxis with either prednisolone via feeding tube (if already present) or methylprednisolone via IV infusion to prevent or suppress potential immune responses associated with BBP-812 administration. Participants will continue glucocorticoid prophylaxis for the duration of their hospital stay. On the day of BBP-812 dosing, participants will receive antihistamine prophylaxis to prevent infusion reactions. At discharge, participants will receive glucocorticoid prophylaxis with prednisolone (by feeding tube, if already present, or orally) for the first month after BBP-812 dosing. Alternative glucocorticoids may be administered per the investigator's discretion and if medically indicated after consultation with the medical monitor. At the day 28 post-dose visit, the investigator will decide whether to begin glucocorticoid tapering according to protocol-defined guidelines.

[0192] Dosing: Treatment will be administered as follows: BBP-812 Cohort 1: 1.32 × 10 14 Vector genomes (vg) / kilogram (kg) body weight; BBP-812 cohort 2: 3.0 × 10 14 vg / kg body weight. If necessary, lower or intermediate doses of BBP-812 may be selected by the sponsor based on review of cumulative safety and activity data. Participants will be assigned sequentially to treatment cohort 1 or 2 depending on the date of confirmation of treatment eligibility. BBP-812 will be administered at sponsor-designated treatment centers as a single IV infusion on day 0 of the study. The duration of the infusion will depend on the dose level administered.

[0193] Medication may be administered up to and including the day before the participant turns 31 months of age.

[0194] Placebo: No placebo will be administered in this study.

[0195] Duration of treatment and study participation Each treatment-eligible participant will receive a single dose of BBP-812 administered by IV infusion on day 0 of the study.

[0196] The maximum total duration of participation in this study will be at least 5 years.

[0197] Screening Period: Up to 42 days (time may be extended if necessary due to transient participant illness, unavoidable logistical challenges, or other factors as determined by the investigator)

[0198] Treatment and acute follow-up period: 1 year

[0199] Long-term follow-up: at least 4 years

[0200] statistical methods Analysis Populations: All participants who received any amount of study drug will be included in the safety analysis set. All participants who received any amount of study drug and have at least one post-baseline efficacy assessment will be included in the modified ITT analysis set. Participants who discontinue will be included in the mITT analysis set, even if they do not have a post-baseline efficacy assessment. All participants in the mITT analysis set without major protocol violations will be included in the per-protocol analysis set.

[0201] Typical presentation of data tabulations will be by age stratification. Continuous variable summaries will include total number (N), mean, median, standard deviation, minimum, maximum, and two-sided 95% confidence intervals (CI). Categorical data summaries will include counts and percentages with two-sided 95% CI. Kaplan-Meier methods will be used to summarize time-to-event endpoints including the 25th, 50th (median), and 75th percentiles with associated two-sided 95% CI.

[0202] Analysis of efficacy parameters will be primarily based on all participants in each analysis population. However, because efficacy may depend on the participant's age at diagnosis or on the time from diagnosis to treatment, additional exploratory analyses will be conducted, whereby participants will be stratified into appropriate categories, such as age groups and time from diagnosis (e.g., ≤12 months and >12-30 months; ≤12 months and >12 months from diagnosis). Comparisons with natural disease history data from study CVN-101 will be performed using matched patient data, and specific details of matching methods, such as pair matching, propensity scoring group matching, etc., will be specified in the statistical analysis plan.

[0203] The baseline for assessment of AEs related to BBP-812 or glucocorticoid dosing is defined as immediately before dosing initiation, and in general, designation of AEs as treatment-emergent (i.e., treatment-emergent adverse events [TEAEs]) begins on the date / time of treatment initiation. The baseline for analysis of changes in all other variables / endpoints is defined as the closest value before formulation infusion. Thus, baseline is considered as either study day -1, -2, -3, -4, -5, -6, -7, -8, -9, -10, or screening for a particular variable. Longitudinal data (collected continuously over time in the trial and follow-up) are presented at appropriate time intervals, such as monthly, quarterly, etc., depending on the nature of the data.

[0204] Participant placement and demographic characteristics Participant disposition will include the number of participants enrolled in each age group and the number and percentage of participants in each analysis population by age group. Also summarized will be the frequency and percentage of participants who discontinued the study early and the primary reasons for discontinuation. Demographic and baseline characteristics including age, sex, race, ethnicity, weight, length, and body mass index (BMI), as well as time from diagnosis and onset of clinical signs will be summarized by age group.

[0205] Safety analysis Statistical methods for safety analyses will be primarily descriptive in nature and will be performed on the safety analysis set. Imputation of missing safety data will not be performed other than for missing or partial dates and will be described in more detail in the statistical analysis plan. Participants will be stratified by age.

[0206] Code all AEs using The Medical Dictionary for Regulatory Activities version 23.1 (or later, as appropriate). Adverse events, including SAEs and TEAEs, will be summarized by age group and overall, by systemic organ class and preferred terminology. A TEAE is defined as any AE occurring during or after administration of BBP-812. All TEAE summaries will include the number and percentage of participants who experienced the event, and the number of AEs experienced by the participant. Percentages are based on the number of participants in the safety analysis set within each age group. Potential delayed safety effects of BBP-812 will be evaluated as part of the planned long-term follow-up safety evaluations. Summarize AEs of grade 3 or grade 4 severity, and AEs with an incidence of 5% or greater. Summarize the relationship of TEAEs to BBP-812 and glucocorticoids.

[0207] Glucocorticoids For all eligible participants, glucocorticoid prophylaxis will be administered to prevent or suppress potential immune responses associated with BBP-812 administration. The dosing regimen is shown in Figure 15. Antigen-specific T cell responses to AAV vectors have been reported in pediatric patients 2-4 weeks after receiving gene therapy (Novartis 2021, Mendell 2017).

[0208] Prophylaxis: On day -1, beginning at least 24 hours prior to dosing with BBP-812 and continuing upon admission, participants will receive glucocorticoid prophylaxis with prednisolone via feeding tube (if already present) or methylprednisolone via IV infusion. At discharge, participants will receive glucocorticoid prophylaxis by oral prednisolone via feeding tube (if already present) or orally until at least the 28-day post-treatment visit. Investigators may choose a different glucocorticoid if clinically indicated. Safety clinical trials and clinical evaluation of participants will be used to guide whether glucocorticoid prophylaxis should be tapered, continued at the current dose, or increased (up to a maximum dose of 2.0 mg / kg / D prednisolone or equivalent). See below for currently recommended dosing and tapering regimens.

[0209] Participants will receive stress doses of steroids for any intervening febrile illness or physiological stress, using a regimen based on the investigator's assessment of the individual participant's clinical status and local best practice. Adrenocorticotropic hormone (ACTH) stimulation tests will be performed to determine whether the hypothalamic-pituitary-adrenal (HPA) axis is intact and whether the use of stress steroids is no longer necessary. The glucocorticoid regimen in this study is not expected to cause long-term adrenal insufficiency.

[0210] Based on a review of safety data from the first dosed participants (see Example 4), the DSMC recommended moving to a higher starting dose of prednisolone, as well as a longer taper and increased monitoring, as outlined below. ●2.0mg / kg / day until day 7 - 1.5 mg / kg / day until day 29 ● 1.0 mg / kg / day (Mo2) until day 56 Begin taper at a rate of no more than 15% reduction per week for at least 6 weeks. -Monitor safety laboratories with liver function tests at least weekly, or more frequently if relevant trends are identified. For events of elevated AST / ALT, obtain at least one creatine kinase level to evaluate for potential nonhepatic causes.

[0211] The above regimen was used for participants who received the second dose (see Example 4) and reflects the current base case, however, is subject to additional modifications based on the natural accrual of further clinical experience with BBP-812.

[0212] Tapering In the absence of clinically significant laboratory or other findings consistent with hepatic inflammation after 1 month of BBP-812 administration, glucocorticoid prophylaxis may be tapered according to the investigator's judgment and local clinical practice over a period of at least 28 days. If liver abnormalities are noted at the Day 28 Clinical Trial Visit, in consultation with the Medical Monitor, glucocorticoid prophylaxis should be continued or increased to a maximum equivalent to a total daily dose of 2.0 mg / kg / day oral prednisolone until resolution of abnormalities, then tapered appropriately based on dose level and duration of treatment according to the investigator and local clinical practice.

[0213] Safety clinical trials and clinical evaluation of participants during glucocorticoid taper are recommended.

[0214] An ACTH stimulation test will be performed to determine if the HPA axis is intact. Until restoration of normal HPA axis function is confirmed by the above tests, participants will receive stress dose steroids for any intervening febrile illness or physiologic stress, using a regimen based on the investigator's assessment of the individual participant's clinical status and local best practice. The protocol-mandated glucocorticoid dosing regimen is not expected to result in long-term adrenal insufficiency.

[0215] Additionally, regardless of glucocorticoid dose or duration, ACTH stimulation testing will be performed during the later phase of the taper and prior to discontinuation of glucocorticoid prophylaxis to determine whether the HPA axis is intact and whether glucocorticoid prophylaxis can be completely stopped. If HPA axis function has not fully recovered, the patient will be maintained on the current glucocorticoid dose and a second ACTH stimulation test will be performed at the next scheduled study visit (approximately 4 weeks or more later). ACTH stimulation testing will continue at study visits until HPA function normalizes, after which the patient may discontinue glucocorticoids and no longer require stress steroid coverage (see below). The specific methodology, reference ranges, collection times, and other details for testing should be in accordance with local standards of practice and the investigator's medical judgment. Until recovery of normal HPA axis function is confirmed by the above tests, participants will receive stress dose steroids for any intervening febrile illness or physiological stress, using a regimen based on the investigator's assessment of the individual participant's clinical status and local best practice. The protocol-mandated glucocorticoid dosing regimen is not expected to result in long-term adrenal insufficiency.

[0216] Antihistamines For all eligible participants, antihistamine prophylaxis will be used for prevention of infusion reactions to BBP-812. Approved antihistamines include diphenhydramine (IV), hydroxyzine (intramuscular [IM]), and chlorpheniramine (IV), or the corresponding oral formulations (if already present) administered via a feeding tube. Selection of antihistamine, dose, and route of administration will be based on the investigator's medical judgment and local site practice.

[0217] Other preventive and rescue medications In case of anaphylactic reactions, prepare the following therapeutic agents according to the treatment center's standard institutional procedures: epinephrine, crystalloids, antihistamines (e.g., diphenhydramine, hydroxyzine, or chlorpheniramine), and hydrocortisone. To address the potential development of CRS, antibodies against cytokines (e.g., tocilizumab, IL-6R antibody) are readily available. In addition, complement inhibitors (e.g., eculizumab, an antibody that blocks C5 cleavage, and recombinant or plasma-derived C1 esterase inhibitors such as Berinert®) should also be readily available, as episodes consistent with complement-mediated thrombotic microangiopathy have been reported with other systemic AAV9-mediated gene therapy products (Pfizer 2019, Solid 2018, Solid 2019), some of which require anti-complement therapy. For more information on these drugs, please refer to the prescribing information for tocilizumab (Actemra® [Genentech 2019]), eculizumab (Soliris® [Alexion 2020]), and C1 esterase inhibitors (Berinert® [CSL Behring 2019]).

[0218] Of note, eculizumab has been associated with serious meningococcal infections and therefore can only be ordered through restricted programs under the Risk Assessment and Mitigation Strategy (Alexion 2020). For information on meningococcal prophylaxis and registration requirements to obtain eculizumab, please refer to the prescribing information for Soliris® (Alexion 2020). Consultation with a medical professional experienced in the use of eculizumab and management of complement activation syndrome / thrombotic microangiopathy is recommended.

[0219] Clinically significant changes in physical examination findings, laboratory assessments, vital signs, and ECGs will be reported as AEs. Laboratory and vital sign data will be summarized by age group. All laboratory data and vital signs will be listed.

[0220] Concomitant medication data will be listed and summarized by age group. Investigational drug exposure will be summarized by age group.

[0221] Pharmacodynamic activity analysis There are several basic aspects of the analysis of NAA levels in urine and CNS (PD markers). Changes in NAA levels after BBP-812 administration, particularly a substantial decrease from baseline, that are distinguishable from inherent patient variability over time, can be considered as markers of physiological effectiveness of BBP-812 gene therapy, i.e., validation that NAA is a biomarker of effectiveness. In these analyses, changes from baseline can be evaluated using longitudinal data analysis methods. In addition, analyses of absolute values ​​and baseline to post-treatment changes in clinical activity measures to evaluate correlations with decreases in NAA levels, as well as analyses to evaluate potential differences in clinical outcome changes over time that may be related to baseline NAA levels, are performed.

[0222] Statistical analysis of changes in NAA levels from baseline to specific time points will be performed using standard methods such as paired t-tests, with the primary time point analysis being changed to 12 months after treatment with BBP-812. Changes from baseline can also be assessed using longitudinal repeated measures data analysis methods. Additionally, analyses of absolute values ​​and changes in clinical activity measures from baseline to post-treatment will be performed to assess correlations with declines in NAA levels, as well as analyses to evaluate potential differences in clinical outcome changes over time that may be related to baseline NAA levels. Details of PD and clinical activity assessments will be provided in a formal statistical analysis plan.

[0223] Clinical Activities: Motor, cognitive, and language development and function; adaptive behavior The mITT analysis set is considered primary for the evaluation of clinical efficacy, and participants who discontinue the study without a post-baseline assessment of clinical efficacy are considered not responding to treatment. Several imputation methods for the analysis of such completely missing outcomes can also be used, including assignment of the worst value across all treated participants (worst-case analysis) and excluding such participants from the analysis (best-case analysis). For all clinical efficacy analyses, participants are stratified by age group and time since diagnosis.

[0224] Clinical analyses will be based on both absolute data and changes from baseline over time in the developmental studies. Clinical activity data may be compared to similar data collected in the natural history database of Canavan disease participants in Study CVN-101 to detect potential efficacy signals and evaluate the clinical importance of these absolute values ​​and changes from baseline.

[0225] Other assessments: imaging, caregiver questionnaire The mITT analysis set will be considered primary for the assessment of imaging and caregiver questionnaires. Participants who discontinue the study without post-baseline imaging assessment or questionnaire completion will be considered as not responding to treatment. For all imaging and questionnaire analyses, participants will be stratified by age group. Descriptive statistics will be used to present data by age group.

[0226] Analyses will involve baseline brain MRI and brain MRS, as well as baseline questionnaire responses and changes from baseline.

[0227] Sample size Enrollment in the trial will be for participants receiving a maximum of 18 doses. A sample size of up to 15 participants treated at the selected doses would be appropriate for the evaluation of PD effects on NAA, based on similar but somewhat conservative assumptions as used in a previously published protocol (Leone 2012), where a standard deviation of 0.493 mmol in change from baseline was determined and it was considered important to detect a difference of 2–3 mmol from baseline. Using these assumptions for a change from baseline in NAA with a more conservative estimated standard deviation of 1.0 mmol and a clinically relevant difference of 2.0 mmol, a sample size of 15 is more than sufficient for a power of greater than 90%.

[0228] Research Schedule The timeline of events for the screening and long-term follow-up periods are shown in Tables 13 and 14, respectively. [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4] [Table 13-5] [Table 13-6] [Table 13-7] [Table 13-8] [Table 13-9] [Table 13-10] [Table 13-11] [Table 13-12] [Table 13-13] [Table 13-14] [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5]

[0229] Example 4: Interim Results of a Phase 1 / 2 Open Label Clinical Trial Two patients were administered BBP-812 as described in Example 3. Using magnetic resonance spectroscopy (MRS) imaging, we observed robust and durable post-treatment reductions in N-acetylaspartate (NAA) in urine, cerebrospinal fluid (CSF), and brain tissue. Signs of de novo myelination as measured by magnetic resonance imaging (MRI) were also observed. The reduction in brain NAA is an early signal suggesting that intravenously (IV) administered BBP-812 has reached its intended target behind the blood-brain barrier and expresses functional aspartoacylase (ASPA) enzyme. There is evidence in the scientific literature that lower NAA levels are associated with milder disease. More time is needed to see how those reductions in NAA translate into clinical outcomes.

[0230] Intravenous infusion of BBP-812 was well tolerated, and thus far, no participant has experienced treatment-related serious adverse events. At 6 months after treatment, participant 1 showed a 77% reduction in NAA in CSF, a 15% reduction in NAA in brain white matter measured by MRS imaging, and a nearly 50% reduction in urinary NAA. At 3 months after treatment, participant 2 showed an 89% reduction in NAA in CSF, a greater than 50% reduction in NAA in brain white matter measured by MRS imaging, and an 81% reduction in urinary NAA. These observed biochemical changes suggest that BBP-812 is reaching cells critical to the Canavan disease process, a milestone in the disease. These data represent preliminary results, and the final safety and efficacy profile of the investigational gene therapy has not been fully established. *******

[0231] All articles, publications, and patents cited herein are incorporated by reference herein as if each individual article, publication, or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the cited publications are disclosed. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be construed as, an admission or any suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0232] The embodiments illustrated and discussed herein are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. The above-described embodiments of the invention may be modified or changed as understood by those skilled in the art in light of the above teachings without departing from the invention. It is therefore understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described. Enumerated Embodiments Embodiment 1. A method comprising administering to a subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, the rAAV vector comprising: (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); (ii) a non-AAV nucleotide sequence encoding aspartoacylase (ASPA), the non-AAV nucleotide sequence being operably linked to a promoter; The therapeutically effective amount is about 10 13 vg / kg~about 10 15 vg / kg range. Embodiment 2. A method in a subject in need of expression of aspartoacylase (ASPA), comprising administering to said subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, said rAAV vector comprising: (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); (ii) a non-AAV nucleotide sequence encoding ASPA, the non-AAV nucleotide sequence being operably linked to a promoter; The therapeutically effective amount is about 10 13 vg / kg~about 10 15 vg / kg range, thereby expressing ASPA in the subject. Embodiment 3. A method in a subject in need of increasing the level of aspartoacylase (ASPA), comprising administering to said subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, said rAAV vector comprising: (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); (ii) a non-AAV nucleotide sequence encoding ASPA, the non-AAV nucleotide sequence being operably linked to a promoter; The therapeutically effective amount is about 10 13 vg / kg~about 10 15 vg / kg range, thereby increasing the level of ASPA in the subject. Embodiment 4. The method of any one of embodiments 1 to 3, wherein the subject is in need of expression of ASPA. Embodiment 5. The method of any one of embodiments 1 to 4, wherein the subject has reduced levels and / or function of ASPA. Embodiment 6. The method of any one of embodiments 1-5, wherein the subject has a leukodystrophy. Embodiment 7. A method of treating a leukodystrophy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV vector is (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); (ii) a non-AAV nucleotide sequence encoding ASPA, the non-AAV nucleotide sequence being operably linked to a promoter; The therapeutically effective amount is about 10 13 vg / kg~about 10 15 vg / kg range, thereby treating leukodystrophy in the subject. Embodiment 8. The method of embodiment 6 or embodiment 7, wherein the leukodystrophy is associated with a condition selected from the group consisting of Canavan disease, adrenomyeloneuropathy, Alexander disease, cerebrotendinous xanthomatosis, Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, and Refsum disease. Embodiment 9. The method of embodiment 8, wherein the leukodystrophy is associated with Canavan disease. Embodiment 10. The method of any one of embodiments 6 to 9, further comprising selecting a subject with a leukodystrophy prior to administering the rAAV vector. Embodiment 11. A method of treating Canavan disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV vector is (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); (ii) a non-AAV nucleotide sequence encoding ASPA, the non-AAV nucleotide sequence being operably linked to a promoter; The therapeutically effective amount is about 10 13 vg / kg~about 10 15 vg / kg range, thereby treating Canavan disease in the subject. Embodiment 12. The method of embodiment 11, further comprising selecting the subject for Canavan disease prior to administering the rAAV vector. Embodiment 13. The method of any one of embodiments 1 to 12, wherein administration of the rAAV vector results in expression of ASPA in a tissue of the subject. Embodiment 14. The method of embodiment 13, wherein the tissue is a peripheral tissue or a central nervous system (CNS) tissue. Embodiment 15. The method of any one of embodiments 1 to 14, wherein the subject has been determined to have a metabolic imbalance involving a shift from glycolysis to beta-oxidation. Embodiment 16 The method of embodiment 15, wherein said method further comprises detecting said metabolic imbalance by assessing levels of one or more glycolytic and / or beta-oxidation factors. Embodiment 17. The method of embodiment 16, wherein the level of one or more glycolytic and / or beta-oxidation factors is assessed using central nervous system (CNS) fluid obtained from the subject. Embodiment 18. The method further comprises: (a) obtaining a CNS fluid from the subject; (b) detecting an increase in beta-oxidation in said CNS fluid; and The method of any one of embodiments 15 to 17, further comprising: (c) administering the rAAV to the subject based on the detection in (b). Embodiment 19. The method of any one of embodiments 15 to 18, wherein the method further comprises: (a) measuring a metabolic profile of a biological sample obtained from the subject; and (b) identifying a metabolic imbalance comprising a shift from glycolysis to beta-oxidation based on the metabolic profile. Embodiment 20. The method of embodiment 19, wherein measuring the metabolic profile comprises assaying the biological sample using liquid chromatography (LC), mass spectrometry (MS), liquid chromatography / mass spectrometry (LC / MS), or ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). Embodiment 21 The method of embodiment 19 or embodiment 20, wherein the biological sample comprises blood, serum, CNS tissue, or cerebrospinal fluid (CSF). Embodiment 22 The method of embodiment 21, wherein the CNS tissue is brain tissue. Embodiment 23. The method of any one of embodiments 19 to 22, wherein the metabolic profile comprises the level of a first biomarker selected from the group consisting of glucose, glucose-6-phosphate, 3-phosphoglycerate, pyruvate, lactate, and phosphoenolpyruvate. Embodiment 24. The method of any one of embodiments 19 to 23, wherein the metabolic profile comprises the level of a second biomarker selected from the group consisting of carnitine, malonylcarnitine, myristoylcarnitine, palmitoylcarnitine, malonylcarnitine, and beta-hydroxybutyrate. Embodiment 25. The therapeutically effective amount is about 10 14 vg / kg ~ approx. 5×10 14 The method of any one of embodiments 1-24, wherein the dose is in the range of vg / kg. Embodiment 26. The therapeutically effective amount is about 1.32×10 14 26. The method of embodiment 25, wherein the total amount of the serotonin receptor agonist is 100 mg / kg. Embodiment 27. The therapeutically effective amount is about 3×10 14 26. The method of embodiment 25, wherein the total amount of the serotonin receptor agonist is 100 mg / kg. Embodiment 28 The method of any one of embodiments 1 to 27, wherein the rAAV is administered by injection. Embodiment 29 The method of embodiment 28, wherein the rAAV is administered by intravenous infusion. Embodiment 30 The method of any one of embodiments 1 to 27, wherein the rAAV is administered by injection. Embodiment 31 The method of embodiment 30, wherein the injection is selected from the group consisting of intravenous injection, intravascular injection, and intraventricular injection. Embodiment 32. The method of any one of embodiments 1 to 31, wherein the subject is 30 months of age or younger. Embodiment 33 The method of any one of embodiments 1 to 32, wherein ASPA comprises human ASPA protein. Embodiment 34. The method of any one of embodiments 1 to 33, wherein ASPA comprises an amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:6. Embodiment 35. The method of any one of embodiments 1 to 34, wherein the promoter is an astrocyte-specific promoter, a glial fibrillary acidic protein (GFAP) promoter, or an enhanced avian β-actin promoter. Embodiment 36. The method of any one of embodiments 1 to 35, wherein the promoter is a cytomegalovirus / β-actin hybrid promoter or a PGK promoter. Embodiment 37. The method of embodiment 36, wherein the cytomegalovirus / β-actin hybrid promoter is a CAG promoter, a CB6 promoter, or a CBA promoter. Embodiment 38. The method of any one of embodiments 1 to 37, wherein the non-AAV nucleotide sequence encoding ASPA comprises or consists of human ASPA cDNA. Embodiment 39. The method of any one of embodiments 1 to 38, wherein the non-AAV nucleotide sequence encoding ASPA comprises or consists of a codon-optimized nucleotide sequence. Embodiment 40. The method of any one of embodiments 1 to 39, wherein the non-AAV nucleotide sequence encoding ASPA comprises or consists of SEQ ID NO:1. Embodiment 41. The method of any one of embodiments 1 to 40, wherein the non-AAV nucleotide sequence encodes an amino acid sequence of SEQ ID NO:6 or an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:6. Embodiment 42. The method of any one of embodiments 1 to 41, wherein the nucleic acid molecule comprises a cytomegalovirus immediate-early enhancer. Embodiment 43. The method of any one of embodiments 1 to 42, wherein the nucleic acid molecule comprises a rabbit β-globin polyA signal. Embodiment 44. The method of any one of embodiments 1 to 43, wherein the nucleic acid molecule comprises a Kozak sequence. Embodiment 45. The method of any one of embodiments 1 to 44, wherein the nucleic acid molecule comprises a miR-122 binding site. Embodiment 46. The method of any one of embodiments 1 to 45, wherein the ITR is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, or rh74 serotype ITR. Embodiment 47. The method of any one of embodiments 1 to 46, wherein the rAAV is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, or rh74 serotype rAAV. Embodiment 48. The method of any one of embodiments 1 to 47, wherein the rAAV is an AAV9 serotype rAAV. Embodiment 49. The method of any one of embodiments 1 to 48, wherein the rAAV is a self-complementary rAAV (scAAV). Embodiment 50. The method of any one of embodiments 1 to 49, wherein the rAAV is a single-stranded rAAV (ssAAV). EMBODIMENT 51. Approximately 1.32×10 14 Administering to the subject by intravenous infusion 100 mg / kg of a recombinant self-complementary adeno-associated virus 9 (scAAV9) vector, wherein the rAAV9 vector comprises: (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR), a cytomegalovirus immediate early enhancer, a Kozak sequence, and a rabbit β-globin polyA signal; (ii) a non-AAV nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, said non-AAV nucleotide sequence being operably linked to a CB6 promoter that directs expression of ASPA in the subject; The method, wherein the subject is 30 months of age or younger, and the subject has Canavan disease. EMBODIMENT 52. Approximately 3×10 14 Administering to the subject by intravenous infusion 100 mg / kg of a recombinant self-complementary adeno-associated virus 9 (scAAV9) vector, wherein the rAAV9 vector comprises: (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR), a cytomegalovirus immediate early enhancer, a Kozak sequence, and a rabbit β-globin polyA signal; (ii) a non-AAV nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, said non-AAV nucleotide sequence being operably linked to a CB6 promoter that directs expression of ASPA in the subject; The method, wherein the subject is 30 months of age or younger, and the subject has Canavan disease. Embodiment 53. The method of any one of embodiments 1 to 52, further comprising: (a) administering to the subject a small molecule metabolic regulator; (b) prescribing to the subject a dietary intervention, wherein the dietary intervention promotes glycolysis and / or reduces beta-oxidation in the subject; and / or (c) administering to the subject an immunosuppressant. Embodiment 54. The method of any one of embodiments 1 to 53, further comprising administering to the subject a therapeutically effective amount of a glucocorticoid. Embodiment 55. The method of embodiment 54, wherein the glucocorticoid is administered before, simultaneously with, and / or after administration of the rAAV vector. Embodiment 56 The method of embodiment 54 or embodiment 55, wherein the glucocorticoid is prednisolone, methylprednisolone, or a combination thereof. Embodiment 57. The method of any one of embodiments 1 to 56, further comprising administering to the subject a therapeutically effective amount of an antihistamine. Embodiment 58. The method of embodiment 57, wherein the antihistamine is administered before, simultaneously with, and / or after administration of the rAAV vector. Embodiment 59. The method of embodiment 57 or embodiment 58, wherein the antihistamine is diphenhydramine, hydroxyzine, chlorpheniramine, or any combination thereof. Embodiment 60. The method of any one of embodiments 1-59, wherein after said administration, N-acetylaspartic acid (NAA) levels are reduced in urine, cerebrospinal fluid (CSF), and / or brain tissue. Embodiment 61. The method of embodiment 60, wherein after said administration, urinary NAA levels are reduced. Embodiment 62. The method of embodiment 61, wherein the urinary NAA level is reduced by at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Embodiment 63. The method of embodiment 61 or 62, wherein the urinary NAA levels remain reduced compared to pre-treatment levels for at least 6 months, 9 months, 12 months, 18 months, 24 months, or more. Embodiment 64. The method of any one of embodiments 60 to 63, wherein after said administration, NAA levels in the CSF are reduced. Embodiment 65. The method of embodiment 64, wherein the NAA level in the CSF is reduced by at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Embodiment 66. The method of embodiment 64 or 65, wherein NAA levels in the CSF remain reduced compared to pre-treatment levels for at least 6 months, 9 months, 12 months, 18 months, 24 months, or more. Embodiment 67. The method of any one of embodiments 60 to 66, wherein after said administration, NAA levels in brain tissue are reduced. Embodiment 68. The method of embodiment 67, wherein the NAA level in the brain tissue is reduced by at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Embodiment 69. The method of embodiment 67 or 68, wherein NAA levels in the brain tissue remain reduced compared to pre-treatment levels for at least 6 months, 9 months, 12 months, 18 months, 24 months, or more. Embodiment 70. The method of any one of embodiments 1 to 69, wherein after said administration, neomyelination is observable using magnetic resonance imaging (MRI).

Claims

1. A composition for use in a method comprising a recombinant adeno-associated virus (rAAV) vector, the method comprising administering a therapeutically effective amount of the recombinant adeno-associated virus (rAAV) vector to a subject, wherein the rAAV vector (i) A nucleic acid molecule containing at least one AAV inverted terminal repeat (ITR), (ii) A non-AAV nucleotide sequence encoding aspartoacylase (ASPA), which is operably linked to a promoter, comprising: The aforementioned effective therapeutic dose is approximately 10 １３ vg / kg ~ approx. 10 １５ A composition in the range of vg / kg.

2. A composition for use in a method for expressing aspartoacylase (ASPA) in a subject requiring such expression, comprising a recombinant adeno-associated virus (rAAV) vector, wherein the method comprises administering a therapeutically effective amount of the recombinant adeno-associated virus (rAAV) vector to the subject, the rAAV vector is (i) A nucleic acid molecule containing at least one AAV inverted terminal repeat (ITR), (ii) A non-AAV nucleotide sequence encoding ASPA, which is operably linked to a promoter, comprising the non-AAV nucleotide sequence, The aforementioned effective therapeutic dose is approximately 10 １３ vg / kg ~ approx. 10 １５ A composition having a concentration in the range of vg / kg, thereby causing ASPA to be expressed in the subject.

3. A composition for use in a method for increasing the level of aspartoacylase (ASPA) in a subject requiring an increase in the level of aspartoacylase (ASPA), comprising a recombinant adeno-associated virus (rAAV) vector, wherein the method comprises administering a therapeutically effective amount of the recombinant adeno-associated virus (rAAV) vector to the subject, the rAAV vector is (i) A nucleic acid molecule containing at least one AAV inverted terminal repeat (ITR), (ii) A non-AAV nucleotide sequence encoding ASPA, which is operably linked to a promoter, comprising the non-AAV nucleotide sequence, The aforementioned effective therapeutic dose is approximately 10 １３ vg / kg ~ approx. 10 １５ A composition having a range of vg / kg, thereby increasing the level of ASPA in the subject.

4. The composition according to any one of claims 1 to 3, wherein the subject requires the expression of ASPA.

5. The composition according to any one of claims 1 to 3, wherein the subject has a reduced level and / or function of ASPA.

6. The composition according to any one of claims 1 to 3, wherein the subject has leukodystrophy.

7. A composition for use in a method of treating a subject requiring treatment for leukodystrophy, comprising a recombinant adeno-associated virus (rAAV) vector, wherein the method comprises administering a therapeutically effective amount of the recombinant adeno-associated virus (rAAV) vector to the subject, the rAAV vector is (i) A nucleic acid molecule containing at least one AAV inverted terminal repeat (ITR), (ii) A non-AAV nucleotide sequence encoding ASPA, which is operably linked to a promoter, comprising the non-AAV nucleotide sequence, The aforementioned effective therapeutic dose is approximately 10 １３ vg / kg ~ approx. 10 １５ The range is vg / kg, A composition for treating leukodystrophy in the subject.

8. A composition according to any one of claims 1 to 3, or a composition according to claim 7, wherein the subject has a leukodystrophy, and the leukodystrophy is related to a condition selected from the group consisting of Canavan disease, adrenal spinal neuropathy, Alexander disease, cerebral tendon xanthomatous disease, Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, and Refsum disease.

9. The composition according to claim 8, wherein the leukodystrophy is related to Canavan disease.

10. A composition according to any one of claims 1 to 3, or a composition according to claim 7, wherein the subject has leukodystrophy, the method further comprises selecting a subject having leukodystrophy before administering the rAAV vector.

11. A composition for use in a method of treating Canavan disease in a subject requiring treatment, comprising a recombinant adeno-associated virus (rAAV) vector, wherein the method comprises administering a therapeutically effective amount of the recombinant adeno-associated virus (rAAV) vector to the subject, the rAAV vector is (i) A nucleic acid molecule containing at least one AAV inverted terminal repeat (ITR), (ii) A non-AAV nucleotide sequence encoding ASPA, which is operably linked to a promoter, comprising the non-AAV nucleotide sequence, The therapeutically effective amount is about 10 １３ vg / kg to about 10 １５ vg / kg, and A composition for treating Canavan disease in the subject.

12. The composition according to claim 11, further comprising selecting a subject with Canavan disease before administering the rAAV vector.

13. The composition according to any one of claims 1 to 3, 7, or 11, wherein administration of the rAAV vector results in the expression of ASPA in the target tissue.

14. The composition according to claim 13, wherein the tissue is peripheral tissue or central nervous system (CNS) tissue.

15. The composition according to any one of claims 1 to 3, 7, or 11, characterized in that a metabolic imbalance, including a transition from glycolysis to beta-oxidation, is determined to exist in the subject.

16. The composition according to claim 15, further comprising detecting the metabolic imbalance by evaluating the levels of one or more glycolytic and / or beta-oxidation factors.

17. The composition according to claim 16, wherein the level of one or more glycolytic and / or beta-oxidizing factors is evaluated using a central nervous system (CNS) fluid obtained from the subject.

18. The method described above is (a) Obtaining CNS liquid from the subject, (b) To detect an increase in beta oxidation in the CNS solution, (c) The composition according to claim 15, further comprising administering the rAAV to the subject based on the detection in (b).

19. The composition according to claim 15, wherein the method further comprises (a) measuring the metabolic profile of a biological sample obtained from the subject, and (b) identifying a metabolic imbalance, including a transition from glycolysis to beta-oxidation, based on the metabolic profile.

20. The composition according to claim 19, wherein measuring the metabolic profile comprises assaying the biological sample using liquid chromatography (LC), mass spectrometry (MS), liquid chromatography / mass spectrometry (LC / MS), or ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS).

21. The composition according to claim 19, wherein the biological sample comprises blood, serum, CNS tissue, or cerebrospinal fluid (CSF).

22. The composition according to claim 21, wherein the CNS tissue is brain tissue.

23. The composition according to claim 19, wherein the metabolic profile includes levels of a first biomarker selected from the group consisting of glucose, glucose-6-phosphate, 3-phosphoglyceric acid, pyruvate, lactate, and phosphoenolpyruvic acid.

24. The composition according to claim 19, wherein the metabolic profile includes the level of a second biomarker selected from the group consisting of carnitine, malonylcarnitine, myristoylcarnitine, palmitoylcarnitine, malonylcarnitine, and beta-hydroxybutyrate.

25. The aforementioned effective therapeutic dose is approximately 10 １４ vg / kg ~ approx. 5×10 １４ A composition according to any one of claims 1 to 3, 7, or 11, wherein the concentration is in the range of vg / kg.

26. The aforementioned effective therapeutic dose is approximately 1.32 × 10⁻⁶ １４ The composition according to claim 25, wherein the concentration is vg / kg.

27. The aforementioned effective therapeutic dose is approximately 3 × 10 １４ The composition according to claim 25, wherein the concentration is vg / kg.

28. The composition according to any one of claims 1 to 3, 7, or 11, characterized in that the composition is administered by injection.

29. The composition according to claim 28, characterized in that the composition is administered by intravenous injection.

30. The method according to any one of claims 1 to 3, 7, or 11, characterized in that the composition is administered by injection.

31. The composition according to claim 30, wherein the injection is selected from the group consisting of intravenous injection, intravascular injection, and intraventricular injection.

32. The composition according to any one of claims 1 to 3, 7, or 11, wherein the subject is 30 months of age or younger.

33. The composition according to any one of claims 1 to 3, 7, or 11, wherein ASPA comprises human ASPA protein.

34. The composition according to any one of claims 1 to 3, 7, or 11, wherein ASPA comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with respect to SEQ ID NO:

6.

35. The composition according to any one of claims 1 to 3, 7, or 11, wherein the promoter is an astrocyte-specific promoter, a glial fibrillary acidic protein (GFAP) promoter, or an enhanced tri-β-actin promoter.

36. The composition according to any one of claims 1 to 3, 7, or 11, wherein the promoter is a cytomegalovirus / β-actin hybrid promoter or a PGK promoter.

37. The composition according to claim 36, wherein the cytomegalovirus / β-actin hybrid promoter is a CAG promoter, a CB6 promoter, or a CBA promoter.

38. The composition according to any one of claims 1 to 3, 7, or 11, wherein the non-AAV nucleotide sequence encoding ASPA includes or consists of human ASPA cDNA.

39. The composition according to any one of claims 1 to 3, 7, or 11, wherein the non-AAV nucleotide sequence encoding ASPA includes or consists of a codon-optimized nucleotide sequence.

40. The composition according to any one of claims 1 to 3, 7, or 11, wherein the non-AAV nucleotide sequence encoding ASPA includes or consists of SEQ ID NO:

1.

41. The composition according to any one of claims 1 to 3, 7, or 11, wherein the non-AAV nucleotide sequence codes for the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with respect to SEQ ID NO:

6.

42. The composition according to any one of claims 1 to 3, 7, or 11, wherein the nucleic acid molecule comprises a cytomegalovirus pre-initial enhancer.

43. The composition according to any one of claims 1 to 3, 7, or 11, wherein the nucleic acid molecule comprises a rabbit β-globin polyA signal.

44. The composition according to any one of claims 1 to 3, 7, or 11, wherein the nucleic acid molecule comprises a Kozak sequence.

45. The composition according to any one of claims 1 to 3, 7, or 11, wherein the nucleic acid molecule includes a miR-122 binding site.

46. The composition according to any one of claims 1 to 3, 7, or 11, wherein the ITR is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, or rh74 serotype ITR.

47. The composition according to any one of claims 1 to 3, 7, or 11, wherein the rAAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, or rh74 serotype rAAV.

48. The composition according to any one of claims 1 to 3, 7, or 11, wherein the rAAV is AAV9 serotype rAAV.

49. The composition according to any one of claims 1 to 3, 7, or 11, wherein the rAAV is a self-complementary rAAV (scAAV).

50. The composition according to any one of claims 1 to 3, 7, or 11, wherein the rAAV is a single-stranded rAAV (ssAAV).

51. A composition for use in a method comprising a recombinant self-complementary adeno-associated virus 9 (scAAV9) vector, wherein the method is Approximately 1.32×10 １４ The procedure includes administering the recombinant autocomplementary adeno-associated virus 9 (scAAV9) vector at a dose of vg / kg to a subject by intravenous infusion, wherein the rAAV9 vector is (i) At least one AAV inverted terminal repeat (ITR), cytomegalovirus prodromal A nucleic acid molecule containing an enhancer, a Kozak sequence, and a rabbit β-globin polyA signal, (ii) A non-AAV nucleotide sequence encoding the amino acid sequence of Sequence ID No. 1, which is operably linked to a CB6 promoter that directs the expression of ASPA in the subject, comprising: A composition wherein the subject is 30 months of age or younger and has Canavan disease.

52. A composition for use in a method comprising a recombinant self-complementary adeno-associated virus 9 (scAAV9) vector, wherein the method is Approximately 3×10 １４ The procedure includes administering the recombinant autocomplementary adeno-associated virus 9 (scAAV9) vector at a dose of vg / kg to a subject by intravenous infusion, wherein the rAAV9 vector is (i) A nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR), a cytomegalovirus pre-initial enhancer, a Kozak sequence, and a rabbit β-globin polyA signaling molecule, (ii) A non-AAV nucleotide sequence encoding the amino acid sequence of Sequence ID No. 1, which is operably linked to a CB6 promoter that directs the expression of ASPA in the subject, comprising: A composition wherein the subject is 30 months of age or younger and has Canavan disease.

53. The composition according to any one of claims 1 to 3, 7, 11, 51 or 52, wherein the method further comprises (a) administering a low molecular weight metabolic modifier to the subject, (b) prescribing a dietary intervention to the subject, wherein the dietary intervention promotes glycolysis and / or reduces beta-oxidation in the subject, and / or (c) administering an immunosuppressant to the subject.

54. The composition according to any one of claims 1 to 3, 7, 11, 51 or 52, characterized in that the composition is administered to the subject in combination with a glucocorticoid.

55. The composition according to claim 54, wherein the glucocorticoid is administered before, simultaneously with, and / or after the administration of the rAAV vector.

56. The composition according to claim 54, wherein the glucocorticoid is prednisolone, methylprednisolone, or a combination thereof.

57. The composition according to any one of claims 1 to 3, 7, 11, 51 or 52, characterized in that the composition is administered to the subject in combination with a therapeutically effective amount of an antihistamine.

58. The composition according to claim 57, wherein the antihistamine is administered before, simultaneously with, and / or after the administration of the rAAV vector.

59. The composition according to claim 57, wherein the antihistamine is diphenhydramine, hydroxyzine, chlorpheniramine, or any combination thereof.

60. The composition according to any one of claims 1 to 3, 7, 11, 51, or 52, wherein, after administration, levels of N-acetylaspartic acid (NAA) in urine, cerebrospinal fluid (CSF), and / or brain tissue decrease.

61. The composition according to claim 60, wherein the level of NAA in the urine decreases after the aforementioned administration.

62. The composition according to claim 61, wherein the NAA level in the urine is reduced by at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.

63. The composition according to claim 61, wherein the NAA level in the urine remains reduced compared to the pre-treatment level for at least 6 months, 9 months, 12 months, 18 months, 24 months, or longer.

64. The composition according to claim 60, wherein the NAA level in CSF decreases after the aforementioned administration.

65. The composition according to claim 64, wherein the NAA level in the CSF is reduced by at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.

66. The composition according to claim 64, wherein the NAA level in the CSF remains reduced compared to the pre-treatment level for at least 6 months, 9 months, 12 months, 18 months, 24 months, or longer.

67. The composition according to claim 60, wherein the NAA level in brain tissue decreases after the aforementioned administration.

68. The composition according to claim 67, wherein the NAA level in the brain tissue is reduced by at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.

69. The composition according to claim 67, wherein the NAA level in the brain tissue remains reduced compared to the pre-treatment level for at least 6 months, 9 months, 12 months, 18 months, 24 months, or longer.

70. The composition according to any one of claims 1 to 3, 7, 11, 51, or 52, wherein, after the administration, new myelin formation is observable using magnetic resonance imaging (MRI).

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