Method for treating metachromatic leukodystrophy

Abstract

An expression cassette for expressing a transgene in hepatocytes is provided herein, wherein the transgene encodes an ARSA polypeptide. Also provided is a method of treating metachromatic leukodystrophy (MLD). Further provided herein are vectors (e.g., rAAV vectors), viral particles, pharmaceutical compositions, and kits for expressing the ARSA polypeptide in an individual in need thereof.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 342,590, filed on May 16, 2022, and U.S. Provisional Patent Application No. 63 / 459,564, filed on April 14, 2023, the entire contents of each of which are incorporated herein by reference.

[0002] Reference to Electronic Sequence Listing The content of the electronic sequence listing (159792018440seqlist.xml; size: 36,314 bytes; created on May 8, 2023) is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to a method of treating metachromatic leukodystrophy (MLD) in a patient in need thereof, the method comprising administering to the patient's cerebrospinal fluid (CSF) viral particles of a recombinant adeno - associated virus (rAAV) comprising a vector encoding arylsulfatase A (ARSA).

Background Art

[0004] Metachromatic leukodystrophy (MLD) is an autosomal recessive neurodegenerative disorder caused by mutations in the enzyme arylsulfatase A (ARSA). A decrease in ARSA activity levels results in the toxic accumulation of sulfatides, which is characterized by the degeneration of myelin - forming cells (oligodendrocytes and Schwann cells) in the central and peripheral nervous systems. This leads to demyelination, dysfunction, neuronal degeneration, and neuroinflammation (astrocytosis, microglial activation). Clinical symptoms are mainly in the nervous system, resulting in intellectual disability, emotional and behavioral problems, loss of motor abilities (mobility, speech, swallowing), decreased muscle function and paralysis, blindness, deafness, and seizures.

[0005] Current treatments for MLD include autologous hematopoietic stem cell transplantation (HSCT), including allogeneic bone marrow transplantation.

Summary of the Invention

Means for Solving the Problem

[0006] In some embodiments, the present invention provides a method for treating metachromatic leukodystrophy (MLD) in a patient in need thereof, the method comprising administering to the cerebrospinal fluid (CSF) of the patient viral particles of a recombinant adeno-associated virus (rAAV) comprising a vector encoding arylsulfatase A (ARSA) (also referred to herein as the ARSA polypeptide). In some embodiments, the viral particles are administered directly to the CSF by intracerebroventricular (ICV) administration, direct cisterna magna (dCM) administration, or intrathecal microcatheter (IT-CM).

[0007] The present invention is based, at least in part, on the development of rAAV viral particles comprising (a) an rAAV vector comprising an expression cassette encoding an arylsulfatase A (ARSA) polypeptide, and (b) a capsid capable of transducing cells of the central nervous system (CNS). Administration of the viral particles to the CSF provides high-level expression of the ARSA protein in the brain. In certain embodiments, the expression cassette of the viral particles can promote transgene expression for treating MLD in the central and peripheral nervous systems. In some embodiments, the expression cassette comprises the ARSA gene of SEQ ID NO: 2. In some embodiments, the ARSA gene expresses an ARSA polypeptide having SEQ ID NO: 1.

[0008] In some embodiments, the invention provides rAAV particles comprising a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV vector comprises an expression cassette for expressing an ARSA polypeptide in the central nervous system and the peripheral nervous system, the expression cassette comprises a promoter and a transgene operably linked, optionally, to an enhancer, the transgene encodes an ARSA polypeptide, and the AAV viral particles comprise a capsid protein capable of transducing cells of the central nervous system (CNS). In some embodiments, the AAV capsid protein is an AAV9 capsid protein (SEQ ID NO: 9). In some embodiments, the capsid of the rAAV is a modified AAV9 capsid protein. In some embodiments, the capsid of the rAAV is an AAV.rh10 capsid protein. In some embodiments, the ARSA gene expresses an ARSA polypeptide having SEQ ID NO: 1.

[0009] In some embodiments, the present disclosure provides rAAV particles for intracerebrospinal fluid (CSF) administration of an ARSA polypeptide, comprising a modified AAV9 capsid protein comprising a targeting peptide that targets the brain to rAAV particles. Such modified AAV9 capsids are described in International Publication No. WO 2021 / 102234A1, which is hereby incorporated by reference in its entirety. In certain embodiments, the targeting peptide of the modified AAV9 capsid is inserted after residue 588 of the AAV9 structural protein. In some embodiments, the targeting peptide has SEQ ID NO: 10. In some embodiments, the targeting peptide has linker sequences flanking the N-terminus and C-terminus of the targeting peptide. In some embodiments, the N-terminal linker sequence has SEQ ID NO: AAA. In some embodiments, the C-terminal linker sequence is AS. In some embodiments, the complete sequence inserted after residue 588 of the AAV9 capsid structural protein has SEQ ID NO: 11. In some embodiments, the fully modified AAV9 capsid structural protein (VP1) has SEQ ID NO: 12. The capsid protein having SEQ ID NO: 12 is referred to herein as AAV1999. In some embodiments, the fully modified AAV9 capsid structural protein is at least 90% (e.g., at least 92%, at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.5%, or at least 99.8%) identical to SEQ ID NO: 12, wherein the modified AAV9 capsid comprises the targeting peptide of SEQ ID NO: 10.

[0010] The rAAV particles containing the modified AAV9 capsid protein disclosed in this specification contain three structural capsid proteins, VP1, VP2, and VP3. The three capsid proteins are alternative splicing variants. The full-length VP1 protein has the sequence of SEQ ID NO: 9. The VP2 protein contains amino acids 138 to 736 of SEQ ID NO: 9. The VP3 protein contains amino acids 203 to 736 of SEQ ID NO: 9. In some embodiments, the targeting peptide (SEQ ID NO: 10) is inserted into the AAV9 VP3 capsid protein in the rAAV particle. In some embodiments, the targeting peptide (SEQ ID NO: 10) is inserted into the AAV9 VP2 capsid protein in the rAAV particle. In some embodiments, the targeting peptide (SEQ ID NO: 10) is inserted into the AAV9 VP1 capsid protein in the rAAV particle. In some embodiments, the targeting peptide (SEQ ID NO: 10) is inserted into the VP1, VP2, and VP3 capsid proteins within the rAAV particle. In some embodiments, the targeting peptide in the modified AAV9 capsid protein has linker sequences adjacent to the N-terminus and C-terminus of the targeting peptide. In some embodiments, the linker sequence on the N-terminal side has the sequence AAA. In some embodiments, the linker sequence on the C-terminal side is AS.

[0011] rAAV particles containing a modified AAV9 capsid protein comprising the targeting peptide of SEQ ID NO: 10 have been found to exhibit very high levels of transgene expression after administration to the brain of a subject (e.g., intracerebrospinal fluid (CSF) administration). For example, rAAV particles comprising a modified VP1 capsid protein having SEQ ID NO: 12 (and / or a modified VP2 capsid protein having SEQ ID NO: 13 and / or a modified VP3 capsid protein having SEQ ID NO: 14) exhibit higher levels of transgene expression in the brain and spinal cord than rAAV particles comprising other capsids capable of transducing cells of the CNS, such as AAV9 and AAV.rh10, after intracerebrospinal fluid (CSF) administration of the rAAV particles. Conversely, after intracerebrospinal fluid (CSF) administration of rAAV particles comprising a modified AAV9 capsid comprising the targeting peptide of SEQ ID NO: 10, the levels of transgene expressed in other organs such as the heart and liver are substantially lower. Furthermore, rAAV particles comprising a modified AAV9 capsid comprising the targeting peptide of SEQ ID NO: 10 can achieve high levels of expression at lower doses compared to rAAV particles comprising other capsids capable of transducing cells of the CNS, such as AAV9 and AAV.rh10. Thus, rAAV particles comprising a modified AAV9 capsid protein comprising the targeting peptide of SEQ ID NO: 10 have an improved therapeutic index compared to rAAV particles comprising an AAV9 capsid or an AAV.rh10 capsid.

[0012] Accordingly, in one aspect, the present invention provides a method of treating metachromatic leukodystrophy (MLD) in a patient in need thereof, the method comprising administering to the patient's cerebrospinal fluid (CSF) viral particles of a recombinant adeno-associated virus (rAAV) comprising (1) a vector encoding an ARSA polypeptide and (2) a modified AAV9 capsid protein, wherein the modified rAAV capsid protein comprises the targeting peptide of SEQ ID NO: 10. In some embodiments, the modified AAV9 capsid protein comprising SEQ ID NO: 10 is at least 90% (e.g., at least 92%, at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.5%, or at least 99.8%) identical to the capsid protein having SEQ ID NO: 12. In some embodiments, the capsid protein has SEQ ID NO: 12.

[0013] In some embodiments of the above aspect, the rAAV particles comprise a vector comprising an expression cassette flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the expression cassette is flanked by two AAV ITRs. In some embodiments, the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAV.rh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or murine AAV serotype ITRs. In some embodiments, the AAV ITR is an AAV2 ITR. In some embodiments, the vector is a self-complementary vector. In some embodiments, the vector comprises a first nucleic acid sequence encoding an ARSA polypeptide and a second nucleic acid sequence encoding a complement of the ARSA polypeptide, and the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence over most or all of its length. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutant AAV ITR, the mutant AAV ITR comprising a deletion in the D region and a mutation in the terminal resolution sequence.

[0014] In some aspects, the present invention provides a composition comprising any of the rAAV particles described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0015] In some aspects, the present invention provides a cell comprising any of the rAAV particles described herein. In some aspects, the present invention provides a method of producing an ARSA polypeptide, the method comprising culturing a cell described herein under conditions that produce the ARSA polypeptide. In some embodiments, the method further comprises a step of purifying the ARSA polypeptide.

[0016] In some aspects, the present invention provides a method of treating MLD in an individual in need thereof, the method comprising administering to the individual an rAAV particle described herein. In some aspects, the present invention provides a method of treating MLD in an individual in need thereof, the method comprising administering to the individual a composition described herein. In some embodiments, the present invention provides a method of treating MLD in an individual in need thereof, the method comprising administering to the individual a cell described herein. In some embodiments, the individual lacks ARSA activity.

[0017] In some embodiments, the present invention provides a method of increasing ARSA activity in an individual in need of an increase in ARSA activity by at least about 5%, the method comprising administering to the individual the rAAV particles described herein. In other embodiments, the present invention provides a method of increasing ARSA activity in an individual in need of an increase in ARSA activity by at least about 10%, the method comprising administering to the individual the rAAV particles described herein. In other embodiments, the present invention provides a method of increasing ARSA activity in an individual in need of an increase in ARSA activity by at least about 20%, the method comprising administering to the individual the rAAV particles described herein. In other embodiments, the present invention provides a method of increasing ARSA activity in an individual in need of an increase in ARSA activity by at least about 30%, the method comprising administering to the individual the rAAV particles described herein. In other embodiments, the present invention provides a method of increasing ARSA activity in an individual in need of an increase in ARSA activity by at least about 50%, the method comprising administering to the individual the rAAV particles described herein.

[0018] Administration of the rAAV particles can be effected via a variety of routes. In some embodiments, administration includes direct spinal cord injection and / or intracerebral administration. In some embodiments, administration is made to a site selected from the cerebrum, medulla, pons, cerebellum, cranial cavity, meninges surrounding the brain, dura mater, arachnoid mater, pia mater, cerebrospinal fluid (CSF) in the subarachnoid space surrounding the brain, deep cerebellar nuclei of the cerebellum, ventricular system of the cerebrum, subarachnoid space, striatum, cortex, septum, thalamus, hypothalamus, and brain parenchyma. In some embodiments, administration includes intracerebroventricular injection into at least one lateral ventricle. In some embodiments, administration includes intrathecal injection in the cervical, thoracic, and / or lumbar regions. In some embodiments, administration includes intrastriatal injection. In some embodiments, administration includes intrathalamic injection.

[0019] In some embodiments of the above aspects, the rAAV particles are administered by direct injection into the spinal cord, by intrathecal injection, and / or by cisterna magna injection. In some embodiments, the rAAV particles are administered at multiple positions in the spinal cord or cisterna magna. In some embodiments, the rAAV particles are administered at multiple positions in the spinal cord. In some embodiments, the rAAV particles are administered to one or more of the lumbar subarachnoid space, thoracic subarachnoid space, and cervical subarachnoid space of the spinal cord. In some embodiments, the rAAV particles are administered to the cisterna magna.

[0020] In some embodiments of the above aspects, the rAAV particles are administered to a patient in need thereof only once. In some embodiments, the rAAV particles are administered to a patient in need thereof multiple times (e.g., over a period of one month or more or one year or more). In other embodiments, the rAAV particles are administered to a patient in need thereof once a year. In other embodiments, the rAAV particles are administered to a patient in need thereof twice a year.

[0021] In some embodiments, the present invention provides a kit comprising any of the rAAV particles, compositions, or cells described herein. In some embodiments, the kit further comprises instructions for use; a buffer and / or a pharmaceutically acceptable excipient; and / or a bottle, vial, and / or syringe.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0023] In some embodiments, the present invention provides an expression cassette comprising a transgene encoding an ARSA polypeptide, a recombinant adeno-associated virus (rAAV) vector, and viral particles and pharmaceutical compositions. In further embodiments, the present invention provides a method of treating metachromatic leukodystrophy (MLD), for example, by increasing ARSA activity, decreasing sulfatide accumulation in the brain, spinal cord, liver, plasma, and CSF, increasing and normalizing myelination in the corpus callosum, increasing the oligodendrocyte population in the brain, and improving CNS pathologies resulting from sulfatide clearance in brain regions. In yet another embodiment, the present invention provides a kit for treating MLD in an individual with the expression cassette of the present disclosure.

[0024] Definitions As used herein, "vector" refers to a recombinant plasmid or virus containing nucleic acid that is delivered to a host cell in vitro or in vivo.

[0025] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, with no limit to the minimum length. Such a polymer of amino acid residues may contain natural or unnatural amino acid residues and includes, but is not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. This definition includes both full-length proteins and fragments thereof. The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Further, for the purposes of the present disclosure, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions (generally conservative) relative to the native sequence, as long as the protein maintains the desired activity. These modifications can be intentional, such as by site-directed mutagenesis, or can be accidental, such as due to mutations in the host producing the protein or errors resulting from PCR amplification.

[0026] "Recombinant viral vector" refers to a recombinant polynucleotide vector containing one or more heterologous sequences (i.e., nucleic acid sequences not of viral origin). In the case of a recombinant AAV vector, the recombinant nucleic acid has at least one, and in some embodiments, two, inverted terminal repeat sequences (ITRs) adjacent thereto.

[0027] "Recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., nucleic acid sequences not of AAV origin) with at least one, and in some embodiments, two, inverted terminal repeat sequences (ITRs) adjacent thereto. Such an rAAV vector is present in a host cell infected with a suitable helper virus (or expressing a suitable helper function) and, when expressing the AAV Rep and Cap gene products (i.e., the AAV Rep and Cap proteins), can replicate and be packaged into infectious virus particles. When the rAAV vector is integrated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), the rAAV vector can be referred to as a "provirus" that can be "rescued" by replication and capsid formation in the presence of the AAV packaging function and a suitable helper function. The rAAV vector can take various forms, such as, but not limited to, a plasmid, a linear artificial chromosome, one complexed with a lipid, one encapsulated within a liposome, and one encapsulated within a virus particle, particularly an AAV particle. The rAAV vector can be packaged into an AAV viral capsid to generate "recombinant adeno-associated virus particles (rAAV particles)".

[0028] "Heterologous" means that it is derived from an entity that has a different genotype from the remainder of the entity to which it is being compared, or into which it is being introduced or incorporated. For example, a polynucleotide introduced into different cell types by genetic engineering techniques is a heterologous polynucleotide (which, when expressed, can encode a heterologous polypeptide). Similarly, a cellular sequence (e.g., a gene or a portion thereof) incorporated into a viral vector is a heterologous nucleotide sequence with respect to the vector.

[0029] The term "transgene" refers to a polynucleotide that is introduced into a cell, transcribed into RNA, and optionally can be translated and / or expressed under appropriate conditions. In an embodiment, it confers a desired property to the cell into which it is introduced or otherwise results in a desired therapeutic or diagnostic outcome.

[0030] The "chicken β-actin (CBA) promoter" refers to a polynucleotide sequence derived from the chicken β-actin gene (e.g., Gallus beta-actin represented by GenBank Entrez Gene ID 396526). As used herein, the "chicken β-actin promoter" can refer to the cytomegalovirus (CMV) immediate enhancer element, the promoter and first exon and intron of the chicken β-actin gene, and the splice acceptor of the rabbit beta-globin gene, such as the sequence described in Miyazaki, J. et al. (1989) Gene 79(2):269-77. As used herein, the term "CAG promoter" can be used interchangeably. As used herein, the term "CMV immediate enhancer / chicken beta-actin (CAG) promoter" can be used interchangeably.

[0031] The terms "genomic particles (gp)", "genomic equivalents", or "genomic copies" as used with respect to viral titer refer to the number of virions containing the recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genomic particles in a particular vector preparation can be measured by procedures as described in the Examples herein, or by procedures such as those described in Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

[0032] The term "vector genome (vg)" as used herein can refer to one or more polynucleotides comprising a set of polynucleotide sequences of a vector, such as a viral vector. The vector genome can be encapsulated in viral particles. Depending on the particular viral vector, the vector genome can comprise single-stranded DNA, double-stranded DNA, or single-stranded RNA, or double-stranded RNA. The vector genome can include endogenous sequences associated with the particular viral vector, and / or any heterologous sequences inserted into the particular viral vector by recombinant techniques. For example, a recombinant AAV vector genome can include at least one ITR sequence flanked by a promoter, a stuffer, a sequence of interest (e.g., RNAi), and a polyadenylation sequence. A complete vector genome can include the complete set of polynucleotide sequences of the vector. In some embodiments, the nucleic acid titer of a viral vector can be measured in vg / mL. Suitable methods for measuring this titer are known in the art (e.g., quantitative PCR).

[0033] The terms "infectious unit (iu)", "infectious particle", or "replication unit", when used with respect to viral titer, refer to the number of recombinant AAV vector particles having infectivity and replication ability, measured by an infectious center assay, also known as a replication center assay, as described, for example, in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

[0034] The term "transducing unit (tu)" as used with respect to viral titer refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product as measured in a functional assay such as described in the examples herein or, for example, in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0035] The term "inverted terminal repeat" or "ITR" sequence is a term well understood in the art and refers to a relatively short sequence found at the ends of the viral genome in opposite orientations.

[0036] The term "AAV inverted terminal repeat (ITR)" sequence, which is well understood in the art, is a sequence of approximately 145 nucleotides that exists at both ends of the native single-stranded AAV genome. The outermost 125 nucleotides of the ITR can exist in either of two alternative orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several short regions of self-complementarity (designated regions A, A', B, B', C, C', and D), which allow for intrastrand base pairing within this portion of the ITR.

[0037] The "terminal resolution sequence" or "trs" is a sequence in the D region of the AAV ITR that is cleaved by the AAV rep protein during viral DNA replication. Mutant terminal resolution sequences are resistant to cleavage by the AAV rep protein.

[0038] "AAV helper function" refers to a function that enables AAV to be replicated and packaged by a host cell. The AAV helper function can be provided in various forms that assist in the replication and packaging of AAV, such as, but not limited to, either a helper virus or a helper virus gene. Other AAV helper functions, such as genotoxic substances, are known in the art.

[0039] The "helper virus" of AAV refers to a virus that enables AAV (which is a defective parvovirus) to be replicated and packaged by a host cell. The helper virus provides a "helper function" that enables the replication of AAV. Many such helper viruses have been identified, including adenoviruses, herpesviruses, and poxviruses such as vaccinia virus and baculovirus. Adenoviruses include several different subgroups, but adenovirus type 5 (Ad5) of subgroup C is most commonly used. Many adenoviruses of human, non-human mammalian, and avian origin are known and are available from depositories such as ATCC. Examples of herpes family viruses that are also available from depositories such as ATCC include herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV). Examples of adenovirus helper functions for the replication of AAV include the E1A function, the E1B function, the E2A function, the VA function, and the E4orf6 function. An example of a baculovirus available from a depository is Autographa californica nuclear polyhedrosis virus.

[0040] Preparations of rAAV have a ratio of infectious AAV particles to infectious helper virus particles of at least about 10 2 :1; at least about 10 4:l 、 at least about 10 6 :1; or at least about 10 8:lIn the above case, the helper virus is said to be "substantially free". In some embodiments, the preparation is also free of equivalent amounts of helper virus protein (i.e., the protein that would be present as a result of such levels of helper virus if the above helper virus particle impurities were in a disrupted form). Contamination by viral and / or cellular proteins can generally be observed as the presence of Coomassie-stained bands on an SDS gel (e.g., the appearance of bands other than those corresponding to the AAV capsid proteins VP1, VP2, and VP3).

[0041] "Effective amount" means an amount sufficient to produce a beneficial or desired result, including clinical results (e.g., improvement of symptoms, achievement of clinical endpoints, etc.). An effective amount can be administered in one or more administrations. With respect to a disease state, an effective amount is an amount sufficient to improve, stabilize, or delay the progression of the disease.

[0042] "Individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0043] As used herein, "treatment" is an approach for obtaining a beneficial or desired clinical result. For the purposes of the present disclosure, beneficial or desired clinical results include, but are not limited to, reduction of symptoms, reduction of the extent of the disease, stabilization of the disease state (e.g., not worsening), prevention of spread of the disease (e.g., metastasis), delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete), whether or not detectable. "Treatment" can also mean an increase in lifespan as compared to the expected lifespan in the absence of treatment.

[0044] As used herein, the term "preventive treatment" refers to treatment of an individual who is known or suspected to have or be at risk of a disorder but who has no symptoms or only mild symptoms of the disorder. An individual undergoing preventive treatment can be treated before symptoms occur.

[0045] References in this specification to "about" values or parameters include (and describe) embodiments that are directed to the value or parameter itself. For example, a description that refers to "about X" includes a description of "X".

[0046] As used herein, the singular forms of the articles "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[0047] ) It is understood that the aspects and embodiments of the disclosure described herein include aspects and embodiments "comprising", "consisting of", and / or "consisting essentially of".

[0048] Expression cassette In some embodiments, the transgene encoding the ARSA polypeptide has codons that are optimized. In some embodiments, the transgene encoding the ARSA polypeptide has codons optimized for expression in specific cells such as eukaryotic cells. Eukaryotic cells can be cells of a specific organism such as a mammal including, but not limited to, human, mouse, rat, rabbit, dog, or non-human primate or cells derived therefrom. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a target host cell by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit specific biases for specific codons of a particular amino acid. Codon usage tables are readily available, for example, in the "Codon Usage Database", and these tables can be adapted in various ways (see, for example, Nakamura, Y. et al. (2000) Nucleic Acids Res. 28:292). Computer algorithms for codon-optimizing a specific sequence for expression in a particular host cell, such as Gene Forge (Aptagen; Jacobus, Pa.), DNA2.0, GeneArt (GA) or Genscript (GS) and the GS algorithm combined with a reduction in CpG content are also available. In some embodiments, the transgene encoding the ARSA polypeptide is codon-optimized using the GA algorithm. In some embodiments, the transgene encoding the ARSA polypeptide has the sequence of SEQ ID NO: 1.

[0049] In some embodiments, the expression cassette further includes an intron. Various introns for use in the present invention are known to those skilled in the art and include MVM intron, FIX truncated intron 1, β-globin SD / immunoglobulin heavy chain SA, adenovirus SD / immunoglobulin SA, SV40 late SD / SA (19S / 16S), and hybrid adenovirus SD / IgG SA. (Wu et al. 2008, Kurachi et al., 1995, Choi et al. 2014, Wong et al., 1985, Yew et al. 1997, Huang and Gorman (1990). In some embodiments, the intron is a chicken β-actin (CBA) / rabbit β-globin hybrid intron. In some embodiments, the intron is a chicken β-actin (CBA) / rabbit β-globin hybrid promoter and intron in which all ATG sites have been removed to minimize incorrect translation start sites. In some embodiments, the intron is an MVM intron, FIX truncated intron 1, β-globin SD / immunoglobulin heavy chain SA, adenovirus SD / immunoglobulin SA, SV40 late SD / SA (19S / 16S), or hybrid adenovirus SD / IgG SA. In some embodiments, the intron is a chicken β-actin (CBA) / rabbit β-globin hybrid intron.

[0050] In some embodiments, the expression cassette further includes a polyadenylation signal. In some embodiments, the polyadenylation signal is a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or HSV TK pA. In some embodiments, the polyadenylation signal is a synthetic polyadenylation signal as described in Levitt, N et al. (1989), Genes Develop. 3:1019-1025.

[0051] In some embodiments, the expression cassette comprises a stuffer nucleic acid. In some embodiments, the stuffer nucleic acid may comprise a sequence encoding a reporter polypeptide. As will be appreciated by those skilled in the art, the stuffer nucleic acid may be located in various regions within the nucleic acid and may be composed of a continuous sequence (e.g., a single stuffer nucleic acid at a single position) or multiple sequences (e.g., multiple stuffer nucleic acids at multiple positions (e.g., two positions, three positions, etc.)) within the nucleic acid. In some embodiments, the stuffer nucleic acid may be located downstream of the transgene encoding the ARSA polypeptide. In embodiments, the stuffer nucleic acid may be located upstream of the transgene encoding the ARSA polypeptide (e.g., between the promoter and the transgene). Also, as will be appreciated by those skilled in the art, various nucleic acids may be used as the stuffer nucleic acid. In some embodiments, the stuffer nucleic acid comprises all or a portion of the human alpha-1-antitrypsin (AAT) stuffer sequence or the C16 P1 chromosome 16 P1 clone (human C16) stuffer sequence. In some embodiments, the stuffer sequence comprises all or a portion of a gene. For example, the stuffer sequence comprises a portion of the human AAT sequence. Those skilled in the art will recognize that various portions of a gene (e.g., the human AAT sequence) can be used as a stuffer fragment. For example, the stuffer fragment can be derived from the 5' end of the gene, the 3' end of the gene, the middle of the gene, the non-coding portion of the gene (e.g., an intron), the coding region of the gene (e.g., an exon), or a mixture of the non-coding and coding portions of the gene. Those skilled in the art will also recognize that all or a portion of the stuffer sequence can be used as the stuffer sequence. In some embodiments, the stuffer sequence is modified to remove internal ATG codons.

[0052] In some embodiments, the expression cassette is incorporated into a vector. In some embodiments, the expression cassette is incorporated into a viral vector. In some embodiments, the viral vector is the rAAV vector described herein.

[0053] Vectors and virus particles In certain embodiments, an expression cassette for expressing an ARSA polypeptide (e.g., a wild-type human ARSA polypeptide) is included in a vector. In some embodiments, the invention contemplates the use of a recombinant viral genome for introducing a nucleic acid sequence encoding an ARSA polypeptide for packaging into virus particles, such as the virus particles described below. The recombinant viral genome can include any element for establishing expression of the ARSA polypeptide, such as a promoter, ITR, ribosome binding element, terminator, enhancer, selectable marker, intron, polyA signal, and / or origin of replication. Exemplary viral genome elements and methods of delivering virus particles are described in more detail below.

[0054] Non-viral delivery systems Conventional non-viral gene delivery methods can also be used to introduce nucleic acids into cells or target tissues. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids complexed to delivery systems. For example, the vector can be complexed with a lipid (e.g., a cationic or neutral lipid), liposome, polycation, nanoparticle, or an agent that enhances cellular uptake of the nucleic acid. The vector can be complexed with an agent suitable for any of the delivery methods described herein. In some embodiments, the nucleic acid includes one or more viral ITRs (e.g., AAV ITRs).

[0055] Virus particles In some embodiments, the vector containing the expression cassette for expressing an ARSA polypeptide (e.g., wild-type human ARSA polypeptide) is a recombinant viral vector. Some examples of recombinant viral vectors include AAV, lentivirus, and adenovirus. In one aspect, the viral vector is a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the expression cassette for expressing an ARSA polypeptide (e.g., wild-type human ARSA polypeptide) is flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the viral particle is a recombinant AAV particle containing an expression cassette for expressing an ARSA polypeptide flanked by one or two ITRs. In some embodiments, the expression cassette for expressing an ARSA polypeptide is flanked by two AAV ITRs.

[0056] In some embodiments, an expression cassette for expressing an ARSA polypeptide of the present disclosure includes components operably linked in the direction of transcription, a transcription initiation sequence, and a transcription termination sequence, thereby including a control sequence that forms the expression cassette. The expression cassette has at least one functional AAV ITR sequence adjacent to its 5' end and 3' end. A "functional AAV ITR sequence" means that the ITR sequence functions for the purpose of rescue, replication, and packaging of AAV virions. See Davidson et al., PNAS, 2000, 97(7) 3428-32; Passini et al., J. Virol., 2003, 77(12):7034-40; and Pechan et al., Gene Ther., 2009, 16:10-16 (all of these references are hereby incorporated by reference in their entirety). To implement some aspects of the present invention, the recombinant vector includes at least all of the AAV sequences essential for capsid formation and the physical structure for infection by rAAV. The AAV ITRs for use in the vectors of the present invention do not have to have a wild-type nucleotide sequence (as described, for example, in Kotin, Hum. Gene Ther., 1994, 5:793-801), and may be modified by nucleotide insertions, deletions, or substitutions, or may be derived from any of several AAV serotypes. More than 40 AAV serotypes are currently known, and new serotypes and variants of existing serotypes continue to be identified. See Gao et al., PNAS, 2002, 99(18):11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossis et al., J. Virol., 2003, 77(12):6799-810.

[0057] The use of any AAV serotype is contemplated to be within the scope of the present invention. In some embodiments, the rAAV vector is a vector derived from an AAV serotype such as, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAV.rh10, AAV11, AAV12, caprine AAV, bovine AAV, or murine AAV. In some embodiments, the nucleic acid in the AAV comprises ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, caprine AAV, bovine AAV, or murine AAV. In certain embodiments, the AAV ITR is the AAV2 ITR.

[0058] In some embodiments, the vector may comprise a stuffer nucleic acid. In some embodiments, the stuffer nucleic acid may encode a green fluorescent protein (GFP). In some embodiments, the stuffer nucleic acid may be located 3' of the expression cassette for expressing the ARSA polypeptide of the present disclosure.

[0059] In some embodiments, the invention provides viral particles comprising a recombinant self-complementary genome. In some embodiments, the vector is a self-complementary vector. AAV viral particles having a self-complementary genome and methods of use of self-complementary AAV genomes are described in U.S. Patent Nos. 6,596,535; 7,125,717; 7,765,583; 7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457; and Wang Z., et al., (2003) Gene Ther 10:2105-2111, each of which is incorporated herein by reference in its entirety. rAAV comprising a self-complementary genome rapidly forms double-stranded DNA molecules by virtue of its partially complementary sequences (e.g., the complementary coding and non-coding strands of the transgene). In some embodiments, the invention provides AAV viral particles comprising an AAV genome, wherein the rAAV genome comprises a first heterologous polynucleotide sequence (e.g., the coding strand of the ARSA polypeptide of the invention) and a second heterologous polynucleotide sequence (e.g., the non-coding or antisense strand of the ARSA polypeptide of the present disclosure), and the first heterologous polynucleotide sequence can form intra-strand base pairs with the second polynucleotide sequence along most or all of its length.

[0060] In some embodiments, the first heterologous polynucleotide sequence and the second heterologous polynucleotide sequence are linked by a sequence that promotes intrastrand base pairing, such as a hairpin DNA structure. Hairpin structures are known in the art, for example, in siRNA molecules. In some embodiments, the first heterologous polynucleotide sequence and the second heterologous polynucleotide sequence are linked by a mutant ITR (e.g., a right ITR). The mutant ITR includes a deletion of the D region that includes the terminal resolution sequence. As a result, when replicating the AAV viral genome, the rep protein does not cleave the viral genome at the mutant ITR, and thus, a recombinant viral genome comprising, in order from 5' to 3': an AAV ITR, a first heterologous polynucleotide sequence comprising regulatory sequences, a mutant AAV ITR, a second heterologous polynucleotide in the reverse orientation to the first heterologous polynucleotide, and a third AAV ITR is packaged into the viral capsid.

[0061] In some embodiments, the first heterologous nucleic acid sequence and the second heterologous nucleic acid sequence are linked by a mutant ITR (e.g., a right ITR). In some embodiments, the ITR comprises the polynucleotide sequence 5'-CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG-3' (SEQ ID NO: 15). This mutant ITR includes a deletion of the D region that includes the terminal resolution sequence. As a result, when replicating the AAV viral genome, the rep protein does not cleave the viral genome at the mutant ITR, and thus, a recombinant viral genome comprising, in order from 5' to 3': an AAV ITR, a first heterologous polynucleotide sequence comprising regulatory sequences, a mutant AAV ITR, a second heterologous polynucleotide in the reverse orientation to the first heterologous polynucleotide, and a third AAV ITR is packaged into the viral capsid.

[0062] In some embodiments, the vector is encapsulated in viral particles. In some embodiments, the viral particles are recombinant AAV viral particles comprising a recombinant AAV vector. A variety of AAV serotypes are used to optimize transduction of specific target cells or target specific cell types within a particular target tissue (e.g., the brain or spinal cord). The rAAV particles can contain viral proteins and viral nucleic acids of the same serotype or a mixed serotype. For example, in some embodiments, the rAAV particles can contain a modified AAV1999 capsid protein and at least one AAV2 ITR, or a modified AAV1999 capsid protein and at least one AAV1 ITR. Combinations of AAV serotypes for generating rAAV particles are provided herein as if each combination were expressly described herein.

[0063] AAV capsids (e.g., AAV9, AAV1999, etc.) are known to contain three capsid proteins: VP1, VP2, and VP3. These proteins contain a significant amount of overlapping amino acid sequences and unique N-terminal sequences. The AAV9 capsid contains 60 subunits arranged by icosahedral symmetry. AAV9 contains VP1, VP2, and VP3 capsid proteins in an approximate ratio of 5:5:50. The VP proteins of AAV9 are the products of an open reading frame (referred to as cap) that encodes the structural proteins of the genome, the minor capsid proteins VP1 (approximately 82 kDa) and VP2 (approximately 73 kDa), and the major capsid protein VP3 (approximately 61 kDa). Upon expression, due to the use of both alternative splicing and read-through, the individual VPs share a C-terminus that encompasses the entire VP3, and VP1 and VP2 extend the N-terminal VP3. VP1 and VP2 share a region of approximately 73 amino acids, and in VP1, an additional approximately 137 amino acids, called the VP1 unique region (VP1u), extend from this. See Penzes et al., (2021), Journal of Virology 95(19)e0084321. In some embodiments of the modified AAV9 capsid proteins disclosed herein, the targeting peptide (e.g., SEQ ID NO: 10) is incorporated into VP1. In some embodiments of the modified AAV9 capsid proteins disclosed herein, the targeting peptide (e.g., SEQ ID NO: 10) is incorporated into VP2. In some embodiments of the modified AAV9 capsid proteins disclosed herein, the targeting peptide (e.g., SEQ ID NO: 10) is incorporated into VP3. In some embodiments of the modified AAV9 capsid proteins disclosed herein, the targeting peptide (e.g., SEQ ID NO: 10) is incorporated into VP1, VP2, and VP3.

[0064] In some embodiments, the present disclosure provides rAAV particles for intracerebrospinal fluid (CSF) administration of an ARSA polypeptide, comprising a modified AAV9 capsid protein comprising a targeting peptide that targets the brain. In certain embodiments, the targeting peptide of the modified AAV9 capsid is inserted after residue 588 of the AAV9 structural protein. In some embodiments, the targeting peptide has SEQ ID NO: 10. In some embodiments, the targeting peptide has linker sequences flanking the N-terminus and C-terminus of the targeting peptide. In some embodiments, the N-terminal linker sequence has the sequence AAA. In some embodiments, the C-terminal linker sequence is AS. In some embodiments, the complete sequence inserted after residue 588 of the AAV9 capsid structural protein has SEQ ID NO: 11.

[0065] In some embodiments, the fully modified AAV9 capsid structural protein (VP1) has the sequence of SEQ ID NO: 12. In some embodiments, the fully modified AAV9 capsid structural protein (VP1) is at least 90% (e.g., at least 92%, at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.5%, or at least 99.8%) identical to SEQ ID NO: 12, wherein the modified AAV9 structural capsid comprises the targeting peptide of SEQ ID NO: 10.

[0066] In some embodiments, the modified VP2 capsid of AAV9 has the sequence of SEQ ID NO: 13. In some embodiments, the modified VP2 capsid of AAV9 has a sequence that is at least 90% (e.g., at least 92%, at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.5%, or at least 99.8%) identical to SEQ ID NO: 13, wherein the modified AAV9 structural capsid comprises the targeting peptide of SEQ ID NO: 10.

[0067] In some embodiments, the modified VP3 capsid of the AAV9 capsid has the sequence of SEQ ID NO: 14. In some embodiments, the modified VP3 capsid of AAV9 has a sequence that is at least 90% (e.g., at least 92%, at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.5%, or at least 99.8%) identical to SEQ ID NO: 14, wherein the modified AAV9 structural capsid comprises the targeting peptide of SEQ ID NO: 10.

[0068] Production of AAV particles In this technical field, there are numerous methods known for the production of rAAV vectors, including transfection, the production of stable cell lines, and infectious hybrid virus production systems including adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, JE et al., (1997) J. Virology 71(11):8780-8789), and baculovirus-AAV hybrids (Urabe, M. et al., (2002) Human Gene Therapy 13(16):1935-1943; Kotin, R. (2011) Hum Mol Genet. 20(R1):R2-R6). All rAAV production cultures for producing rAAV virus particles require 1) a suitable host cell, 2) suitable helper virus functions, 3) AAV rep and CAP genes and gene products, 4) a nucleic acid flanked by at least one AAV ITR sequence (e.g., an AAV genome encoding an ARSA polypeptide), and 5) a suitable medium and medium components to support rAAV production. In some embodiments, the suitable host cell is a primate host cell. In some embodiments, the suitable host cell is a human-derived cell line such as HeLa cells, A549 cells, 293 cells, or Perc.6 cells. In some embodiments, the suitable helper virus functions are provided by wild-type or mutant adenoviruses (e.g., temperature-sensitive adenoviruses), herpesviruses (HSV), baculoviruses, or plasmid constructs that provide helper functions. In some embodiments, the AAV rep and cap gene products can be derived from any AAV serotype. Generally, although not essential, the AAV rep gene product is of the same serotype as the ITRs of the rAAV vector genome as long as the rep gene product can function to replicate and package the rAAV genome. Suitable media known in the art can be used for the production of rAAV vectors.These media include, but are not limited to, modified Eagle's medium (MEM), Dulbecco's modified Eagle's medium (DMEM), custom formulations such as those described in U.S. Patent No. 6,566,118, and media manufactured by HyClone Laboratories and JRH, such as Sf-900 II SFM medium as described in U.S. Patent No. 6,723,551, each of which is hereby incorporated by reference in its entirety, particularly with respect to custom medium formulations for the production of recombinant AAV vectors. In some embodiments, the AAV helper function is provided by adenovirus or HSV. In some embodiments, the AAV helper function is provided by baculovirus, and the host cell is an insect cell (e.g., Spodoptera frugiperda (Sf9) cells).

[0069] One method for producing rAAV particles is the triple transfection method. Briefly described, plasmids containing the rep gene and the capsid gene can be transfected into a cell line (e.g., HEK-293 cells) together with a helper adenovirus plasmid (e.g., using the calcium phosphate method), and the virus can be collected and optionally purified. Thus, in some embodiments, rAAV particles are produced by triple transfection of a host cell with a nucleic acid encoding an rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding an AAV helper virus function, and transfection of this nucleic acid into the host cell generates a host cell capable of producing rAAV particles.

[0070] In some embodiments, rAAV particles can be produced by the producer cell line method (see Martin et al., (2013) Human Gene Therapy Methods 24:253-269; U.S. Patent Application Publication No. 2004 / 0224411; and Liu, X.L. et al. (1999) Gene Ther. 6:293-299). Briefly described, a cell line (e.g., HeLa, 293, A549, or Perc.6 cell line) can be stably transfected with a plasmid containing a vector genome that includes a rep gene, a capsid gene, and a promoter heterologous nucleic acid sequence (e.g., an ARSA polypeptide). The cell line is screened to select a lead clone for rAAV production, which is then expanded into a production bioreactor and infected with a helper virus (e.g., adenovirus or HSV) to initiate rAAV production. The virus can then be harvested, the adenovirus inactivated and / or removed (e.g., by heat), and the rAAV particles purified. Thus, in some embodiments, rAAV particles are produced by a producer cell line that contains one or more nucleic acids encoding the rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding AAV helper virus functions. As described herein, the producer cell line method can be advantageous for the production of rAAV particles with large genomes compared to the triple transfection method.

[0071] In some embodiments, the nucleic acids encoding the AAV rep and cap genes, and / or the rAAV genome, are stably maintained in the production cell line. In some embodiments, the nucleic acids encoding the AAV rep and cap genes, and / or the rAAV genome, are introduced into the cell line with one or more plasmids to generate a production cell line. In some embodiments, the AAV rep, AAV cap, and rAAV genome are introduced into the cell with the same plasmid. In other embodiments, the AAV rep, AAV cap, and rAAV genome are introduced into the cell with different plasmids. In some embodiments, the cell line stably transfected with the plasmid maintains the plasmid over multiple passages of the cell line (e.g., 5, 10, 20, 30, 40, 50, or more than 50 passages of the cells). For example, the plasmid can replicate with cell replication, or the plasmid can integrate into the cell genome. Various sequences have been identified that allow plasmids to replicate autonomously in cells (e.g., human cells) (see, e.g., Krysan, P.J. et al. (1989) Mol. Cell Biol. 9:1026-1033). In some embodiments, the plasmid can include a selectable marker (e.g., an antibiotic resistance marker) that allows for the selection of cells that maintain the plasmid. Selectable markers commonly used in mammalian cells include, but are not limited to, blasticidin, G418, hygromycin B, zeocin, puromycin, and derivatives thereof. Methods for introducing nucleic acids into cells are known in the art and include, but are not limited to, viral introduction, cationic transfection (e.g., using cationic polymers such as DEAE-dextran or cationic lipids such as lipofectamine), calcium phosphate transfection, microinjection, particle bombardment, electroporation, and nanoparticle transfection (for details, see, e.g., Kim, T.K. and Eberwine, J.H. (2010) Anal. Bioanal. Chem. 397:3173-3178).

[0072] In some embodiments, the nucleic acids encoding the AAV rep and cap genes, and / or the rAAV genome, are stably integrated into the genome of the producer cell line. In some embodiments, the nucleic acids encoding the AAV rep and cap genes, and / or the rAAV genome, are introduced into the cell line with one or more plasmids to generate a producer cell line. In some embodiments, the AAV rep, AAV cap, and rAAV genome are introduced into the cell with the same plasmid. In other embodiments, the AAV rep, AAV cap, and rAAV genome are introduced into the cell with different plasmids. In some embodiments, the plasmid may include a selection marker (e.g., an antibiotic resistance marker) that allows for the selection of cells that maintain the plasmid. Methods for stably integrating nucleic acids into various host cell lines are known in the art. For example, iterative selection (e.g., by use of a selection marker) can be used to select cells that have incorporated a nucleic acid containing the selection marker (as well as the AAV cap and rep genes, and / or the rAAV genome). In other embodiments, the nucleic acid can be site-specifically integrated into the cell line to generate a producer cell line. Some site-specific recombination systems are known in the art, such as FLP / FRT (see, e.g., O’Gorman, S. et al. (1991) Science 251:1351-1355), Cre / loxP (see, e.g., Sauer, B. and Henderson, N. (1988) Proc. Natl. Acad. Sci. 85:5166-5170), and phi C31-att (see, e.g., Groth, A. C. et al. (2000) Proc. Natl. Acad. Sci. 97:5995-6000).

[0073] In some embodiments, the production cell line is derived from a primate cell line (e.g., a non-human primate cell line such as the Vero or FRhL-2 cell line). In some embodiments, the cell line is derived from a human cell line. In some embodiments, the production cell line is derived from HeLa, 293, A549, or PERC.6® (Crucell) cells. For example, prior to introducing the nucleic acids encoding the AAV rep and cap genes, and / or the oversized rAAV genome into the cell line and / or generating the production cell line by stably maintaining / integrating them, the cell line is the HeLa, 293, A549, or PERC.6® (Crucell) cell line, or a derivative thereof.

[0074] In some embodiments, the production cell line is adapted for growth in suspension. As is known in the art, anchorage-dependent cells typically cannot grow in suspension without a substrate such as microcarrier beads. Adapting a cell line to grow in suspension can include, for example, growing the cell line in a stirred culture using a paddle stirrer, using a culture medium lacking calcium and magnesium ions (and optionally an antifoaming agent) to prevent aggregation, using a culture vessel coated with a silicone-based compound, and selecting cells in culture (not large clumps or the sides of the vessel) at each passage. For further explanation, see, for example, the ATCC Frequently Asked Questions document (available at www.atcc.org / Global / FAQs / 9 / 1 / Adapting%20a%20monolayer%20cell%20line%20to%20suspension-40.aspx) and the references cited therein.

[0075] In some embodiments, a method for producing rAAV particles disclosed herein is provided, comprising: (a) culturing a host cell under conditions under which rAAV particles are produced, wherein the host cell comprises (i) one or more AAV packaging genes, wherein each of the AAV packaging genes encodes an AAV replication and / or capsid-forming protein; (ii) an rAAV vector encoding a heterologous nucleic acid described herein, wherein at least one AAV ITR is adjacent thereto; and (iii) an AAV helper function; and (b) recovering the rAAV particles produced by the host cell. In some embodiments, the at least one AAV ITR is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, caprine AAV, bovine AAV, or murine AAV serotype ITRs. For example, in some embodiments, the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In certain embodiments, the nucleic acid in AAV comprises an AAV2 ITR. In some embodiments, the capsid-forming protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV1999, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, caprine AAV, AAV1 / AAV2 chimera, bovine AAV, murine AAV capsid, rAAV2 / HBoV1 serotype, AAV-XL32, or AAV-XL32.1 capsid protein, or variants thereof. In some embodiments, the capsid-forming protein is an AAV8 capsid protein. In some embodiments, the rAAV particles comprise a recombinant genome comprising an AAV9 capsid and an AAV2 ITR, and a nucleic acid encoding a therapeutic transgene / nucleic acid (e.g., an expression cassette for expressing an ARSA polypeptide).In some embodiments, the rAAV particles comprise a recombinant genome comprising an AAV1999 capsid and AAV2 ITRs, and a nucleic acid encoding a therapeutic transgene / nucleic acid (e.g., an expression cassette for expressing an ARSA polypeptide).

[0076] The preferred rAAV production medium of the present invention can be supplemented with serum or serum-derived recombinant proteins at levels of 0.5% - 20% (v / v or w / v). Alternatively, as is known in the art, rAAV vectors can be produced under serum-free conditions, also referred to as media that do not contain animal-derived products. One of ordinary skill in the art can understand that commercially available or custom media designed to support the production of rAAV vectors can also be supplemented with one or more cell culture components known in the art (e.g., including but not limited to glucose, vitamins, amino acids, and / or growth factors) to increase the titer of rAAV in the production culture.

[0077] The rAAV production cultures can be grown under various conditions (a wide range of temperatures, various lengths of time, etc.) suitable for the particular host cells utilized. As is known in the art, rAAV production cultures include adherent-dependent cultures that can be cultured in suitable adherent-dependent vessels such as, for example, roller bottles, hollow fiber filters, microcarriers, and packed or fluidized bed bioreactors. The rAAV vector production cultures can also include suspension-compatible host cells such as HeLa, 293, and SF-9 cells that can be cultured in various ways including, for example, disposable systems such as spinner flasks, stirred tank bioreactors, and Wave bag systems.

[0078] The rAAV vector particles of the present invention can be recovered from the rAAV production culture by lysis of the host cells of the production culture or by recovery of the spent medium from the production culture, provided that the cells are cultured under conditions known in the art and on the condition that the rAAV particles are released into the medium from the intact cells, as more fully described in U.S. Patent No. 6,566,118. Suitable methods of cell lysis are also known in the art and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals such as detergents and / or proteases.

[0079] In a further embodiment, the rAAV particles are purified. As used herein, the term "purified" includes the preparation of rAAV particles that lack at least some of the other components that may be present naturally or where the rAAV particles were first prepared. Thus, for example, isolated rAAV particles can be prepared using purification techniques that can concentrate a source mixture such as a culture lysate or production culture supernatant. Concentration can be measured in a variety of ways, for example, by the ratio of DNase-resistant particles (DRP) or genomic copies (gc) present in the solution, or by infectivity, or with respect to a second potentially interfering substance present in the source mixture such as contaminants in the production culture or process contaminants such as helper virus, medium components, etc.

[0080] In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters including, for example, a Grade DOHC Millipore Millistak+HC pod filter, a Grade A1HC Millipore Millistak+HC pod filter, and a 0.2 μm filter Opticap XL1O Millipore Express SHC hydrophilic membrane filter. Clarification can also be achieved by various other standard techniques known in the art, such as centrifugation, or filtration through any cellulose acetate filter with a pore size of 0.2 μm or greater known in the art.

[0081] In some embodiments, the rAAV production culture harvest is further treated with Benzonase® to digest high molecular weight DNA present in the production culture. In some embodiments, the Benzonase® digestion is carried out under standard conditions known in the art, for example, at a temperature in the range of ambient to 37 °C, for 30 minutes to several hours, at a final concentration of 1 to 2.5 units / ml of Benzonase®.

[0082] rAAV particles can be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) to concentrate rAAV particles; apatite chromatography for rAAV capture; heat inactivation of helper virus; hydrophobic interaction chromatography for rAAV capture; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and rAAV capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps can be used alone, in various combinations, or in different orders. In some embodiments, the method includes all steps in the order described below. Methods for purifying rAAV particles are described, for example, in Xiao et al., (1998) Journal of Virology 72:2224-2232; U.S. Patent Nos. 6,989,264 and 8,137,948; and International Publication No. WO 2010 / 148143 pamphlet.

[0083] Treatment method Certain aspects of the present disclosure relate to methods of treating metachromatic leukodystrophy (MLD) and / or increasing the level of arylsulfatase A (ARSA) polypeptide in an individual in need thereof. In some embodiments, the invention provides a method of treating MLD by administering an effective amount of an expression cassette (e.g., an expression cassette delivered by rAAV particles) for expressing the ARSA polypeptide of the present disclosure. In some embodiments, the ARSA polypeptide is a wild-type ARSA polypeptide. The expression cassette for expressing the ARSA polypeptide (e.g., an expression cassette delivered by rAAV particles) can be administered via various routes. In some embodiments, the administration includes direct spinal cord injection and / or intracerebral administration. In some embodiments, the administration is made to a site selected from the cerebrum, medulla, pons, cerebellum, cranial cavity, meninges surrounding the brain, dura mater, arachnoid mater, pia mater, cerebrospinal fluid (CSF) in the subarachnoid space surrounding the brain, deep cerebellar nuclei of the cerebellum, ventricular system of the cerebrum, subarachnoid space, striatum, cortex, septum, thalamus, hypothalamus, and brain parenchyma. In some embodiments, the administration includes intracerebroventricular injection into at least one lateral ventricle. In some embodiments, the administration includes intrathecal injection in the cervical, thoracic, and / or lumbar regions. In some embodiments, the administration includes intrastriatal injection. In some embodiments, the administration includes intrathalamic injection.

[0084] Depending on the purpose of treatment, an effective amount of rAAV (in some embodiments, in the form of particles) is administered. For example, if a desired therapeutic effect can be achieved with a low rate of transduction, the purpose of treatment is generally to meet or exceed this level of transduction. In some examples, this level of transduction can be achieved by transduction of only about 1-5% of the target cells of the desired tissue type, in some embodiments at least about 20% of the cells of the desired tissue type, in some embodiments at least about 50%, in some embodiments at least about 80%, in some embodiments at least about 95%, and in some embodiments at least about 99% of the cells of the desired tissue type. The rAAV composition can be administered by one or more administrations during the same procedure or at intervals of days, weeks, months, or years. Any one or more of the administration routes described herein can be used. In some embodiments, multiple vectors can be used to treat a human.

[0085] In some embodiments of the above aspects, rAAV is administered by direct injection into the spinal cord, by intrathecal injection, or by intracisternal injection. In some embodiments, rAAV is administered at multiple locations in the spinal cord or cisterna magna. In some embodiments, rAAV is administered at multiple locations in the spinal cord. In some embodiments, rAAV is administered to one or more of the lumbar subarachnoid space, thoracic subarachnoid space, and cervical subarachnoid space of the spinal cord. In some embodiments, rAAV is administered to the cisterna magna.

[0086] Methods for identifying cells transduced by AAV viral particles are known in the art and can detect, for example, immunohistochemically or using a marker such as a highly sensitive green fluorescent protein, the introduction of viral particles, such as viral particles containing an rAAV capsid with one or more substitutions of amino acids.

[0087] In some embodiments, an effective amount of rAAV particles is administered simultaneously or sequentially at multiple sites. In other embodiments, an effective amount of rAAV particles is administered multiple times (e.g., repeatedly) at a single site. In some embodiments, multiple injections of rAAV viral particles are performed at intervals of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, or 24 hours or less.

[0088] In some embodiments, the present invention provides a method of treating a human suffering from MLD by administering an effective amount of a pharmaceutical composition comprising a recombinant viral vector encoding an ARSA polypeptide of the present disclosure. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

[0089] In some embodiments, the method comprises administering an effective amount of a pharmaceutical composition comprising a recombinant viral vector encoding an ARSA polypeptide of the present disclosure to treat MLD in an individual in need thereof. In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 10×10 12 , 11×10 12 , 15×10 12 , 20×10 12 , 25×10 12 , 30×10 12 , or 50×10 12 genome copies / mL. In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is about 5×10 12 ~6×10 12 , 6×10 12 ~7×10 12 , 7×10 12 ~8×10 12 , 8×10 12 ~9×10 12 , 9×10 12 ~10×10 12 , 10×10 12~11×10 12 , 11×10 12 ~15×10 12 , 15×10 12 ~20×10 12 , 20×10 12 ~25×10 12 , 25×10 12 ~30×10 12 , 30×10 12 ~50×10 12 , or 50 x 10 12 ~100×10 12 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is about 5×10 12 ~10×10 12 , 10×10 12 ~25×10 12 , or 25 x 10 12 ~50×10 12 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 10×10 9 , 11×10 9 , 15×10 9 , 20×10 9 , 25×10 9 , 30×10 9 , or 50 x 10 9 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is about 5×10 9 ~6×10 9 , 6×10 9 ~7×10 9 , 7×10 9 ~8×10 9 , 8×10 9 ~9×10 9 , 9×10 9 ~10×10 9 , 10×10 9 ~11×10 9 , 11×109 ~15×10 9 、15×10 9 ~20×10 9 、20×10 9 ~25×10 9 、25×10 9 ~30×10 9 、30×10 9 ~50×10 9 or 50×10 9 ~100×10 9 Any of the transduction units / mL. In some embodiments, the viral titer of viral particles (e.g., rAAV particles) is about 5×10 9 ~10×10 9 、10×10 9 ~15×10 9 、15×10 9 ~25×10 9 、or 25×10 9 ~50×10 9 Any of the transduction units / mL. In some embodiments, the viral titer of viral particles (e.g., rAAV particles) is at least about 5×10 10 、6×10 10 、7×10 10 、8×10 10 、9×10 10 、10×10 10 、11×10 10 、15×10 10 、20×10 10 、25×10 10 、30×10 10 、40×10 10 、or 50×10 10 Any of the infectious units / mL. In some embodiments, the viral titer of viral particles (e.g., rAAV particles) is at least, about 5×10 10 ~6×10 10 、6×10 10 ~7×10 10 、7×10 10 ~8×10 10 、8×10 10 ~9×10 10 、9×10 10 ~10×10 10, 10×10 10 ~11×10 10 , 11×10 10 ~15×10 10 , 15×10 10 ~20×10 10 , 20×10 10 ~25×10 10 , 25×10 10 ~30×10 10 , 30×10 10 ~40×10 10 , 40×10 10 ~50×10 10 , or 50×10 10 ~100×10 10 Any of the infectious units / mL. In some embodiments, the viral titer of viral particles (e.g., rAAV particles) is at least about 5×10 10 ~10×10 10 , 10×10 10 ~15×10 10 , 15×10 10 ~25×10 10 , or 25×10 10 ~50×10 10 Any of the infectious units / mL. In some embodiments, the viral particles are rAAV particles. In some embodiments, the rAAV particles contain the AAV1999 capsid protein.

[0090] In some embodiments, the dose of viral particles administered to an individual is at least about 1×10 8 ~about 6×10 13 genome copies per kg of body weight. In some embodiments, the dose of viral particles administered to an individual is about 1×10 8 ~about 6×10 13 genome copies per kg of body weight. In some embodiments, the dose of viral particles administered to an individual is about 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×1010 , 8×10 10 , 9×10 10 , 1×10 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1×10 12 , 2×10 12 , 13×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , or 1×10 13 is any of the genomic copies.

[0091] In some embodiments, the total amount of viral particles administered to the individual is at least about 1×10 9 ~ about 1×10 14 is any of the genomic copies. In some embodiments, the total amount of viral particles administered to the individual is about 1×10 9 ~ about 1×10 14 is any of the genomic copies. In some embodiments, the total amount of viral particles administered to the individual is about 1×10 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1×10 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 2×1013 、 13 × 10 13 、 4 × 10 13 、 5 × 10 13 、 6 × 10 13 、 7 × 10 13 、 8 × 10 13 、 9 × 10 13 、 or 1 × 10 14 is any of the genomic copies.

[0092] The compositions of the present invention (e.g., recombinant viral particles comprising a vector encoding an ARSA polypeptide of the present disclosure) can be used alone or in combination with one or more additional therapeutic agents for treating MLD. The intervals between sequential administrations can be in units of at least (or less than) minutes, hours, or days.

[0093] For therapeutic purposes, an effective amount of (in some embodiments, in particulate form) rAAV is administered. For example, if a desired therapeutic effect can be achieved with a low rate of transduction, the therapeutic goal generally is to meet or exceed this level of transduction. In some examples, this level of transduction can be achieved by transduction of only about 1 - 5%, in some embodiments at least about 20% of the cells of the desired tissue type, in some embodiments at least about 50%, in some embodiments at least about 80%, in some embodiments at least about 95%, in some embodiments at least about 99% of the cells of the desired tissue type. The rAAV composition can be administered by one or more administrations during the same procedure or at intervals of days, weeks, months, or years. In some embodiments, multiple vectors can be used to treat a mammal (e.g., a human).

[0094] In some embodiments, the rAAV compositions of the present disclosure can be used for administration to humans. In some embodiments, the rAAV compositions of the present disclosure can be used for administration to pediatric patients. In some embodiments, an effective amount of rAAV (in some embodiments, in particle form) is administered to a patient who is less than 1 month, less than 2 months, less than 3 months, less than 4 months, less than 5 months, less than 6 months, less than 7 months, less than 8 months, less than 9 months, less than 10 months, less than 11 months, less than 1 year, less than 13 months, less than 14 months, less than 15 months, less than 16 months, less than 17 months, less than 18 months, less than 19 months, less than 20 months, less than 21 months, less than 22 months, less than 2 years, less than 3 years, less than 5 years, or less than 7 years old.

[0095] In some embodiments, the rAAV compositions of the present disclosure can be used for administration to young adults. In some embodiments, an effective amount of rAAV (in some embodiments, in particle form) is administered to a patient who is less than 12 years, less than 13 years, less than 14 years, less than 15 years, less than 16 years, less than 17 years, less than 18 years, less than 19 years, less than 20 years, less than 21 years, less than 22 years, less than 23 years, less than 24 years, or less than 25 years old.

[0096] Kits and Products The expression cassettes (e.g., expression cassettes for expressing an ARSA polypeptide such as a wild-type human ARSA polypeptide), rAAV vectors, particles, and / or pharmaceutical compositions described herein can be included in a kit or product (e.g., a product) designed for use in one of the methods of the invention described herein.

[0097] Generally, the system includes a cannula, one or more syringes (e.g., 1, 2, 3, 4 or more), and one or more fluids (e.g., 1, 2, 3, 4 or more) suitable for use in the methods of the invention.

[0098] The syringe can be any suitable syringe as long as it can be connected to a cannula for delivering fluid. In some embodiments, the system has one syringe. In some embodiments, the system has two syringes. In some embodiments, the system has three syringes. In some embodiments, the system has four or more syringes. Suitable fluids for use in the methods of the present invention include those described herein, for example, one or more fluids each containing an effective amount of one or more vectors described herein, and one or more fluids containing one or more therapeutic agents.

[0099] In some embodiments, the kit contains a single fluid (e.g., a pharmaceutically acceptable fluid containing an effective amount of a vector). In some embodiments, the kit contains two fluids. In some embodiments, the kit contains three fluids. In some embodiments, the kit contains four or more fluids. The fluid can contain a diluent, buffer, excipient, or any other liquid described herein or known in the art suitable for delivering, diluting, stabilizing, buffering, or transporting an expression cassette for expressing the ARSA polypeptide or rAAV vector composition of the present disclosure. In some embodiments, the kit contains one or more buffers, such as an aqueous pH buffer. Examples of buffers can include, but are not limited to, buffers of phosphate, citrate, Tris, HEPES, and other organic acids.

[0100] In some embodiments, the kit contains a container. Suitable containers can include, for example, vials, bags, syringes, and bottles. The container can be made from one or more materials such as glass, metal, or plastic. In some embodiments, the container is used to hold the rAAV composition of the present disclosure. In some embodiments, the container can also hold fluids and / or other therapeutic agents.

[0101] In some embodiments, the kit includes an additional therapeutic agent, along with the rAAV composition of the present disclosure. In some embodiments, the rAAV composition and the additional therapeutic agent can be mixed. In some embodiments, the rAAV composition and the additional therapeutic agent can be stored separately. In some embodiments, the rAAV composition and the additional therapeutic agent can be present in the same container. In some embodiments, the rAAV composition and the additional therapeutic agent can be present in different containers. In some embodiments, the rAAV composition and the additional therapeutic agent can be administered simultaneously. In some embodiments, the rAAV composition and the additional therapeutic agent can be administered on the same day. In some embodiments, the rAAV composition can be administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months after administration of the additional therapeutic agent.

[0102] In some embodiments, the kit includes a therapeutic agent for temporarily suppressing the immune system prior to AAV administration. In some embodiments, the patient is temporarily immunosuppressed immediately before and after viral injection to inhibit the T cell response to AAV particles (see, e.g., Ferreira et al., Hum. Gene Ther. 25:180-188, 2014). In some embodiments, the kit further provides cyclosporine, mycophenolate mofetil, and / or methylprednisolone.

[0103] The rAAV particles and / or compositions of the invention can be packaged in a kit that further includes instructions for use. In some embodiments, the kit further includes a device for delivery of the composition of rAAV particles (e.g., any type of parenteral administration described herein). In some embodiments, the instructions for use include instructions according to one of the methods described herein. In some embodiments, the instructions are printed on a label provided on (e.g., affixed to) the container. In some embodiments, the instructions for use include instructions for administering to an individual (e.g., a human) an effective amount of rAAV particles, e.g., to treat the MLD of the individual.

Example

[0104] The present disclosure will be understood more deeply by referring to the following examples. However, the following examples should not be construed as limiting the scope of the present invention. The examples and embodiments described herein are for illustrative purposes only, and in light of this, various modifications or changes will be suggested to those skilled in the art, and it is understood that they are included within the spirit and scope of the present application and the scope of the appended embodiments.

[0105] General method Homogenization of tissue Tissue was homogenized with cold 10 mM Tris 1 mM EDTA buffer added to a 1.4 mm ceramic bead homogenization tube in a cooled Omni bead ruptor for -20 second cycles at 4.7 vibrations / second. After homogenization, aliquots were made for subsequent assays.

[0106] Solubilization of tissue for sulfatase activity Nonidet P-40 was added to the homogenized tissue to a final concentration of 0.1%, and it was solubilized at 4°C for 1.5 hours on an orbital shaker and then centrifuged at 18,000 × g for 20 minutes. The supernatant was removed and transferred to an Eppendorf tube on ice.

[0107] BCA assay Using 10 μL of the supernatant diluted in water, the total protein concentration was determined by a BCA (bicinchoninic acid) assay (Thermo Scientific 23227). Colorimetric detection of the first copper cation (Cu 1+ ) by bicinchoninic acid (BCA) based on absorbance at 562 nm. A 96-well microtiter plate was read using a Molecular Devices SpectraMax 340PC-384 equipped with SoftMax Pro version 5.4.4 software.

[0108] Sulfatase activity The total sulfatase activity of 10 microliters of clarified supernatant was assayed using a sulfatase activity assay for the hydrolysis of 4-nitrocatechol (PNC) from the 4-nitrocatechol sulfate (PNCS) substrate. (Abcam Ab204731). Activity was measured by the hydrolysis of 4-nitrocatechol (PNC) of the sample against a PNC standard curve, and the absorbance at 515 nm was read. Sulfatase activity was reported as mU / mg (nmol / min / mg). A 96-well microtiter plate was read using a Molecular Devices SpectraMax 340PC-384 equipped with SoftMax Pro version 5.4.4 software.

[0109] Lipid extraction An aliquot of tissue homogenate (or fluid) was extracted with 20 - 100× extraction solution (5 mM ammonium formate, 0.2% formic acid in acetonitrile:methanol (70:30) supplemented with 10 ng / ml C17-sulfatide). After vigorously mixing for 10 minutes, the sample was allowed to stand for 5 minutes and then vortexed again quickly (30 seconds). All samples were centrifuged at 8,400 rpm for 10 minutes at 4°C. An aliquot of the supernatant (200 uL) was transferred to an inactivated Q-Sert vial for LC-MS analysis.

[0110] Sulfatides having sulfatides of C16, C18, C24 and C24:1 chain lengths (Matreya) and C18 2R-OH sulfatide and lysosulfatide (Avanti) were used to create a standard curve (linear range 0.03 - 1000 ng / mL, two-fold serial dilution) with the same extraction solution.

[0111] LC-MS analysis of sulfatides The Waters Acquity UPLC system (Milford, MA) was coupled to a Qtrap 6500 mass spectrometer system (Framingham, MA) equipped with an ESI source operating in negative ion mode with the following parameters: curtain gas 25.0; ion spray voltage -4.5 kV; temperature 500 °C; ion source gas: 50 and 70; declustering potential -80 V; entrance potential -10 V; collision energy -155 V; and collision cell exit potential -15 V.

[0112] Sulfide species were separated on a Waters Acquity UPLC BEH amide (1.7 μm, 2.1×100 mM, P / N: 186004801). The autosampler and column oven were maintained at 10 °C and 20 °C, respectively. The mobile phase consisted of solvent A: acetonitrile: water (95:5) with 5 mM ammonium formate: water, and solvent B: methanol: water (90:10) with 5 mM ammonium formate.

[0113] The gradient program was started at 0% B, held for 2 minutes, followed by a linear curve from 0% B to 100% B. After holding at 100% B for an additional 1 minute, it was re-equilibrated at 0% B. All sulfide species were normalized to the internal standard of C17-sulfide and calculated by the standard curve of the corresponding standard sulfide.

[0114] Nitroblue tetrazolium staining Transfer the sections to a 12-well plate and perform all steps with free-floating sections. Wash the sections in TBA using a shaker (2 times, 1 minute). Incubate with nitroblue solution (Sigma catalog number N5374) for 1 hour and rinse in TBA using a shaker (2 times, 5 minutes). Incubate the sections in DAB staining solution (Sigma kit catalog number D4293) for 1 hour and rinse with RO water (2 times, 1 minute). Wash the sections with PBS for 1 minute, transfer to PBS and mount on air-dried slides. Counterstain with neutral red solution for 2 minutes and rinse with RO water (2 times, 2 minutes). Rinse the slides with 95% reagent alcohol for 1 minute, 100% reagent alcohol 2 times, 2 minutes each, xylene 2 times, 2 minutes each, and cover slip using Acrytol mounting agent.

[0115] GFP ELISA The measurement of GFP protein was completed using the Abcam GFP SimpleStep ELISA® kit (ab171581). Since some reagent concentrations vary between different lots, especially the standards, it is essential to read the protocol insert supplied with each kit lot.

[0116] Before starting, all kit components were equilibrated to room temperature (18 - 25°C) for at least 30 minutes. An aliquot of the protein - treated tissue homogenate was thawed on ice. Reagents and working standards were freshly prepared according to the kit instructions. Samples were diluted with complete cell extraction buffer as follows: gray matter 1:5, spinal cord 1:5, heart: 1:5, liver 1:20. The diluted samples and standards were added to the appropriate wells, followed by the addition of the antibody cocktail to each well. The plate was sealed and incubated at room temperature for 1 hour with shaking at 400 RPM. The wells were washed three times with wash buffer, ensuring complete removal of the liquid. TMB solution was added to each well and incubated in the dark at room temperature for 10 minutes with shaking at 400 RPM. Immediately after the TMB incubation, stop solution was added to each well and mixed on a plate shaker for 1 minute. The plate was read at 450 nm using Softmax software on a SpectraMax plate reader (Molecular Devices).

[0117] FLAG IHC NHP brain FFPE slides were treated in EDTA solution (pH 9.0) at 90°C for 20 minutes for antigen retrieval, followed by blocking with 3% hydrogen peroxide for 5 minutes. The slides were then incubated at room temperature for 1 hour with Flag DDK antibody (Abcam ab205606) diluted 1:200 with antibody diluent (Cell Signaling catalog number 8112), followed by incubation with anti - rabbit HRP (Abcam ab6721) secondary antibody. The Flag DDK signal was developed by incubating the slides in DAB solution at room temperature for 3 minutes, and then the slides were counterstained with hematoxylin for nuclear staining.

[0118] GFP IHC NHP brain FFPE slides were treated in an EDTA solution (pH 9.0) at 90 °C for 20 minutes for antigen retrieval, then blocked with 3% hydrogen peroxide for 10 minutes at room temperature, followed by 5% horse serum for 45 minutes. The slides were then incubated with a GFP antibody (ThermoFisher A-11122) diluted 1:500 with an antibody diluent (Cell Signaling catalog number 8112) for 1 hour at room temperature, followed by incubation with an anti-rabbit HRP (Abcam ab6721) secondary antibody. The GFP signal was developed by incubating the slides in a DAB solution for 3 minutes at room temperature, and the slides were counterstained with hematoxylin for nuclear staining.

[0119] Cross-correction analysis Adjacent 5-μm FFPE sections were processed for either ARSA IHC and DAPI or WPRE ISH and DAPI. Individual tiles were then stitched together to obtain sagittal sections of both the IHC image and the ISH image. These images were then globally registered with a transformation matrix (rotation and translation parameters) to align the IHC image and the ISH image. Individual tiles within the sagittal section were then locally registered using DAPI to ensure good alignment between individual IHC tiles and ISH tiles. In each tile, IHC-positive cells and ISH-positive cells were estimated using empirically determined threshold parameters across the entire sagittal section. For ISH, IHC-positive cells had to have at least 20% overlap with the nucleus to be counted as true ISH signals. For the IHC image, threshold parameters were determined using a negative control without ARSA staining. The cross-correction factor, which is the ratio of IHC-positive cells to ISH-positive cells, was estimated for each tile and represented as a heatmap.

[0120] Single-nucleus sequencing Nuclei Isolation: For nuclei isolation, the dissected brains were transferred to microcentrifuge tubes, snap-frozen in a slurry of dry ice and ethanol, and stored at -80 °C until use. To isolate nuclei, the frozen mouse brains were placed in a nuclear lysis buffer containing 0.1% Triton-100 (Sigma-Aldrich), 1 mM DTT (Sigma-Aldrich), and 0.2 U / μl RNase inhibitor (Sigma-Aldrich) in a 1 ml Dounce homogenizer (Wheaton). The tissue was homogenized by hitting it 10 times with a loose Dounce pestle followed by 10 times with a tight pestle and incubated on ice for 15 minutes. The resulting homogenate was passed through a 30 μm cell strainer (Miltenyi Biotech) and centrifuged at 500 × g for 5 minutes to pellet the nuclei. The nuclei were resuspended in a buffer containing 1×PBS (Thermo Fisher), 1% nuclease-free BSA (Sigma-Aldrich), 1 mM DTT (Sigma-Aldrich), and 0.2 U / μl RNase inhibitor (Sigma-Aldrich). Mouse anti-NeuN conjugated to PE (EMD Millipore) was added to the preparation at a 1:500 dilution and the samples were incubated at 4 °C for 30 minutes. The samples were then centrifuged at 500 × g for 5 minutes to pellet the nuclei and the pellet was resuspended in 1×PBS, 1% BSA, 1 mM DTT, and 0.2 U / μl RNase inhibitor. DAPI was added at a concentration of 0.1 μg / ml. Single nuclei sorting was performed using a 100 μm nozzle on an Influx-83 (BD Biosciences). The nuclei were gated based on DAPI and NeuN signals (PE).

[0121] Library Preparation and NovaSeq Sequencing Libraries were prepared according to the 10xGenomics protocol for the Chromium Single Cell 3’ Gene Expression V3.1 kit. Briefly, immediately after sorting, the nuclei were mixed with 90% NeuN- and 10% NeuN+ nuclei. The GEM generation step in the 10XGenomics gene expression kit (10xGenomics) was performed. After GEM generation, the samples were -20 0It was retained in C and further steps were carried out. All samples were processed together up to cDNA amplification, library construction, and library sequencing. The quantification of cDNA and libraries was solid for all samples. The library was sequenced on an illumine Novaseq 6000. The library was sequenced at a median depth of approximately 50K reads / nucleus. A UMI count matrix was generated by Cell Ranger V5.

[0122] Data preprocessing: The generated count matrix was loaded into Partek Flow along with the nuclear barcodes and gene labels. For quality control (QC), nuclei were filtered according to a standard protocol based on the inspection of violin plots. The detailed cutoffs were 250 < nFeature_RNA < 6000 and nCount_RNA < 30000. Genes encoding mitochondrial proteins were less than 2%. After quality filtering, a total of 147313 nuclei remained for further analysis.

[0123] Vector genome evaluation HT gDNA isolation: The QIAmp 96 DNA QIAcube HT kit (catalog number 51331) was used according to the manufacturer's protocol "QIAamp® 96 DNA QIAcube® HT Handbook" to isolate gDNA from 50 μl of NHP tissue homogenate. The tissue homogenate was first treated with proteinase K at 56 °C overnight, then transferred to the S block, the sample was placed in the QIAcub HT device, and gDNA was isolated using the QIAcub HT Prep Mange software. Then, the gDNA concentration was measured with a NANODROP 8000 (Thermo Fisher Scientific).

[0124] dPCR via QIAcuty: The vector genome was determined by dPCR via QIAcuty manufactured by QIAGEN. 7 μg of gDNA isolated from NHP tissue, 1× Probe PCR Master Mix (Catalog No. 250103); 1× Primer-Probe Mix 1 (BGH) and Mix 2 (housekeeping gene); and 5 μl of master reaction mix containing 0.25 U of HindIII restriction enzyme were mixed in a standard PCR plate and then transferred to a nanoplate. The total reaction volume was 12 μl per well. The nanoplate was then placed in a QIAcuity device and dPCR was performed using the recommended manufacturing cycle. DNA copies for each sample and each gene were automatically analyzed by software. The vector genome per cell was then calculated. The 2× BGH copy divides the housekeeping gene copy.

[0125] Example 1. ARSA Expression In Vivo Using Two Different Expression Cassettes The vector payload was designed to express the human codon-optimized arylsulfatase A (ARSA) gene (SEQ ID NO: 2) driven by a constitutive chimeric CMV - chicken β-actin (CBA) promoter. Transgene expression was further enhanced by the addition of the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). SEQ ID NO: 3 shows a representative complete DNA sequence of a plasmid for rAAV packaging containing the 5’ AAV2 ITR (SEQ ID NO: 4) and 3’ AAV2 ITR (SEQ ID NO: 5), CMV enhancer element (SEQ ID NO: 6), chicken β-actin promoter (SEQ ID NO: 7), and WPRE element (SEQ ID NO: 8). A schematic diagram of the map of a specific plasmid for rAAV particle production is shown in Figure 17. Specific elements of the plasmid are listed in Table 1.

[0126] [Table 1]

[0127] On the second day after birth, ARSA - / -Mice were administered PBS or AAV9 (2e11 VG; 2e14 VG / kg) rAAV viral particles expressing human ARSA via IV (bilateral retro-orbital injection), regardless of the presence or absence of the WPRE element. Two expression cassettes are illustrated in FIG. 1A. FIG. 1B shows Western blot analysis of hARSA expression in the forebrain, midbrain, and hindbrain after administration of the viral particles. The expression levels were compared with those in mice and mice expressing normal levels of ARSA (i.e., WT mice). - / - Compare the expression levels in mice expressing normal levels of ARSA (i.e., WT mice).

[0128] As shown in FIG. 1B, the expression levels increase after administration of the viral particles containing the two expression cassettes. The inclusion of the WPRE element in the expression cassette increases the expression level of ARSA compared to the expression cassette without the WPRE element.

[0129] Next, LC-MS was used to measure the C24-ST levels. The ST levels were normalized to tissue weight. FIG. 1C shows the CST levels in the forebrain, midbrain, and hindbrain after administration of the viral particles containing the two expression cassettes. Error bars represent the mean including the standard deviation. One-way ANOVA with Tukey's multiple comparison test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ELN: 20200205-188, 20200305-152, 20200205-006. ARSA containing an expression cassette without the WPRE element - / - The C24-ST levels after administration of AAV9 particles to mice were - / - significantly decreased compared to the C24-ST levels in mice. ARSA containing an expression cassette with the WPRE element - / - A further decrease in the C24-ST levels was observed after administration of AAV9 particles to mice.

[0130] Example 2. Evaluation of sulfatase activity in ARSA mice administered AAV.rh10-CBA-ARSA-WPRE - / - Assessment of sulfatase activity in mice Late stage (13 months) ARSA - / -Mice were administered AAV.rh10-CBA-ARSA-WPRE (1.97e11 VG per animal). As a control, age-matched WT mice and ARSA - / - mice were injected with the formulation buffer. Three months after administration, the mice were euthanized and the brain, spinal cord, and liver were collected. ARSA-mediated sulfatase activity was measured using a sulfatase activity assay kit (ab204731), and the data were normalized to the total protein measured by the BCA assay. Error bars represent the mean including the standard deviation. Two-way ANOVA with Tukey's multiple comparison test was used. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ELN: 20201205-001, 20210225-107, 20210211-117. As shown in Figure 2, late-stage MLD (ARSA - / - ) mice treated with AAV.rh10-CBA-ARSA-WPRE showed an increase in ARSA-mediated sulfatase activity in the brain, spinal cord, and liver.

[0131] Example 3. Evaluation of sulfatide in ARSA - / - mice administered AAV.rh10-CBA-ARSA-WPRE The deposition of sulfatide in MLD patients is a major factor promoting toxicity and neurodeath. Short-chain fatty acids (C16, C18) mainly accumulate in neurons and astrocytes, while long-chain fatty acid sulfatides (C24:1, C24) accumulate in myelin-forming cells. Lysosulfatide, a deacylated sulfatide, accumulates in all tissues of MLD patients.

[0132] Late-stage (13 months) ARSA - / - (ARSA KO) mice were administered AAV.rh10-CBA-ARSA-WPRE. As a control, age-matched WT mice and ARSA - / -The formulation buffer was injected into the mice. Three months after administration, the mice were euthanized and the brain and spinal cord were collected. Sulfatide levels were measured using LC-MS. The data were normalized to tissue weight and converted from ng / mL (50 μL) to μg / g. Error bars represent the mean including the standard deviation. Two-way ANOVA with Tukey's multiple comparison test was used.

[0133] LC-MS and ARSA administered with AAV.rh10-CBA-ARSA-WPRE - / - Sulfatide levels measured using both brain and spinal cord samples from the mice showed a significant decrease at all sulfatide isoform levels (Figure 3).

[0134] Example 4. ARSA administered with AAV.rh10-CBA-ARSA-WPRE - / - Evaluation of myelination in mice MLD patients show a significant loss of myelination in the white matter tracts. Late stage (13 months) ARSA - / - To address the effect of gene therapy on mice, AAV.rh10-CBA-ARSA-WPRE was administered to the mice. As controls, age-matched WT mice and ARSA - / - The formulation buffer was injected into the mice. Sagittal brain hemisections taken 3 months after administration were stained with nitroblue and the signal intensity, particularly in the corpus callosum, was quantified. Error bars represent the mean including the standard deviation. One-way ANOVA with Tukey's multiple comparison test was used. Late stage MLD (ARSA - / - ) mice treated with AAV.rh10-CBA-ARSA-WPRE showed improved myelination in the corpus callosum compared to the controls (Figure 4).

[0135] Example 5. ARSA administered with AAV.rh10-CBA-ARSA-WPRE - / - Evaluation of myelination and mature oligodendrocyte cell number in mice Late stage (13 months) MLD ARSA - / - AAV.rh10-CBA-ARSA-WPRE was administered to the mice. As controls, age-matched WT mice and ARSA- / - The formulation buffer was injected into the mice. The cerebral hemispheres were collected and snap-frozen, and the cerebellum, brainstem, and olfactory bulbs were dissected. The remaining part of the brain was processed for single-nucleus RNAseq to measure the relative abundance of different cell types. Error bars represent the mean including the standard deviation. Two-way ANOVA with Tukey's multiple comparisons. Figure 5 shows ARSA treated with AAV.rh10-CBA-ARSA-WPRE - / - in mice, untreated ARSA - / - in mice, showing normalization of oligodendrocyte cell numbers compared to untreated ARSA mice.

[0136] Example 6. ARSA protein cross-correction test Late-stage (13 months) ARSA - / - AAV.rh10-CBA-ARSA-WPRE was administered to mice. Three months after administration, ARSA-mRNA in situ hybridization (ISH) and ARSA-protein immunohistochemistry (IHC) were performed on corresponding sagittal brain hemisections. The sections were imaged and the signal overlays were analyzed. (Figure 6A) Adjacent sagittal sections from the brains of mice were treated for WPRE ISH or ARSA IHC with nuclear DAPI staining. The sagittal sections were then analyzed as individual 1024×1024 pixel tiles. In each tile, ISH-positive cells and IHC-positive cells were determined by empirically determined threshold parameters across the entire sagittal image. The cross-correction coefficient, which is the ratio of IHC+ cells to ISH+ cells, is represented as a heatmap with highly cross-corrected tiles shown in red (6B). The number of ISH+ cells versus IHC+ cells for each tile was plotted as a scatter plot, with the line y = x shown in red. Tiles above the y = x line indicate cross-corrected cells. In late-stage neuropathic mice, 10-fold more cells were cross-corrected compared to infected cells, highlighting that extensive cross-correction was achieved with this strategy.

[0137] Example 7. Evaluation of AAV.rh10-hARSA-WPRE-mediated gene replacement in early (6 months) neuropathological stage MLD mice (Arsa - / - ) resulted in improvement of MLD-related pathology Early (6 months) ARSA - / - Mice were administered AAV.rh10-CBA-ARSA-WPRE. As controls, age-matched WT mice and ARSA - / - mice were injected with formulation buffer. Four months after administration, extensive ARSA expression in sagittal brain sections was observed by IHC for human ARSA (Figure 7A). In the same animals, the brain, spinal cord, DRG, and liver showed a significant increase in ARSA activity (7B). Thus, early MLD (ARSA - / - ) mice treated with AAV.RH10-CBA-ARSA-WPRE show extensive CNS human ARSA expression and increased ARSA-mediated sulfatase activity in the brain, spinal cord, DRG, and liver.

[0138] Example 8. Evaluation of sulfatide levels in AAV.rh10-hARSA-WPRE-mediated gene replacement (ARSA- / -) of MLD mice (Arsa - / - ) at an early (6 months) neuropathological stage Early (6 months) ARSA - / - Mice were administered AAV.rh10-CBA-ARSA-WPRE. As controls, age-matched WT mice and ARSA - / - mice were injected with formulation buffer. Four months after administration, the animals were euthanized and (A) brain and spinal cord, (B) liver, (C) plasma, and (D) CSF were collected. Sulfatide levels were measured using LC-MS. Data were normalized to tissue weight and converted from ng / mL (50 μL) to μg / g. Fluids: Data were converted from ng / mL (30 μL) to ng / mL and from ng / mL (5 μL) to ng / μL. Figures 8A-D show sulfatide levels in the brain (Figure 8A), liver (Figure 8B), plasma (Figure 8C), and CSF (Figure 8D). Sulfatide levels increased significantly in the brain, spinal cord, liver, plasma, and CSF after administration of AAV.rh10-CBA-ARSA-WPRE.

[0139] Example 9: Studies using non-human primates (NHP) - Sulfatase activity Cynomolgus monkeys (male, 2-year-old, Mauritius, 2 - 3 kg) seronegative for AAVrh.10 were intrathecally administered AAV.rh10-CBA-ARSA-WPRE at the cervical levels 1 - 2 junction using an intrathecal catheter with a port inserted into the lumbar region. To animals in the Trendelenburg position, 2.5 mL was administered by infusion twice at 0.125 mL / min at approximately 6-hour intervals. The following two doses were administered: 7.5e12 VG / NHP (@1e11 VG / g brain) and 2.75e13 VG / NHP (@3.3e11 VG / g brain). Twenty-nine days after administration, the animals were euthanized and the ARSA activity of the following samples was evaluated: 59 tissue punches from the brain and 7 tissue punches from the spinal cord. A significant increase in sulfatase activity was observed in the brain and spinal cord, and 59% and 86% of the whole brain punches showed at least a 10% increase in activity compared to the background at the low and high doses, respectively (Figure 9A). This exceeded the inventors' target criterion of 50% of punches showing >10% increase in activity. Similarly, increases in sulfatase activity of 25% and 32% were observed in the spinal cords of NHPs treated with the low and high doses, respectively (Figure 9B). Extensive ARSA biodistribution was observed in the brains of treated NHPs by IHC against the FLAG antibody (Figure 9C). These results indicate that AAV.rh10-CBA-ARSA-WPRE-mediated gene replacement is a feasible approach to achieve broad therapeutic levels of ARSA in the central nervous system (CNS) and peripheral nervous system (PNS).

[0140] Example 10: Study Using Non-Human Primates (NHP) - Comparison of Transgene Expression Levels after Administration of Particles Containing AAV1999 or AAV.rh10 Serum - negative cynomolgus monkeys (male, 2 years old, 2 - 3 kg from Mauritius) of AAVrh10 and AAV1999 (VP1 with SEQ ID NO: 12, VP2 with SEQ ID NO: 13; and VP3 with SEQ ID NO: 14) were intrathecally administered at the cervical level 1 - 2 junction using an intrathecal catheter with a port inserted into the lumbar region. To animals in the Trendelenburg position, 2.5 mL was administered by infusion twice at 0.125 mL / min at intervals of about 6 hours. One dose of AAV1999 / rh10 / Myo - CBA - EGFP was administered at 2.75e13 VG / NHP (3.3e11 VG per gram of brain). Two weeks after administration, the animals were euthanized, and the expression of green fluorescent protein (GFP) in the samples was evaluated by ELISA.

[0141] A total of 41 brain punches (gray and white matter) representing 16 different brain regions showed significantly higher GFP expression from AAV1999 compared to AAVrh.10 (Figure 10A). In all samples, GFP expression in the brains of AAV1999 - treated NHPs was 93% - 123% higher compared to AAVrh.10 - treated NHPs (Figure 10B). This is shown by the heatmap, with 38 out of 41 punches (average of 3 NHPs) in AAV1999 - treated NHPs showing a minimum 10% increase in GFP expression compared to AAVrh10 - treated NHPs (Figure 11). Representative matching brain sections stained with an antibody to eGFP showed strong expression in AAV1999 - treated NHPs, indicating superior biodistribution compared to AAVrh10 (Figure 12). Furthermore, GFP expression in the spinal cord and DRG of AAV1999 - treated NHPs was 79% and 22% higher, respectively, compared to AAVrh.10 - treated NHPs. Figure 13 shows GFP expression in the spinal cord and DRG of NHPs. AAV1999 - GFP expression in the heart and liver was lower compared to AAVrh10 - GFP - treated NHPs (Figure 14).

[0142] The AAV1999 vector genome load was 10-fold lower in the brain and 3-fold lower in the spinal cord compared to AAVrh.10 (Figure 15a). This was prominent in the “left shift” in the correlation between GFP expression (y-axis) and tissue vector genome (x-axis), and the left shift in the correlation of AAV1999 (red) indicated higher protein expression of the vector and lower (by about 10-fold) tissue dose (Figure 16). Furthermore, DRG, liver, heart, lung, and kidney also showed lower AAV1999 vector genomes compared to AAVrh.10 (Figure 15B). The spleen was the only tissue tested that showed a higher AAV1999 vector genome compared to AAVrh.10 (Figure 15B). These results indicate that AAV1999 is superior to AAVrh.10, resulting in a broader biodistribution and higher transgene expression at a lower dose.

[0143] Example 11: Long-Term Pharmacology, Efficacy, and Durability Tests in a Pre-Neuropathic Arsa KO Mouse Model Using AAV1999-ARSA This study was designed to evaluate the long-term pharmacology and efficacy of AAV1999-ARSA in an Arsa KO mouse model. The study design is summarized in Table 2. Two-month-old Arsa KO mice were administered AAV1999-ARSA (lot number VP091321) at 1.6e11 VG per mouse (3.3e11 VG per gram of brain weight) (bilateral ICV injection; 4 μL per hemisphere). As a control, age-matched WT mice and Arsa KO mice were injected with formulation buffer. Mice were euthanized 13 months after administration, and samples were collected.

[0144]

Table 2

[0145] Strong hARSA mRNA expression was observed in the brains and spinal cords of ARSA KO mice treated with AAV1999-ARSA (Figure 18). Sulfatase activity was measured using a sulfatase activity assay kit (Abcam, ab204731). Arsa KO mice administered AAV1999-ARSA showed normal (WT-equivalent) levels of sulfatase activity in the brain, spinal cord, DRG, and sciatic nerve (Figure 19). A measurable increase above WT sulfatase activity levels was observed in the livers of treated mice (Figure 19). Sulfatide deposition in MLD patients is a major factor promoting toxicity and neurodeath. Lysosulfatide (lysoST), a deacylated sulfatide, accumulates in all tissues and is a prominent feature of MLD-related pathology in patients. In Arsa KO mice treated with AAV1999-ARSA, the levels of lysoST in the brain and spinal cord returned to normal (similar to WT) (Figure 20). A significant decrease in lysoST levels was also observed in the DRG, sciatic nerve, and liver, but total sulfatide levels were significantly decreased in the plasma and CSF of treated animals (Figure 20). As a result of sulfatide clearance in the brain and spinal cord, Arsa KO mice treated with AAV1999-ARSA showed a significant improvement in the neuroinflammatory markers (Gfap and Aif1) and Lamp1 expression (lysosome health marker) in the brain and spinal cord (Figure 21). Furthermore, a significant decrease in plasma NF-L levels was observed in Arsa KO treated with AAV1999-ARSA compared to Arsa KO mice treated with buffer (Figure 22).

[0146] MLD patients often present with hearing impairment. Auditory brainstem response (ABR) testing provides functional information regarding the inner ear (cochlea) and the central auditory pathways. ABR reflects the electrical responses of both the spiral ganglion neurons and the nuclei of the central auditory conduction pathway to sound stimuli (Zhou et al., 2006; Burkard et al., 2007). ABR is recorded by electrodes placed on the scalp of anesthetized animals. The ABR threshold refers to the lowest sound pressure level (SPL) capable of generating distinguishable electrical response waves. Arsa KO shows progressive hearing loss over time, which is reversed by treatment with AAV1999-ARSA (Figure 23). Partial reversal of the phenotype is observed at an early time point of 4 months after administration (the earliest time point tested after administration) (Figure 23).

[0147] Vacuolation and neurodegeneration were observed in the brains (usually the brainstem and / or cerebellum) of all Arsa KO mice. In treated mice, histological changes were detected in only 4 out of 10 mice, and the severity was lower than that in Arsa KO mice (Figure 24A). All Arsa KO mice showed mild severity vacuolation in the spinal cord, but no changes were detected in WT mice or AAV1999-ARSA-treated ARSA KO mice (Figure 24B). Finally, no pathological changes were detected in the DRG in any group (except for 1 out of 10 animals in the KO+AAV group) (Figure 24C). Therefore, treatment of Arsa KO mice with AAV1999-ARSA significantly minimized and / or prevented MLD-related pathology in the brain and prevented MLD-related pathology in the spinal cord.

[0148] Example 12: Long-Term Pharmacology, Efficacy, and Durability Testing in an Early Neurodegenerative ARSA KO Mouse Model Using AAV1999 This study was designed to evaluate target engagement and reversal of MLD-related pathological and biochemical phenotypes in the Arsa KO model over 6 months. Early neuropathy (at dosing, 6 months) was selected as most relevant to the target patient population. The study design is summarized in Table 3. Six-month-old Arsa KO mice were administered AAV1999-ARSA (lot number VP091321) at 5e10 VG per mouse (1e11 VG per gram of brain weight) (bilateral ICV injection; 4 μL per hemisphere). Age-matched WT and Arsa KO control mice were injected with formulation buffer. Mice were euthanized at 1, 2, 3, and 6 months post-dose and samples were collected.

[0149] [[Table 3]]

[0150] AAV1999-ARSA vector genome exposure in the brain was maintained consistently over 6 months post-dose (Figure 25A). This led to the persistence of ARSA-mediated sulfatase activity (Figure 25B), with sulfatase activity peaking at 1 month post-dose (Figure 25C). At each time point, treated mice showed a significant decrease in lysosomal, C16-, and C18-sulfatides (Figure 26A–C). Importantly, clearance proceeded and sulfatide levels (especially lysosomal ST) returned to normal at 6 months post-dose (Figure 26D–F). Additionally, total sulfatide levels in CSF and plasma were significantly decreased in AAV1999-ARSA-treated ARSA KO mice (Figure 27). Finally, at 6 months post-dose of AAV1999-ARSA, the initial increase in plasma Nf-L levels was decreased compared to the KO+FB group (Figure 28).

[0151] Changes in neurons and neutrophils in the brains of Arsa KO mice were observed in the brainstem and / or cerebellar nuclei. Notably, in Arsa KO mice treated with AAV1999-ARSA, a decrease in the incidence and severity of these changes was observed 2 months after administration (Figures 29A-B). In the brains of some AAV1999-ARSA-treated mice (usually near the hippocampus), a mild severity of local or multifocal perivascular infiltration of mononuclear cells was observed (Figure 29C). In addition, in 1-2 animals per group, there was mild degeneration / necrosis of neurons mainly in the CA3 region of the hippocampus (Figure 29D).

[0152] In the dorsal root ganglia of some AAV1999-ARSA-treated mice, an increase in the cell density of glial cells and / or mononuclear cells with usually mild severity was observed (Figure 30A), and some treated Arsa KO mice showed slight degeneration of nerve axons (Figures 30B-C). Microscopic findings were observed in the spinal cords of animals in all groups and were considered random or incidental (Figure 30D). Most Arsa KO mice showed histological changes (very mild or mild axonal degeneration) in the sciatic nerve, and in AAV1999-ARSA-treated Arsa KO mice, the incidence and severity sometimes increased to mild / mild or mild (Figure 30E). The sciatic nerves of WT mice also had a rare and very mild background of incidental axonal degeneration (Figure 30E). No specific findings of the test substance were observed in the liver.

[0153] In summary, these data indicate that AAV1999-ARSA-mediated gene replacement in ARSA KO mice is highly tolerated to the same extent in both male and female mice, and the MLD-related phenotype is reversed.

[0154] Example 13: NHP Pharmacology and Dose Range Exploration Test for Evaluating AAV1999-ARSA Briefly stated, naive male / female cynomolgus monkeys (Cambodian, 2 - 3 years old, 2.6 - 3.1 kg) NHP that were seronegative for AAV1999 neutralizing antibodies and purpose-bred were administered by single direct cisterna magna (ICM) injection. During administration, the animals were placed in the Trendelenburg position. The dosing paradigm included a single 2.5 mL injection of AAV1999-ARSA (lot number VP050322) at 0.125 mL / min, followed by a flush with 250 μL of formulation buffer. Five weeks after dosing, the animals were euthanized and samples were evaluated for vector biodistribution (dPCR) and hARSA mRNA (RTdPCR), along with safety assessment. Samples represented 64 punches from the brain representing 19 different gray matter regions and 7 distinct white matter regions, 8 sections of spinal cord with adjacent DRG, peripheral nerves, and viscera. The study design is summarized in Table 4.

[0155]

Table 4

[0156] A dose-dependent increase in the biodistribution of the AAV1999-ARSA vector was observed in the NHP brain using ICM administration, in both gray matter regions (19 distinct brain regions) and white matter regions (7 distinct brain regions) (Figure 31). The AAV1999-ARSA treatment also resulted in a broad dose-dependent increase in hARSA mRNA (Figure 32), as well as protein (Figure 33) levels at two top doses: 7.5e12 VG (1e11 VG / gm brain weight) and 2.5e13 VG (3.3e11 VG / gm brain weight). Along the rostrocaudal axis of the spinal cord, uniform and dose-dependent vector biodistribution and hARSA expression were observed in the DRG and spinal cord (Figure 34). Among the viscera, the liver, spleen, and cervical lymph nodes showed a dose-dependent increase in vector biodistribution (Figure 35).

[0157] To determine whether administration of AAV1999-ARSA resulted in meaningful human ARSA expression in the NHP brain, levels of AAV-derived human ARSA protein were evaluated and compared to 1) endogenous cynomolgus monkey cyARSA protein (hARSA / cyARSA in 19 brain regions measured in the same samples), and 2) human ARSA protein measured in the brains of 7 healthy human organ donors between 3 and 8 years old (12 brain regions). At doses of 1e11 VG / gm brain weight and 3.3e11 VG / gm brain weight, the mean human ARSA protein levels across the brain were approximately 63% and approximately 546% of native cyARSA (Figure 36). Additionally, at doses of 1e11 VG / gm brain weight and 3.3e11 VG / gm brain weight, the mean human ARSA protein levels across the brain were approximately 51% and approximately 416% of the human ARSA protein levels from healthy control organ donors (Figure 37). Thus, AAV1999-ARSA treatment in NHP results in meaningful ARSA protein expression in the CNS at brain weights of 1e11 VG / gm and 3.3e11 VG / gm brain weight.

[0158] Compared to pre-treatment testing, no clinical signs (functional or behavioral deficits) were observed in NHPs at 1 week or 5 weeks (necropsy) post-administration in any of the dosing groups (Figure 38). Intra-CM injection was well tolerated; as expected, CSF Nf-L levels were significantly increased by ICM treatment, and a further increase was observed in AAV1999-ARSA treated NHPs compared to vehicle-treated NHPs (Figure 39A). However, a dose-dependent increase was not observed (Figure 39A). No significant changes in plasma cytokine concentrations were observed in NHPs treated with AAV1999-ARSA at any dose (Figure 39B), and no cellular immune response to the AAV1999 capsid or hARSA protein was detected in NHPs treated with AAV1999-ARSA at two high doses by IFN-γ ELISpot (Figure 39C). Additionally, no gross findings related to the study items were observed in the animals at necropsy.

[0159] Cerebrospinal fluid changes consisted of a slight increase in the number of nucleated cells (mainly monocytes) in most animals at 1e10 VG / gm brain weight and above, and were dose-independent. In addition, albumin and / or total protein increased in most animals at 3.3e10 VG / gm brain weight and above 5 weeks after vector administration. A small number of individual animals at 3.3e10 or 3.3e11 VG / gm brain weight had varying amounts of basophilic to eosinophilic granular material and were morphologically compatible with nerve material. These changes correlated with neurodegeneration and mononuclear infiltration observed in various parts of the central nervous system.

[0160] Hematological changes were a transient slight increase in the number of reticulocytes in most males at 1e10 VG / gm brain weight and above and a few sporadic females at 1E11 VG / gm brain weight on day 7, with a slight decrease in erythrocyte mass parameters simultaneously in a few individuals. An increase in white blood cell count associated with an increase in the number of neutrophils, lymphocytes, and / or monocytes was seen in some males and females at 3.3e10 VG / gm brain weight, and this was still observed in some animals (mainly males) administered 3.3e11 VG / gm brain weight. No test substance-related effects on coagulation or clinical chemistry were observed.

[0161] The main test substance effects consisted of neurodegeneration (affecting the brain, lumbar spinal cord, and DRG), increased glial cell response (cerebellum and spinal cord), nerve fiber degeneration (affecting the white matter of the spinal cord and nerve fibers of the peripheral nerves), and mononuclear cell infiltration (including perivascular distribution) affecting the brain (cerebellum), DRG, and spinal cord (Figs. 40 - 42). Test substance-related findings were not observed in the viscera (heart, liver, gallbladder, spleen, pancreas, adrenal glands, lungs, bone, sternum / bone marrow, ovaries, duodenum, testes, epididymis, thymus, eyes, uterus including the neck, kidneys) at all doses. For some changes in some locations such as neurodegeneration (cerebellum, lumbar DRG at the highest dose), gliosis (other than the cerebral cortex, cerebellum, spinal cord), and mononuclear cell infiltration (cerebral cortex, lumbar DRG at the highest dose), a dose response was observed between the lowest dose (1e10 VG / gm brain weight) and the highest dose (3.3e11 VG / gm brain weight) (Figs. 40 - 42).

[0162] In conclusion, single administration of AAV1999-ARSA directly into the cisterna magna (ICM) was well tolerated in cynomolgus monkeys (both male and female) at dosing levels of 1e10, 3.3e10, 1E11, and 3.3e11 VG / gm brain weight, and 1E11 and 3.3e11 VG / gm brain weight were considered effective doses.

[0163] Sequence ARSA polypeptide sequence

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Claims

1. (1) A recombinant adeno-associated virus (rAAV) vector comprising an expression cassette for expressing an arylsulfatase A (ARSA) polypeptide, wherein the expression cassette comprises a gene encoding the ARSA polypeptide operably linked to a promoter and optionally to an enhancer; and (2) an rAAV particle comprising a modified AAV9 capsid protein containing a targeting peptide having the sequence of SEQ ID NO:

10.

2. The rAAV particle according to claim 1, wherein the ARSA polypeptide comprises the sequence of Sequence ID No.

1.

3. The rAAV particle according to claim 1, wherein the targeting peptide has linker sequences adjacent to its N-terminus and C-terminus, and the combined targeting peptide and linker sequence includes SEQ ID NO:

11.

4. The rAAV particle according to claim 1, wherein the modified AAV9 capsid protein comprises a sequence that is at least 98.5% identical to the sequence of SEQ ID NO: 12, and optionally, the modified AAV9 capsid protein comprises the sequence of SEQ ID NO:

12.

5. The rAAV particle according to claim 1, wherein the gene encoding the ARSA polypeptide is a codon-optimized ARSA gene containing the sequence of Sequence ID No.

2.

6. The rAAV vector includes the 5'AAV2 ITR sequence of sequence number 4 and the 3'AAV2 ITR sequence of sequence number 5; The expression cassette includes a CMV enhancer element containing the sequence of sequence number 6; The expression cassette includes a chicken β-actin promoter containing the sequence of SEQ ID NO: 7; and / or The rAAV particle according to claim 1, wherein the rAAV vector further comprises a WPRE element containing the sequence of sequence number 8.

7. The rAAV particle according to claim 1, wherein the rAAV vector includes the sequence of sequence number 16.

8. A composition for use in the treatment of metachromatic leukodystrophy (MLD), comprising an effective amount of rAAV particles according to any one of claims 1 to 7, and formulated for administration into cerebrospinal fluid (CSF).

9. The composition according to claim 8, wherein the composition is formulated for direct administration to CSF ​​by intraventricular (ICV) administration, direct administration of cisterna magna (dCM), or direct administration to CSF ​​via intrathecal microcatheter (IT-CM).

10. The composition according to claim 8, wherein the composition is formulated for a single dose throughout a lifetime or for a single dose per year.

11. The composition according to claim 8, formulated to increase ARSA activity by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, or at least 50% upon administration.

12. A composition for use in increasing the expression and / or activity of ARSA, comprising an effective amount of rAAV particles according to any one of claims 1 to 7, and formulated for administration to CSF.

13. A plasmid containing the sequence of Sequence ID No.

3.

14. A codon-optimized human ARSA sequence containing the sequence of sequence number 2.

15. An expression cassette comprising the codon-optimized human ARSA sequence described in claim 14.

16. The expression cassette according to claim 15, further comprising a chicken β-actin promoter containing the sequence of SEQ ID NO: 7 and / or a CMV enhancer element containing the sequence of SEQ ID NO:

6.

17. A vector comprising an expression cassette according to claim 15 or 16 and a WPRE element containing the sequence of SEQ ID NO:

8.

18. A pharmaceutical composition comprising rAAV particles according to any one of claims 1 to 7.

19. A kit comprising the pharmaceutical composition described in claim 18.

20. rAAV particles according to any one of claims 1 to 7 for use in the treatment of MLD.

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