By “engineered” is meant that the nucleic acid sequences encoding a functional NAGLU protein described herein are assembled and placed into any suitable genetic element, e.g, naked DNA, phage, transposon, cosmid, episome, etc., which transfers the NAGLU sequences carried thereon to a host cell, e.g., for generating non-viral delivery systems (e.g., RNA-based systems, naked DNA, or the like), or for generating viral vectors in a packaging host cell, and/or for delivery to a host cells in a subject. In one embodiment, the genetic element is a vector. In one embodiment, tire genetic element is a plasmid. The methods used to make such engineered constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g.. Green and Sambrook. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press. Cold Spring Harbor, NY (2012).

The term “percent (%) identity”, “sequence identity”, “percent sequence identity", or “percent identical” in tire context of nucleic acid sequences and/or amino acid sequences refer to the residues in the two sequences which are the same when aligned for correspondence, often with corrections for missing or additional bases or amino acids as compared to a reference sequence. With respect to nucleic acids, the length of sequence identity may be specified to be over the full-length of the genome, tire full-length of a gene coding sequence. In certain embodiments, a fragment of at least about 500 to 5000 nucleotides, or smaller fragments, e.g.. of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be selected. Similarly, for amino acids, tire identity may be over the full-length of a protein, or a specified peptide, polypeptide or region. A suitable amino acid fragment may be at least about 7 amino acids in length, and may be up to about 700 amino acids.

As used herein, tire phrase “at least X% identity” includes tire value of X and greater values. For example, at least 95% identity includes 95% or greater, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9%, up to 100% or 95% to 100%. and values therebetween. In this example. 95% may also include decimals rounded to the nearest value of 95% in conformance with principles of direct rounding to an integer, including but not limited to round-toward-zero, round-away from zero, round to the nearest integer, round up, round down. E.g., if the decimal point of the integer starts with 5, 6, 7, 8, 9, the integer is rounded up to next full integer (i.e., 95.7% to 96%), and if the decimal point of the integer starts with 0, 1, 2, 3, 4, the integer is rounded down to next full integer (i.e., 95.3% to 95%).

Multiple sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include, “Clustal Omega”, “Clustal W”, “MUSCLE”, “CAP Sequence Assembly”, “BLAST”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG Version 10.1. Fasta™ provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using Fasta™ with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 10. 1, herein incorporated by reference.

Percent identity’ may be readily determined for amino acid sequences over tire full- length of a protein, polypeptide, about 32 amino acids, about 330 amino acids, or a peptide fragment thereof or the corresponding nucleic acid sequence coding sequences. A suitable amino acid fragment may be at least about 8 amino acids in length, and may be up to about 700 amino acids. Generally, when referring to “identity”, “homology”, or “similarity” between two different sequences, “identity”, “homology” or “similarity” is determined in reference to "aligned" sequences. ‘"Aligned"’ sequences or “alignments” refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence.

Identity may be determined by preparing an alignment of the sequences and through the use of a variety of algorithms and/or computer programs known in the art or commercially available (e.g., BLAST, ExPASy; Clustal Omega; FASTA; using, e.g., Needleman-Wunsch algorithm. Smith-Waterman algorithm). Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Multiple sequence alignment programs are available for nucleic acid sequences. Examples of such programs include, “Clustal Omega”, “Clustal W”, “MUSCLE”, “CAP Sequence Assembly”, “BLAST”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG Version 10.1. Fasta™ provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using Fasta™ with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 10. 1, herein incorporated by reference. Sequence alignment programs are also available for amino acid sequences, e.g., the “Clustal Omega”, “Clustal X”, “MUSCLE”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

As used herein, “a desired function” refers to an NAGLU enzyme activity at least 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%. about 60%. about 65%. about 70%. about 75%. about 80%, about 85%, about 90%, about 95%, about 100%, or greater than 100% of a healthy control. As used herein, the phrases “ameliorate a symptom’; “improve a symptom” or any grammatical variants thereof refer to reversal of an MPS IIIB-related symptoms, showdown or prevention of progression of an MPS IITB-related symptoms. Tn one embodiment, the amelioration or improvement refers to the total number of symptoms in a patient after administration of tire described composition(s) or use of the described method, which is reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%. about 80%. about 90%, about 95% compared to that before the administration or use. In another embodiment, the amelioration or improvement refers to the severity or progression of a symptom after administration of the described composition(s) or use of the described method, which is reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% compared to that before the administration or use.

It should be understood that the compositions in the functional NAGLU protein and NAGLU coding sequence described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

2. Expression Cassette

In one aspect, provided is an expression cassette comprising an engineered nucleic acid sequence encoding a functional hNAGLU, and operably linked thereto regulatory sequences which direct expression of the hNAGLU. In one embodiment, an expression cassette comprising an engineered nucleic acid sequence as described herein which encodes a functional hNAGLU, and regulatory sequences which enable expression thereof. In certain embodiments, the regulatory sequences comprise a promoter. In certain embodiment, the regulatory sequences comprise one or more intron(s). one or more enhancer(s), and a polyadenylation (polyA) signal sequence.

In one embodiment, the promoter is a chicken -actin (also referred to as chicken beta-actin, CB or CBA) promoter. A variety of chicken beta-actin promoters have been described alone, or in combination with various enhancer elements (e.g., CB7 is a chicken beta-actin promoter with cytomegalovirus enhancer elements, a CAG promoter, which includes the promoter, the first exon and first intron of chicken beta actin, and the splice acceptor of the rabbit beta-globin gene), a CBh promoter [S J Gray et al. Hu Gene Ther, 201 1 Sep; 22(9): 143-1 153], In certain embodiments, the promoter is cytomegalovirus (CMV) promoter. In certain, embodiments, the CB promoter comprises nucleic acid sequence of SEQ ID NO: 4.

In a further embodiment, the promoter is a CB7 (also referred to as hybrid CB7) promoter comprising a cytomegalovirus immediate-early (CMV IE) enhancer and tire chicken -actin promoter, optionally with spacer sequence, optionally with a chimeric intron comprising chicken beta actin intron and further comprising a chicken beta-actin splicing donor (including the exon sequence, chicken beta actin intron) and rabbit beta-globin splicing acceptor. In certain embodiments, the CMV IE enhancer comprises nucleic acid sequence of SEQ ID NO: 3. In certain embodiments, the promoter is a CB7 hybrid promoter comprising a CMV IE enhancer, a chicken beta-actin promoter, and a chimeric intron comprising a chicken beta actin intron (CI). In certain embodiments, the regulatory sequence further comprises a chicken beta-actin intron. In certain embodiments, the chicken beta-actin intron comprises the nucleic acid sequence of SEQ ID NO: 5. In certain embodiments, the hybrid CB7 promoter comprise nucleic acid sequence of SEQ ID NO: 31. See, e.g., cytomegalovirus (CMV) immediate early enhancer (260 bp, C4; GenBank # K03104.1). Chicken beta-actin promoter (281 bp; CB; GenBank # X00182.1). In certain embodiments, the regulatory sequences further comprise a rabbit globin poly A (also referred to as rabbit beta globin or RBG polyA). In certain embodiments, the rabbit globin poly A comprises the nucleic acid sequence of SEQ ID NO: 6.

In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 7 or a sequence at least 95%, at least 96%, at least 97%. at least 98%, at least 99 to at least 100% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 7 or a sequence at least 99% identical thereto. In one embodiment, the hNAGLU coding sequence is at least 99% identical to SEQ ID NO: 1 (hNAGLUcoV3). In a further embodiment, tire hNAGLU coding sequence is SEQ ID NO: 1 (hNAGLUcoV3). In certain embodiments, the hNAGLU coding sequence comprises nucleic acid sequence of SEQ ID NO: 1 or a sequence at least 99 to at least 100% identical thereto.

As used herein, the term "expression” or “gene expression” refers to the process by which information from a gene is used in the synthesis of a functional gene product. The gene product may be a protein, a peptide, or a nucleic acid polymer (such as a RNA, a DNA or a PNA). As used herein, an "‘expression cassete⁷' refers to a nucleic acid molecule which comprises a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme or other useful gene product, mRNA, etc.) and regulatory sequences operably linked thereto which control, direct, enable or modulate transcription, translation, and/or expression of the nucleic acid sequence and its gene product. As used herein, “operably linked” sequences include both regulator}’ sequences that are contiguous or non-contiguous with the nucleic acid sequence and one or more regulatory elements may act in cis or trans with nucleic acid sequence. Such regulatory sequences may include, e.g., one or more of a promoter, an enhancer, an intron, a Kozak sequence, a polyadenylation sequence, and a TATA signal. The regulatory' sequences may include, e.g., one or more of transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance nucleic acid or protein stability; and when desired, sequences that enhance protein processing and/or secretion. Many varied expression control sequences, including native and non-native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized herein, depending upon the ty pe of expression desired. The expression cassette may contain regulatory sequences upstream (5’ to) of the gene sequence, e.g., one or more of a promoter, an enhancer, an intron, etc., and one or more of an enhancer, or regulatory’ sequences downstream (3’ to) a gene sequence, e.g., 3‘ untranslated region (‘3 UTR) comprising a polyadenylation site, among other elements. In certain embodiments, the regulatory sequences are operably linked to the nucleic acid sequence of a gene product, wherein the regulatory sequences are separated from nucleic acid sequence of a gene product by an intervening nucleic acid sequences, i.e., 5 ’-untranslated regions (5’UTR). In certain embodiments, the expression cassette comprises nucleic acid sequence of one or more of gene products. In some embodiments, the expression cassete can be a monocistronic or a bicistronic expression cassette. In other embodiments, the term “transgene” refers to one or more DNA sequences from an exogenous source which arc inserted into a target cell. Typically, such an expression cassete for generating a viral vector contains the coding sequence for the gene product described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein. In certain embodiments, a vector genome may contain two or more expression cassetes. As used herein, the term "‘regulatory sequence”, or “regulatory control sequences “or “expression control sequence” refers to nucleic acid sequences, such as initiator sequences, enhancer sequences, promoter sequences, intron sequences, and polyA signal sequences which direct, enable, induce, repress, or otherwise control the transcription, translation and/or expression of protein encoding nucleic acid sequences to which they are operably linked.

The term “exogenous” as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein does not naturally occur in the position in which it exists in a chromosome, or host cell. An exogenous nucleic acid sequence also refers to a sequence derived from and inserted into the same host cell or subject, but w hich is present in a nonnatural state, e.g. a different copy number, or under the control of different regulatory elements.

The term “heterologous” as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein w as derived from a different organism or a different species of the same organism than the host cell or subject in which it is expressed. The tenn “heterologous” when used with reference to a protein or a nucleic acid in a plasmid, expression cassette, or vector, indicates that the protein or the nucleic acid is present with another sequence or subsequence which with w hich the protein or nucleic acid in question is not found in the same relationship to each other in nature.

In one embodiment, the regulatory sequences comprise a promoter. In one embodiment, the promoter is a chicken 0-actin promoter. In a further embodiment, the promoter is a hybrid of a cytomegalovirus immediate-early enhancer and the chicken 0-actin promoter. In a further embodiment, the promoter is a CB7 (also referred to as hybrid CB7) promoter comprising a cytomegalovirus immediate-early (CMV IE) enhancer and the chicken 0-actin promoter, optionally with spacer sequence, optionally with a chimeric intron comprising chicken beta actin intron and further comprising a chicken beta-actin splicing donor (including the exon sequence, chicken beta actin intron) and rabbit beta-globin splicing acceptor. In another embodiment, a suitable promoter may include without limitation, an elongation factor 1 alpha (EFl alpha) promoter (see, e.g., Kim DW et al, Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Gene. 1990 Jul 16:91(2):217-23), a Synapsin 1 promoter (see, e.g., Kugler S et al, Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 2003 Feb;10(4):337-47), a neuron-specific enolase (NSE) promoter (see, e.g., Kim J et al, Involvement of cholesterol-rich lipid rafts in interleukin-6-induced neuroendocrine differentiation of LNCaP prostate cancer cells. Endocrinology. 2004 Feb;145(2):613-9. Epub 2003 Oct 16), or a CB6 promoter (see, e.g., Large-Scale Production of Adeno-Associated Viral Vector Serotype-9 Carrying tire Human Survival Motor Neuron Gene, Mol Biotechnol. 2016 Jan;58(l):30-6. Doi: 10.1007/sl2033-015-9899-5).

In one embodiment, the expression cassette is designed for expression and secretion in a human subject. In one embodiment, the expression cassette is designed for expression in the central nervous system (CNS), including the cerebral spinal fluid and brain. In a further embodiment, the expression cassette is useful for expression in both the CNS and in the liver. Suitable promoters may be selected, including but not limited to a constitutive promoter, a tissue-specific promoter or an induciblc/regulatory promoter. Example of a constitutive promoter is chicken beta-actin promoter. A variety’ of chicken beta-actin promoters have been described alone, or in combination with various enhancer elements (e.g.. CB7 is a chicken beta-actin promoter with cytomegalovirus enhancer elements; a CAG promoter, which includes the promoter, the first exon and first intron of chicken beta actin, and the splice acceptor of tire rabbit beta-globin gene; a CBh promoter, SJ Gray et al, Hu Gene Ther, 2011 Sep: 22(9): 1143- 1153). Examples of promoters that are tissue-specific are well known for liver (albumin, Miyatake et al., (1997) J. Virol., 71:5124-32; hepatitis B virus core promoter, Sandig et al., (1996) Gene Ther., 3: 1002-9; alpha-fetoprotein (AFP), Arbuthnot et al., (1996) Hum. Gene Ther., 7: 1503-14), neuron (such as neuron-specific enolase (NSE) promoter, Andersen et al., (1993) Cell. Mol. Neurobiol., 13:503-15; neurofilament light-chain gene, Piccioli et al., (1991) Proc. Natl. Acad. Sci. USA. 88:5611-5; and the neuron-specific vgf gene. Piccioli et al., ( 1995) Neuron. 15:373-84), and other tissues. Alternatively, a regulatable promoter may be selected. See, e.g., WO 2011/126808B2, incorporated by reference herein.

In one embodiment, the regulatory sequence further comprises an enhancer. In one embodiment, the regulatory sequence comprises one enhancer. In another embodiment, tire regulatory sequence contains two or more enhancers. These enhancers may be the same or may be different. For example, an enhancer may include an Alpha mic/bik enhancer or a CMV enhancer (e.g.. CMV IE enhancer). This enhancer may be present in two copies which are located adjacent to one another. Alternatively, the dual copies of the enhancer may be separated by one or more sequences. In one embodiment, the regulatory sequence further comprises an intron. In a further embodiment, the intron is a chicken beta-actin intron. In a further embodiment, the intron is a chimeric intron comprising chicken beta-actin intron. In a further embodiment, the intron is chimeric intron comprising chicken beta actin intron and further comprising a chicken betaactin splicing donor (including the exon sequence, chicken beta actin intron) and rabbit betaglobin splicing acceptor. Other suitable introns include those known in the art, and may include a human 0-globulin intron, and/or a chimeric intron commercially available from Promega®, and those described in WO 2011/126808.

In one embodiment, the regulatory sequence further comprises a Polyadenylation signal (polyA). In a further embodiment, the polyA is a rabbit globin poly A. See, e.g., WO 2014/151341. Alternatively, another polyA, e.g., a human growth hormone (hGH) polyadenylation sequence, an SV40 polyA, or a synthetic polyA may be included in an expression cassette.

In certain embodiments, the regulatory' sequence comprises a hybrid promoter comprising a CMV IE enhancer, a chicken beta-actin promoter, and a chimeric intron comprising a chicken beta actin intron. In one embodiment, the regulatory sequence comprises a promoter element comprising a chicken beta actin promoter having the sequence of SEQ ID NO: 4 or a sequence at least 99.9% identical thereto. In one embodiment, the regulatory sequence comprises an enhancer element comprising a CMV IE enhancer having the sequence of SEQ ID NO: 3 or a sequence at least 99.9% identical thereto. In a further embodiment, the regulatory' sequence comprises an intron comprising a chicken beta actin intro having the sequence of SEQ ID NO: 5 or a sequence at least 99.9% identical thereto. In one embodiment, the regulatory sequence further comprises a rabbit beta globin poly A having the sequence of SEQ ID NO: 6 or a sequence at least 99.9% identical thereto. In certain embodiments, the AAV vector genome comprises expression cassette comprising the sequence of SEQ ID NO: 7 (CB7.CI.hNAGLUcoV3.RBG). In certain embodiments, the AAV vector genome comprises the sequence of SEQ ID NO: 8 (AAV.CB7.CI.hNAGLUcoV3.RBG).

It should be understood that the compositions in the expression cassette described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification. 3. Vector

In one aspect, provided herein is a vector (e.g.., a recombinant adeno-associated viral vector having an AAV capsid) comprising an engineered nucleic acid sequence encoding a functional human NAGLU and a regulator ' sequence which direct expression thereof in a target cell (e.g., in a vector genome packaged in an rAAV capsid). In one embodiment, the hNAGLU coding sequence is at least 99% identical to SEQ ID NO: 1 (hNAGLUcoV3). In a further embodiment, the hNAGLU coding sequence is SEQ ID NO: 1 (hNAGLUcoV3).

A "vector'’ as used herein is a biological or chemical moiety comprising a nucleic acid sequence which can be introduced into an appropriate target cell for replication or expression of said nucleic acid sequence. Examples of a vector includes but not limited to a recombinant virus, a plasmid, Lipoplexes, a Polymersome, Polyplexes, a dendrimer, a cell penetrating peptide (CPP) conjugate, a magnetic particle, or a nanoparticle. In one embodiment, a vector is a nucleic acid molecule into which an exogenous or heterologous or engineered nucleic acid encoding a functional hNAGLU may be inserted, which can then be introduced into an appropriate target cell. Such vectors preferably have one or more origin of replication, and one or more site into which the recombinant DNA can be inserted. Vectors often have means by which cells with vectors can be selected from those without, e.g., they encode drag resistance genes. Common vectors include plasmids, viral genomes, and "artificial chromosomes". Conventional methods of generation, production, characterization or quantification of the vectors are available to one of skill in the art.

In one embodiment, the vector is a non-viral plasmid that comprises an expression cassette described thereof, e.g., “naked DNA'’, “naked plasmid DNA'’, RNA, and mRNA; coupled with various compositions and nano particles, including, e.g., micelles, liposomes, cationic lipid - nucleic acid compositions, poly-glycan compositions and other polymers, lipid and/or cholesterol-based - nucleic acid conjugates, and other constructs such as are described herein. See, e.g., X. Su et al, Mol. Pharmaceutics, 2011, 8 (3), pp 774-787; web publication: March 21, 2011; WO2013/182683, WO 2010/053572 and WO 2012/170930, all of which arc incorporated herein by reference.

In certain embodiments, the vector described herein is a “replication-defective virus" or a “viral vector” which refers to a synthetic or artificial viral particle in which an expression cassette containing a nucleic acid sequence encoding a functional hNAGLU is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells. In one embodiment, tire genome of the viral vector does not include genes encoding the enzymes required to replicate (the genome can be engineered to be "gutless" - containing only the nucleic acid sequence encoding NAGLU flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Therefore, it is deemed safe for use in gene therapy since replication and infection by progeny virions cannot occur except in the presence of tire viral enzyme required for replication.

As used herein, a recombinant virus vector is an adeno-associated virus (AAV), an adenovirus, a bocavirus, a hybrid AAV/bocavirus, a herpes simplex virus or a lentivirus.

As used herein, the term ‘'host cell” may refer to the packaging cell line in which a vector (e.g., a recombinant AAV) is produced. A host cell may be a prokaryotic or eukaryotic cell (e.g., human, insect, or yeast) that contains exogenous or heterologous DNA that has been introduced into tire cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. Examples of host cells may include, but are not limited to an isolated cell, a cell culture, an Escherichia coli cell, a yeast cell, a human cell, a non-human cell, a mammalian cell, a non-mammalian cell, an insect cell, an HEK-293 cell, a liver cell, a kidney cell, a cell of the central nervous system, a neuron, a glial cell, or a stem cell.

As used herein, the term “target cell” refers to any target cell in which expression of the functional NAGLU is desired. In certain embodiments, the term "target cell" is intended to reference the cells of the subject being treated for MPS IIIB. Examples of target cells may include, but are not limited to, a liver cell, a kidney cell, a cell of the central nervous system, a neuron, a glial cell, and a stem cell. In certain embodiments, the vector is delivered to a target cell ex vivo. In certain embodiments, the vector is delivered to the target cell in vivo.

It should be understood that the compositions in the vector described herein are intended to be applied to other compositions, regimens, aspects, embodiments and methods described across the Specification.

4. Adeno-associated Virus (AAV)

In one aspect, provided herein is a recombinant AAV (rAAV) comprising an AAV capsid and a vector genome packaged therein. The rAAV is for use in the treatment of Mucopolysaccharidosis III B (MPS IIIB). In one aspect provided herein is a recombinant AAV (rAAV) comprising an AAV capsid and a vector genome, wherein the vector genome is a nucleic acid molecule comprising expression cassette comprising an engineered nucleic acid sequence encoding a functional human N-acetyl-alpha-glucosaminidase (hNAGLU) operably linked to regulatory sequences which directs expression of hNAGLU in a target cell, wherein the hNAGLU coding sequence is SEQ ID NO: 1 or a sequence at least 99% identical to SEQ ID NO: 1 (hNAGLUcoV3). In certain embodiment, the rAAV comprises the vector genome comprising an AAV 5^? inverted terminal repeat (ITR). an expression cassette comprising an engineered nucleic acid sequence encoding a functional hNAGLU as described herein, regulatory sequences which direct expression of hNAGLU in a target cell, and an AAV 3’ ITR. In certain embodiment, the rAAV comprises the vector genome comprising an AAV 5’ inverted terminal repeat (ITR), an expression cassette comprising an engineered nucleic acid sequence encoding a functional hNAGLU as described herein, operably linked thereto regulatory sequences which direct expression of hNAGLU in a target cell, and an AAV 3’ ITR.

In another aspect, provided herein is a recombinant nucleic acid molecule comprising a vector genome comprising an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR), and expression cassette, and an AAV 3' ITR, wherein the expression cassette comprises nucleic acid sequence of SEQ ID NO: 7. In certain embodiments, the vector genome comprises nucleic acid sequence of SEQ ID NO: 8. In certain embodiments, the recombinant nucleic acid molecule is a plasmid.

In one embodiment, tire hNAGLU coding sequence is at least 99% identical to SEQ ID NO: 1 (hNAGLUcoV3). In a further embodiment, the hNAGLU coding sequence is SEQ ID NO: 1 (hNAGLUcoV3). In one embodiment, the regulatory sequences comprise a promoter. In a further embodiment, the regulatory sequenced further comprise an enhancer. In one embodiment, the regulatory sequenced further comprise an intron. In one embodiment, the regulatory sequences further comprise a poly A. In certain embodiments, the AAV vector genome comprises expression cassette comprising the sequence of SEQ ID NO: 7 (CB7.CI.hNAGLUcoV3.RBG), which encodes tire hNAGLU protein of SEQ ID NO: 2.1n certain embodiments, the AAV vector genome comprises tlie sequence of SEQ ID NO: 8 (AAV.CB7.CI.hNAGLUcoV3.RBG), which encodes the hNAGLU protein of SEQ ID NO: 2. In one embodiment, the AAV capsid is an AAVhu68 capsid with the coding sequence SEQ ID NO: 9 and/or the amino acid sequence SEQ ID NO: 10. In one embodiment, the AAV capsid is an AAVhu95 capsid with the coding sequence SEQ ID NO: 13 and/or the amino acid sequence SEQ ID NO: 14. In another embodiment, the AAV capsid is an AAVhu96 capsid with the coding sequence SEQ ID NO: 15 and/or the amino acid sequence SEQ ID NO: 16. In one embodiment, tire AAV capsid is an AAV9 capsid with the coding sequence SEQ ID NO: 17 and/or the amino acid sequence SEQ ID NO: 18. In yet another embodiment, the AAV capsid is an AAVrh91 capsid with the coding sequence SEQ ID NO: 11 and/or the amino acid sequence SEQ ID NO: 12. In one embodiment, tire rAAV described herein is for use in the treatment of Mucopolysaccharidosis III B (MPS IIIB). See also, International Patent Application No. PCT/US2021/055436. filed October 18, 2021, now publication No. WO 2022/082109, International Patent Application No.

PCT/US2022/077315, filed September 30, 2022, now publication No. WO 2023/056399, and International Patent Application No. PCT/US2021/045945, filed August 13, 2021, now publication No. WO 2022/036220 are incorporated herein by reference in their entireties.

In one embodiment, the regulatory sequences are as described above.

In one embodiment, provided is a rAAV comprising an AAV serotype hu68 (AAVhu68) capsid and a vector genome comprising a CB7 promoter expressing an engineered version of hNAGLU with a rabbit beta-globin (rBG) polyA sequence. In a further embodiment, the rAAV vector genome comprises the sequence of SEQ ID NO: 8 (CB7.CI.hNAGLUcoV3.rBG). In one embodiment, tire rAAV comprises an AAVhu68 capsid and a vector genome comprising expression cassette comprising the sequence of SEQ ID NO: 7, wherein the rAAV is represented as AAVhu68.CB7.CI.hNAGLUcoV3.rBG. In one embodiment, the rAAV comprises an AAVhu68 capsid and a vector genome comprising the sequence of SEQ ID NO: 8, wherein the rAAV is represented as AAVhu68.CB7.Cl.hNAGLUcoV3.rBG

In one embodiment, provided is a rAAV comprising an AAV serotype hu95 (AAVhu95) capsid and a vector genome comprising a CB7 promoter expressing an engineered version of hNAGLU with a rabbit beta-globin (rBG) polyA sequence. In a further embodiment, the rAAV vector genome comprises the sequence of SEQ ID NO: 8 (AAV.CB7.CI.hNAGLUcoV3.rBG). In one embodiment, the rAAV comprises an AAVhu95 capsid and a vector genome comprising the sequence of SEQ ID NO: 8, wherein the rAAV is represented as AAVhu95.CB7.CI.hNAGLUcoV3.rBG.

In one embodiment, provided is a rAAV comprising an AAV serotype hu96 (AAVhu96) capsid and a vector genome comprising a CB7 promoter expressing an engineered version of hNAGLU with a rabbit beta-globin (rBG) polyA sequence. In a further embodiment, the rAAV vector genome comprises the sequence of SEQ ID NO: 8 (.CB7.CI.hSNAGLUcoV3.rBG). In one embodiment, the rAAV comprises an AAVhu96 capsid and a vector genome comprising the sequence of SEQ ID NO: 8, wherein the rAAV is represented as AAVhu96.CB7.CI.hNAGLUcoV3.rBG.

In one embodiment, provided is a rAAV comprising an AAV serotype 9 (AAV9) capsid and a vector genome comprising a CB7 promoter expressing an engineered version of hNAGLU with a rabbit beta-globin (rBG) polyA sequence. In a further embodiment, the rAAV vector genome comprises the sequence of SEQ ID NO: 8 (CB7.CI.hSNAGLUcoV3.rBG). In one embodiment, the rAAV comprises an AAV9 capsid and a vector genome comprising the sequence of SEQ ID NO: 8, wherein the rAAV is represented as AAV9.CB7.CI.hSNAGLUcoV3.rBG.

In one embodiment, provided is a rAAV comprising an AAV serotype rh91(AAVrh91) capsid and a vector genome comprising a CB7 promoter expressing an engineered version of hNAGLU with a rabbit beta-globin (rBG) polyA sequence. In a further embodiment, the rAAV vector genome comprises tire sequence of SEQ ID NO: 8 (CB7.CI.hSNAGLUcoV3.rBG). In one embodiment, the rAAV comprises an AAVrh91 capsid and a vector genome comprising the sequence of SEQ ID NO: 8, wherein the rAAV is represented as AAVrh91.CB7.CI.hSNAGLUcoV3.rBG.

As used herein, the term “vector genome” refers to a nucleic acid molecule which is packaged in a viral capsid, for example, an AAV capsid, and is capable of being delivered to a host cell or a cell in a patient. In certain embodiments, the vector genome comprises terminal repeat sequences (e.g., AAV inverted terminal repeat sequences (ITRs) necessary for packaging the vector genome into the capsid at the extreme 5’ and 3’ end and containing therebetween an expression cassette comprising a nucleic acid sequence encoding a functional NAGLU as described herein operably linked to regulatory sequences which direct expression thereof. In one example, a vector genome contains, at a minimum, from 5’ to 3’, an AAV2 5’ ITR, a nucleic acid sequence encoding a functional NAGLU, and an AAV2 3’ ITR. However, ITRs from a different source AAV other than AAV2 may be selected. Further, other ITRs may be used. Further, tire vector genome contains regulatory sequences which direct expression of the functional NAGLU.

The AAV sequences of the vector typically comprise the cis-acting AAV 5’ and AAV 3’ inverted terminal repeat (ITR) sequences (See, e g., B. J. Carter, in “Handbook of Parvoviruses", ed.. P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are about 145 base pairs (bp) in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al, “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J. Virol., 70:520 532 (1996)). An example of such a molecule employed in the present invention is a “cisacting” plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5’ and 3’ AAV ITR sequences (also referred to as “AAV 5’ ITR”, “5’ ITR”, “AAV 5' ITR”, or “5' ITR”, “AAV 3^: ITR”, “3’ ITR”, “AAV 3' ITR”, or “3' ITR”). In one embodiment, the ITRs are from an AAV different than that supplying a capsid. In one embodiment, the ITR sequences are from AAV2.

How ever, ITRs from other AAV sources may be selected. A shortened version of the 5 ’ ITR, termed AITR, has been described in which the D-sequence and terminal resolution site (trs) are deleted. In certain embodiments, the vector genome includes a shortened AAV2 ITR of 130 base pairs, wherein the external A elements is deleted. Without wishing to be bound by theory, it is believed that the shortened ITR reverts back to the wild-type (WT) length of 145 base pairs during vector DNA amplification using the internal (A’) element as a template. In other embodiments, full-length AAV 5’ and 3’ ITRs are used. Where the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be termed pseudotyped. However, other configurations of these elements may be suitable.

In certain embodiments, the provided herein is rAAV comprising a nucleic acid molecule comprising a vector genome comprising at least one AAV ITR at the extreme 5' and/or extreme 3' end of the nucleic acid molecule which is the vector genome and an expression cassette. In certain embodiments, the vector genome is a nucleic acid molecule which comprises a 5' - AAV ITR, the expression cassette and a 3' - AAV ITR.

In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising, 5' to 3', AAV- 5' ITR - an optional enhancer - a promoter - an optional intron - coding sequence - polyadenylation (poly A) signal sequence - AAV3' - ITR. In other embodiments, tire orientation of the ITRs may change from the orientation presented in the vector genome of the nucleic acid used in production (e.g., a plasmid). Thus, in certain embodiments, the rAAV may comprise a vector genome flanked by 3' and 5' AAV ITRs, respectively. In certain embodiments, the rAAV may comprise a vector genome flanked by two 5' AAV ITRs. In certain embodiments, the rAAV may comprise a vector genome flanked by two 3' AAV ITRs. In other embodiments, an rAAV as provided herein may be partially truncated such that the 5' AAV ITR and/or the 3' AAV ITR is not detectable in the vector genome packaged in a final rAAV product.

The term “AAV” as used herein refers to naturally occurring adeno-associated viruses, adeno-associated viruses available to one of skill in the art and/or in light of the composition(s) and method(s) described herein, as well as artificial AAVs. An adeno- associated virus (AAV) viral vector is an AAV Dnase-resistant particle having an AAV protein capsid into which is packaged expression cassette flanked by AAV inverted terminal repeat sequences (ITRs) for delivery to target cells. An AAV capsid is composed of 60 capsid (cap) protein subunits, VP1, VP2, and VP3, that are arranged in an icosahedral symmetry in a ratio of approximately 1 : 1 : 10 to 1:1 :20, depending upon the selected AAV. Various AAVs may be selected as sources for capsids of AAV viral vectors as identified above. See, e.g., US Published Patent Application No. 2007-0036760-Al; US Published Patent Application No. 2009-0197338-Al; EP 1310571. See also, WO 2003/042397 (AAV7 and other simian AAV), US Patent 7790449 and US Patent 7282199 (AAV8), WO 2005/033321 and US 7,906,111 (AAV 9), and WO 2006/110689, and WO 2003/042397 (rh. 10). These documents also describe other AAV which may be selected for generating AAV and are incorporated by reference. Among the AAVs isolated or engineered from human or non-human primates (NHP) and well characterized, human AAV2 is the first AAV that was developed as a gene transfer vector; it has been widely used for efficient gene transfer experiments in different target tissues and animal models. Unless otherwise specified, the AAV capsid. ITRs. and other selected AAV components described herein, may be readily selected from among any AAV, including, without limitation, the AAVs commonly identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8 and AAVAnc80, variants of any of the known or mentioned AAVs or AAVs yet to be discovered or variants or mixtures thereof. See, e.g., WO 2005/033321, which is incorporated herein by reference.

In certain embodiments, tire AAV capsid is Clade F AAV capsid, wherein the Clade F AAV capsid is selected from an AAVhu68 capsid [See. e.g., US2020/0056159; PCT/US21/55436; PCT/US 18/19992, filed February 27, 2018, now published WO2018/160582, which is incorporated by reference herein], an AAVhu95 capsid [See, e.g., US Provisional Application No. 63/251,599, filed October 2, 2201, International Patent Application No. PCT/US2022/077315. filed September 30. 2022], or an AAVhu96 capsid [See, e.g., US Provisional Application No. 63/251,599, filed October 2, 2201, and International Patent Application No. PCT/US2022/077315, filed September 30, 2022], AAV9 capsid. In certain embodiments, the AAV capsid is a Clade A capsid, such as AAVrh91 capsid. See, PCT/US20/030266, filed April 29, 2020, now published WO2020/223231, which is incorporated by reference herein and International Application No. PCT/US21/45945, filed August 13, 2021 which are incorporated herein by reference.

In certain embodiment, the AAV capsid is an AAVhu68 capsid. In certain embodiments tire AAV capsid is an AAV9 capsid. In certain embodiments the AAV capsid is an AAVhu95 capsid. In certain embodiments, the AAV capsid is an AAVhu96 capsid.

In certain embodiments, the AAV capsid for the compositions and methods described herein is chosen based on the target cell. In certain embodiment, the AAV capsid transduces a CNS cell and/or a PNS cell. In certain embodiments, other AAV capsid may be chosen. The AAV capsid is selected from a cy02 capsid, a rh43 capsid, an AAV8 capsid, a rhOl capsid, an AAV9 capsid, an rh8 capsid, a rhlO capsid, a bbOl capsid, a hu37 capsid, a rh02 capsid, a rh20 capsid, a rh39 capsid, a rh64 capsid, an AAV6 capsid, an AAV1 capsid, a hu44 capsid, a hu48 capsid, a cy05 capsid a hul 1 capsid, a hu32 capsid, a pi2 capsid, or a variation thereof. In certain embodiments, the AAV capsid is a Clade F capsid, such as AAV9 capsid, AAVhu68 capsid, hu31 capsid, hu32 capsid, or a variation thereof. See, e.g., WO 2005/033321 published April 14, 2015, WO 2018/160582, and US 2015/0079038, each of which is incorporated herein by reference in its entirety. In certain embodiments, tire AAV capsid is a non-clade F capsid, for example a Clade A, B, C, D, or E capsid. In certain embodiment, the non-Clade F capsid is an AAV 1 or a variation thereof. In certain embodiment, the AAV capsid transduces a target cell other than tire nervous system cells. In certain embodiments, the AAV capsid is a Clade A capsid (e.g., AAV1, AAV6, AAVrh91), a Clade B capsid (e.g., AAV 2), a Clade C capsid (e.g., hu53), a Clade D capsid (e.g., AAV7), or a Clade E capsid (e.g., rhlO).

An AAV capsid is an assembly of a heterogeneous population of vpl, a heterogeneous population of vp2, and a heterogeneous population of vp3 proteins. As used herein when used to refer to vp capsid proteins, the term "‘heterogeneous" or any grammatical variation thereof, refers to a population consisting of elements that are not the same, for example, having vpl, vp2 or vp3 monomers (proteins) with different modified amino acid sequences. As used herein when used to refer to vp capsid proteins, the term "‘heterogeneous” or any grammatical variation thereof, refers to a population consisting of elements that are not the same, for example, having vp 1 , vp2 or vp3 (also referenced as VP1 , VP2, VP3, or Vpl , Vp2, Vp3) monomers (proteins) with different modified amino acid sequences. The term “heterogeneous population” as used in connection with vpl, vp2 and vp3 proteins (alternatively termed isoforms), refers to differences in the amino acid sequence of the vpl, vp2 and vp3 proteins within a capsid. The AAV capsid contains subpopulations within tire vpl proteins, within the vp2 proteins and within the vp3 proteins which have modifications from the predicted amino acid residues. These subpopulations include, at a minimum, certain deamidated asparagine (N or Asn) residues. For example, certain subpopulations comprise at least one, two, three or four highly deamidated asparagines (N) positions in asparagine - glycine pairs and optionally further comprising other deamidated amino acids, wherein the deamidation results in an amino acid change and other optional modifications.

In certain embodiments, AAV capsids are provided which have a heterogeneous population of AAV capsid isoforms (i.e., VP1, VP2, VP3) which contain multiple highly deamidated “NG” positions. In certain embodiments, the highly deamidated positions are in the locations identified below, with reference to the predicted full-length VP 1 amino acid sequence. In other embodiments, the capsid gene is modified such that the referenced “NG” is ablated and a mutant “NG” is engineered into another position.

As used herein, relating to AAV, the term “variant” means any AAV sequence which is derived from a known AAV sequence, including those with a conservative amino acid replacement, and those sharing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or greater sequence identity over the amino acid or nucleic acid sequence. In another embodiment, the AAV capsid includes variants which may include up to about 10% variation from any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% identity to about 99.9 % identity, about 95% to about 99% identity' or about 97% to about 98% identity' to an AAV capsid provided herein and/or known in tire art. In one embodiment, the AAV capsid shares at least 95% identity with an AAV capsid. When determining the percent identity of an AAV capsid, the comparison may be made over any of the variable proteins (e g., vpl, vp2, or vp3).

The ITRs or other AAV components may be readily isolated or engineered using techniques available to those of skill in the art from an AAV. Such AAV may be isolated, engineered, or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection. Manassas, VA). Alternatively, tire AAV sequences may be engineered through synthetic or other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank, PubMed, or the like. AAV viruses may be engineered by conventional molecular biology techniques, making it possible to optimize these particles for cell specific delivery of nucleic acid sequences, for minimizing immunogenicity, for tuning stability and particle lifetime, for efficient degradation, for accurate delivery to the nucleus, etc.

As used herein, the terms “rAA V" and "‘artificial AAV used interchangeably, mean, without limitation, a AAV comprising a capsid protein and a vector genome packaged therein, wherein the vector genome comprising a nucleic acid heterologous to the AAV. In one embodiment, the capsid protein is a non-naturally occurring capsid. Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a vp 1 capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV, non-contiguous portions of the same AAV, from a non-AAV viral source, or from a non-viral source. An artificial AAV may be. w ithout limitation, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAV capsid. Pseudotyped vectors, wherein the capsid of one AAV is replaced with a heterologous capsid protein, are useful in the invention. In one embodiment, AAV2/5 and AAV2/8 are exemplary' pseudotyped vectors. The selected genetic element may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. The methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g.. Green and Sambrook. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

In one embodiment, the rAAV as described herein is a self-complementary AAV. “Self-complementary' AAV” refers a construct in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intra-molccular double-stranded DNA template. Upon infection, rather than w aiting for cell mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. See, e.g., D M McCarty et al, “Self-complementary recombinant adeno- associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis". Gene Therapy, (August 2001), Vol 8, Number 16. Pages 1248-1254. Self- complementary AAVs are described in, e.g., U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety.

In certain embodiments, the rAAV described herein is nuclease-resistant. Such nuclease may be a single nuclease, or mixtures of nucleases, and may be endonucleases or exonucleases. A nuclease-resistant rAAV indicates that the AAV capsid has fully assembled and protects these packaged genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids which may be present from the production process. In many instances, tire rAAV described herein is Dnase resistant.

The recombinant adeno-associated virus (AAV) described herein may be generated using techniques which are known. See, e.g., WO 2003/042397; WO 2005/033321, WO 2006/110689; US 7588772 B2; and WO 2017/136500, which is incorporated by reference herein. Such a method involves culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette as described herein flanked by AAV inverted terminal repeats (ITRs); and sufficient helper functions to pennit packaging of the expression cassette into the AAV capsid protein. Also provided herein is the host cell which contains a nucleic acid sequence encoding an AAV capsid; a functional rep gene; a vector genome as described; and sufficient helper functions to permit packaging of the vector genome into the AAV capsid protein. In one embodiment, the host cell is a HEK 293 cell. These methods are described in more detail in W02017160360 A2, which is incorporated by reference herein.

In one embodiment, a production cell culture useful for producing a recombinant AAV having a capsid selected from AAVhu68. AAVrh91, AAVhu95 or AAVhu96 is provided. Such a cell culture contains a nucleic acid which expresses the AAVhu68 capsid protein in the host cell (e.g., SEQ ID NO: 9 or SEQ ID NO: 10); a nucleic acid molecule suitable for packaging into the AAVhu68 capsid, e.g., a vector genome which contains AAV ITRs and a non-AAV nucleic acid sequence encoding a gene operably linked to regulator}’ sequences which direct expression of the gene in a host cell; and sufficient AAV rep functions and adenovirus helper functions to permit packaging of the vector genome into the recombinant AAVhu68, or AAVrh91 capsid (e.g., SEQ ID NO: 11 or SEQ ID NO: 12). AAVhu95 capsid (e.g., SEQ ID NO: 13 or SEQ ID NO: 14), AAVhu96 capsid (e.g., SEQ ID NO: 15 or SEQ ID NO: 16). In one embodiment, the cell culture is composed of mammalian cells (e.g., human embryonic kidney 293 cells, among others) or insect cells (e.g.. Spodoptera frugiperda (Sf9) cells). In certain embodiments, baculovirus provides the helper functions necessary for packaging the vector genome into the recombinant AAVhu68, AAVrh91, AAVhu95 or AAVhu96 capsid.

Other methods of producing rAAV available to one of skill in the art may be utilized. Suitable methods may include without limitation, baculovirus expression system or production via yeast. See, e.g., Robert M. Kotin, Large-scale recombinant adeno-associated virus production. Hum Mol Genet. 2011 Apr 15; 2O(R1): R2-R6. Published online 2011 Apr 29. Doi: 10. 1093/hmg/ddrl41: Aucoin MG et al., Production of adeno-associated viral vectors in insect cells using triple infection: optimization of baculovirus concentration ratios. Biotechnol Bioeng. 2006 Dec 20:95(6): 1081-92; SAMI S. THAKUR, Production of Recombinant Adeno-associated viral vectors in yeast. Thesis presented to tire Graduate School of the University of Florida, 2012; Kondratov O et al. Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells, Mol Ther. 2017 Aug 10. Pii: S1525-0016(17)30362-3. Doi:

10. 1016/j.ymthe.2017.08.003. [Epub ahead of print]; Mietzsch M et al, OneBac 2.0: Sf9 Cell Lines for Production of AAVL AAV2, and AAV8 Vectors with Minimal Encapsidation of Foreign DNA. Hum Gene Ther Methods. 2017 Feb:28( l): 15-22. Doi:

10. 1089/hgtb.2016. 164.; Li L et al. Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer. PloS One. 2013 Aug l;8(8):e69879. Doi: 10. 1371/joumal. pone.0069879. Print 2013; Galibert L et al, Latest developments in the large-scale production of adeno-associated virus vectors in insect cells toward the treatment of neuromuscular diseases. J Invertebr Pathol. 2011 Jul;107 Suppl:S80-93. Doi: 10. 1016/j.jip.2011.05.008; and Kotin RM. Large- scale recombinant adeno-associated virus production. Hum Mol Genet. 2011 Apr 15;20(Rl):R2-6. Doi: 10.1093/hmg/ddrl41. Epub 2011 Apr 29.

A two-step affinity chromatography purification at high salt concentration followed by anion exchange resin chromatography arc used to purify the vector drug product and to remove empty capsids. These methods are described in more detail in International Patent Application No. PCT/US2016/065970, filed December 9, 2016, and US 11,098,286 B2, entitled “Scalable Purification Method for AAV9”, which are incorporated by reference. Purification methods for AAV8, International Patent Application No. PCT/US2016/065976, filed December 9, 2016, and US 1 1,015,174 B2, entitled “Scalable Purification Method for AAV8", which are incorporated herein by reference. Purification methods for rhlO. International Patent Application No. PCT/US16/066013, filed December 9, 2016, and US 1 1,028,372 B2, entitled “Scalable Purification Method for A AVrh I O". which are incorporated herein by reference. Purification methods for AAV1, International Patent Application No. PCT/US2016/065974, filed December 9, 2016, and US 11,015,173 B2, entitled “Scalable Purification Method for AAV1”, which are incorporated herein by reference. See also, International Patent Application No. PCT/US2018/019992, filed February 27 , 2018. now published WO 2018/160582. and International Patent Application No. PCT/US2021/055436, filed October 18, 2021, now published WO 2022/082109, which are incorporated herein by reference in their entireties. Other suitable methods may be selected.

Conventional methods for characterization or quantification of rAAV are available to one of skill in the art. To calculate empty and full particle content, VP3 band volumes for a selected sample (e.g., in examples herein an iodixanol gradient-purified preparation where # of GC = # of particles) are plotted against GC particles loaded. The resulting linear equation (y = mx+c) is used to calculate the number of particles in the band volumes of tire test article peaks. The number of particles (pt) per 20 pL loaded is then multiplied by 50 to give particles (pt) /mL. Pt/mL divided by GC/mL gives the ratio of particles to genome copies (pt/GC). Pt/mL-GC/mL gives empty⁷ pt/mL. Empty pt/mL divided by pt/mL and x 100 gives the percentage of empty⁷ particles. Generally, methods for assaying for empty capsids and AAV vector particles with packaged genomes have been known in the art. See, e.g.. Grimm et al., Gene Therapy (1999) 6: 1322-1330; Sommer et al., Molec. Ther. (2003) 7: 122-128. To test for denatured capsid, the methods include subjecting the treated AAV stock to SDS- polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel containing 3-8% Tris-acetate in the buffer, then running the gel until sample material is separated, and blotting the gel onto nylon or nitrocellulose membranes, preferably nylon. Anti-AAV capsid antibodies are then used as the primary⁷ antibodies that bind to denatured capsid proteins, preferably an anti-AAV capsid monoclonal antibody, most preferably tire Bl anti-AAV-2 monoclonal antibody (Wobus et al.. J. Viral. (2000) 74:9281-9293). A secondary antibody is then used, one that binds to the primary antibody and contains a means for detecting binding with tire primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound to it, most preferably a sheep anti-mouse IgG antibody⁷ covalently linked to horseradish peroxidase. A method for detecting binding is used to semi-quantitatively determine binding between the primary and secondary antibodies, preferably a detection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit. For example, for SDS-PAGE, samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing reducing agent (e.g., DTT), and capsid proteins were resolved on pre-cast gradient polyacrylamide gels (e g., Novex). Silver staining may be performed using SilverXpress (Invitrogen, CA) according to tire manufacturer's instructions or other suitable staining method, i.e. SYPRO ruby or coomassie stains. In one embodiment, the concentration of AAV vector genomes (vg) in column fractions can be measured by quantitative real time PCR (Q-PCR). Samples are diluted and digested with Dnase I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the samples are further diluted and amplified using primers and a TaqMan™ Anorogenic probe specific for tire DNA sequence between the primers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 Sequence Detection System. Plasmid DNA containing identical sequences to that contained in the AAV vector is employed to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) values obtained from the samples are used to determine vector genome titer by normalizing it to the Ct value of the plasmid standard curve. Endpoint assays based on the digital PCR can also be used.

In one aspect, an optimized q-PCR method is used which utilizes a broad spectrum serine protease, e.g., proteinase K (such as is commercially available from Qiagen). More particularly, the optimized qPCR genome titer assay is similar to a standard assay, except that after the Dnase 1 digestion, samples are diluted with proteinase K buffer and treated with proteinase K followed by heat inactivation. Suitably samples are diluted with proteinase K buffer in an amount equal to the sample size. The proteinase K buffer may be concentrated to 2 fold or higher. Typically, proteinase K treatment is about 0.2 mg/mL, but may be varied from 0. 1 mg/mL to about 1 mg/mL. The treatment step is generally conducted at about 55 °C for about 15 minutes, but may be performed at a lower temperature (e.g., about 37 °C to about 50 °C) over a longer time period (e.g., about 20 minutes to about 30 minutes), or a higher temperature (e.g., up to about 60 °C) for a shorter time period (e.g., about 5 to 10 minutes). Similarly, heat inactivation is generally at about 95 °C for about 15 minutes, but the temperature may be lowered (e.g., about 70 to about 90 °C) and the time extended (e g., about 20 minutes to about 30 minutes). Samples are then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in the standard assay.

Additionally, or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for determining single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2): 115-25. Doi: 10. 1089/hgtb.2013. 131. Epub 2014 Feb 14.

Methods for determining the ratio among vpl, vp2 and vp3 of capsid protein are also available. See. e.g., Vamseedhar Rayaprolu et al. Comparative Analysis of Adeno- Associated Virus Capsid Stability and Dynamics, J Virol. 2013 Dec: 87(24): 13150-13160; Buller RM, Rose JA. 1978. Characterization of adenovirus-associated virus-induced polypeptides in KB cells. J. Virol. 25:331-338; and Rose JA, Maizel JV, Inman JK, Shatkin AJ. 1971. Structural proteins of adenovirus-associated viruses. J. Virol. 8:766-770.

As used herein, the term “treatment” or “treating” refers to composition(s) and/or method(s) for the purposes of amelioration of one or more symptoms of MPS IIIB, restore of a desired function of NAGLU, or improvement of biomarker of disease. In some embodiments, the term “treatment” or “treating” is defined encompassing administering to a subject one or more compositions described herein for the purposes indicated herein. “Treatment” can thus include one or more of reducing onset or progression of MPS IIIB, preventing disease, reducing the severity of tire disease symptoms, retarding their progression, removing tire disease symptoms, delaying progression of disease, or increasing efficacy of therapy in a given subject.

It should be understood that tire compositions in the rAAV described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

5. Pharmaceutical Composition

In one aspect, provided herein is a pharmaceutical composition comprising a vector as described herein in a formulation buffer. In one embodiment, the pharmaceutical composition is suitable for co-administering with a functional hNAGLU protein or a protein comprising a functional hNAGLU. In one embodiment, provided is a pharmaceutical composition comprising a rAAV as described herein in a formulation buffer. In one embodiment, the rAAV is formulated at about 1 x 10⁹ genome copies (GC)/mL to about 1 x 10¹⁴ GC/mL. In a further embodiment, the rAAV is formulated at about 3 x 10⁹ GC/mL to about 3 x 10¹³ GC/mL. Tn yet a further embodiment, the rAAV is formulated at about 1 x 10⁹ GC/mL to about 1 x 10¹³ GC/mL. In one embodiment, the rAAV is formulated at least about 1 x 10" GC/mL.

Provided herein also is a composition comprising an rAAV as described herein and an aqueous suspension media. In certain embodiments, the suspension is formulated for intravenous delivery, intrathecal administration, or intracerebroventricular administration. In one aspect, the compositions contain at least one rAAV stock and an optional carrier, excipient and/or preservative.

As used herein, a “stock” of rAAV refers to a population of rAAV. Despite heterogeneity in their capsid proteins due to deamidation, rAAV in a stock are expected to share an identical vector genome. A stock can include rAAV having capsids with, for example, heterogeneous deamidation patterns characteristic of tire selected AAV capsid proteins and a selected production system. The stock may be produced from a single production system or pooled from multiple runs of the production system. A variety of production systems, including but not limited to those described herein, may be selected.

Tn one embodiment, the formulation further comprises a surfactant, preservative, excipients, and/or buffer dissolved in the aqueous suspending liquid. In one embodiment, the buffer is PBS. In another embodiment, the buffer is an artificial cerebrospinal fluid (aCSF), e.g., Eliott's formulation buffer; or Harvard apparatus perfusion fluid (an artificial CSF with final Ion Concentrations (in mM): Na 150; K 3.0; Ca 1.4; Mg 0.8; P 1.0; Cl 155). Various suitable solutions are known including those which include one or more of: buffering saline, a surfactant, and a physiologically compatible salt or mixture of salts adjusted to an ionic strength equivalent to about 100 mM sodium chloride (NaCl) to about 250 mM sodium chloride, or a physiologically compatible salt adjusted to an equivalent ionic concentration.

Suitably, the formulation is adjusted to a physiologically acceptable pH, e.g., in the range of pH 6 to 8, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. As the pH of the cerebrospinal fluid is about 7.28 to about 7.32, for intrathecal delivery, a pH within this range may be desired; whereas for intravenous delivery, a pH of 6.8 to about 7.2 may be desired. However, other pHs within tire broadest ranges and these subranges may be selected for other route of delivery. A suitable surfactant, or combination of surfactants, may be selected from among non-ionic surfactants that are nontoxic. In one embodiment, a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.g., such as Pluronic® F68 [BASF], also known as Poloxamer 188, which has a neutral pH, has an average molecular weight of 8400. Other surfactants and other Poloxamers may be selected, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly (propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly (ethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Polyoxy capryllic glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are commonly named with the letter “P” (for poloxamer) followed by three digits: the first tw o digits x 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content. In one embodiment Poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension.

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

In other embodiments, the formulation may contain one or more permeation enhancers. Examples of suitable permeation enhancers may include, e.g., mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium capry late, sodium caprate, sodium laury l sulfate, polyoxycthylcnc-9-laurcl ether, or EDTA.

Additionally provided is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a vector comprising a nucleic acid sequence encoding a functional NAGLU as described herein. As used herein, “carrier’ includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active Ingredients can also be incorporated into the compositions. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present invention into suitable host cells. In particular, the rAAV vector may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like. In one embodiment, a therapeutically effective amount of said vector is included in the pharmaceutical composition. The selection of the carrier is not a limitation of the present invention. Other conventional pharmaceutically acceptable carrier, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

The phrase “phamiaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

As used herein, the term ‘'dosage” or "amount” can refer to the total dosage or amount delivered to the subject in the course of treatment, or the dosage or amount delivered in a single unit (or multiple unit or split dosage) administration.

Also, the replication-defective virus compositions can be formulated in dosage units to contain an amount of replication-defective virus that is in the range of about 1.0 x 10⁹ GC to about 1.0 x 10¹⁶ GC (to treat an average subject of 70 kg in body weight) including all integers or fractional amounts within the range, and preferably 1.0 x 10¹² GC to 1.0 x 10¹⁴ GC for a human patient. In one embodiment, the compositions are formulated to contain at least IxlO⁹, 2xl0⁹, 3xl0⁹, 4xl0⁹, 5xl0⁹, 6xl0⁹, 7xl0⁹, 8xl0⁹, or 9xl0⁹ GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO¹⁰, 2xlO¹⁰, 3xl0^1C1, 4xlO¹⁰, 5xl0¹⁰, 6xlO¹⁰, 7xlO¹⁰, 8xl0¹⁰, or 9xlO¹⁰ GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO¹ ', 2x10”, 3x10”, 4x10”. 5x10”, 6x10”, 7x10”, 8x10”, or 9x10” GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO¹², 2xl0¹², 3xl0¹², 4xl0¹², 5xl0¹², 6xl0¹², 7xl0¹², 8xl0¹², or 9x10¹² GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO¹³, 2xl0¹³, 3xl0¹³, 4xl0¹³, 5xl0¹³, 6xl0¹³, 7xl0¹³, 8xl0¹³, or 9xl0¹³ GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO¹⁴, 2xl0¹⁴, 3xl0¹⁴, 4xl0¹⁴, 5xl0¹⁴, 6xl0¹⁴, 7xl0¹⁴, 8xl0¹⁴, or 9x10¹⁴ GC per dose including all integers or fractional amounts within the range. In another embodiment, tire compositions are formulated to contain at least IxlO¹⁵, 2xl0¹⁵, 3xl0¹⁵, 4xl0¹⁵. 5xl0¹⁵, 6xl0¹⁵, 7xl0¹⁵, 8xl0¹⁵, or 9xl0¹⁵ GC per dose including all integers or fractional amounts within the range. In one embodiment, for human application the dose can range from IxlO¹⁰ to about IxlO¹² GC per dose including all integers or fractional amounts within the range.

In certain embodiments, the rAAV compositions can be formulated in dosage units to contain an amount of rAAV that is in the range of about I x lO⁹ GC per gram of brain mass to about I x lO¹³ genome copies (GC) per gram (g) of brain mass, including all integers or fractional amounts within tire range and tire endpoints. In another embodiment, the dosage is 1 x 1O^1CI GC per gram of brain mass to about 1 x 10¹³ GC per gram of brain mass. In specific embodiments, the dose of the vector administered to a patient is at least about 1.0 x 10⁹ GC/g, about 1.5 x 10⁹ GC/g, about 2.0 x 10⁹ GC/g, about 2.5 x 10⁹ GC/g, about 3.0 x 10⁹ GC/g, about 3.5 x 10⁹ GC/g, about 4.0 x 10⁹ GC/g, about 4.5 x 10⁹ GC/g, about 5.0 x 10⁹ GC/g, about 5.5 x 10⁹ GC/g, about 6.0 x 10⁹ GC/g, about 6.5 x 10⁹ GC/g, about 7.0 x 10⁹ GC/g, about 7.5 x 10⁹ GC/g, about 8.0 x 10⁹ GC/g, about 8.5 x 10⁹ GC/g, about 9.0 x 10⁹ GC/g, about 9.5 x 10⁹ GC/g, about 1.0 x 10¹⁰ GC/g. about 1.5 x 10¹⁰ GC/g, about 2.0 x 10¹⁰ GC/g, about 2.5 x 10¹⁰ GC/g. about 3.0 x 10¹⁰ GC/g, about 3.5 x 10¹⁰ GC/g. about 4.0 x 10^lu

GC/g, about 4.5 x 10¹⁰ GC/g. about 5.0 x 10^lu GC/g, about 5.5 x 10¹⁰ GC/g. about 6.0 x 10^lu

GC/g, about 6.5 x 10¹⁰ GC/g. about 7.0 x 10^lu GC/g, about 7.5 x 10¹⁰ GC/g. about 8.0 x 10^lu

GC/g, about 8.5 x 10¹⁰ GC/g, about 9.0 x 10¹⁰ GC/g, about 9.5 x 10¹⁰ GC/g, about 1.0 x 10"

GC/g, about 1.5 x 10¹¹ GC/g, about 2.0 x 10¹¹ GC/g, about 2.5 x 10¹¹ GC/g, about 3.0 x 10¹¹

GC/g, about 3.5 x 10¹¹ GC/g, about 4.0 x 10¹¹ GC/g, about 4.5 x 10¹¹ GC/g, about 5.0 x 10¹¹

GC/g, about 5.5 x 10¹¹ GC/g, about 6.0 x 10¹¹ GC/g, about 6.5 x 10¹¹ GC/g, about 7.0 x 10¹¹

GC/g, about 7.5 x 10¹¹ GC/g. about 8.0 x 10¹¹ GC/g, about 8.5 x 10¹¹ GC/g. about 9.0 x 10¹¹

GC/g, about 9.5 x 10¹¹ GC/g. about 1.0 x 10¹² GC/g, about 1.5 x 10¹² GC/g. about 2.0 x 10¹²

GC/g, about 2.5 x 10¹² GC/g. about 3.0 x 10¹² GC/g, about 3.5 x 10¹² GC/g. about 4.0 x 10¹²

GC/g, about 4.5 x 10¹² GC/g, about 5.0 x 10¹² GC/g, about 5.5 x 10¹² GC/g, about 6.0 x 10¹²

GC/g, about 6.5 x 10¹² GC/g, about 7.0 x 10¹² GC/g, about 7.5 x 10¹² GC/g, about 8.0 x 10¹² GC/g, about 8.5 x 10¹² GC/g. about 9.0 x 10¹² GC/g, about 9.5 x 10¹² GC/g. about 1.0 x 10¹³

GC/g, about 1.5 x 10¹³ GC/g, about 2.0 x 10¹³ GC/g, about 2.5 x 10¹³ GC/g, about 3.0 x 10¹³

GC/g, about 3.5 x 10¹³ GC/g, about 4.0 x 10¹³ GC/g, about 4.5 x 10¹³ GC/g, about 5.0 x 10¹³

GC/g, about 5.5 x 10¹³ GC/g, about 6.0 x 10¹³ GC/g, about 6.5 x 10¹³ GC/g, about 7.0 x 10¹³

GC/g, about 7.5 x 10¹³ GC/g, about 8.0 x 10¹³ GC/g, about 8.5 x 10¹³ GC/g, about 9.0 x 10¹³

GC/g, about 9.5 x 10¹³ GC/g, or about 1.0 x 10¹⁴ GC/g brain mass.

In one embodiment, the pharmaceutical composition comprising a rAAV as described herein is administrable at a dose of about 1 x 10⁹ GC per gram of brain mass to about 1 x 10¹⁴ GC per gram of brain mass.

The aqueous suspension or pharmaceutical compositions described herein are designed for delivery to subjects in need thereof by any suitable route or a combination of different routes. In one embodiment, the pharmaceutical composition is formulated for delivery via intracerebroventricular (ICV), intrathecal (IT), or intracistemal injection. In one embodiment, the compositions described herein are designed for delivery to subjects in need thereof by intravenous injection. Alternatively, other routes of administration may be selected (e.g., oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intramuscular, and other parenteral routes). In certain embodiments, the pharmaceutical composition is delivered intrathecally, optionally via intra-cistema magna (ICM) injection. In certain embodiments, the composition is delivered via intraparenchymal administration. In certain embodiments, the composition is delivered via Ommaya Reservoir delivery system.

As used herein, the terms “intrathecal deliver}’’' or “intrathecal administration refer to a route of administration for drugs via an injection into the spinal canal, more specifically into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF). Intrathecal delivery may include lumbar puncture, intraventricular, suboccipital/intracistemal, and/or Cl -2 puncture. For example, material may be introduced for diffusion throughout the subarachnoid space by means of lumbar puncture. In another example, injection may be into the cisterna magna. Intracistemal delivery may increase vector diffusion and/or reduce toxicity’ and inflammation caused by tire administration. Sec, e.g., Christian Hinderer et al, Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cistema magna, Mol Ther Methods Clin Dev. 2014; 1: 14051. Published online 2014 Dec 10. doi:

10. 1038/mtm.2014.51. In certain embodiment, a rAAV, vector, or composition as described herein is administrated to a subject in need via the intrathecal administration. In certain embodiments, the intrathecal administration is performed as described in US Patent Publication No. 2018/0339065 Al, published November 29, 2019, which is incorporated herein by reference in its entirety. In certain embodiments, the CNS administration is performed using Ommaya Reservoir (also referred to as Ommaya device or Ommaya system).

As used herein, tire terms “intracistemal delivery” or “intracistemal administration” refer to a route of administration for drugs directly into the cerebrospinal fluid of the brain ventricles or within the cistema magna cerebellomedularis, more specifically via a suboccipital puncture or by direct injection into the cistema magna or via permanently positioned tube.

As used herein, tire term “intraparenchymal”, “dentate nucleus” or IDN refers to a route of administration of a composition directly into dentate nuclei. IDN allows for targeting of dentate nuclei and/or cerebellum. In certain embodiments, the IDN administration is performed using ClearPoint® Neuro Navigation System (MRI Interventions, Inc., Memphis, TN) and ventricular cannula, which allows for MRI-guided visualization and administration. Alternatively, other devices and methods may be selected.

It should be understood that the compositions in the pharmaceutical composition described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

6. Method of Treatment

In one aspect, provided herein is a method of treating a human subject diagnosed with MPS IIIB. In one aspect, provided herein is a method of treating a human subject having a hNAGLU associated disorder or having a disorder associated with defect in hNAGLU. Currently, when there is a clinical suspicion of MPS 111. the first step is the request of a quantitative test to detect the presence of GAGs in urine through spectrophotometric methods using dimethylmethylene blue (DMB). The DMB test is based on the union of GAGs to the dimethylmethylene blue and the quantification of the GAG- DMB complex with a spectrophotometer. The sensitivity of this test is 100%, with a specificity of 75-100%. A negative result when detecting GAGs in urine does not rule out the existence of MPS III due to the fact that in some patients with attenuated forms of the disease, the levels of GAGs excretion with healthy controls can overlap and the increased excretion of heparan sulfate in the MPS III can be ignored. The current gold standard technique for diagnosis is the determination of enzyme activity in cultured skin fibroblasts, leukocytes, plasma or serum. The specific diagnosis of MPS IIIB is confirmed by showing a decrease or absence of one of the NAGLU enzymatic activities involved in the degradation of heparan sulfate in the patient’s leukocytes or fibroblasts; the reduction should be less than 10% when compared to the activity in healthy individuals, with normalcy in other sulfatases. Because the disease due to deficiency in multiple sulfatases also shows a reduction in the activity of the heparan N-sulfatase , N-acetylglucosamine 6-sulfatase and other sulfatases, biochemical analysis of at least other sulfatase is required to confirm the diagnosis of MPS III and thus rule out multiple sulfatases deficiency. However, the method of diagnosis is not a limitation of the present invention and other suitable methods may be selected.

The method comprises administering to a subject a suspension of a vector as described herein. In one embodiment, the method comprises administering to a subject a suspension of a rAAV as described herein in a formulation buffer at a dose of about 1 x 10⁹ GO per gram of brain mass to about 1 x 10¹⁴ GC per gram of brain mass.

The composition(s) and method(s) provided achieve efficacy in treating a subject in need with MPS IIIB. Efficacy of the method in a subject can be shown by assessing (a) an increase in NAGLU enzymatic activity; (b) amelioration of a MPS IIIB symptom; (c) improvement of MPS IIIB-related biomarkers, e.g., GAG levels polyamine (e.g., spermine) levels in the cerebrospinal fluid (CSF), serum, urine and/or other biological samples; or (e) facilitation of any treatment(s) for MPS IIIB. In certain embodiments, efficacy may be detennined by monitoring cognitive improvement and/or anxiety correction, gait and/or mobility improvement, reduction in tremor frequency and/or severity, reduction in clasping/spasms. improvements in posture, improvements in corneal opacity. Examples of suitable scoring, which is hereby incorporated in this section. Additionally or alternatively, efficacy of the method may be predicted based on an animal model. One example of a suitable murine model is described in Example 1. In another embodiment, a multiparameter grading scale was developed to evaluate disease correction and response to the MPSIIIA vector therapy described herein in an animal model. Animals are assigned a score based on an assessment of a combination of tremor, posture, fur quality, clasping, corneal clouding, and gait/mobility. In certain embodiments, any combination of one or more of these factors may be used to demonstrate efficacy, alone, or in combination with other factors. See, Burkholder et al. Curr Protoc Mouse Biol. June 2012, 2: 145-65; Tumpey et al. J Virol. May 1998, 3705-10: and Guyenet et al. J Vis Exp. May 2010, 39; 1787). Cognitive improvement and anxiety correction of treated animals is evaluated by assessing movement in an open field (i.e. beam break measurement as described, e.g., in Tatem et al. J Vis Exp, 2014, (91):51785) and the elevated plus maze assay (as described, e.g., in Waif and Frye, Nat Protoc, 2007, 2(2): 322-328).

As used herein, “facilitation of any treatment(s) for MPS IIIB” or any grammatical variant thereof, refers to a decreased dosage or a lower frequency of a treatment of MPS IIIB in a subject other than the composition(s) or method(s) which is/are firstly disclosed in the invention, compared to that of a standard treatment without administration of the described composition(s) and use of the described method(s).

Examples of suitable treatment facilitated by the composition(s) or method(s) described herein might include, but not limited to,

(a) medications used to relieve symptoms (such as seizures and sleep disturbances) and improve quality of life:

(b) hematopoietic stem cell transplantation, such as bone marrow transplantation or umbilical cord blood transplantation (see, e.g., Vellodi A. Young E, New M, Pot-Mees C, Hugh-Jones K. Bone marrow transplantation for Sanfilippo disease type B. J Inherit Metab Dis. 1992; 15 : 91 1-8; Garbuzova-Davis, S, Willing, AE, Desjarlais, T, et al. Transplantation of human umbilical cord blood cells benefits an animal model of Sanfilippo syndrome type B. Stem Cells Dev. 2005; 14:384-394; and Garbuzova-Davis, S, Klasko, SK, and Sanberg, PR. Intravenous administration of human umbilical cord blood cells in an animal model of MPS III B. J Comp Neurol. 2009; 515:93-101.);

(c) enzyme replacement therapies (ERT) (e.g., via intravenous administration or intracerebroventricular infusion, see. e.g., Aoyagi-Scharber M et al, Clearance of Heparan Sulfate and Attenuation of CNS Pathology by Intracerebroventricular BMN 250 (NAGLU- IGF2) in Sanfilippo Type B Mice, Mol Ther Methods Clin Dev. 2017 Jim 6:6:43-53. doi:

10. 1016/j.omtm.2017.05.009. eCollection 2017 Sep 15; and Alexion Pharmaceuticals. Safety, Phannacokinctics, and Pharmacodynamics/Efficacy of SBC- 103 in MPS IIIB. In: ClinicalTrialsgov [Internet]. Bethesda: National Library of Medicine (US). 2000, Available from: clinicaltrials.gov/show/NCT02324049. NLM identifier: NCT02324049.);

(d) substrate reduction therapy (e.g., treatment with genistein. Delgadillo V et al. Genistein supplementation in patients affected by Sanfilippo disease. J Inherit Metab Dis. 201 1 Oct;34(5): 1039-44. doi: 10. 1007/s 10545-01 1-9342-4. Epub 201 1 May 10; Piotrowska E et al. Two-year follow-up of Sanfilippo Disease patients treated with a geni stein-rich isoflavone extract: assessment of effects on cognitive functions and general status of patients. Med Sci Monit. 201 1 Apr; 17(4):CR 196-202; and Piotrowska, E et al. Genistin-rich soy isoflavone extract in substrate reduction therapy for Sanfilippo syndrome: an open label, pilot study in 10 pediatric patients. Curr. Ther. Res. Clin. Exp. 2008;69: 166-179);

(e) chaperone therapy (see, IGF2 in Kan SH, Troitskaya LA, Sinow CS, Haitz K, Todd AK. Di Stefano A, et al. Insulin-like growth factor II peptide fusion enables uptake and lysosomal delivery of alpha-N-acctylghicosaminidasc to mucopolysaccharidosis type IIIB fibroblasts. Biochem J. 2014;458:281-9; HIRMAb in Boado RJ, Lu JZ, Hui EK, Lin H, Pardridge WM. Insulin Receptor Antibody alpha-N-Acetylglucosaminidase Fusion Protein Penetrates the Primate Blood-Brain Barrier and Reduces Glycosoaminoglycans in Sanfilippo Type B Fibroblasts. Mol Phann. 2016; 13: 1385-92; CpGH89 inhibitor in Ficko- Blean, E, Stubbs, KA, Nemirovsky, O, et al. Structural and mechanistic insight into tire basis of mucopolysaccharidosis IIIB. Proc Natl Acad Sci U S A. 2008; 105:6560- 6565; and Zhao, KW and Neufeld, EF. Purification and characterization of recombinant human alpha-N- acetylglucosaminidase secreted by Chinese hamster ovary cells. Protein Expr Purif. 2000; 19:202-211); and

(f) any combination thereof.

In one embodiment, the described method results in the subject demonstrating an improvement of biomarkers related to MPS IIIB.

An “increase in NAGLU enzymatic activity ” is used interchangeably with tire term “increase in desired NAGLU function”, and refers to a NAGLU activity at least about 5%, 10%, 15%. 20%. about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%. about 70%. about 75%. about 80%. about 85%. about 90%, about 95%, or about 100% of the NAGLU enzyme range for a healthy patient. The NAGLU enzymatic activity might be measured by an assay as described herein. In one embodiment, the NAGLU enzy matic activity' might be measured in the serum, plasma, blood, urine, CSF, or another biological sample. In one embodiment, administration of the composition as described herein, or use of tire method as described herein, result in an increase in NAGLU enzymatic activity in serum, plasma, saliva, urine or other biological samples. Alternatively. CSF GAG levels and other CSF biomarkers such as spermine levels may be measured to determine therapeutic effect. See. e.g., WO 2017/136533. Neurocognition can be determined by conventional methods, See. e.g., WO 2017/136500 Al, which is hereby incorporated by reference in its entirety. Prevention of neurocognitive decline refers to a slowdown of a neurocognitive decline of the subject administered with the composition described herein or received the method described herein by at least about 5%, at least about 20%, at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%. about 90%. about 95%, or about 100% compared to that of a MPS IIIB patient.

As used herein, the terms “biomarker” or "MPS UlB-related biomarker’’ refer to presence, concentration, expression level or activity of a biological or chemical molecular in a biological sample of a subject which correlates to progression or development of MPS IIIB in a positive or negative matter. In one embodiment, the biomarker is GAG levels in the cerebrospinal fluid (CSF), serum, urine, skin fibroblasts, leukocytes, plasma, or any other biological samples. In another embodiment, the biomarker is assessed using clinical chemistry. In yet another embodiment, the biomarker is liver or spleen volumes. In one embodiment, the biomarker is the activity’ of the heparan N-sulfatase. N-acetylglucosamine 6-sulfatase and other sulfatases. In another embodiment, the biomarker is spermine level in CSF, serum, or another biological sample. In yet another embodiment, the biomarker is lysosomal enzy me activity in serum, CSF, or another biological sample. In one embodiment, the biomarker is assessed via magnetic resonance imaging (MRI) of brain. In another embodiment, the biomarker is a neurocognitive score measured by a neurocognitive developmental test. The phrase “improvement of biomarker” as used herein means a reduction in a biomarker positively correlating to the progression of the disease, or an increase in a biomarker negatively correlating to the progression of the disease, wherein the reduction or increase is at least about 5%, at least about 20%, at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% compared to that before administration of tire composition as described herein or use of the method as described herein.

In one embodiment, the method further comprises detecting or monitoring biomarkers related to MPS IIIB in the subject prior to initiation of therapy with therapy provided herein. In one aspect, the method comprises detection of a biomarker which is a polyamine (such as spermine) in a sample from a subject (see WO/2017/136533, which is incorporated herein by reference). Thus, in one embodiment, the method comprises detecting spermine in a patient sample for purposes of diagnosing a patient with MPSIIIB. In another embodiment, spermine concentration levels in a patient sample are detected to monitor the effectiveness of a treatment for MPSIIIB using the vector as described herein. Currently, patients with MPSIIIB are not considered candidates for bone marrow transplantation (BMT), Substrate Reduction Therapy (SRT) or enzyme replacement therapy (ERT).

However, in certain embodiments, a gene therapy patient treated with a vector expressing the NAGLU described herein has, at a minimum, sufficient enzyme expression levels that any sub-normal range enzyme levels can be treated with ERT or SRT. Such ERT may be a cotherapy in which the dose of the ERT is monitored and modulated for months or years postvector dosing. Additionally or alternatively, a SRT may be a co-therapy in which the dose of the SRT is monitored and modulated for months or years post-vector dosing. Additionally or alternatively, a chaperone therapy may be a co-therapy in which the dose of the chaperone therapy is monitored and modulated for months or years post-vector dosing.

Thus, in one embodiment, the suspension is suitable for co-administering with a functional hNAGLU protein or a recombinant protein comprising a functional NAGLU. In one embodiment, tire recombinant protein is a NAGLU fused with insulin-like growth factor 2 (IGF2).

In one embodiment, the suspension is delivered into the subject in need intracerebroventricularly, intrathecally, intracistemaly or intravenously.

In one embodiment, the suspension has a pH of about 6 to about 8.

As used herein, an enzyme replacement therapy (ERT) is a medical treatment that consists in replacing an enzyme in patients where a particular enzyme is deficient or absent. The enzyme is usually produced as a recombinant protein and administrated to the patient. In one embodiment, the enzyme is a functional NAGLU. In another embodiment, the enzyme is a recombinant protein comprising a functional NAGLU. In one embodiment, the enzyme is a recombinant protein comprising a functional NAGLU and an insulin-like growth factor 2 (IGF2).Aoyagi-Scharbcr M ct al, Clearance of Heparan Sulfate and Attenuation of CNS Pathology by Intracerebroventricular BMN 250 in Sanfilippo Type B Mice, Mol Ther Methods Clin Dev. 2017 Jun 6:6:43-53. doi: 10. 1016/j.omtm.2017.05.009. eCollection 2017 Sep 15; and WO2017132675A1. Systemic, intrathecal, intracerebroventricular or intracistemal delivery can be used for ERT or SRT co-therapy. As used herein, a Substrate Reduction Therapy (SRT) refers to a therapy using a small molecule drug to partially inhibit the biosynthesis of the compounds, which accumulate in the absence of NAGLU. In one embodiment, the SRT is a therapy via genistein. See, e.g., Ritva Tikkanen et al, Less Is More: Substrate Reduction Therapy for Lysosomal Storage Disorders. Int J Mol Sci. 2016 Jul; 17(7): 1065. Published online 2016 Jul 4. doi: 10.3390/ijmsl7071065; Delgadillo V et al, Epub 2011 May 10; and de Ruijter J et al, Genistein in Sanfilippo disease: a randomized controlled crossover trial. Ann Neurol. 2012 Jan;71(l): 110-20. doi: 10. 1002/ana.22643. NAGLU.

As used herein, a chaperone therapy refers to a therapy using a small molecule drug to helps folding and/or secretion of NAGLUE. In one embodiment, the chaperone therapy is a therapy via IGF2. See, e.g., Kan SH, Troitskaya LA, Sinow CS, Haitz K, Todd AK, Di Stefano A, et al. Insulin-like growth factor II peptide fusion enables uptake and lysosomal delivery of alpha-N-acetylglucosaminidase to mucopolysaccharidosis type IIIB fibroblasts. Biochem J. 2014;458:281-9; and HIRMAb in Boado RJ. Lu JZ, Hui EK, Lin H, Pardridge WM. Insulin Receptor Antibody alpha-N-Acetylglucosaminidase Fusion Protein Penetrates the Primate Blood-Brain Barrier and Reduces Glycosoaminoglycans in Sanfilippo Type B Fibroblasts. Mol Pharm. 2016;13: 1385-92. In another embodiment, the chaperone therapy is a therapy via CpGH89 inhibitor. See, e.g., Ficko-Blean, E, Stubbs, KA, Nemirovsky, O, et al. Structural and mechanistic insight into the basis of mucopolysaccharidosis IIIB. Proc Natl Acad Sci U S A. 2008; 105:6560- 6565. In yet another embodiment, the chaperone therapy is a therapy disclosed in Zhao, KW and Neufeld, EF. Purification and characterization of recombinant human alpha-N-acetylglucosaminidase secreted by Chinese hamster ovary cells. Protein Expr Purif. 2000; 19:202-211.

Suitable volumes for delivery of these doses and concentrations may be determined by one of skill in the art. For example, volumes of about 1 pL to 150 mL may be selected, with the higher volumes being selected for adults. Typically, for newborn infants a suitable volume is about 0.5 mL to about 10 mL, for older infants, about 0.5 mL to about 15 mL may be selected. For toddlers, a volume of about 0.5 mL to about 20 mL may be selected. For children, volumes of up to about 30 mL may be selected. For pre-teens and teens, volumes up to about 50 mL may be selected. In still other embodiments, a patient may receive an intrathecal administration in a volume of about 5 mL to about 15 mL are selected, or about 7.5 mL to about 10 mL. Other suitable volumes and dosages may be determined. The dosage will be adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed.

In one embodiment, the rAAV as described herein is administrable at a dose of about 1 x 10⁹ GC per gram of brain mass to about 1 x 10¹⁴ GC per gram of brain mass. In certain embodiments, the rAAV is co-administered systemically at a dose of about 1 x 10⁹ GC per kg body weight to about 1 x 10¹³ GC per kg body weight.

In one embodiment, the subject is delivered a therapeutically effective amount of the vectors described herein. As used herein, a “therapeutically effective amount’⁷ refers to the amount of the composition comprising the nucleic acid sequence encoding a functional NAGLU which delivers and expresses in the target cells an amount of enzyme sufficient to achieve efficacy. In one embodiment, the dosage of the vector is about 1 x 10⁹ GC per gram of brain mass to about 1 x 10¹³ genome copies (GC) per gram (g) of brain mass, including all integers or fractional amounts within tire range and the endpoints. In another embodiment, the dosage is 1 x 10'° GC per gram of brain mass to about 1 x 10¹³ GC per gram of brain mass. In specific embodiments, the dose of the vector administered to a patient is at least about 1.0 x 10⁹ GC/g, about 1.5 x 10⁹ GC/g, about 2.0 x 10⁹ GC/g, about 2.5 x 10⁹ GC/g, about 3.0 x 10⁹ GC/g, about 3.5 x 10⁹ GC/g, about 4.0 x 10⁹ GC/g, about 4.5 x 10⁹ GC/g, about 5.0 x 10⁹ GC/g, about 5.5 x 10⁹ GC/g, about 6.0 x 10⁹ GC/g, about 6.5 x 10⁹ GC/g, about 7.0 x 10⁹ GC/g, about 7.5 x 10⁹ GC/g, about 8.0 x 10⁹ GC/g, about 8.5 x 10⁹ GC/g, about 9.0 x 10⁹ GC/g, about 9.5 x 10⁹ GC/g, about 1.0 x 10¹⁰ GC/g, about 1.5 x 10^1C1 GC/g, about 2.0 x 10¹⁰ GC/g, about 2.5 x 10¹⁰ GC/g, about 3.0 x 10¹⁰ GC/g, about 3.5 x 10¹⁰ GC/g, about 4.0 x IO¹⁰ GC/g, about 4.5 x 10¹⁰ GC/g, about 5.0 x IO¹⁰ GC/g, about 5.5 x 10¹⁰ GC/g, about 6.0 x 10¹⁰ GC/g, about 6.5 x 10¹⁰ GC/g, about 7.0 x 10¹⁰ GC/g, about 7.5 x 10¹⁰ GC/g, about 8.0 x IO¹⁰ GC/g. about 8.5 x 10¹⁰ GC/g, about 9.0 x IO¹⁰ GC/g, about 9.5 x 10¹⁰ GC/g, about 1.0 x 10¹¹ GC/g, about 1.5 x 10¹¹ GC/g, about 2.0 x 10¹¹ GC/g, about 2.5 x 10¹¹ GC/g, about 3.0 x 10¹¹ GC/g, about 3.5 x 10¹¹ GC/g, about 4.0 x 10¹¹ GC/g, about 4.5 x 10¹¹ GC/g, about 5.0 x 10” GC/g, about 5.5 x 10¹¹ GC/g, about 6.0 x 10” GC/g, about 6.5 x 10¹¹ GC/g, about 7.0 x 10¹¹ GC/g, about 7.5 x 10” GC/g, about 8.0 x 10¹¹ GC/g, about 8.5 x 10” GC/g, about 9.0 x 10” GC/g, about 9.5 x 10” GC/g, about 1.0 x 10¹² GC/g, about 1.5 x 10¹² GC/g, about 2.0 x 10¹² GC/g, about 2.5 x 10¹² GC/g, about 3.0 x 10¹² GC/g, about 3.5 x 10¹² GC/g, about 4.0 x 10¹² GC/g, about 4.5 x 10¹² GC/g, about 5.0 x 10¹² GC/g, about 5.5 x 10¹² GC/g, about 6.0 x 10¹² GC/g, about 6.5 x 10¹² GC/g, about 7.0 x 10¹² GC/g, about 7.5 x 10¹² GC/g, about 8.0 x 10¹² GC/g, about 8.5 x 10¹² GC/g, about 9.0 x 10¹² GC/g, about 9.5 x 10¹² GC/g, about 1.0 x 10¹³ GC/g, about 1.5 x 10¹³ GC/g, about 2.0 x 10¹³ GC/g, about 2.5 x 10¹³ GC/g, about 3.0 x 10¹³ GC/g, about 3.5 x 10¹³ GC/g, about 4.0 x 10¹³ GC/g, about 4.5 x 10¹³ GC/g, about 5.0 x 10¹³ GC/g, about 5.5 x 10¹³ GC/g, about 6.0 x 10¹³ GC/g, about 6.5 x 10¹³ GC/g, about 7.0 x 10¹³ GC/g, about 7.5 x 10¹³ GC/g, about 8.0 x 10¹³ GC/g, about 8.5 x 10¹³ GC/g, about 9.0 x 10¹³ GC/g, about 9.5 x 10¹³ GC/g, or about 1.0 x 10¹⁴ GC/g brain mass.

Prior to treatment, the MPS IIIB patient can be assessed for neutralizing antibodies (Nab) to the AAV serotype used to deliver the hNAGLU gene. Such Nabs can interfere with transduction efficiency and reduce therapeutic efficacy. In one embodiment, the method further comprises the subject receives an immunosuppressive co-therapy. Without wishing to be bound by theory, immune suppression co-therapy does one or more of the following: induces anergy or immunologic tolerance to the rAAV and/or Transgene; blocks an immune response to optimize efficacy; minimize de novo immune response against transgene; minimize impact of pre-existing immune response to transgene: minimize impact of pre-existing immune response to AAV; prevent immune medicated toxicity: minimize destruction of TG expressing cells; reduce axonopathy/DRG neurotoxicity in NHPs.

In certain embodiments, the method further comprises a combination therapy, such as transient co-treatment with an immunosuppressant before and/or during treatment with rAAV. Optionally, immunosuppressive co-therapy may be used as a precautionary measure without prior assessment of neutralizing antibodies to the AAV vector capsid and/or other components of the formulation. Prior immunosuppression therapy may be desirable to prevent potential adverse immune reaction to tire hNAGLU transgene product i.e., where the transgene product may be seen as “foreign.” Results of non-clinical studies in NHPs described infra are consistent with the development of an immune response to hNAGLU (See. Example 6). While a similar reaction may not occur in human subjects, as a precaution immunosuppression therapy is recommended or may be used for all recipients of rAAV comprising vector genome encoding hNAGLU.

Immunosuppressants for such co-therapy include, but are not limited to, a glucocorticoid, steroids or corticosteroids, antimetabolites, T-cell inhibitors, a macrolide (c.g., a rapamycin or rapalog), and cytostatic agents including an alkylating agent, an antimetabolite. a cytotoxic antibiotic, an antibody, or an agent active on immunophilin. The immune suppressant may include a nitrogen mustard, nitrosourea, platinum compound, methotrexate, azathioprine. mycophenolate mofetil , methotrexate, leflunomide (Arava), cyclophosphamide, chlorambucil (Leukeran), a chloroquine (e.g., hydroxychloroquine), quinine sulfate, mefloquine, a combination of atovaquone and proguanil, sulfasalazine, mercaptopurine, fluorouracil, dactinomycin, an anthracycline, mitomycin C, bleomycin, mithramycin, IL-2 receptor- (CD25-) or CD3-directed antibodies, anti-IL-2 antibodies, abatacept (Orencia), adalimumab (Humira), anakinra (Kineret), certolizumab (Cimzia), etanercept (Enbrel), golimumab (Simponi), infliximab (Remicade), rituximab (Rituxan), tocilizumab (Actemra) and tofacitinib (Xeljanz), cyclosporine, tacrolimus, sirolimus, IFN-0, IFN-y. an opioid, or TNF-a (tumor necrosis factor-alpha) binding agent, and combinations of these drugs .

In certain embodiments, the immunosuppressive therapy may be started 0, 1, 2, 7, or more days prior to the gene therapy administration. Such therapy may involve coadministration of two or more drugs, the (e.g., prednelisone, micophenolate mofetil (MMF) and/or sirolimus (i.e., rapamycin)) on the same day. One or more of these drugs may be continued after gene therapy administration, at the same dose or an adjusted dose. Such therapy may be for about 1 week (7 days), about 60 days, or longer, as needed. In one embodiment, the two or more drugs may be, e.g., one or more corticosteroids (e.g., a prednelisone or prednisone) and optionally, MMF and/or a calcinuerin inhibitor (e.g., tacrolimus or sirolimus (i.e., rapamycin)). In one embodiment, the two or more drugs are micophenolate mofetil (MMF) and/or sirolimus. In another embodiment, the two or more drugs may be, e.g., methylprednisolone, prednisone, tacrolimus, and/or sirolimus. In certain embodiments, the drugs are MMF and tacrolimus for 0 to 15 days pre-vector delivery and maintaining for about 8 weeks with MMF and/or throughout follow-up appointments with tacrolimus. One or more of these drugs may be continued after gene therapy administration, at the same dose or an adjusted dose. In certain embodiments, patients are dosed initially with an IV steroid (e.g., methylprednisolone) to load the dose, followed by with an oral steroid (e.g., prednisolone) that is gradually tapered down so that the patient is off steroids by week 12. The corticosteroid treatment is supplemented by tacrolimus (for 24 weeks) and/or sirolimus (for 12 weeks), and can be further supplemented with MMF. When using both tacrolimus and sirolimus, the dose of each should be a low dose adjusted to maintain a blood trough level of about 4 ng/mL to about 8 ng/ml, or a total of about 8 ng/mL to about 16 ng/mL. In certain embodiments, when only one of these agents is used, the total dose for tacrolimus and/or sirolimus may be in the range of about 16 ng/mL to about 24 ng/mL. If only one of the agents is used, the label dose (higher dose) should be employed: e.g., tacrolimus at 0. 15-0.20 mg/kg/day given as two divided doses every 12 hours; and sirolimus at 1 mg/m2/day; the loading dose should be 3 mg/m2. If MMF is added to the regimen, the dose for tacrolimus and/or sirolimus can be maintained since the mechanisms of action differ. These and other therapies may be started at about day - 14 to day -1 (e.g., day -2, day 0, etc.), and continue to about to up to about 1 week (7 days), or up to about 60 days, or up to about 12 weeks, or up to about 16 weeks, or up to about 24 weeks, or up to about 48 weeks, or longer, as needed. In certain embodiments, a tacrolimus-free regimen is selected.

In certain embodiments, patients will receive immune suppression (IS) as follows: corticosteroids: methylprednisolone 10 mg/kg IV once on Day 1 predose and oral prednisone starting at 0.5 mg/kg/day on Day 2 with gradual tapering and discontinuation by Week 12; Tacrolimus: 1 mg BID by mouth Day 2 to Week 24 with tapering over 8 weeks between Week 24 and 32; Sirolimus: (a loading dose on Day -2 and then sirolimus 0.5 mg/m2/day divided in BID dosing until Week 48. In certain embodiments, IS therapy is discontinued at Week 48 post dosing with the rAAV.

In certain embodiments, the method further comprises administering subject with intramuscular steroid or corticosteroid prior to and/or post administration of rAAV. In certain embodiments, the method further comprises administering subject with oral steroid or corticosteroid prior to and/or post administration with rAAV.

In certain embodiments, the immunosuppressive therapy regimen is as follows: Corticosteroids:

In the morning of vector administration (Day 1 predose), patients receive methylprednisolone lOmg/kg IV (maximum of 500 mg) over at least 30 minutes. The methylprednisolone is administered before the lumbar puncture and intrathecal (1C) injection of rAAV. Premedication with acetaminophen and an antihistamine is optional.

On Day 2, oral prednisone is started with the goal to discontinue prednisone by Week 12. The dose of prednisone is as follows: Day 2 to the end of Week 2: 0.5 mg/kg/day. Week 3 and 4: 0.35 mg/kg/day. Week 5-8: 0.2 mg/kg/day. Week 9-12: 0.1 mg/kg.

Prednisone is discontinued after Week 12. The exact dose of prednisone can be adjusted to tire next higher clinically practical dose.

Sirolimus: 2 days prior to vector administration (Day -2): a loading dose of sirolimus 1 mg/m2 every 4 hours x 3 doses is administered. From Day -1 : sirolimus 0.5 mg/m2/day divided in twice a day dosing with target blood level of 4-8 ng/ml. Sirolimus is discontinued after the Week 48 visit.

Tacrolimus: Tacrolimus is started on Day 2 (the day following rAAV) at a dose of 1 mg twice daily and adjusted to achieve a blood level 4-8 ng/mL for 24 Weeks. Starting at Week 24 visit, tacrolimus is tapered off over 8 weeks. At week 24 the dose is decreased by approximately 50%. At Week 28 the dose is further decreased by approximately 50%. Tacrolimus is discontinued at Week 32.

In one embodiment, the method further comprises administering to a subject anti- AAV neutralizing antibodies (NAb) to reduce peripheral transduction, and mitigate the potential risk of NAGLU-induced toxicity. In certain embodiments, the method further comprises detect the presence of systemic AAV NAb prior to treating with anti-AAV NAb, wherein patients with levels of anti-AAV NAb in excess of a predetermined level against the rAAV capsid (or a sero-crossreactive capsid) do not require pretreatment. Such levels may be, e.g., in excess of about 1:10, about 1:20, about 1:50, about 1: 100, about 1:250, or higher or lower levels. In certain embodiments, the method further comprises intravenously administering human anti-AAV polyclonal antibodies (e.g.. plasma-derived, pooled human immunoglobulin (I VIG)), an anti-AAV monoclonal antibody, or a cocktail of anti-AAV antibodies, to a patient about 1 day to about 2 hours before treatment with a rAAV as described herein.

In certain embodiments, a combination regimen is provided for preventing off-target delivery rAAV, the regimen comprising (a) pretreating the patient by systemically administering a composition comprising anti-AAV capsid neutralizing antibodies directed against an AAV capsid in a recombinant AAV vector, and (b) administering to the central nervous system (CNS) rAAV as described herein (e.g.. rAAV). See also, US Provisional Patent Application No. 63/328,227, filed April 6, 2022, and International Patent Application No. PCT/US2023/065422, filed April 6, 2023, now Publication No. WO2023/196892A1 which are incorporated herein by reference in their entirety.

As used herein, tire term “NAb titer” a measurement of how much neutralizing antibody (e.g., anti-AAV Nab) is produced which neutralizes the physiologic effect of its targeted epitope (e.g., an AAV). Anti-AAV NAb titers may be measured as described in, e.g., Calcedo, R.. et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno- Associated Viruses. Journal of Infectious Diseases, 2009. 199(3): p. 381-390, which is incorporated by reference herein. In certain embodiments, a combination regimen is provided including an anti-IgG enzymes, which have been described as being useful for depleting anti- AAV antibodies (and thus may permit administration to patients testing above a threshold level of antibody for the selected AAV capsid), and/or delivery of anti-FcRN antibodies and/or one or more of a) a steroid or combination of steroids and/or (b) an IgG-cleaving enzyme, (c) an inhibitor of Fc- IgE binding; (d) an inhibitor of Fc-IgM binding; (e) an inhibitor of Fc-IgA binding; and/or (I) gamma interferon. anti-FcRN antibodies include, e.g., rozanolixizumab (UCB7665) (UCB SA); IMVT-1401, RVT-1401 (HL161), HBM9161 (all form HanAll BioPhrma Co. Ltd). Nipocalimab (M281) (Momenta Pharmaceuticals Inc), ARGX-113 (efgartigimod) (Argenx S.E.), orilanolimab (ALXN 1830, SYNT001, Alexion Pharmaceuticals Inc), SYNT002, ABY-039 (Affibody AB), or DX-2507 (Takeda Pharmaceutical Co. Ltd). In certain embodiments, a combinations of anti-FcRN antibodies is administered. In certain embodiments, an anti-FcRN antibody is administered in combination with a suitable anti- FcRn ligand (i.e., a peptide or protein construct binding human FcRn so as to inhibit IgG binding).

In one embodiment, a combination regimen for treating a patient with MPSIIIB is provided, wherein the regiment includes administering a vector describe herein in combination with a ligand which inhibits binding of human FcRn and pre-existing patient neutralizing antibodies (e.g., IgG). In certain embodiments, the patient may be naive to any therapeutic treatment with a vector and may have pre-existing immunity due to prior infections with a wild-type virus. In other embodiments, the patient may have neutralizing antibodies as a result of a prior treatment or vaccination. In certain embodiments, the patient may have neutralizing antibodies 1: 1 to 1:20. or in excess of 1:2, in excess of 1:5. in excess of 1: 10, in excess of 1:20, in excess of 1:50, in excess of 1: 100. in excess of 1:200, in excess of 1 :300 or higher. In certain embodiments, a patient has neutralizing antibodies in the range of 1: 1 to 1:200, or 1:5 to 1: 100, or 1:2 to 1: 20, or 1:5 to 1: 50, or 1:5 to 1 :20. In certain embodiments, a patient receives a single anti-FcRn ligand (e.g., anti-FcRn antibody) as the sole agent to modulate FcRn-IgG binding and to permit effective vector delivery. In other embodiments, a patient may receive a combination of one or more anti-FcRn ligands and a second component (e.g., an Fc receptor down-regulator (e.g., interferon gamma), an IgG enzyme, or another suitable component). Such combinations may be particularly desirable for patients having particularly high neutralizing antibody levels (e.g., in excess of 1:200). In certain embodiments, an anti-FcRn ligand(s) (e.g., antibodies) is administered to a patient having neutralizing antibodies prior to and, optionally, concurrently with a selected viral vector. In certain embodiments, continued expression of an anti-FcRn ligand post administration of tire gene therapy vector may desired on a short-term (transient basis), e.g., until such time as the viral vector clears from tire patient. In certain embodiments, persistent expression of an anti-FcRn ligand may be desired. Optionally, in this embodiment, the ligand may be delivered via a viral vector, including, e.g., in the viral vector expressing the therapeutic transgene. However, this embodiment is not desirable where the therapeutic gene being delivered is an antibody or antibody construct or another construct comprising an IgG chain. In such embodiments, where an antibody construct having an IgG chain is being delivered via a viral vector to a patient having pre-existing immunity, the anti-FcRn ligand is delivered or dosed transiently so that the amount of anti-FcRn ligand in the circulation is cleared from tire sera before effective levels of vector-mediated transgene product are expressed.

In certain embodiments, the FcRn ligand is delivered one to seven days prior to administration of the vector (e.g.. rAAV). In certain embodiments, the FcRn ligand is delivered daily. In certain embodiments, the FcRn ligand (e.g., immunoglobulin construct(s)) is delivered on the same day as the vector is administered. In certain embodiments, the FcRn ligand (e.g., immunoglobulin construct(s)) is delivered at least one day to four weeks post- rAAV administration. In certain embodiments, the ligand is delivered for four weeks to six months post-rAAV administration. In certain embodiments, tire ligand is dosed via a different route of administration than the rAAV. In certain embodiments, the ligand is dosed orally, intravenously, or intraperitoneally. See also. International Patent Application No. PCT/US2021/037575, filed June 16, 2021, and now published WO 2021/257668 Al, which is incorporated herein by reference in its entirety.

In certain embodiment, the method comprises measurement of serum anti-hNAGLU antibodies. Suitable assays of measuring anti-hNAGLU antibody are available, See, e.g., Example 1.

In one embodiment, the rAAV as described herein is administrated once to the subject in need. In another embodiment, the rAAV is administrated more than once to the subject in need. It should be understood that tire compositions in the method described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

7. Kit

In certain embodiments, a kit is provided which includes a concentrated vector suspended in a formulation (optionally frozen), optional dilution buffer, and devices and components required for intrathecal, intracerebroventricular or intracistemal administration. In another embodiment, the kit may additional or alternatively include components for intravenous delivery. In one embodiment, the kit provides sufficient buffer to allow for injection. Such buffer may allow for about a 1: 1 to a 1:5 dilution of the concentrated vector, or more. In other embodiments, higher or lower amounts of buffer or sterile water are included to allow for dose titration and other adjustments by the treating clinician. In still other embodiments, one or more components of the device arc included in the kit. Suitable dilution buffer is available, such as, a saline, a phosphate buffered saline (PBS) or a glycerol/PBS.

It should be understood that tire compositions in kit described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

8. Device

In one aspect, the vectors provided herein may be administered intrathecally via tire method and/or the device described, e.g., in WO 2017/136500, which is incorporated herein by reference in its entirety. Alternatively, other devices and methods may be selected. In summary, the method comprises the steps of advancing a spinal needle into the cistema magna of a patient, connecting a length of flexible tubing to a proximal hub of the spinal needle and an output port of a valve to a proximal end of the flexible tubing, and after said advancing and connecting steps and after permitting the tubing to be self-primed with the patient’s cerebrospinal fluid, connecting a first vessel containing an amount of isotonic solution to a flush inlet port of the valve and thereafter connecting a second vessel containing an amount of a pharmaceutical composition to a vector inlet port of the valve. After connecting the first and second vessels to the valve, a path for fluid flow is opened between the vector inlet port and the outlet port of the valve and the pharmaceutical composition is injected into the patient through the spinal needle, and after injecting the pharmaceutical composition, a path for fluid flow is opened through the flush inlet port and the outlet port of the valve and tire isotonic solution is injected into tire spinal needle to flush the pharmaceutical composition into the patient. This method and this device may each optionally be used for intrathecal delivery of the compositions provided herein. Alternatively, other methods and devices may be used for such intrathecal delivery.

It should be understood that tire compositions in the device described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

Examples

The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these examples but rather should be construed to encompass any and all variations drat become evident as a result of the teaching provided herein.

Example 1 : Methods

A. Vector Genome Design

A comparison of different engineered hNAGLU coding sequences was performed in WT C57BL6 mice following IV administration. Mice are administered with rAAV having an AAVhu68 capsid and comprising vector genome which comprises various hNAGLU coding sequences, including the wild-type sequences encoding the native (reference) protein (hNAGLU) and an R737G missense variant. These amino acid sequences compared include: hNAGLUcoVl (SEQ ID NO: 21); hNAGLUcoVl-R737G (SEQ ID NO: 22); hNAGLUcoV2 (SEQ ID NO: 23); hNAGLUcoV2-R737G (SEQ ID NO: 24); hNAGLUcoV3 (SEQ ID NO: 2); and hNAGLUcoV3-R737G (SEQ ID NO: 25). The sequence variants tested are administered at a dose of IxlO¹⁰ GC and IxlO¹¹ GC. Control mice are administered PBS. NAGLU activity is measured in the liver and plasma.

The injection is performed on Day 0 of the study. Plasma samples are collected on Day 7. Necropsy is performed, during which liver tissue is collected. The plasma samples from day 7 post injection are collected and stored at -80 °C. Necropsy samples are collected and stored at -80 °C. The stored samples, including plasma and liver tissue, are analyzed for NAGLUE enzyme activity. Additionally, samples of liver tissue are fixed in formalin and transferred to pathology.

The engineered sequence achieving the highest expression of the transgene based on enzyme activity readout is selected for further studies. As illustrated in FIG. 1A - FIG. 1C. co-variant 3. lacking the R737G mutation (AAVhu68.hNAGLUcoV3). demonstrates statistically significant higher transgene expression based on enzyme activity than the other co-variants, when administered at a dose of IxlO¹¹ GC. Co-variant 1, lacking the R737G mutation (AAVhu68.hNAGLUcoVl) also demonstrates higher levels of transgene expression when administered at a dose of 1x10¹¹ GC, as compared to the other variants; although not as great as co-variant 3. Co-variant 2 produces minimal to no transgene expression at either dose, without or without the R737G mutation. Immunohistochemical microscopy analysis is performed of liver tissue collected from mice administered with AAVhu68.hNAGLUcoV3 at a dose of 1 x 10¹¹ GC, at a dose of 1 x 10¹⁰ GC or treated with PBS (data not shown). These results confirm tire hNAGLUcoV3 expression in liver tissue.

Overall, AAVhu68.hNAGLUcoV3 outperforms all other co-variants and is selected moving forward.

B. Vector - AAVhu68.CB7.CI. hNAGLUcoV3.rBG

A hNAGLU engineered sequence as shown in SEQ ID NO: 1 (hNAGLUcoV3) is cloned into an expression construct containing a CB7 promoter (a hybrid of a cytomegalovirus immediate-early enhancer, the chicken P-actin promoter and chicken -actin intron (CI)) and rabbit beta globin (rBG) polyadenylation sequence. The cis plasmid further contains the full vector genome, i.e., the expression cassette flanked by AAV2 inverted terminal repeats. For triple transfection, a trans plasmid encoding AAV2 rep proteins and the AAVhu68 VP1 capsid coding sequence is used, together with a plasmid providing helper functions containing required adenoviral genes not contained in the packaging host cell. The packaging i.e.. HEK293 cells expressing adenovirus Ela and Elb gene functions.

AAV vectors are manufactured with iodixanol gradient method. See, Lock, M., et al., Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale. Human Gene Therapy, 2010. 21(10): p. 1259-1271. The purified vectors are titrated with classic qPCR for MPS IIIB by Penn Vector Core.

Dubelco’s phosphate buffer saline (dPBS) without calcium and magnesium is used as control article (vehicle control) and diluent for vector. The test article is diluted with sterile phosphate buffered saline (PBS) to the appropriate concentration for each dose group. Diluted vector is kept on wet ice and injected to the animals within 4 hours after dilution.

C. Vector and Vehicle Administration

MPS IIIB mice vector doses are IxlO¹⁰, IxlO¹¹ or 5xlO¹⁰ GC per mouse at an average age of 18 weeks. It is noted that ddPCR (Lock, M., et al.. Absolute Determination of Single-Stranded and Self-Complementary Adeno-Associated Viral Vector Genome Titers by Droplet Digital PCR. Human Gene Therapy Methods, 2014. 25(2): p. 115-125) gives titers that are approximately 3 fold higher than the classic qPCR method. Mice are anesthetized with Isoflurane. Each anesthetized mouse is grasped firmly by the loose skin behind the head and injected free hand anterior and lateral to tire bregma with a Hamilton syringe fitted with a 27-gauge needle, which is adjusted to be inserted 3 mm deep.

D. Neurobehavioral Assessment

Rocking rotarod is performed to assess coordination and balance 2 months pi (MPS IIIB). Mice are habituated to the rotarod during 2 trials at a constant low speed (5 rpm) for 120 seconds. After 2 minutes rest, mice are placed back on the rotarod and submitted to a rocking paradigm where tire rod rotates at a constant speed of 10 rpm with reversal of the rotation direction every other rotation. Three trials are performed with intertrial rest of 2 minutes. Results are expressed as the average latency to fall from the rod: the longer the latency, the better the coordination.

E. Histology

Mice are euthanized by cardiac puncture exsanguination under ketamine/xylazine anesthesia 3 months post injection. Tissues are promptly collected, half are snap-frozen on dry ice (enzyme activity), and half are immersion-fixed in 10% neutral formalin and embedded in paraffin for histology. Collected tissues are brain, spinal cord, liver, and heart.

Hematoxylin & eosin (H&E) staining is perfomred according to standard protocols on paraffin sections. Histopathology is scored in brain and spinal cord by a board-certified veterinary Pathologist blinded to the treatment. Brain score is the cumulative sum of 4-grade severity scores of glial cell vacuolation in brain, neuronal vacuolation in brain cortex, neuronal vacuolation in brainstem and hindbrain, perivascular mononuclear cell infiltration mononuclear cell infiltration (maximum score of 20). Cumulative scores are analyzed by one-way Anova Kruskall Wallis test with post hoc Dunn’s multiple comparison test, alpha 0.05.

F. Enzyme Activity and Glycosaminoglycan Storage

For enzyme activity assays and GAGs content, proteins arc extracted by mechanical homogenization (Qiagen TissueLizer) in an acidic lysis solution (0.2% triton, 0.9% NaCl, adjusted to pH 4). Samples are freeze -thawed and clarified by centrifugation. Protein was quantified by BCA assay.

NAGLU activity is measured by incubating 10 pL sample with 20 pL of 2mM 4- MU-2-Acetamido-2-deoxy-alpha-D-glucopyranoside (Toronto Research Chemicals) dissolved in sodium acetate 0.1M pH 3.58; NaCl 150 mM; Triton X100 0.05%. After incubating for 2 h at 37°C, the mixture is diluted in glycine NaOH buffer, pH 10.6, and released 4-MU was quantified by fluorescence (excitation 365 nm, emission 450 nm) compared with standard dilutions of free 4-MU and nonnalized by the protein content.

GAGs content in tissue extract is measured using dye-binding method with a commercial kit used per manufacturer recommendations (Blyscan Biocolor GAGs kit).

G. Anti-transgene antibodies

Blood for measurement of serum anti-hNAGLU antibodies is collected at several in vivo timepoints by submandibular bleeding as well as at terminal necropsy by cardiac puncture. Serum is separated and frozen on dry ice and stored at -80°C until analyzed. Polystyrene plates are coated overnight with recombinant human NAGLU (R&D Systems), 5 pg/mL in PBS. titrated to pH 5.8. Plates are washed and blocked 1 hour in 2% bovine serum albumin (BSA) in neutral PBS. Plates are then incubated with serum samples diluted 1: 1000 in PBS. Bound antibody is detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Abeam) diluted 1: 10,000 in PBS with 2% BSA. The assay is developed using tetramethylbenzidine substrate and stopped with 2N sulfuric acid before measuring absorbance at 450 nm.

H. Pharmacology Study in Mice

Transgene Expression

A study is performed to assess transgene product expression following ICV administration of AAVhu68.CB7.CI.hNAGLUcoV3.RBG to adult C57BL6 (wild type) mice and MPS IIIB mice. The aim of this study is to assess cellular transgene product expression in disease-relevant target tissues of the brain (a key target organ) and in the periphery (serum and liver) following ICV administration of AAVhu68.CB7.CI.hNAGLUcoV3.RBG in an MPS TUB mouse model.

Wild type mice receive a single ICV administration of PBS as a control. MPS TUB mice receive a single ICV administration of AAVhu68.CB7.CI.hNAGLUcoV3.RBG at a dose of IxlO¹⁰ GC or 5xlO¹⁰ GC. MPS IIIB mice also receive a single ICV administration of PBS as a control. On day 7, serum is collected for evaluation of transgene produce expression (NAGLU enzyme activity). On day 28, serum is collected for anti-NAGLU titer. Brain and liver are also collected at necropsy to evaluate transgene product expression (NAGLU enzyme activity or NAGLU immunofluorescence [IF]).

FIG. 2A - FIG. 2E illustrates the dose dependent expression of the transgene in the brain of MPS IIIB mice administered the low dose (lxlO^loGC) and high dose (5xlO¹⁰ GC) of AAVhu68.CB7.CI.hNAGLUcoV3.RBG. MPS IIIB mice administered the low dose and high dose demonstrate statistically significant higher transgene expression in the brain as compared to the control. Anti-NAGLU antibody titer was examined using ELISA.

FIG. 6A and FIG. 6B provide a comparison of different engineered sequences in WT C57BL6 mice following IV administration based on enzyme activity readout. FIG. 6A demonstrates NAGLU activity in the liver and FIG. 6B demonstrates NAGLU activity in plasma. The natural cDNA (hNAGLUwt) is compared with three engineered sequences (hNAGLUco, hNAGLUcoV3 and hNAGLUcoVl) and assessed for expression of tire transgene. The wt sequence and three engineered sequences were administered at a dose of 3x10" GC. hNAGLUcoV3 demonstrates the highest enzyme activity and outperforms the other constructs and the natural cDNA.

Cumulatively, ICV administration of AAVhu68.CB7.CI.hNAGLUcoV3.RBG to MPS IIIB mice at a dose of lxlO^luGC or 5xlO¹⁰ GC leads to transgene product expression (NAGLU enzyme activity and NAGLU protein expression) in a disease-relevant target tissue (brain).

Reduction of Lysosomal Pathology

LAMP- 1 IHC is performed to evaluate lysosomal storage lesions in the brain of untreated MPS IIIB mice, AAVhu68.CB7.CLhNAGLUcoV3.RBG treated MPS IIIB mice and wild type controls. An increase in LAMP -1 -positive area indicates an increase in lysosomal storage. Untreated MPS IIIB mice demonstrate increased LAMP-1 staining in the cortex and hippocampus compared to MPS IIB mice treated with AAVhu68.CB7.CI.hNAGLUcoV3.RBG and wild type controls (FIG. 3A - FIG. 3C). MPS IIIB mice are treated administered AAVhu68.CB7.CI.hNAGLUcoV3.RBG at a dose of IxlO¹⁰ GC or 5xlO¹⁰ GC. FIG. 3A shows percent LAMP-1 area in the cortex and FIG. 3B shows percent LAMP-1 area in tire hippocampus.

Quantification of LAMP- 1 IHC staining using image analysis software confirms that untreated MPS IIIB mice exhibit more LAMP- 1 -positive staining (indicated by a larger average LAMP- 1 -positive area) throughout the brain (cortex and hippocampus) compared to MPS 11B mice treated with AAVhu68.CB7.CI.hNAGLUcoV3.RBG and wild type controls.

LAMP-1 immunohistochemical staining is performed on deparaffinized paraffin sections. Briefly, antigen retrieval is performed by boiling slides at 100°C for 6 minutes in 10 mM citrate buffer (pH 6.0). Slides are then incubated with 2% hydrogen peroxide for 15 minutes, blocked using avidin/biotin reagents for 15 minutes each (Vector Laboratory; Catalog number: SP-2001), and incubated with 1% donkey serum in phosphate buffered saline (PBS) with 0.2% Triton-X for 10 minutes at room temperature. Slides are then incubated with a rat anti -mouse LAMP-1 primary antibody (Abeam, Catalog # Ab25245) at 37°C for 1 hour. Slides are washed and then incubated with a biotinylated donkey anti-rabbit IgG secondary antibody (Jackson; Catalog number: 711 -065- 152) for 45 minutes at room temperature. Slides are washed and then incubated with Vectastain ABC reagent (Vector Laboratories; Catalog number: PK-6100). Colorimetric development is performed using a 3, 3 'Diaminobenzidine (DAB) kit (Vector Laboratories; Catalog number: SK-4100) followed by counterstaining with hematoxylin and coverslipping for evaluation.

Cumulatively, there is a dose dependent reduction of LAMP- 1 staining in the brain, which indicates improvement of lysosomal pathology in MPS IIIB mice when treated with A AVhu68.CB7. CI . hNAGLUco V3 RBG.

Reduction of Substrate

Next, a glycosaminoglycan (GAG) (HS) accumulation/reduction assay is performed. GAG (HS) accumulation/reduction was examined using LC-MS/MS quantitation of disaccharide breakdown products from butanolysis of HS in the brain and liver. The assay was performed on untreated MPS IIIB mice, AAVhu68.CB7.CI.hNAGLUcoV3. RBG treated MPS IIIB mice and wild type controls. Treated MPS IIIB mice were administered AAVhu68.CB7.CI.hNAGLUcoV3.RBG at a dose of IxlO¹⁰ GC or 5xl0¹⁰ GC. FIG. 4A illustrates a statistically significant dose dependent GAG reduction in tire brain of MPS IIIB mice treated with AAVhu68.CB7.CI.hNAGLUcoV3.RBG. FIG. 4B illustrates a statistically significant dose dependent GAG reduction in the liver of MPS IIIB mice treated with AAVhu68.CB7.CI.hNAGLUcoV3.RBG. FIG. 4A and FIG. 4B show GAG levels plotted as ng GAG(HS) per mg protein.

These results demonstrate a dose dependent reduction of the disease-relevant biomarker heparan sulfate (HS) in brain tissue, showing target engagement.

Example 2: Determination of Minimum Effective Dose (MED) in a Murine Model of MPSIIIb

Experiments arc performed to evaluate the expression, bioactivity, and minimum effective dose (MED) of a single intracerebroventricular (ICV) administration of AAVhu68.CB7.CI.hNAGLUcoV3.RBG, an AAVhu68 vector expressing human N-acetyl- a-D-glucosaminidase (NAGLU), in a murine model of MPSIIIb.

AAVhu68.CB7.CI.hNAGLUcoV3.RBG is administered through the ICV route to MPS Illb mice, average age of 4 months (n=10 per group) at four dose levels(detennined by qPCR tittering of tire vector) on Day 0 with a 3 month post-injection (pi) obser ation period. Vehicle treated MPS IIIB and heterozygous littennates serve as controls (n=10 per group).

Bioactivity is assessed by measuring the NAGLU activity at 3 months pi in the brain, spinal cord, liver, serum and heart. Efficacy and MED are determined by measuring performance on a rocking rotarod at 2 months pi as well as brain and spinal cord lysosomal storage and histopathology at 3 month pi.

Example 3: Pharmacology /Toxicology Study in Rhesus Macaque

Experiments are performed to evaluate the safety of intrathecal administration of three doses of AAVhu68.CB7.CI.hNAGLUcoV3.RBG.

The control article is administered via suboccipital puncture to 3 macaques (both genders) in Group 1. The vector of AAVhu68.CB7.CI.hNAGLUcoV3.RBG is administered via suboccipital puncture to 6 rhesus macaques randomized to Groups 2-3. Macaques in Group 2 receive test article high dose (3xlO¹³ GC) (N=3); macaques in Group 3 receive test article low dose (IxlO¹³ GC) (N=3). Blood and cerebrospinal fluid are collected as part of a general safety panel. Serum and peripheral blood mononuclear cells (PBMC) are collected to investigate humoral and cellular immune response to the capsid and transgene.

Following completion of the in-life phase of these studies at 90 ± 3 days post-vector administration, macaques are necropsied with tissues harvested for a comprehensive histopathological examination. Lymphocytes are harvested from spleen, and bone marrow to examine the presence of CTLs in these organs at the time of necropsy.

Example 4: Long Tenn Effects of AAV.hNAGLU administration

Experiments are perfonned to investigate the long-tenn effects of AAV.hNAGLUcoV3 on MPS Illb mice. Twenty MPS Illb mice are injected with a high dose of AAV.CB7.CLhNAGLUcoV3.RBG (9xlO¹⁰ GC, ICV) at 2 months of age. An additional twenty MPS Illa mice and twenty wild-type mice receive PBS control injections. The mice are monitored for 7 months post injection, during which they are assigned clinical scores weekly and undergo behavioral and cognitive testing.

A multiparameter grading scale was developed to evaluate disease correction and response to treatment for the duration of the study. A score is assigned to individual mice based on an assessment of a combination of tremor, posture, fur quality, clasping, comeal clouding, and gait/mobility. The clinical scoring system was adapted based on previously described methods (see, e.g., Burkholder et al. Curr Protoc Mouse Biol. June 2012, 2: 145- 65; Tumpey et al. J Virol. May 1998, 3705-10; and Guyenet et al. J Vis Exp, May 2010, 39; 1787).

Example 5: Long-Term Survival Minimum Effective Dose Study

Experiments are perfonned to investigate the long-term survival of AAV.hNAGLUcoV3 in MPSIIIb mice in a minimum effective dose study. Five groups of mice are studied, each containing ten males and ten females. The groups include: WT control mice, MPSIIIB control mice and MPS IIIB mice administered AAVhu68.hNAGLUcoV3 at doses of L3xlO¹⁰ GC, 4.5xlO¹⁰ GC or 1.3xl0ⁿ GC. All groups are dosed between 2-3 months old. Bodyweight is measured weekly. Open field testing is perfonned at day 210. Elevated zero maze testing is performed at day 180. Blood is drawn on day 7, day 30, day 60. day 90. day 150 and terminally. Necropsy is performed at humane endpoints. The study includes analysis of behavioral, biomarker and survival data, including clinical pathology data, cerebrospinal fluid and brain GAG levels as well as serum, brain and liver NAGLU activity.

Clinical scores in male and female WT and MPS TUB mice as compared to MPS IITB mice administered AAVhu68.hNAGLUcoV3 at doses of 1.3xlO¹⁰ GC, 4.5xlO¹⁰ GC or 1.3x 10" GC are shown in FIG. 7. Higher clinical scores indicate a worse phenotype. All of the mice treated with AAVhu68.hNAGLUcoV3 show similar clinical scores to WT mice, regardless of the dose. Survival curves of WT and MPS IIIB mice as compared to MPS IIIB mice administered AAVhu68.hNAGLUcoV3 at doses of 1.3xl0^lu GC, 4.5xlO¹⁰ GC or 1.3X10¹¹ GC are compared in FIG. 8. The probability of survival is shown. As compared to the untreated MPSIIIB mice, survival rescue is shown in all of the MPSIIIB mice treated with AAVhu68.hNAGLUcoV3, regardless of the dose.

Example 6: Evaluation of Pharmacology and Safety' in Rhesus Macaques

Experiments are performed to evaluate the safety of intra-cistema magna injection of of AAVhu68.CB7.CI.hNAGLUcoV3.RBG at a dose of 3.3x10” GC/g brain. Three animals were administered the treatment. Following completion of the in-life phase of these studies at 90 ± 3 days post-vector administration, two of the NHPs were necropsied with tissues harvested for examination (FIG. 9A to 9E). Treatment was well-tolerated in these tw o animals and typical mild to moderate DRG pathology was shown. FIG. 9A illustrates brain slice “5” taken from the cortex and periventricular region, and FIG. 9B illustrates brain slice "9" taken from the occipital cortex. FIGs. 9C to 9E illustrates hNAGLU expression by in situ hybridization.

The third NHP developed neurotoxicity starting from day 15 post- vector administration. Steroids were provided at day 16, via intramuscular injection, and the animal was later switched to oral prednisone. An emergency necropsy was performed on this animal at 42 days. The animal showed a cytotoxic immune response to hNAGLU (non selfresponse) with neurological signs and exaggerated DRG pathology’ due to a robust T cell response to an hNAGLU epitope. Additional studies were performed to confirm responses to sub-pools of interest (FIG. 10A-FIG. 10C, Table A and Table B). The predicted immunodominant epitope is LAPEDPIFPI (SEQ ID NO: 33). from the pool sample which included peptides: Peptide 55 - SEQ ID NO: 34; Peptide 56 - SEQ ID NO: 35; Peptide 57 - SEQ ID NO: 36; Peptide 58 - SEQ ID NO: 37; Peptide 59 - SEQ ID NO: 38). Table A, below, summarizes the spot forming units (SFUs)/million cells. DMSO and Pool B were tested in four replicates. An asterisk indicates a sample was tested as a single sample replicate. Groups Pool B, B. 1, B.6, B.7, B.8, B.9, Peptide 56, and Peptide 57 indicated a positive response. Groups Pool B, B. 1, B.6, B.7, B.8, B.9, and Peptide 56, 57, and 58 indicated the value is just below 3x DMSO confirmatory cut point.

Table B, below, summarizes positive sub-pools based on liver lymphocyte data collected. As asterisk indicates tire value is just below 3x DMSO response.

Responses to individual peptides were evaluated within the sub-pools that generated positive IFN-y responses. IFN-y responses to individual peptides identified one immune- dominant epitope within Peptide Pool B. The predicted immunodominant epitope is LAPEDPIFPI (SEQ ID NO: 33). As there is no known mutation in this region, it is believed human patients would be tolerant to this epitope.

All publications cited in this specification are incorporated herein by reference in their entireties. US Provisional Patent Application No. 63/507,586, filed June 12, 2023, is incorporated herein by reference in its entirety. Similarly, the SEQ ID NOs which are referenced herein and which appear in the appended Sequence Listing are incorporated by reference. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

CLAIMS:

1 . A recombinant AAV (rAAV) comprising a vector genome in an AAV capsid, wherein the vector genome is a nucleic acid molecule which comprises an expression cassette comprising an engineered nucleic acid sequence encoding a functional human N-acetyl-alpha-glucosaminidase (hNAGLU) operably linked to regulatory sequences for the hNAGLU, wherein the hNAGLU coding sequence is SEQ ID NO: 1 or a sequence at least 99% identical to SEQ ID NO: 1 (hNAGLUcoV3).

2. The rAAV according to claim 1. wherein the hNAGLU coding sequence is SEQ ID N0 1 (hNAGLUcoV3).

3. The rAAV according to claim 1 or claim 2, wherein the regulatory sequences comprise a hybrid promoter comprising a cytomegalovirus immediate early (CMV IE) enhancer, a chicken beta-actin promoter, and a chimeric intron comprising a chicken beta actin intron.

4. The rAAV according to any one of claims 1 to 3, wherein the regulatory sequences comprise a promoter element comprising a chicken beta actin promoter having the sequence of SEQ ID NO: 4 or a sequence at least 99.9% identical to SEQ ID NO: 4.

5. The rAAV according to any one of claims 1 to 4, wherein the regulatory⁷ sequences comprise an enhancer element comprising a CMV IE enhancer having the sequence of SEQ ID NO: 3 or a sequence at least 99.9% identical to SEQ ID NO: 3.

6. The rAAV according to any one of claims 1 to 5, wherein the regulatory⁷ sequences comprise an intron comprising a chicken beta actin intron having the sequence of SEQ ID NO: 5 or a sequence at least 99.9% identical to SEQ ID NO: 5.

7. The rAAV according to any one of claims 1 to 6, wherein the regulatory sequences further comprise a rabbit beta globin poly A having the sequence of SEQ ID NO: 6 or a sequence at least 99.9% identical to SEQ ID NO: 6.

8. The rAAV according to any one of claims 1 to 7, wherein the expression cassette comprises the sequence of SEQ ID NO: 7 (CB7.CI.hNAGLUcoV3.RBG).

9. The rAAV according to any one of claims 1 to 8, wherein the vector genome comprises the sequence of SEQ ID NO: 8 (CB7.CI.hNAGLUcoV3.RBG).

10. The rAAV according to any one of claims 1 to 9, wherein the AAV capsid is an AAVhu68 capsid.

11. The rAAV according to any one of claims 1 to 9, wherein the AAV capsid is an AAVhu95 capsid, an AAVhu96 capsid, an AAV9 capsid, or an AAVrh91 capsid.

12. The rAAV according to any one of claims 1 to 11, which is for use in the treatment of Mucopolysaccharidosis III B (MPS IIIB), a disorder associated with a defect in hNAGLU, and/or for improving gait or mobility, reducing tremors, reducing spasms, improving posture, or reducing the progression of vision loss in a subject having an hNAGLU associated disorder.

13. A pharmaceutical composition comprising a rAAV according to any one of claims 1 to 11 in a formulation buffer.

14. The pharmaceutical composition according to claim 13. which is suitable for co-administering with a functional hNAGLU protein.

15. The pharmaceutical composition according to claim 13 or claim 14, which is formulated for administration via intracerebroventricular (ICV), intrathecal (IT), intraci sternal magna (ICM) or intravenous (IV) injection.

16. The pharmaceutical composition according to any one of claims 13 to 15, which is administrable at a dose of 1 x 10⁹ Genome Copies (GC) per gram (GC/g) of brain mass to about 1 x 10¹³ GC/g of brain mass.

17. The pharmaceutical composition according to any one of claims 13 to 16, which is formulated to have a pH of 6 to 8.

18. A method of treating a human subj ect diagnosed with MPS IIIB, a disorder associated with a defect in hNAGLU, and/or improving gait or mobility, reducing tremors, reducing spasms, improving posture, or reducing the progression of vision loss in a subj ect having an hNAGLU associated disorder, comprising administering to the subject a suspension of a rAAV according to any one of claims 1 to

11 in a formulation buffer at a dose of 1 x 10⁹ GC/g of brain mass to about 1 x 10¹³ GC/g of brain mass.

19. The method according to claim 18, wherein the suspension is suitable for co-administering with the functional hNAGLU protein.

20. The method according to claim 18 or claim 19, wherein the suspension is administered to the subject in need intracerebroventricularly, intrathecally, intravenously or via intracistemal magna injection.

21. The method according to any one of claims 18 to 20, wherein the suspension is administered via an Ommaya device.

22. The method according to any one of claims 18 to 21, wherein the suspension has a pH of 6 to 8.

23. The method according to any one of claims 19 to 21, wherein (a) the subject receives an enzyme replacement therapy at a decreased dosage or with a lower frequency compared to a standard treatment via the enzyme replacement therapy only; and/or

(b) the subject demonstrates an improvement of biomarkers related to MPS IIIB.

24. The method according to any one of claims 18 to 23, wherein the rAAV is administered once to the subj ect in need.

25. The method according to any one of claims 18 to 23, wherein the rAAV is administered more than once to the subject in need.

26. A recombinant adeno-associated virus (rAAV) according to any one of claims 1 to 11 or a pharmaceutical composition according to any one of claims 13 to 17 for use in preparing a medicament for treatment of MPS IIIB, a disorder associated with a defect in hNAGLU and/or improving gait or mobility, reducing tremors, reducing spasms, improving posture, or reducing the progression of vision loss in a subject having an hNAGLU associated disorder.