Adeno-Associated Virus Vector Delivery Of Cystathionine Beta-Synthase (CBS)

Enzyme For Treating CBS Deficiency

Field

The present disclosure is directed, in part, to enzyme replacement therapy using gene therapy vectors, such as adeno-associated virus (AAV) vectors, expressing Cystathionine Beta- Synthase (CBS) to reduce the amount of serum homocysteine (Hey) and increase the amount of downstream metabolites, such as cystathionine and cysteine (Cys), which can be used for treatment of diseases such as homocystinuria and homocysteine remethylation disorders.

Background

CBS, a central enzyme in the transsulfuration pathway, plays an essential role in Hey metabolism in eukaryotes (Mudd et al., The Metabolic and Molecular Basis of Inherited Disease, 2001, 8 Ed., p. 2007-2056, McGraw-Hill, New York). CBS catalyzes Hey condensation with L- serine to form cystathionine. When CBS activity is dramatically reduced or absent, as a result of certain genetic mutations, Hey builds up in tissues and blood. CBS enzyme catalyzes a pyridoxal-5'-phosphate (PLP; Vitamin B ₆ )-dependent condensation of serine and homocysteine to form cystathionine, which is then used to produce cysteine by another PLP-dependent enzyme, cystathionine g-lyase. In mammalian cells that possess the transsulfuration pathway, CBS occupies a key regulatory position between the re-methylation of Hey to methionine or its alternative use in the biosynthesis of Cys.

In healthy normal individuals, CBS-mediated conversion of Hey to cystathionine is the rate-limiting intermediate step of methionine (Met) metabolism to Cys. Vitamin B ₆ is an essential coenzyme for this process. In patients with certain genetic mutations in the CBS enzyme, the conversion of Hey to cystathionine is slowed or absent, resulting in elevations in the serum concentrations of the enzymatic substrate (Hey) and a corresponding decrease in the serum concentrations of the enzymatic product (cystathionine). The clinical condition of an elevated serum level of Hey, and its concomitant excretion into the urine, is collectively known as homocystinuria.

The estimates on the prevalence of homocystinuria vary widely. Data from newborn screening and clinical ascertainment provide a range of 1 :200,000 to 1 :335,000 live births (Mudd et al., The Metabolic and Molecular Basis of Inherited Disease, 2001, 8 Ed., p. 2007-2056, McGraw-Hill, New York). Recent evidence from DNA screening studies of newborns in Denmark, Germany, Norway and the Czech Republic found that the true incidence may be as high as about 1 :6,000 (Gaustadnes et al., N. Engl. J. Med., 1999, 1340, 1513; Linnebank et al., Thromb. Haemost., 2001, 85, 986; Refsum et al., Clin. Chem., 2004, 50, 3; and Sokolova et al., Hum. Mutat., 2001, 18, 548). Additionally, recent work has indicated that CBS-deficient homocystinuria (CBSDH) patients exist with either psychiatric or cardiovascular complications, but are currently undiagnosed due to a lack of the characteristic connective tissue defects that are typically instrumental in diagnosis (Li and Stewart, Pathol., 1999, 31, 221; Linnebank et al., J. Inherited Metabol. Dis., 2003, 26, 509; and Maclean et al., Hum. Mutat., 2002, 19, 641). Primary health problems associated with CBSDH include cardiovascular disease with a predisposition to thrombosis, resulting in a high rate of mortality in untreated and partially treated patients;

connective tissue problems affecting the ocular system with progressive myopia and lens dislocation (ectopia lentis); connective tissue problems affecting the skeleton characterized by marfanoid habitus, osteoporosis, and scoliosis; and central nervous system problems, including mental retardation and seizures.

Currently, three potential treatment options exist for the treatment of CBSDH: 1) increase of residual activity of CBS activity using pharmacologic doses of Vitamin B ₆ in Vitamin B ₆ -responsive patients; 2) lowering of serum Hey by a diet with a strict restriction of the intake of Met; and 3) detoxification by betaine-mediated conversion of Hey into Met, thus lowering serum Hey concentration. Each of these three therapies is aimed at lowering serum Hey concentration. The standard treatment for individuals affected with Vitamin B ₆ non-responsive CBSDH consists of a Met-restricted diet supplemented with a metabolic formula and Cys (which has become a conditionally essential amino acid in this condition). Intake of meat, dairy products and other food high in natural protein is prohibited. Daily consumption of a poorly palatable, synthetic metabolic formula containing amino acids and micronutrients is required to prevent secondary malnutrition. Supplementation with CYSTADANE ^® (betaine) is also a standard therapy. Betaine serves as a methyl donor for the remethylation of Hey to Met catalyzed by betaine-homocysteinemethyltransf erase in the liver (Wilcken et al., N. Engl. J. Med., 1983, 309, 448-53). Dietary compliance generally has been poor, even in those medical centers where optimal care and resources are provided, and this non-compliance has major implications on the development of life-threatening complications of homocystinuria. Accordingly, novel approaches are needed in homocystinuria treatment. Summary

The present disclosure provides recombinant AAV nucleic acid molecules comprising a CMV early enhancer/chicken beta actin (CAG) promoter operably linked to an exogenous nucleic acid sequence encoding a human Cystathionine b-synthase (hCBS) polypeptide.

The present disclosure also provides methods of preparing the recombinant AAV nucleic acid molecules comprising: amplifying the exogenous nucleic acid sequence encoding the hCBS polypeptide from a source containing the exogenous nucleic acid sequence using a pair of primers; and cloning the amplified exogenous nucleic acid sequence into a pAAV-CAG- containing nucleic acid molecule.

The present disclosure also provides viral vectors comprising the recombinant AAV nucleic acid molecules.

The present disclosure also provides methods of producing a recombinant AAV vector comprising: co-transfecting a host cell with CAG-hCBS DNA surrounded by AAV inverted terminal repeats (ITRs) and a helper nucleic acid molecule that comprises the AAV Rep and Cap sequences and adenovirus helper functions E4, E2a and VA; and culturing the host cell for a period of time sufficient to produce the recombinant AAV vector.

The present disclosure also provides methods of producing a recombinant AAV vector comprising: co-transfecting a host cell with CAG-hCBS DNA surrounded by AAV ITRs and two helper nucleic acid molecules, the first helper nucleic acid molecule comprising the AAV Rep and Cap sequences, and the second helper nucleic acid molecule comprising the adenovirus helper functions E4, E2a and VA; and culturing the host cell for a period of time sufficient to produce the recombinant AAV vector.

The present disclosure also provides methods of producing a recombinant AAV vector comprising: transfecting a host cell with CAG-hCBS DNA surrounded by AAV ITRs, wherein the host cell expresses AAV Cap and Rep proteins and adenoviral replication proteins E2, E4, and VA; and culturing the host cell for a period of time sufficient to produce the recombinant AAV vector.

The present disclosure also provides compositions comprising a recombinant AAV nucleic acid molecule comprising CAG-hCBS DNA surrounded by AAV ITRs and a

pharmaceutically acceptable carrier.

The present disclosure also provides compositions comprising an AAV vector encapsidating a recombinant AAV nucleic acid molecule comprising CAG-hCBS DNA surrounded by AAV ITRs and a pharmaceutically acceptable carrier. The present disclosure also provides methods of preventing or treating a disease, disorder, or condition associated with elevated homocysteine in a subject in need thereof, comprising administering to the subject a composition comprising a recombinant AAV nucleic acid molecule comprising CAG-hCBS DNA surrounded by AAV ITRs encapsidated in a viral vector and a pharmaceutically acceptable carrier.

The present disclosure also provides uses of a composition comprising a recombinant AAV nucleic acid molecule or a viral vector and a pharmaceutically acceptable carrier for the preparation of a medicament for the prevention or treatment of a disease, disorder, or condition associated with elevated homocysteine in a human subject.

The present disclosure also provides uses of a composition comprising a recombinant AAV nucleic acid molecule or a viral vector and a pharmaceutically acceptable carrier for the prevention or treatment of a disease, disorder, or condition associated with elevated

homocysteine in a human subject.

Brief Description Of The Drawings

Figure 1A shows a map of pAAVrh. lO-CAG-hCBS; interspersed tandem repeat (ITR) sequences are required for viral genome replication and packaging; all elements located between the ITR sequences are packaged into the virus.

Figure IB shows the experimental scheme of a representative study; time of injection is shown by the red up arrow; ticks above the line show days in which blood was collected;

downward arrow shows day of tissue collection for mice with indicated route of injection and dosage; a summary of age, sex, and the method of injection of the mice are shown in the table underneath.

Figure 2A shows the effect of AAVrh.lO-CAG-hCBS on serum tHcy and methionine in mice at -7, 7, 14, and 21 days after injection with indicated dose of virus; IP and IV injections were combined; asterisks indicate P<0.05 compared to D-7 value.

Figure 2B shows the effect of AAVrh. lO-CAG-hCBS on liver and kidney CBS activity in mice 21 days after infection with indicated viral dose; error bar shows SEM; IP and IV injections were combined; control -/- bar indicates saline injected Tg-1287T Cbs ^_/ mice, while +/+ control bar is activity in wild-type mice.

Figure 2C shows a comparison of IP versus IV injection on tHcy and methionine; each line represents a single mouse; control mice were saline injected.

Figure 2D shows a summary of individual mice treatment for experiments in Figure 2C. Figure 2E shows data from Figure 2C (top) in table form; the middle panel shows percent reduction in tHcy comparing D-7 with D7 and D14 for mock and AAVrh.10-CAG-hCBS injected animals; the bottom panel shows statistical significance of these changes.

Figure 2F (top) shows data from Figure 2C (bottom) in table form; the middle panel shows percent reduction in tHcy comparing D-7 with D7 and D14 for mock and AAVrh.10- CAG-hCBS injected animals; the bottom panel shows statistical significance of these changes.

Figure 3 A shows the long-term effects of AAVrh.10-CAG-hCBS on serum tHcy and methionine in treated and control Tg-I278T Cbs mice; error bars show SE; asterisk indicates P<0.005.

Figure 3B shows data from Figure 3 A in table form.

Figure 3C shows a Western blot showing liver CBS levels at D78 in control and treated mice; endogenous CBS shows leaky background expression from mutant I278T CBS expressing transgene detected by antibody to an HA tag.

Figure 4A shows liver and kidney CBS enzyme activity at D78 measured in lysates.

Figure 4B shows a comparison of alopecia in treated vs. control mice.

Figure 5A shows an experimental scheme timeline; upticks show days in which blood was collected; red arrow indicates time at which AAVl-CMV-hCBS was delivered into Tg-

I278T Cbs mice via a single injection; down arrows show numbers of mice from each group euthanized on each date and the method of AAV injection.

Figure 5B shows Tg-I278T Cbs mice were injected with PBS or variable numbers of virus by IP, IM or IV; serum tHcy was measured at before (D-7) and a week after injection (D7); asterisks indicate P<0.05 compared D-7 to D7.

Figure 5C shows mice injected with PBS, 4xl0 ¹² , or 8xl0 ¹² virus by IP and CBS activity was measured in the liver and kidney lysates collected at D17; asterisk indicates P < 0.05 compared to buffer-injected mice; error bars show SE.

Figure 6 shows a summary of individual mice treatment with AAVl-CMV-hCBS.

Figure 7A shows a representative summary and results of a second experiment in which mice on either a CH3 or C57BL6 strain background were either injected or not injected with AAVrh.10. CAG-hCBS; the transgenic mice express the indicated allele CBS mutations.

Figure 7B shows CBS levels at D27 in control and treated mice; top panel shows total CBS expression; mouse CBS runs slightly slower than hCBS; middle panel shows reactivity using anti -HA antibody; this only expresses transgene expressed CBS; in some control animals, low levels of transgene CBS was detected even though the mice were not on zinc. Figure 8 shows a representative summary of individual treatment of Tg-I278T Cbs ^_/ mice in Figures 9A, 9B, and 9C.

Figure 9A shows the effect of AAVrh.lO-CAG-hCBS on weight in Tg-I278T Cbs ^_/ mice.

Figure 9B shows the effect of AAVrh. lO-CAG-hCBS on serum tHcy in Tg-I278T Cbs ^/_ mice.

Figure 9C shows the effect of AAVrh. lO-CAG-hCBS on serum methionine in Tg-I278T Cbs ^/_ mice.

Figure 10 shows a comparison of alopecia and whiskers in treated vs. control mice.

Description Of Embodiments

Various terms relating to aspects of the present disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order.

Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-expressed basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

As used herein, the singular forms“a,”“an” and“the” include plural referents unless the context clearly dictates otherwise.

As used herein, the term“AAV” is an abbreviation for adeno-associated virus, which is a single-stranded DNA parvovirus or self-complementary double-stranded DNA that grows only in cells in which certain functions are provided by a co-infecting helper virus or plasmid.

As used herein, the phrase“AAV nucleic acid molecule” refers to a nucleic acid molecule comprising one or more polynucleotides of interest (transgenes or exogenous nucleic acid sequences, such as those encoding hCBS) that are flanked by AAV ITRs. Such AAV nucleic acid molecules can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a helper plasmid encoding and expressing AAV Rep and Cap gene products, as well as other helper gene products. As used herein, the phrase“AAV vector” refers to a viral particle composed of an AAV capsid protein and an encapsidated polynucleotide comprising at least one AAV nucleic acid molecule (comprising, for example, a polynucleotide sequence encoding hCBS).

As used herein, the term“comprising” may be replaced with“consisting” or“consisting essentially of’ in particular embodiments as desired.

As used herein, the term“homolog” refers to a protein or peptide which differs from a naturally occurring protein or peptide (e.g., the“wild-type” protein) by modifications to the naturally occurring protein or peptide, but which maintains the basic protein and side chain structure of the naturally occurring form. Such changes include, but are not limited to: changes in one, few, or even several amino acid side chains; changes in one, few or several amino acids; changes in stereochemistry of one or a few atoms; and/or minor derivatizations, including but not limited to: methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol. A homolog can have enhanced, decreased, changed, or essentially similar properties as compared to the naturally occurring protein or peptide. In some embodiments, truncated CBS proteins, for example having C -terminal deletions of the naturally occurring CBS protein, are included.

As used herein, a“nucleic acid,” a“nucleic acid molecule,” a“nucleic acid sequence,” a “polynucleotide,” or an“oligonucleotide” can comprise a polymeric form of nucleotides of any length, can comprise DNA and/or RNA, and can be single-stranded, double-stranded, or multiple stranded. One strand of a nucleic acid also refers to its complement.

As used herein, the phrase“operably linked” refers to both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

As used herein, the term“serotype” refers to a distinction with respect to an AAV having a capsid which is serologically distinct from other AAV serotypes. Serologic

distinctiveness can be determined on the basis of the lack of cross-reactivity between antibodies to the AAV as compared to other AAV. Cross-reactivity is typically measured in a neutralizing antibody assay.

As used herein, the terms“subject” and“patient” are used interchangeably. A subject may include any animal, including mammals. Mammals include, but are not limited to, farm animals (such as, for example, horse, cow, pig), companion animals (such as, for example, dog, cat), laboratory animals (such as, for example, mouse, rat, rabbits), and non-human primates. In some embodiments, the subject is a human. A used herein, the term“tropism” refers to the ability of an AAV vector to infect one or more specified cell types, but can also encompass how the vector functions to transduce the cell in the one or more specified cell types (i.e., preferential entry of the AAV vector into certain cell or tissue type(s) and/or preferential interaction with the cell surface that facilitates entry into certain cell or tissue types, optionally and followed by expression (e.g., transcription and, optionally, translation) of sequences carried by the AAV vector in the cell.

The present disclosure provides recombinant AAV nucleic acid molecules comprising a CAG promoter operably linked to an exogenous nucleic acid sequence encoding an hCBS polypeptide. In some embodiments, the recombinant AAV nucleic acid molecule is present within a plasmid, bacmid, or baculovirus in order to produce a viral vector. In some

embodiments, the recombinant AAV nucleic acid molecule is present within a plasmid. In some embodiments, the recombinant AAV nucleic acid molecule is present within a bacmid. In some embodiments, the recombinant AAV nucleic acid molecule is present within a baculovirus.

In some embodiments, the AAV nucleic acid molecules can contain a full-length AAV 5' ITR and a full-length 3' ITR. A shortened version of the 5' ITR, termed AITR, in which the D- sequence and terminal resolution site (trs) are deleted can also be utilized. In addition, self- complementary AAV (scAAV) nucleic acid molecules can be constructed in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intra molecular double-stranded DNA template. Upon infection, rather than waiting 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. In some embodiments, a single-stranded AAV nucleic acid molecule can be used.

In some embodiments, the ITRs can be selected from a source which differs from the AAV source of the capsid. For example, AAV2 ITRs can be selected for use with an AAV capsid having a particular efficiency for a selected cellular receptor, target tissue, or viral target. In some embodiments, the ITR or AITR sequences from AAV2 can be used for convenience and to accelerate regulatory approval. However, ITRs from other AAV sources can also be selected. Where the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector produced therefrom may be termed pseudotyped. However, other sources of AAV ITRs may be utilized.

In some embodiments, recombinant AAV nucleic acid molecules can comprise the exogenous nucleic acid sequences described herein and one or more flanking AAV ITRs. AAV DNA in the AAV genomes may be from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13.

Production of pseudotyped AAV is disclosed in, for example, PCT Publication No. WO

01/83692. Other types of AAV variants, for example AAV with capsid mutations, are also contemplated (see, Marsic et al., Mol. Therapy, 2014, 22, 1900-1909). In some embodiments, the recombinant AAV nucleic acid molecules are derived from the AAVrh.10 serotype.

In some embodiments, recombinant AAV nucleic acid molecules (such as those used to produce infectious encapsidated AAV particles) comprise an AAV genome. In some

embodiments, the genomes of both AAV lack AAV rep and cap DNA (i.e., there is no AAV rep or cap DNA between the ITRs of the genomes). Examples of AAV that may be constructed to comprise the exogenous nucleic acid sequences described herein are included in PCT Publication No. WO 13/016352.

In some embodiments, the AAV nucleic acid molecules described herein are designed for expressing its gene product in specific cell types (such as, hepatocytes). In addition to the AAV 5' ITR and 3' ITR, the open reading frame(s) may include tissue-specific regulatory elements or constitutive elements.

The AAV nucleic acid molecules described herein typically contain a CAG promoter sequence as part of the expression control sequences, located between the 5' ITR sequence and the coding sequence of the exogenous nucleic acid sequence. In some embodiments, the CAG promoter is upstream of the exogenous nucleic acid sequence encoding the hCBS polypeptide. The CAG promoter is a hybrid promoter containing a CMV/chicken beta-actin/rabbit beta-globin splice acceptor sequences (Miyazaki et al., Gene, 1989, 79, 269-77). Additional promoters include, but are not limited to, human alpha 1 anti-trypsin (AAT), human thyroxin binding globulin (TBG), cytomegalovirus (CMV), human GLUT1 promoter, hybrid liver specific promoter (HLP promoter), LP-1 promoter, LSP-1 promoter, chicken beta actin, and mouse beta- actin, and any other promoter that supports expression in the liver.

In some embodiments, the AAV nucleic acid molecules can also contain one or more other appropriate transcription initiation, termination, enhancer sequences, efficient RNA processing signals, such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. Examples of suitable polyA sequences include, but are not limited to, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic poly As. Examples of suitable enhancers include, but are not limited to, the alpha fetoprotein enhancer, the TTR minimal promoter/enhancer, LSP (TH-binding globulin promoter/alphal- microglobulin/bikunin enhancer), amongst others. In some embodiments, the AAV nucleic acid molecules can comprise one or more expression enhancers. In some embodiments, the AAV nucleic acid molecules can contain two or more expression enhancers. These enhancers may be the same or may differ from one another. For example, an enhancer may include an Alpha mic/bik enhancer. This enhancer may be present in two copies which are located adjacent to one another. Alternately, the dual copies of the enhancer may be separated by one or more sequences. In some embodiments, the AAV nucleic acid molecules can further contain an intron such as, for example, the Promega intron. In some embodiments, one or more sequences may be selected to stabilize mRNA. An example of such a sequence is a modified WPRE sequence, which may be engineered upstream of the polyA sequence and downstream of the coding sequence (see, Zanta-Boussif et al, Gene Therapy, 2009, 16, 605-619). These additional elements are operably linked to the coding sequences.

The amino acid sequence for full-length hCBS comprises 551 amino acids: MPSETPQ AEVGPTGCPHRSGPHSAKGSLEKGSPEDKEAKEPLWIRPDAPSRCTWQLGRPASESPHH HTAPAKSPKILPDILKKIGDTPMVRINKIGKKFGLKCELLAKCEFFNAGGSVKDRISLRM I EDAERDGTLKPGDTIIEPTSGNTGIGLALAAAVRGYRCIIVMPEKMSSEKVDVLRALGAE IVRTPTNARFDSPESHVGVAWRLKNEIPNSHILDQYRNASNPLAHYDTTADEILQQCDG KLDMLVASVGTGGTITGIARKLKEKCPGCRIIGVDPEGSILAEPEELNQTEQTTYEVEGI G YDFIPTVLDRTVVDKWFKSNDEEAFTFARMLIAQEGLLCGGSAGSTVAVAVKAAQELQ EGQRC VVILPD S VRNYMTKFLSDRWMLQKGFLKEEDLTEKKPWWWHLRV QELGL SAP LT VLPTITCGHTIEILREKGFDQ AP VVDE AGVILGMVTLGNML S SLL AGK V QP SDQ VGK VIYKQFKQIRLTDTLGRLSHILEMDHFALVVHEQIQYHSTGKSSQRQMVFGVVTAIDLLN FVAAQERDQK (SEQ ID NO: 1).

In some embodiments, the exogenous nucleic acid sequence encodes an hCBS polypeptide comprising an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 1, or to a biologically active truncation thereof (including non-heme-binding, but catalytically active variants). In some embodiments, the exogenous nucleic acid sequence encodes an hCBS polypeptide comprising an amino acid sequence at least about 70% identical to the amino acid sequence of SEQ ID NO: l . In some embodiments, the exogenous nucleic acid sequence encodes an hCBS polypeptide comprising an amino acid sequence at least about 75% identical to the amino acid sequence of SEQ ID NO: l . In some embodiments, the exogenous nucleic acid sequence encodes an hCBS polypeptide comprising an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: l. In some embodiments, the exogenous nucleic acid sequence encodes an hCBS polypeptide comprising an amino acid sequence at least about 85% identical to the amino acid sequence of SEQ ID NO: l. In some embodiments, the exogenous nucleic acid sequence encodes an hCBS polypeptide comprising an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the exogenous nucleic acid sequence encodes an hCBS polypeptide comprising an amino acid sequence at least about 95% identical to the amino acid sequence of SEQ ID NO: l. In some embodiments, the exogenous nucleic acid sequence encodes an hCBS polypeptide comprising an amino acid sequence at least about 96% identical to the amino acid sequence of SEQ ID NO: l. In some embodiments, the exogenous nucleic acid sequence encodes an hCBS polypeptide comprising an amino acid sequence at least about 97% identical to the amino acid sequence of SEQ ID NO: l. In some embodiments, the exogenous nucleic acid sequence encodes an hCBS polypeptide comprising an amino acid sequence at least about 98% identical to the amino acid sequence of SEQ ID NO:l. In some embodiments, the exogenous nucleic acid sequence encodes an hCBS polypeptide comprising an amino acid sequence at least about 99% identical to the amino acid sequence of SEQ ID NO: l. In some embodiments, the exogenous nucleic acid sequence encodes an hCBS polypeptide comprising an amino acid sequence identical to the amino acid sequence of SEQ ID NO: l. In some embodiments, the exogenous nucleic acid sequence encodes an hCBS polypeptide consisting of an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, an hCBS variant can include any combination of the N-terminal deletions or modifications and the C-terminal deletions described herein or in U.S. Patent No. 8,007,787. In some embodiments, additional modifications can be achieved by modification of other amino acid residues to provide a given percent identity to the wild-type hCBS sequence. In some embodiments, the hCBS variant is a truncated recombinant hCBS (r-hCBSAC)

homodimeric enzyme wherein the C-terminal regulatory region has been removed. In some embodiments, any of the hCBS variants described herein can have no more than one or two non- hCBS amino acid residues at the N-terminus (i.e., the variant comprises no more than one or two amino acid residues at the N-terminus that is/are not a residue of the naturally occurring hCBS amino acid sequence at that position).

The AAV nucleic acid molecules described herein contain an exogenous nucleic acid sequence that encodes an hCBS polypeptide, or a homolog thereof. The nucleic acid sequence encoding hCBS and the amino acid sequence encoded thereby are available through GenBank Accession No. L19501, and these sequences are also disclosed in U.S. Patent No. 5,523,225. A nucleotide sequence coding for hCBS is TCCCGGGCCCGCGACACACGCCCTCGGGGTCG

GTCCTCGAGGACGCGCAGGGCCCCCCACCCACCAGGACGCACGTTTCAAGCTCATC

AGTAAAGGTTCCTTAAATTCCCGAAGGGCAAGAAGTTAACCAAGTAAAACAGCATC

GGAACACCAGGATCCCATGACAGATTCTGTTGTCACGTCTCCTTACAGAGTTTGAGC

GGTGCTGAACTGTCAGCACCATCTGTCCGGTCCCAGCATGCCTTCTGAGACCCCCCA

GGCAGAAGTGGGGCCCACAGGCTGCCCCCACCGCTCAGGGCCACACTCGGCGAAGG

GGAGCCTGGAGAAGGGGTCCCCAGAGGATAAGGAAGCCAAGGAGCCCCTGTGGAT

CCGGCCCGATGCTCCGAGCAGGTGCACCTGGCAGCTGGGCCGGCCTGCCTCCGAGT

CCCCACATCACCACACTGCCCCGGCAAAATCTCCAAAAATCTTGCCAGATATTCTGA

AGAAAATCGGGGAC ACCCCT AT GGTC AGAAT C AAC A AGATT GGGAAGAAGTTCGGC

CTGAAGTGTGAGCTCTTGGCCAAGTGTGAGTTCTTCAACGCGGGCGGGAGCGTGAA

GGACCGCATCAGCCTGCGGATGATTGAGGATGCTGAGCGCGACGGGACGCTGAAGC

CCGGGGACACGATTATCGAGCCGACATCCGGGAACACCGGGATCGGGCTGGCCCTG

GCTGCGGC AGTGAGGGGCT ATCGCTGC AT C ATCGT GAT GCC AGAGAAGAT GAGCTC

CGAGAAGGTGGACGTGCTGCGGGCACTGGGGGCTGAGATTGTGAGGACGCCCACCA

ATGCCAGGTTCGACTCCCCGGAGTCACACGTGGGGGTGGCCTGGCGGCTGAAGAAC

GAAATCCCCAATTCTCACATCCTAGACCAGTACCGCAACGCCAGCAACCCCCTGGCT

CACTACGACACCACCGCTGATGAGATCCTGCAGCAGTGTGATGGGAAGCTGGACAT

GCTGGTGGCTTCAGTGGGCACGGGCGGCACCATCACGGGCATTGCCAGGAAGCTGA

AGGAGAAGTGTCCTGGATGCAGGATCATTGGGGTGGATCCCGAAGGGTCCATCCTC

GCAGAGCCGGAGGAGCTGAACCAGACGGAGCAGACAACCTACGAGGTGGAAGGGA

TCGGCTACGACTTCATCCCCACGGTGCTGGACAGGACGGTGGTGGACAAGTGGTTC

A AG AGC AAC GAT GAGGAGGCGTT C ACCTTT GCCCGC AT GCTGATCGCGC AAGAGGG

GCTGCTGTGCGGTGGCAGTGCTGGCAGCACGGTGGCGGTGGCCGTGAAGGCCGCGC

AGGAGCTGCAGGAGGGCCAGCGCTGCGTGGTCATTCTGCCCGACTCAGTGCGGAAC

T AC ATGACC AAGTTCCTGAGCGAC AGGT GGAT GCTGC AGAAGGGCTTTCTGAAGGA

GGAGGACCTCACGGAGAAGAAGCCCTGGTGGTGGCACCTCCGTGTTCAGGAGCTGG

GCCTGTCAGCCCCGCTGACCGTGCTCCCGACCATCACCTGTGGGCACACCATCGAGA

TCCTCCGGGAGAAGGGCTTCGACCAGGCGCCCGTGGTGGATGAGGCGGGGGTAATC

CTGGGAATGGTGACGCTTGGGAACATGCTCTCGTCCCTGCTTGCCGGGAAGGTGCAG

CCGTCAGACCAAGTTGGCAAAGTCATCTACAAGCAGTTCAAACAGATCCGCCTCAC

GGACACGCTGGGCAGGCTCTCGCACATCCTGGAGATGGACCACTTCGCCCTGGTGGT

GCACGAGCAGATCCAGTACCACAGCACCGGGAAGTCCAGTCAGCGGCAGATGGTGT TCGGGGTGGTCACCGCCATTGACTTGCTGAACTTCGTGGCCGCCCAGGAGCGGGACC

AGAAGTGAAGTCCGGAGCGCTGGGCGGTGCGGAGCGGGCCCGCCACCCTTGCCCAC

TTCTCCTTCGCTTTCCTGAGCCCTAAACACACGCGTGATTGGTAACTGCCTGGCCTG G

CACCGTTATCCCTGCACACGGCACAGAGCATCCGTCTCCCCTCGTTAACACATGGCT

TCCTAAATGGCCCTGTTTACGGCCTATGAGATGAAATATGTGATTTTCTCTAATGTA

ACTTCCTCTT AGGAT GTTTC ACC A AGGAAAT ATT GAGAGAGAAGTCGGCC AGGT AG

GATGAACACAGGCAATGACTGCGCAGAGTGGATTAAAGGCAAAAGAGAGAAGAGT

CCAGGAAGGGGCGGGGAGAAGCCTGGGTGGCTCAGCATCCTCCACGGGCTGCGCCG

TCTGCTCGGGGCTGAGCTGGCGGGAGCAGTTTGCGTGTTTGGGTTTTTTAATTGAGA

TGAAATTCAAATAACCTAAAAATCAATCACTTGAAAGTGAACAATCAGCGGCATTT

AGTACATCCAGAAAGTTGTGTAGGCACCACCTCTGTCACGTTCTGGAACATTCTGTC

ATCACCCCGTGAAGCAATCATTTCCCCTCCCGTCTTCCTCCTCCCCTGGCAACTGCT G

ATCGACTTTGTGTCTCTGTTGTCTAAAATAGGTTTTCCCTGTTCTGGACATTTCATA T

A A AT GG A ATC AC AC A A (SEQ ID NO:2).

In some embodiments, the exogenous nucleic acid sequence comprises a nucleotide sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the exogenous nucleic acid sequence comprises a nucleotide sequence at least about 70% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the exogenous nucleic acid sequence comprises a nucleotide sequence at least about 75% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the exogenous nucleic acid sequence comprises a nucleotide sequence at least about 80% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the exogenous nucleic acid sequence comprises a nucleotide sequence at least about 85% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the exogenous nucleic acid sequence comprises a nucleotide sequence at least about 90% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the exogenous nucleic acid sequence comprises a nucleotide sequence at least about 95% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the exogenous nucleic acid sequence comprises a nucleotide sequence at least about 96% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the exogenous nucleic acid sequence comprises a nucleotide sequence at least about 97% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the exogenous nucleic acid sequence comprises a nucleotide sequence at least about 98% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the exogenous nucleic acid sequence comprises a nucleotide sequence at least about 99% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the exogenous nucleic acid sequence comprises a nucleotide sequence identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the exogenous nucleic acid sequence consists of a nucleotide sequence identical to the nucleotide sequence of SEQ ID NO:2.

Percent sequence identity can be determined by, for example, the Gap program

(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group,

University Research Park, Madison WI), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Any amino acid or nucleotide number calculated as a % identity can be rounded up or down, as the case may be, to the closest whole number. Alternately, optimal alignment of sequences for comparison may be conducted by computerized implementations of algorithms (GAP, BESTFIT, BLAST, FASTA, and

TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection. Additional examples of algorithms for

determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et ak, Nuc. Acids Res., 1977, 25, 3389-3402 and Altschul et ak, J. Mol. Biol., 1990, 215, 403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

The present disclosure also provides methods of preparing the recombinant AAV nucleic acid molecules described herein. In some embodiments, the methods comprise:

amplifying the exogenous nucleic acid sequence encoding the hCBS polypeptide from a source containing the exogenous nucleic acid sequence using a pair of primers; and cloning the amplified exogenous nucleic acid sequence into a pAAV-CAG-containing nucleic acid molecule.

In some embodiments, the source containing the exogenous nucleic acid sequence is pUC AHCBS, which is commercially available or can be obtained through RT-PCR of RNA derived from human tissue, or can be chemically synthesized.

In some embodiments, the pair of primers comprises a first primer comprising the nucleotide sequence 5’ -C AGTCTCGAACTTAAC ATGCCTTCTGAGACCCCC-3’ (SEQ ID NO:3) and a second primer comprising the nucleotide sequence 5’-GGGCCCATTACCGAT ACTTCACTTCTGGTCCGCTCC-3’(SEQ ID NO:4).

In some embodiments, the pAAV-CAG-containing nucleic acid molecule is pAAV- CAG-MCS. The pAAV-CAG-MCS plasmid provides the AAV inverted terminal repeats, as well as the expression regulation sequences (CAG promoter and poly A tail). The MCS is a multiple cloning site that facilitates the insertion of the desired gene to be expressed, in this case hCBS. The pAAV-CAG-MCS plasmid is kanamycin resistant. Additional AAV plasmids can be used

(see, world wide web at“addgene.org/viral-service/penn-vector-core/”). A representative nucleotide sequence of the pAAV-CAG-MCS construct is: CCTGCAGGCAGCTGCGCGCTCG

CTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCC

CGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGG

GGTTCCTGCGGCCGCTCTAGAACTAGTCGACATTGATTATTGACTAGTTATTAATAG

TAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAA

CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCA

ATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG

GTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCA

AGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAG

TACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCT A

TTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCT C

CCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGC G

GGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGC

GGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGT

TTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGG

CGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCG

CGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGA

CGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTT T

CTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAG

CGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCG

CGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGC

AGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCT

GCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGG

TGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGA

GCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCG

TGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCG

GGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGT

CGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCA

GGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCAC

CCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGC

GGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGG

GGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGG CTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTC

TTTTTCCTACAGCTCCTGGGCAaCGTGCTGGTTATTGTGCTGTCTCATCATTTTGGC A

A AGaattcgagctcggtacccggggatcctctagagtcgacctgcagg AATTCGAGCTCCT AGGAT AT C AATT

GTTAATTAAGnnnAATTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCT G

GTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAA

ATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAAT

TTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACAT A

T GGGAGGGC AAAT C ATTT AAAAC ATC AGAAT GAGT ATTTGGTTT AGAGTTTGGC AAC

AT AT GCC C AT AT GC T GGC T GC CAT GA AC A A AGGTT GGC T AT A A AGAGGT CAT C AGT

ATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAG

GTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAAT TTT

CCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAG C

TGTCCCTCTTCTCTTATGGAGATCCCTCGACCTGCAGCCCAAGCTTATCGATACCGT C

GACCTCGAGGGGGGGCCCGGTACCCAGCTTTTGTTCCCTTTGCGGCCGCAGGAACCC

CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGG

CGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCT

GCCTGCAgGACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGC

CGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCG

ACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTC

CCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACC T

GTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTA T

CTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTT

CAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGA

CACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTA

TGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAA

GAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTG

GTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCA

AGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCT

ACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGGG

CCGCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTT

ATGAGCCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATG

GATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCG

ACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGC

A A AGGT AGC GT T GC C A AT GAT GTT AC AG AT GAG AT GGT C AG AC T A A AC T GGC T G AC GGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGG

TTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCT

GATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCG

ATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCG C

AATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATG

GCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGG

ATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGA A

ATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCT

TGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTT T

CAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTC

GATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCAGAGCATTACG

CTGACTTGACGGGACGGCGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGG

ATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTC

AAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCGGGGATCC

TCTAGAGTCGACCTGCAGGCATGCAAGCTTCAGCGGCCCATGACATTAACCTATAAA

AATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAAC

CTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGG

GAGC AGAC AAGCCCGT C AGGGCGCGT C AGCGGGT GTT GGCGGGTGTCGGGGCTGGC

TTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAAAATTGTAAAC

GTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAAC C

AATAGACCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGCCCGAGATAGAG

TTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAAC

GTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACC

CAAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAG

GGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGA

AGGGAAGAAAGCGAAAGGAGCGGGCGCT AAGGCGCTGGC AAGT GT AGCGGT C ACG

CTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTACTAT

GGTTGCTTTGACGTAtGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGC TC

ACATGTC (SEQ ID NO:5).

The present disclosure also provides viral vectors comprising any of the recombinant AAV nucleic acid molecules described herein. In some embodiments, the serotype of the AAV vector is AAVrh.10. In some embodiments, the AAV vectors have restricted tropism (target cell population). In some embodiments, the AAV vectors have liver tropism. The present disclosure also provides methods of producing a recombinant AAV vector. In some embodiments, the methods of producing a recombinant AAV vector comprise: co transfecting a host cell with CAG-hCBS DNA surrounded by AAV ITRs and a helper nucleic acid molecule that comprises the AAV Rep and Cap sequences and adenovirus helper functions E4, E2a and VA; and culturing the host cell for a period of time sufficient to produce the recombinant AAV vector. In some embodiments, the methods of producing a recombinant AAV vector comprise: co-transfecting a host cell with CAG-hCBS DNA surrounded by AAV ITRs and two helper nucleic acid molecules, the first helper nucleic acid molecule comprising the AAV Rep and Cap sequences, and the second helper nucleic acid molecule comprising the adenovirus helper functions E4, E2a and VA; and culturing the host cell for a period of time sufficient to produce the recombinant AAV vector. In some embodiments, the methods of producing a recombinant AAV vector comprise: transfecting a host cell with CAG-hCBS DNA surrounded by AAV ITRs, wherein the host cell expresses AAV Cap and Rep proteins and adenoviral replication proteins E2, E4, and VA; and culturing the host cell for a period of time sufficient to produce the recombinant AAV vector.

In some embodiments, the methods further comprise obtaining a lysate from the cell. In some embodiments, the methods further comprise purifying the viral vector from the lysate. In some embodiments, an affinity capture method as provided herein is performed using an antibody-capture affinity resin. In some embodiments, the solid support is a cross-linked 6% agarose matrix having an average particle size of about 34 pm and having an AAV-specific antibody. An example of one such commercially available affinity resin is AVB Sepharose™ high performance affinity resin using an AAV-specific camelid-derived single chain antibody fragment of llama origin which is commercially available from GE Healthcare (AVB

Sepharose). The manufacturer’s literature further recommends up to a 150 cm/h flow rate and a relatively low loading salt concentration. Other suitable affinity resins may be selected or designed which contain an AAV-specific antibody, AAV1 specific antibody, or other immunoglobulin construct which is an AAV-specific ligand. Such solid supports may be any suitable polymeric matrix material, e.g., agarose, sepharose, sephadex, amongst others. Suitable loading amounts may be in the range of about 2 to about 5x 10 ¹⁵ GC, or less, based on the capacity of a 30-mL column. Equivalent amounts may be calculated for other sized columns or other vessels.

Methods for generating and isolating AAV vectors suitable for delivery to a subject are described in, for example, U.S. Patent Nos. 7,790,449, 7,282,199, and 7,588,772, and PCT Publication Nos. WO 2003/042397, WO 2005/033321, and WO 2006/110689. In general, production of AAV vectors involves the following components present within a single cell (denoted herein as a packaging cell): AAV genome, AAV rep and cap genes separate from (i.e., not in) the AAV genome, and helper virus functions. In some embodiments, the AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than the AAV genome ITRs, including, but not limited to, the AAV serotypes described herein. In some embodiments, a producer cell line is transiently transfected with a nucleic acid molecule that encodes the transgene (e.g., encoding any of the hCBS polypeptides described herein) flanked by ITRs and a nucleic acid molecule that encodes rep and cap. In some embodiments, a packaging cell line that stably supplies rep and cap is transiently transfected with a nucleic acid molecule encoding the transgene (e.g., encoding any of the hCBS polypeptides described herein) flanked by ITRs. In each of these systems, AAV vectors are produced in response to infection with helper adenovirus, El -deleted adenovirus, or herpesvirus, requiring the separation of the AAVs from contaminating virus. More recently, systems have been developed that do not require infection with helper virus to recover the AAV; the required helper functions (i.e., adenovirus El, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are supplied in trans by the system. In these newer systems, the helper functions can be supplied by transient transfection of the cells with nucleic acid molecules that encode the required helper functions, or the cells can be engineered to stably contain genes encoding the helper functions, the expression of which can be controlled at the transcriptional or posttranscriptional level. In some embodiments, the transgene (e.g., encoding any of the hCBS polypeptides described herein) flanked by ITRs and rep/cap genes are introduced into insect cells by infection with baculovirus-based vectors. Methods of making and using these and other AAV production systems are also described in U.S. Patent Nos. 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604;

7,172,893; 7,201,898; 7,229,823; and 7,439,065.

In some embodiments, an AAV Cap for use in the methods described herein can be generated by mutagenesis (i.e., by insertions, deletions, or substitutions) of one of the aforementioned AAV Caps or its encoding nucleic acid molecule. In some embodiments, the AAV Cap is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to one or more of the aforementioned AAV Caps.

In some embodiments, an AAV Cap for use in a AAV composition is engineered to contain a heterologous sequence or other modification. For example, a peptide or protein sequence that confers selective targeting or immune evasion may be engineered into a Cap protein. Alternately or in addition, the Cap may be chemically modified so that the surface of the AAV is polyethylene glycolated (i.e., pegylated), which may facilitate immune evasion. The Cap protein may also be mutagenized (e.g., to remove its natural receptor binding, or to mask an immunogenic epitope).

The present disclosure also provides methods of generating a packaging cell comprising creating a cell line that stably expresses all the necessary components for AAV viral particle production. For example, a plasmid (or multiple plasmids) comprising a AAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the AAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et ah, Proc. Natl. Acad., 1982, 79, 2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et ah, Gene, 1983, 23, 65-73) or by direct, blunt-end ligation (Senapathy & Carter, J. Biol. Chem., 1984, 259, 4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of AAV. Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce AAV genomes and/or rep and cap genes into packaging cells.

In some embodiments, the AAV may be prepared as described in, for example, U.S. Patent Application Publication No. 2009/0275107, which provides an optionally continuous process for producing AAV and isolating from cell culture without requiring cell

permeabilization and/or cell lysis. Alternately, AAVrh. lO-based AAV vectors or AAV with engineered capsids as described herein may be purified using the methods described herein.

In some embodiments, packaging cells that produce infectious AAV are also provided. In some embodiments, the packaging cells are any cell lines that provide adenovirus helper functions required for the transfection method. In some embodiments, packaging cells may be stably transformed cancer cells such as HeLa cells, HEK 293 cells, HEK 293T cells, PerC.6 cells (a cognate 293 line), and SF9 cells, or any other insect or mammalian cell lines transduced to express helper functions. In some embodiments, packaging cells are cells that are not

transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with El of adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).

In some embodiments, the AAV may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying AAV vectors from helper virus are known in the art (see, Clark et ah, Hum. Gene Then, 1999, 10, 1031-1039; Schenpp and Clark, Methods Mol. Med., 2002, 69, 427-443; U.S. Patent No. 6,566,118; and PCT Publication No. WO 98/09657).

Methods of altering the AAV vector may involve a variety of techniques, which techniques are known to those of skill in the art. For example, site directed mutagenesis may be performed at the level of the nucleic acids encoding one or more amino acids to be altered. Alternately, an insertion of one or more amino acids (e.g., 2, 3, 4, 5 or more) may be made at the target region within the AAV capsid.

Additional expression systems can also be used. For example, a baculovirus/insect cell (e.g., Sf9) expression system can be used. Examples of such expression vectors and insect cell expression systems and methods are described in The Baculovirus Expression System: A Laboratory Guide, Linda King, Springer; 2012. Baculovirus promoters such as baculovirus polyhedrin and plO promoters are commercially available (see, e.g., Invitrogen’s“Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques”, 2002 (Life Technologies, Carlsbad, Calif.) and F. J. Haines et al.“Baculovirus Expression Vectors”, undated (Oxford Expression Technologies, Oxford, UK).

The engineered AAV may also be generated using methods described herein, or other methods described in the art, and purified as described (see, Mietzsch et al, Hum. Gene Then, 2014, 25, 212-222; Smith et al, Mol. Then, 2009, 17, 1888-96), describing a simplified baculovirus-AAV vector expression system coupled with one-step affinity purification. For example, lystates or supernatants (e.g., treated, freeze-thaw supernatants or media containing secreted AAV), may be purified using one-step AVB sepharose affinity chromatography using 1 ml prepacked HiTrap columns on an ACTA purifier (GE Healthcare) as described by

manufacturer.

In some embodiments, the methods of producing AAV vectors comprise using host cells that are mammalian cells. In these embodiments, the mammalian cell can be HEK 293 cell, HEK 293T cell, PerC.6 cell, or any other cell line comprising Adenovirus El helper function. In these embodiments, the CAG-hCBS DNA surrounded by AAV ITRs is in pAAV-CAG-hCBS plasmid and the helper nucleic acid molecule is a helper plasmid. Suitable examples of helper plasmids include, but are not limited to, pPAKMArh. lO; pXX6-80 (Aldevron); pRepCap, pAdDeltaF6, pAAV-syn, and pDGM (Addgene); pGMAAV (Genmedi); pRepCap and pHELP (Applied Viromnics); pDP (Karolinska Institute); as well as helper plasmids described in, for example, U.S. Patent Application Publication No. 2004/0235174, PCT Publication No. WO 02/012525, EP 1983057, and U.S. Patent No. 6,846,665. In some embodiments, the helper plasmid is pPAKMArh.10. In some embodiments, the methods of producing AAV vectors comprise using host cells that are insect cells. In these embodiments, the insect cells can be Sf9 cells. In these embodiments, the CAG-hCBS DNA surrounded by AAV ITRs is present in a baculovirus or a Bacmid.

The present disclosure also provides compositions comprising any of the AAV nucleic acid molecules or AAV vectors described herein. In some embodiments, the compositions comprise the AAV nucleic acid molecules or AAV vectors and a pharmaceutically acceptable carrier. The compositions may also comprise other ingredients such as diluents. Acceptable carriers and diluents are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter ions such as sodium; and/or nonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG).

Titers of AAV vectors in the compositions will vary depending, for example, on the particular AAV vector, the mode of administration, the treatment goal, the individual being treated, and the cell type(s) being targeted, and may be determined by methods standard in the art. Titers of AAV vectors may be about 1 ^c 10 ⁶ , about 1 ^c 10 ⁷ , about 1 ^c 10 ⁸ , about 1 ^c 10 ⁹ , about 1 x 10 ¹⁰ , about 1 ^c 10 ¹¹ , about 1 ^c 10 ¹² , about 1 ^c 10 ¹³ , or about 1 ^c 10 ¹⁴ or more particles per ml, such as DNase resistant particles (DRP) per ml.

In some embodiments, quantification of the genome copies (“GC”) may be used as the measure of the dose contained in the compositions. Any method known in the art can be used to determine the GC number. One method for performing AAV GC number titration is as follows: purified AAV vector samples are first treated with DNase to eliminate un-encapsidated AAV genome DNA or contaminating plasmid DNA from the production process. The DNase resistant particles are then subjected to heat treatment to release the genome from the capsid. The released genomes are then quantitated by real-time PCR using primer/probe sets targeting specific region of the viral genome (usually poly A signal). The AAV vectors can be formulated in dosage units to contain an amount of AAV vector that is in the range of about 1 0x 10 ⁷ GC to about 1 0x 10 ¹⁵ GC (to treat an average subject of 70 kg in body weight), and preferably l .Ox 10 ¹² GC to l .Ox lO ¹⁴ GC for a human patient. In some embodiments, the dose is l .Ox lO ⁷ GC, 5.0x l0 ⁷ GC, l .Ox lO ⁸ GC, 5.0x l0 ⁸ GC, l .Ox lO ⁹ GC, 5.0x l0 ⁹ GC, l .Ox lO ¹⁰ GC, 5.0x l0 ¹⁰ GC, l .Ox lO ¹¹ GC, 5.0x l0 ^u GC, l .Ox lO ¹² GC, 5.0x l0 ¹² GC, l .Ox lO ¹³ GC, 5.0x l0 ¹³ GC, l .Ox lO ¹⁴ GC, 5.0x l0 ¹⁴ GC, or l .Ox lO ¹⁵ GC. In some embodiments, dosages may also be expressed in units of GC per kilogram (kg) of bodyweight (i.e., l x lO ¹⁰ GC/kg, l x lO ¹¹ GC/kg, l x l0 ¹² GC/kg, l x lO ¹³ GC/kg,

1 x 10 ¹⁴ GC/kg, l x lO ¹⁵ GC/kg, respectively). Methods for titering AAV are described in Clark et ak, Hum. Gene Ther., 1999, 10, 1031-1039.

The present disclosure also provides methods of preventing or treating a disease, disorder, or condition associated with elevated homocysteine in a subject in need thereof, comprising administering to the subject any of the AAV nucleic acid molecules encapsidated within any of the AAV vectors, or compositions comprising the same, described herein. In some embodiments, the disease, disorder, or condition associated with elevated homocysteine is CBS deficiency. In some embodiments, the disease, disorder, or condition associated with elevated homocysteine is prevented. In some embodiments, the disease, disorder, or condition associated with elevated homocysteine is treated.

In some embodiments, the disease, disorder, or condition associated with elevated homocysteine is associated with particular genes, such as, for example, methylmalonic aciduria and homocystinuria type C (MMACHC (cblC)), methylmalonic aciduria and homocystinuria type D (MMADHC (cblD-combined and cblD-homocystinuria)),

5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR(cblE)), LMBRl domain containing 1 (LMBRD1 (cblF)), 5-methyltetrahydrofolate-homocysteine

methyltransferase (MTR (cblG)), ATP binding cassette subfamily D member 4 (ABCD4 (cblJ)), THAP domain containing 11 (THAPl 1 (cblX-like)), zinc finger protein 143 (ZNF143(cblX- like)), or a hemizygous variant in host cell factor Cl (HCFC1 (cblX)). In some embodiments, the disease, disorder, or condition associated with elevated homocysteine is

methylenetetrahydrofolate reductase (MTHFR) deficiency. In some embodiments, the disease, disorder, or condition associated with elevated homocysteine is low folate, low B6, or low B 12 status. In some embodiments, the disease, disorder, or condition associated with elevated homocysteine is idiopathic hyperhomocystaneimia. In some embodiments, the disease, disorder, or condition associated with elevated homocysteine is in individuals with only slightly elevated tHcy, which may include atherosclerosis, thrombosis, and osteoporosis.

The present disclosure also provides methods of transducing or transfecting a target cell with AAV nucleic acid molecules encapsidated within AAV vectors, or compositions comprising the same, described herein in vivo or in vitro. The in vivo methods comprise the step of administering an effective dose, or effective multiple doses, of a composition comprising an AAV nucleic acid molecule encapsidated within an AAV vector, or composition comprising the same, described herein to an animal (including a human) in need thereof. If the dose is administered prior to development of a disorder/disease, the administration is prophylactic. If the dose is administered after the development of a disorder/disease, the administration is therapeutic. In some embodiments, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder/disease state being treated, that slows or prevents progression to a disorder/disease state, that slows or prevents progression of a disorder/disease state, that diminishes the extent of disease, that results in remission (partial or total) of disease, and/or that prolongs survival.

In some embodiments, combination therapies are provided. Combination treatments includes both simultaneous treatment and sequential treatments. Combination treatments include any of the methods described herein and standard medical treatments (e.g., anethole,

dithiolethione, or betaine). In some embodiments, any of the methods described herein can be used in combination with lifestyle changes, such as relaxation of a protein restricted diet.

Administration of an effective dose of the compositions may be by routes standard in the art including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. Systemic administration is administration into the circulatory system so that the entire body is affected. Systemic administration includes enteral administration such as absorption through the gastrointestinal tract and parental administration through injection, infusion or implantation. In some

embodiments, the composition is administered to the subject by intramuscular injection or intravenous injection.

Administration of an AAV nucleic acid molecule encapsidated within an AAV vector, or composition comprising the same, described herein may also be accomplished by using any physical method that will transport the AAV vector into the target tissue of an animal.

Administration includes, but is not limited to, injection into muscle, the bloodstream and/or directly into the liver. In some embodiments, the compositions can be prepared as injectable formulations or as topical formulations to be delivered to the muscles by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in the practice of the methods described herein.

For purposes of injection, sterile aqueous solutions can be employed. Such aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose. Solutions of an AAV vector as a free acid or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxpropylcellulose. A dispersion of an AAV vector can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

In some embodiments, the pharmaceutical carriers, diluents or excipients suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In some embodiments, the form is sterile and fluid to the extent that easy syringability exists. In some embodiments, the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens,

chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.

In some embodiments, sterile injectable solutions are prepared by incorporating an AAV vector in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and the freeze drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

In some embodiments, transduction or transfection of cells with an AAV nucleic acid molecule encapsidated within an AAV vector may also be carried out in vitro. In some embodiments, desired target muscle cells are removed from the subject, transduced with an AAV nucleic acid molecule encapsidated within an AAV vector and reintroduced into the subject. Alternately, syngeneic or xenogeneic muscle cells can be used where those cells will not generate an inappropriate immune response in the subject.

Suitable methods for the transduction and reintroduction of transduced cells into a subject are known in the art. In some embodiments, cells can be transduced in vitro by combining an AAV nucleic acid molecule encapsidated within an AAV vector with muscle cells, e.g., in appropriate media, and screening for those cells harboring the DNA of interest using conventional techniques such as Southern blots and/or PCR, or by using selectable markers. Transduced cells can then be formulated into pharmaceutical compositions, and the composition introduced into the subject by various techniques, such as by intramuscular, intravenous, subcutaneous and intraperitoneal injection, or by injection into smooth and cardiac muscle, by using, for example, a catheter.

Transduction of cells with the AAV nucleic acid molecules encapsidated within AAV vectors described herein results in sustained expression of CBS. In some embodiments, the AAV-borne CBS transgene is efficiently expressed in transduced cells. Methods to measure protein expression levels of CBS include, but are not limited to, Coomasie blue or silver staining of protein in a separation media, such as gel electrophoresis, Western blotting,

immunocytochemistry, other immunologic-based assays; assays based on a property of the protein including but not limited to, enzyme assays, ligand binding or interaction with other protein partners. Binding assays are also well known in the art. For example, a BIAcore instrument can be used to determine the binding constant of a complex between two proteins. The dissociation constant for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme linked immunoabsorbent assays (ELISA) and radioimmunoassays (RIA), or

determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UV absorption, circular dichrosim, or nuclear magnetic resonance (NMR).

The present disclosure also provides methods of regulating biological processes, including cystathionine production, by regulating the expression and/or activity of CBS. In some embodiments, the methods regulate cystathionine production in an animal or human patient, wherein the patient is protected from or treated for a disease that is amenable to regulation of cystathionine production, such as homocystinuria and conditions/symptoms related thereto (e.g., dislocated optic lenses, skeletal disorders, mental retardation and premature arteriosclerosis and thrombosis). As used herein, the phrase“protected from a disease” refers to reducing the symptoms of the disease; reducing the occurrence of the disease, and/or reducing the severity of the disease. Protecting a patient can refer to the ability of a therapeutic composition, when administered to a patient, to prevent a disease from occurring and/or to cure or to treat the disease by alleviating disease symptoms, signs or causes. As such, to protect a patient from a disease includes both preventing disease occurrence (prophylactic treatment) and treating a patient that has a disease or that is experiencing initial symptoms or later stage symptoms of a disease (therapeutic treatment). More specifically, a therapeutic composition as described herein, when administered to a patient by the methods described herein, preferably produces a result which can include alleviation of the disease (e.g., reduction of at least one symptom or clinical manifestation of the disease), elimination of the disease, alleviation of a secondary disease resulting from the occurrence of a primary disease, or prevention of the disease. In some embodiments, administration of the therapeutic composition can produce a result that can include increased accumulation of downstream metabolites of transsulfuration in a mammal.

The present disclosure also provides uses of any of the compositions described herein for the preparation of a medicament for the prevention or treatment of a disease, disorder, or condition associated with elevated homocysteine in a human subject.

The present disclosure also provides uses of any of the compositions described herein for the prevention or treatment of a disease, disorder, or condition associated with elevated homocysteine in a human subject.

In order that the subject matter disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the claimed subject matter in any manner. Throughout these examples, molecular cloning reactions, and other standard recombinant DNA techniques, were carried out according to methods described in Maniatis et al., Molecular Cloning - A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989), using commercially available reagents, except where otherwise noted.

Examples

Example 1: Materials and Methods

pAAVrh.10-CAG-hCBS plasmid construction:

The hCBS coding sequence from pUCAHCBS (see, Kruger et al., Proc. Natl. Acad.

Sci. USA, 1994, 91, 6614-6618) was PCR amplified using the primers 5'-CAGTCTCGAAC TTAACATGCCTTCTGAGACCCCC-3' (SEQ ID NO:3) and 5'-GGGCCCATTACCGAT ACTTCACTTCTGGTCCGCTCC-3' (SEQ ID NO:4), digested with AF1II and EcoRV-HF and cloned into the vector backbone of pAAV-TBG-FFLUC digested with BstAP 1 and EcoRV-HF via blunt end ligation. Because the orientation of the clone could not be obtained in the correct direction, a subsequent cloning was performed in which the hCBS insert was removed with Msel an Xhol and cloned into pAAV-CAG-MCS digested with Mfel and Sacl via blunt end ligation. The final plasmid, pAAV-CAG-hCBS, was verified by sequencing. The product pAAV-CAG-hCBS can be produced by: 1) using the baculovirus system which uses the insect virus to transduce insect cells with all of the components used to generate the AAV; 2) from a cell line that has been genetically modified to produce AAV-CAG-hCBS; and 3) from a herpes virus system that infect either proviral cell lines that contain an integrated rAAV genome or cells transfected with an rAAV plasmid or infected with rAAV.

pAAVl-CMV-hCBS plasmid construction:

pAAVl-CMV-hCBS was constructed by inserting a Notl fragment containing the hCBS cDNA fragment obtained from the 15ACY6PP plasmid (Invitrogen), which contains a codon optimized CBS gene. The resulting fragment was then cloned into Notl site of the pAAV- GFP vector (Addgene) containing CMV promoter and the AAV inverted terminal repeat sequences (ITR). pAAVl-CMV-hCBS was verified by sequencing.

AA V viral production:

AAVrh.lO-CAG-hCBS was produced essentially as described in Rosenberg et al.

(Human Gene Therapy Clin. Devel., 2014, 25, 164-177). In brief, HEK 293 cells were co transfected with pAAV-CAG-hCBS and pPAKMA.rhlO, which is a helper and packaging plasmid that provides the AAV Rep proteins derived from AAV2 needed for vector replication, the AAVrh.10 viral structural (Cap) proteins, and the Ad helper functions of E2, E4, Va RNA. After 72 hours, cells were harvested and a crude viral lysate was prepared by four freeze/thaw cycles and clarified by centrifugation. AAVrh. lO-CAG-hCBS was purified by iodixanol gradient and QHP anion exchange chromatography, and concentrated using an Amicon Ultra- 15 100K centrifugal filter device. Vector genome titers were determined by quantitative PCR.

Mouse model:

Tg-I278T Cbs ^/_ mice were generated as previously described (see, Wang et al., Hum. Mol. Genet., 2005, 14, 2201-2208). In brief male Tg-I278T Cbs ^/_ mice were mated with female Tg-I278T Cbs ^+/ mice in cages with water bottles containing 25 mM ZnS0 ₄ All pups were genotyped between 10 and 14 days of age. At the time of weaning (around 30 days), mice were put in new cages with non-zinc water. The average age of the mice in this study was 310 days (range 288-365), and an equal number of male (n=10) and females (n=10) were used. Serum was collected from animals by retro-orbital bleed.

Gene delivery and experimental schemes:

Before injection (D-7), all mice had serum collected for baseline tHcy and methionine determination. AAVrh. lO-CAG-hCBS was delivered into Tg-I278T Cbs ^/_ mice (n= 16) via a single lateral tail vein injection (IV) or intraperitoneal injection (IP) at doses of l.lxlO ¹⁰ (n=2), 5.6xl0 ¹⁰ (n=2), l. lxlO ¹¹ (n=2), and 5.6xl0 ^u (n=10) genome copies/mouse. Saline (150 pi) was injected for control mice. Serum was prepared from blood collected at D7, D14, D21, D28, D42, D63 and D78 after injection. All mice were euthanized by isoflurane overdose at either 21 or 78 days after injection. Liver, kidney, and serum were collected at this time.

Immunoblots:

Tissue homogenates from liver were prepared in 10 mM Tris-HCl (pH 7.5)

supplemented with protease inhibitors (Roche). 25 pg of lysate was separated by 4-12% SDS- PAGE (Invitrogen) under reducing conditions and transferred to nitrocellulose. Blots were probed with HA antibody (H9658, Sigma) for endogenous I278T CBS protein, rabbit anti-CBS sera for total CBS protein (see, Kruger, Proc. Natl. Acad. Sci. USA, 1994, 91, 6614-6618), and actin antibody (A5441, Sigma) for loading control. Gel images were captured and quantified using the FluorChem SP system (Alpha Innotech).

Measurement of serum tHcy and tissue CBS activity:

CBS activity in the liver and kidney was measured using a Biochrom 30 amino acid analyzer (Cambridge, UK) as described previously (see, Wang et ah, Hum. Mol. Genet., 2005,

14, 2201-2208). One unit of activity is defined as nmoles of cystathionine formed/mg of tissue lysate protein/hour. Serum and tissue tHcy (a sum total of free and disulfide-bonded

homocysteine) and methionine levels were measure using the Biochrom 30 amino acid analyzer as performed previously (see, Gupta et ah, FASEB J., 2009, 23, 883-893).

Statistical analysis:

Values in text are mean ± SEM. Differences between two groups were analyzed by the Mann-Whitney U test (unpaired and non-parametric test). Significance between more than two groups was determined using one-way ANOVA followed by Tukey’s multiple comparison tests employing GraphPad Prism 6.0 software. Statistical significance was accepted at the value of P < 0.05.

pAAVl-CMV-hCBS plasmid construction and viral production:

An hCBS cDNA fragment was obtained from 15ACY6PP plasmid (Invitrogen) by Notl digestion, and cloned into the Notl site of the pAAV-GFP vector (Addgene) containing the CMV promoter and the AAV inverted terminal repeat sequences (ITR). pAAVl-CMV-hCBS was verified by sequencing.

Gene delivery and experimental schemes:

AAVl-CMV-hCBS was delivered into Tg-I278T Cbs ^/_ mice (n=49) via a single lateral tail vein injection (IV), intraperitoneal injection (IP), or intramuscular injection (IM) at variable dosages of vector number (see, Figure 6). Serum was prepared from blood collected before (D-7) and weekly after injection for tHcy and methionine determination. All mice were euthanized by isoflurane overdose at the end of experiments. Measurement of serum tHcy/Met, Western blot analysis, and tissue CBS activity were performed as previously described herein.

Example 2: Animal Model Studies

In an initial study, an rAAV vector was constructed in which the CMV promoter was placed upstream of the hCBS cDNA. This construct was then packaged in a cell line that produced AAV1 serotype viral particles (rAAVl-CMV-hCBS). Forty-nine Tg-I278T Cbs ^_/ mice were injected with either rAAVl-CMV-hCBS (n=37) or PBS (n=12; see Figure 5A). Mice were injected with four different doses of virus (1.25-8 x 10 ¹² genomes/mouse) using three different routes; intramuscular injection (IM), intraperitoneal injection (IP), or intravascular injection (IV). A modest but statistically significant lowering of tHcy was observed in the IP injected animals treated with either the 4xl0 ¹² or 8xl0 ¹² dose (see, Figure 5B). A small but significant increase in liver CBS activity was also observed (see, Figure 5C).

Another study was carried out using a modified vector (see, Figure 1 A). The promoter in the modified vector was changed from CMV to the CAG promoter. The CAG promoter is a hybrid promoter consisting of the CMV enhancer fused to the b-actin promoter, and has extremely high expression in liver (Nguyen et al., J. Surg. Res., 2008, 148, 60-66; and Sen et al., Scientific Reports, 2013, 3, 1832). In addition, the serotype of the virus was changed from AAV1 to AAVrh.10.

This modified vector was used to produce virus that was injected into a total of 16 Tg- I278T Cbs ^/_ mice either IV or IP at doses ranging from l. lxlO ¹⁰ to 5.6xlO ^u genomes/mouse (see, Figure IB). In addition, four control animals were mock injected. Over the course of the next 21 days a highly significant decrease in mean tHcy was observed that was dosage dependent (see, Figure 2A). Mice injected with the highest dose (5.6xlO ^u genomes/mouse) showed a 97% decrease in tHcy after 14 days. This tHcy level was similar to what was observed in wild-type C57BL6 mice (Esse et al., FASEB J., 2014, 28, 2686-2695). Methionine was not significantly lowered by the treatment, but it should be noted that adult CBS deficient mice, unlike humans, do not show significant hypermethionemia. After 21 days, nine of the mice were euthanized and their livers were assessed for CBS activity. Significant levels of CBS activity were observed in all the treated mice, with the highest amounts of activity observed in the livers of animals treated with the highest dose of virus. In these animals, the amount of activity observed was similar to that observed in wildtype C57BL6 mice. A significant increase in kidney CBS activity was not observed, suggesting that either the AAVrh.10 serotype has poor tropism toward kidney, or the CAG promoter did not function well in kidney. No significant difference has been observed between the IP or IV routes of delivery (see, Figure 2C). Figure 2D shows a summary of individual mice treatment for experiments in Figures 2C.

Seven mice injected with the highest dose were followed for up to 77 days (see, Figure 3A). tHcy showed a >90% decrease from pre-injection levels from D14 through D78.

Examination of AAV-encoded CBS protein indicated that protein levels were similar to those observed at D21 (see, Figure 3B). CBS activity at D78 was similar to that observed in control WT CBS mice (see, Figure 4A). A noticeably reduced alopecia phenotype was observed in the treated animals (see, Figure 4B).

A separate cohort of Tg-I278T ( Vxv ^~ (n=20) and Tg-I278T Cbs ^+/~ (n=9) mice were generated and kept on Zn-water (25 mM ZnS0 ) to induce transgene expression until the termination of the experiment. AAVrh. lO-CAG-hCBS (5.6 x 10 ¹¹ ) (n=10) or saline (n=10) was delivered into Tg-I278T ( Vxs mice via a single lateral vein injection. Three groups of mice, including vector-delivered mice, saline-delivered mice, and Tg-I278T Cbs ^+/ mice, will be monitored for one year. At the indicated time points (see, Figure 8), all mice will be monitored for weight changes, serum tHcy, methionine levels, and phenotypic changes in fur and whiskers. After 119 days, AAVrh. lO-CAG-hCBS treatment did not affect weight (see, Figure 9A).

Moreover, AAVrh. lO-CAG-hCBS treatment resulted in a 94% reduction of serum tHcy levels (20 mM vs. 341 pM, P< 0.0001) at D21 that was maintained until at least D49 (see, Figure 9B) without affecting serum methionine levels (see, Figure 9C). AAVrh. lO-CAG-hCBS also induced correction of phenotypes with thicker whiskers and body hairs, which was maintained for 119 days post-treatment (see, Figure 10).

In addition, the above experiments were repeated in T191M CBS C3H strain having a different genetic background from the previously used C57BL6 strain. AAVrh. lO-CAG-hCBS (5.6 x 10 ¹¹ ) were delivered into lg ^' -l ^' 191M Cbs ^{~ ~} (C3H strain background) mice (n=2) and Tg- negative Cbs ^/_ mice (n=2) via a single lateral vein injection. Serum was prepared from blood collected at D7, D22 and D27 and analyzed for tHcy and methionine (see, Figure 7A). The sera from wild type mice ( Cbs ^+/+ or Cbs ^+A ) and ( Vxv in the indicated strain backgrounds was also analyzed. Mice administered vector had a mean tHcy decrease by 83% (Ave. 56 pM vs. 328 pM; P<0.00004) 27 days after injection. Western blot analysis confirmed the expression of vector- derived hCBS protein in the liver tissue (see, Figure 7B).

In sum, the effectiveness of two different AAV vectors was examined utilizing the Tg- I278T Cbs mouse model of CBS deficiency. Large differences were found in the effectiveness of the different vectors. A virus with the AAVrh.10 serotype containing a CAG promoter driving expression of the hCBS cDNA, however, was highly effective in expressing CBS in the liver and lowering tHcy for an extended period of time. In addition, because the AAVrh.10 serotype is a Rhesus monkey virus, it is expected that it would be less likely to encounter pre-existing neutralizing antibodies in humans.

The data presented herein compares favorably to another approach to treat CBS deficiency, namely enzyme replacement therapy using PEGylated human truncated CBS. Using the exact same Tg-I278T Cbs ^/_ mouse model described here, Majtan et al. found that they could achieve sustained lowering of tHcy from about 320 mM to about 100 mM with IP injection of 7.5 mg/kg PEG-CBS given 3 times a week (Majtan et al., Molecular Therapy: The Journal of the American Society of Gene Therapy, 2018, 26, 834-844). In the studies described here, however, a greater reduction of tHcy was actually observed. At 77 days after injection, the mean tHcy in the AAVrh.10-CAG-hCBS treated animals was only 26 pM. Also, like the PEG-CBS treated mice, a substantial improvement in the facial alopecia phenotype was observed. Thus, the gene therapy approach described here has the advantage of both greater tHcy lowering and the avoidance of multiple injections. Overall, the results presented herein indicate that AAVrh.10- CAG-hCBS may be a promising approach to treat CBS deficiency.

Various modifications of the described subject matter, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, and the like) cited in the present application is incorporated herein by reference in its entirety.