WO2021183895A1

WO2021183895A1 - Treatment of fabry disease with aav gene therapy vectors

Info

Publication number: WO2021183895A1
Application number: PCT/US2021/022117
Authority: WO
Inventors: Shinong Long; Peter Colosi; Hassib AKEEFE; Jon LEBOWITZ
Original assignee: Biomarin Pharmaceutical Inc.
Priority date: 2020-03-13
Filing date: 2021-03-12
Publication date: 2021-09-16

Abstract

Provided herein are compositions and methods of treating an α-galactosidase A deficiency by normalizing levels of α-galactosidase A protein in a subject having Fabry Disease.

Description

TREATMENT OF FABRY DISEASE WITH AAV GENE THERAPY VECTORS

FIELD

[0001] Provided herein are recombinant adeno-associated virus (rAAV) vectors and virus particles for treating Fabry Disease by increasing expression and secretion of a-Galactosidase A to increases clearance of globotriaosylceramide (Gb-3 or GL-3) in target tissues of a subject.

BACKGROUND

[0002] Fabry Disease is a rare X-linked inherited multisystem lysosomal storage disorder caused by the deficiency of a-galactosidase A (a-Gal A), an enzyme responsible for the degradation of globotriaosylceramide (abbreviated as Gb-3 or GL-3), globatriaosylsphingosine (Lyso-Gb3), and other neutral glycosphingolipids. A mutation in the encoding gene, called GLA, can result in the absence of this enzyme or production of inactive enzyme. The classic form of Fabry Disease occurs in male patients who have less than 1% a-Gal A activity. The deficiency of functional enzyme results in the accumulation of neutral glycosphingolipids in the lysosomes of a variety of organs including the liver, kidneys and heart as well as the endothelial and smooth muscle cells of blood vessels. Symptoms include neuropathic pain (e.g., burning, tingling pain), recurrent fever, gastrointestinal problems, and skin abnormalities (e.g., angiokeratomas). This Gb-3 accumulation over time leads to an impairment of organ function leading to progressive kidney failure, cardiac complications, stroke, and reduced life expectancy.

[0003] Enzyme replacement therapy (ERT) is the current standard of care for the treatment of Fabry Disease but does not represent a cure. It requires bi-weekly intravenous administration for the lifetime of the patients. Additionally, 50-55% of patients experience at least one infusion related reaction and a significant number of patients (55-58%) develop neutralizing antibodies to the a-galactosidase A, rendering ERT ineffective.

[0004] The inventors have developed a gene therapy approach using adeno-associated viral vectors (AAV) to increase the expression and secretion of a-galactosidase A enzyme in cells to increase clearance of Gb-3 and/or Lyso-Gb-3. SUMMARY

[0005] The embodiments described herein relate to a vector construct, a recombinant replication deficient AAV particle, cells, and pharmaceutical compositions for delivering functional a-galactosidase A to a subject in need thereof, particularly a subject with Fabry Disease, or a deficiency in functional a-galactosidase A. The embodiments described herein also relate to the use of such AAV particles or such vector constructs to deliver a gene encoding a- galactosidase A to liver, kidney, heart and/or plasma cells of patients (human subjects) diagnosed with Fabry Disease, or a deficiency in functional a-galactosidase A.

[0006] In one aspect, the embodiments described herein provide a vector construct comprising a nucleic acid sequence that encodes a functional a-galactosidase A protein. In one or more embodiments, the functional a-galactosidase A comprises an amino acid sequence at least 90%, 95% or 98% identical to amino acids 32 to 429 of SEQ ID NO: 2 (a human a-galactosidase A, or “alpha-Gal A”). In example embodiments, the nucleic acid sequence encoding the functional a-galactosidase A is a wild-type sequence, of which SEQ ID NO: 1 is one example, or is codon optimized, or is a variant. Alternative codon optimized human a-galactosidase A- encoding sequences are set out as SEQ ID NO: 3-23. In example embodiments, the nucleic acid sequence encoding the functional a-galactosidase A comprises a nucleotide sequence having at least 95% homology to at least 100, 200, 300, 400, or 500 consecutive bases of any of SEQ ID NO: 3, 4 or 8 or at least 96%, 97%, 98% or 99% identical to the full length of any of SEQ ID NO: 3, 4 or 8, and which preferably encodes functional human a-galactosidase A protein at least 95% identical to amino acids 32 to 429 of SEQ ID NO: 2. The coding sequence for a- galactosidase A is, in some embodiments, codon optimized for expression in humans.

[0007] In one or more embodiments, the nucleic acid sequence encoding a-galactosidase A is operably linked to one or more heterologous expression control elements. Preferably, expression of the a-galactosidase A-encoding transgene is controlled by liver-specific expression control elements. Thus, in such embodiments, in the vector constructs described herein, the nucleic acid sequence encoding a-galactosidase A is operably linked to a heterologous liver-specific transcription regulatory region. In some embodiments, in the vector constructs described herein, the expression control elements include one or more of the following: a promoter and/or enhancer; an intron; optionally an exon from the same gene as the intron; and a polyadenylation (poly A) signal. Such elements are further described herein. [0008] The liver-specific transcription regulatory region may comprise one or more liver- specific expression control elements. In one or more embodiments, the liver-specific transcription regulatory region is a synthetic promoter sequence comprising a fragment of a human alpha- 1 -antitrypsin (hAAT) promoter, and/or a fragment of a hepatic control region (HCR) enhancer, and/or a fragment of an apolipoprotein E (ApoE) enhancer. In some embodiments, the liver-specific transcription regulatory region comprises (a) one or more promoters selected from (i) an alpha anti-trypsin (hAAT) proximal promoter sequence at least 90% identical to SEQ ID NO: 60 or a fragment thereof, (ii) an AAT promoter distal X region, or (iii) an AAT promoter distal region; and (b) one or more enhancers, optionally an ApoE/HCR enhancer at least 90% identical to SEQ ID NO: 61. In an example embodiment, the sequence of the liver-specific transcription regulatory region comprises a nucleotide sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO: 59.

[0009] In some embodiments, the vector construct comprises one or more introns. In some embodiments, the intron also enhances expression of the a-galactosidase A-encoding nucleic acid, such that increased levels are detectable in the liver, kidneys, heart and/or plasma. In one or more embodiments, the intron comprises a fragment of a human alpha- 1 antitrypsin (hAAT) intron, and/or a fragment of a hemoglobin intron.

[0010] In one or more embodiments, the intron comprises a nucleotide sequence at least 90% identical to any one of SEQ ID NO: 63-69. In some embodiments, the intron comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 64 or a fragment thereof that retains expression enhancing activity, e.g. an intron that is about 800 to about 1000 nucleotides in length, or a fragment about 600-700 nucleotides in length, about 400-500 nucleotides in length, or about 200-300 nucleotides in length. Such fragments may comprise a nucleotide sequence at least 90% identical to SEQ ID NO: 65 or a nucleotide sequence at least 90% identical to SEQ ID NO: 66. In some embodiments, the fragments comprise a nucleotide sequence at least 90% identical to SEQ ID NO: 66 and is missing at least nucleotides 300-900, or 300-500, of SEQ ID NO: 64.

[0011] In some embodiments, the vector construct may further comprise an exon sequence or fragment thereof, preferably adjacent to an intron sequence, e.g. an hAAT intron adjacent to an hAAT exon and/or a hemoglobin intron adjacent to a hemoglobin exon. [0012] In some embodiments, the vector construct comprises a polyadenylation signal, optionally a bovine growth hormone (bGH) polyA signal (e.g., SEQ I D NO: 72) or preferably a human growth hormone (hGH) polyA signal or fragment thereof (e.g., SEQ ID NO: 73-79). [0013] In any of the foregoing embodiments, the length of the intron and the length of the polyA signal may be adjusted so that the length of the vector insert beginning at one ITR and ending with the second ITR is between about 4kb to about 4.5kb in size.

[0014] The vector construct is preferably a recombinant AAV vector construct. In some embodiments, the vector construct comprises (a) one or both of (i) an AAV 5' inverted terminal repeat (ITR) and (ii) an AAV3’ ITR; (b) a promoter and/or enhancer, e.g. a liver-specific transcription regulatory region; and (c) a nucleic acid sequence encoding a functionally active human a-galactosidase A protein. In some embodiments, the vector construct comprises (a) an AAV 5' inverted terminal repeat (ITR) sequence; (b) a promoter and/or enhancer, e.g. a liver- specific transcription regulatory region; (c) a nucleic acid sequence encoding a functionally active human a-galactosidase A protein; (d) an intron; (e) a polyadenylation signal; and (f) an AAV 3' ITR. The AAV 5' ITR and/or AAV 3' ITR may be from a heterologous AAV pseudotype (which may or may not be modified as known in the art). In some embodiments, the 5’ ITR and 3’ ITR sequences are derived from AAV2 (e.g. SEQ ID NO: 80 and 81). In one or more embodiments, the vector construct is an AAV vector genome about 3 kb to about 5 kb in size, or about 4 kb to about 4.5 kb in size.

[0015] In any of the foregoing embodiments, the vector construct comprises a nucleotide sequence at least 80%, 85%, 90% or 95% identical to any of SEQ ID NOS: 24-58. Such vectors, for example, preferably comprise flanking ITRs, a nucleic acid sequence encoding a functionally active human a-galactosidase A protein, optionally a tag that improves lysosomal uptake, a liver- specific regulatory region, an hAAT intron or hemoglobin intron, and a growth hormone polyA signal.

[0016] In another aspect, provided herein is a recombinant adeno-associated virus (rAAV) particle comprising an AAV capsid and the vector construct as described in one or more of the embodiments herein. In some embodiments, the recombinant AAV (rAAV) particle used for delivering the a-galactosidase A-encoding gene ("rAAV.GLA" or “AAV-GLA”) has tropism for the liver. In such embodiments, the rAAV comprises an AAV capsid with liver tropism, for example, an AAV5 capsid at least 90% identical to SEQ ID NO: 91, or a simian AAV capsid, optionally a baboon-derived AAV capsid, or a variant thereof, that exhibits liver tropism. In one or more embodiments, the AAV capsid is a capsid for which preexisting humoral immunity is similar to AAV5, or reduced compared to AAV5, e.g., when evaluated by IVIG neutralization in vitro.

[0017] In another aspect, provided herein are methods for the production of an AAV particle, useful as a gene delivery vector, the method comprising the steps of: (1) providing an insect cell comprising one or more nucleic acid constructs (a) comprising a vector construct as described herein comprising a nucleic acid as described herein that is flanked by two AAV ITR nucleotide sequences; (b) a nucleotide sequence encoding one or more AAV Rep proteins which is operably linked to a promoter that is capable of driving expression of the Rep protein(s) in an insect cell (c) a nucleotide sequence encoding one or more AAV capsid proteins which is operably linked to a promoter that is capable of driving expression of the capsid protein(s) in the insect cell; wherein (b) and (c) are in the same expression cassette or in two different expression cassettes; and (d) optionally genes encoding AAP and MAAP contained in the VP2/3; (2) culturing the insect cell defined in (1) under conditions conducive to the expression of the Rep and the capsid proteins; and, optionally (3) recovering the AAV particle.

[0018] In yet another aspect, provided herein are pharmaceutical compositions comprising the vector construct described herein or the rAAV particle described herein, and a sterile pharmaceutically acceptable diluent, excipient or carrier.

[0019] In a further aspect, provided herein are methods of delivering a GLA gene to a mammalian subject. Such methods include methods of expressing a-galactosidase A in a mammalian subject comprising administering to the subject a composition comprising the vector construct described herein, the rAAV particle described herein, or the pharmaceutical composition described herein, thereby expressing the encoded a-galactosidase A enzyme in the subject. Preferably, in such methods, the mammal is a human and the a-galactosidase A is functional human a-galactosidase A as described herein. Such methods include a method of expressing a-galactosidase A in the liver of a mammal by administering an amount of the vector construct, rAAV particle or pharmaceutical composition effective to increase the level of a- galactosidase A expression and secretion in the liver of the mammal. Such methods also include a method of increasing the level of functional a-galactosidase A in the blood, e.g. plasma, or tissues or lysosomes of a mammal by administering an amount of the vector construct, rAAV particle or pharmaceutical composition effective to increase the level of functional a- galactosidase A in the blood of a mammal. Such methods also include a method of treating a deficiency in functional a-galactosidase A in a mammal by administering an amount of the vector construct, rAAV particle or pharmaceutical composition effective to increase the level of functional a-galactosidase A in the blood, e.g. plasma, or tissues or lysosomes of a mammal. In some embodiments, the amount of the vector construct, rAAV particle or pharmaceutical composition is effective to increase the level of functional a-galactosidase A activity in blood to at least about 7.8-14.6 nmol/hr/ml (plasma) or higher. In some embodiments, the amount of the vector construct, rAAV particle or pharmaceutical composition is effective to increase the level of functional a-galactosidase A protein in blood to at least about 1 ng/mL or 1.5 ng/mL or 2 ng/mL or 2.5 ng/mL or 3 ng/mL or higher.

[0020] Such methods also include a method of treating Fabry Disease in a mammal, or treating or preventing any symptom thereof, comprising administering a therapeutically effective amount of the vector construct, rAAV particle or pharmaceutical composition. In one or more embodiments, such methods reduce the accumulation of Gb-3 and/or Lyso-Gb-3 in plasma or in tissues such as liver or heart or kidney or brain by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the levels without treatment, or to the levels seen in healthy humans. Normal human ranges of lyso-Gb3 range from about 0-2 ng/ml (plasma) and of Gb3 from about 0.9±0.4 pg/ml (plasma). In one or more embodiments, such methods reduce the frequency or severity of kidney function eGFR decline, heart problems, pain, GI problems, dizziness, vertigo, and in severe cases, stroke, changes in the appearance of the eyes, such as corneal opacities, heart problems such as an irregular or abnormal heart rhythm or beat, hearing loss, tinnitus, abdominal pain, nausea, vomiting, constipation and diarrhea, kidney impairment including cysts or progressive kidney failure, skin abnormalities such as angiokeratoma (small raised dark red spots), absent sweating (anhidrosis), diminished sweating (hypohidrosis), excessive sweating (hyperhidrosis), temperature sensitivity, recurrent fever, and neuropathic pain (e.g., burning or tingling sensations of the skin).

[0021] In any of the methods described herein, the rAAV particle is delivered at a dose of about 1 x 10¹¹ to about 6 x 10¹⁴ vg/kg in an aqueous suspension. In any of the methods described herein, the administration of the vector construct, rAAV particle, or pharmaceutical composition may further comprise administration of prophylactic or therapeutic corticosteroid treatment, and/or may further include administration of a second therapy for treating Fabry Disease. In any of the methods herein, prior to administration of an AAV particle to a patient as described above, the prospective patient may be assessed for the presence of anti-AAV capsid antibodies or anti- AAV neutralizing antibodies that are capable of blocking cell transduction or otherwise reduce the overall efficiency of the treatment.

[0022] Other embodiments will be evident to one skilled in the art upon reading the present specification.

BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 depicts expression levels of various constructs (SEQ ID 24, 29, 32, 39, 41, 42, 43, 44 and 45) in HepG2 cells.

[0024] Figure 2 depicts the rise in a-galactosidase A activity in plasma of GLA knockout mice over time after administration of various AAV5 particles comprising a-galactosidase A- encoding vector constructs, up to 8 weeks after dosing.

[0025] Figures 3, 4, 5 and 6 respectively depict a-galactosidase A activity in plasma, kidney, heart and liver of GLA knockout mice at 8 weeks after administration of various AAV5 particles comprising a-galactosidase A-encoding vector constructs.

[0026] Figures 7A-B, 8A-B, and 9A-B respectively show the levels of total Gb-3 and Lyso- Gb-3 (ng Lyso-Gb-3/mg protein) in kidney, heart and liver of the GLAko mice at 8 weeks after injection of AAV particles.

[0027] Figures 10A-B shows the level of total Gb-3 and Lyso-Gb-3 in plasma of the GLAko mice at 8 weeks.

[0028] Figures 11, 12, 13, 14 and 15 respectively depict a-galactosidase A activity in plasma, kidney, heart, liver and spleen of Rag2 ^/_ mice 8 weeks after administration of various doses of AAV5 particles comprising a codon optimized “JCAT” a-galactosidase A-encoding vector constructs.

[0029] Figure 16 depicts a schematic of the organization of a variety of vector constructs. [0030] Figure 17 depicts the a-galactosidase A activity in plasma of Rag2 ^/_ mice over time after administration of various AAV5 particles comprising different a-galactosidase A-encoding vector constructs with different fragments of introns and different fragments of polyA signals. [0031] Figure 18 depicts a-galactosidase A activity in plasma of GLAko mice 8 weeks after administration of various doses of AAV5 particles comprising one optimal a-galactosidase A- encoding vector construct.

[0032] Figures 19A and 19B show the levels of total Gb-3 (total acyl Gb3/mg protein) and Lyso-Gb-3 (ng Lyso-Gb-3/mg protein) in kidney tissue of GLA knockout mice at 8 weeks after administration of AAV5 particles comprising an a-galactosidase A-encoding vector construct designated GLA-co-4.

[0033] Figures 20, 21 and 22 respectively depict a-galactosidase A activity in kidney, heart and liver of GLA knockout mice at 8 weeks after administration of AAV5 particles comprising an a-galactosidase A-encoding vector construct designated GLA-co-4.

[0034] Figure 23 depicts plasma a-galactosidase A activity of GLA knockout mice at 3, 5 and 8 weeks after administration of a 6el3 vg/kg dose of AAV5 particles comprising a- galactosidase A-encoding vector constructs designated GLA-co-3, GLA-co-4 and GLA-co-5. [0035] Figures 24 and 25 respectively depict a-galactosidase A activity in plasma and kidney tissue of GLA knockout mice at 8 weeks after administration of various doses of AAV5 particles comprising a-galactosidase A-encoding vector constructs designated GLA-co-3, GLA-co-4 and GLA-co-5, with the results from a higher dose of AAV5 particles comprising the codon optimized “JCAT” or wild-type (WT) constructs for comparison.

[0036] Figures 26A and 26B show the levels of total Gb-3 (total acyl Gb3/mg protein) and Lyso-Gb-3 (ng Lyso-Gb-3/mg protein) in kidney tissue of GLA knockout mice at 8 weeks after administration of various doses of AAV5 particles comprising a-galactosidase A-encoding vector constructs designated GLA-co-3, GLA-co-4 and GLA-co-5.

DETAILED DESCRIPTION

[0037] Provided herein are nucleic acids or vector constructs encoding functionally active therapeutic a-galactosidase A protein, AAV vector genomes and replication deficient rAAV particles comprising such vector constructs, and pharmaceutical compositions comprising such vector constructs, vector genomes and AAV particles. The compositions and methods of the invention may provide improved AAV virus production yield and/or simplified purification and/or enhanced expression and/or enhanced a-galactosidase A activity in plasma, or enhanced a-galactosidase A activity or uptake in tissues, particularly in liver, kidney, heart or brain. Also provided herein are methods of making the vector constructs, AAV vector genomes and replication deficient rAAV particles comprising such vector constructs. Further provided herein are methods of treating a deficiency in functional a-galactosidase A, or Fabry Disease.

[0038] Definitions:

[0039] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, e.g. Singleton et ak, Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994); Sambrook et ak, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). For purposes of the present disclosure, the following terms are defined below.

[0040] As used herein, in the context of gene delivery, the term "vector" or "gene delivery vector" may refer to a particle that functions as a gene delivery vehicle, and which comprises nucleic acid (i.e., the vector genome comprising any of the vector constructs described herein) packaged within, for example, an envelope or capsid. A gene delivery vector may be a viral gene delivery vector or a non-viral gene delivery vector. Alternatively, in some contexts, the term "vector" may be used to refer only to the vector genome or vector construct. Viral vectors suitable for use herein may be a parvovirus, an adenovirus, a retrovirus, a lentivirus or a herpes simplex virus. The parvovirus may be an adenovirus-associated virus (AAV).

[0041] As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. There are numerous serotypes of AAV that have been characterized. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Voh 1, pp. 169-228; and Bems, 1990, Virology, pp. 1743-1764, Raven Press, (New York); Gao et ak, 2011, Methods Mol. Biol. 807: 93-118; Ojala et ak, 2018, Mol. Ther. 26(1): 304-19. However, it is fully expected that these same principles will be applicable to additional AAV serotypes since it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to "inverted terminal repeat sequences" (ITRs).

[0042] As used herein, an "AAV vector construct" refers to nucleic acids, either single- stranded or double-stranded, having at least one of (i) an AAV 5' inverted terminal repeat (ITR) sequence and (ii) an AAV 3' ITR, flanking a protein-coding sequence (in one embodiment, a functional therapeutic protein-encoding sequence, e.g. a-galactosidase A-encoding sequence) operably linked to transcription regulatory elements (also called “expression control elements”) that are heterologous to protein-encoding sequence and/or heterologous to the AAV viral genome, i.e., one or more promoters and/or enhancers and, optionally, a polyadenylation sequence and/or one or more introns inserted between exons of the protein-coding sequence. A single-stranded AAV vector refers to nucleic acids that are present in the genome of an AAV virus particle, and can be either the sense strand or the anti-sense strand of the nucleic acid sequences disclosed herein. The size of such single-stranded nucleic acids is provided in bases. A double-stranded AAV vector refers to nucleic acids that are present in the DNA of plasmids, e.g., pUC19, or genome of a double-stranded virus, e.g., baculovirus, used to express or transfer the AAV vector nucleic acids. The size of such double-stranded nucleic acids in provided in base pairs (bp).

[0043] The AAV vector constructs provided herein in single strand form are less than about 7.0 kb in length, or are less than 6.5 kb in length, or are less than 6.4 kb in length, or are less than

6.3 kb in length, or are less than 6.2 kb in length, or are less than 6.0 kb in length, or are less than

5.8 kb in length, or are less than 5.6 kb in length, or are less than 5.5 kb in length, or are less than

5.4 kb in length, or are less than 5.3 kb in length, or are less than 5.2 kb in length or are less than

5.0 kb in length, or are less than 4.8 kb in length, or are less than 4.6 kb in length, or are less than

4.5 kb in length, or are less than 4.4 kb in length, or are less than 4.3 kb in length, or are less than

4.2 kb in length, or are less than 4.1 kb in length, or are less than 4.0 kb in length, or are less than

3.9 kb in length, or are less than 3.8 kb in length, or are less than 3.7 kb in length, or are less than

3.6 kb in length, or are less than 3.5 kb in length, or are less than 3.4 kb in length, or are less than

3.3 kb in length, or are less than 3.2 kb in length, or are less than 3.1 kb in length, or are less than

3.0 kb in length. The AAV vector constructs provided herein in single strand form range from about 5.0 kb to about 6.5 kb in length, or range from about 4.8 kb to about 5.2 k in length, or 4.8 kb to 5.3 kb in length, or range from about 4.9 kb to about 5.5 kb in length, or about 4.8 kb to about 6.0 kb in length, or about 5.0 kb to 6.2 kb in length or about 5.1 kb to about 6.3 kb in length, or about 5.2 kb to about 6.4 kb in length, or about 5.5 kb to about 6.5 kb in length, or range from about 4.0 kb to about 5.0 kb in length, or range from about 3.8 kb to about 4.8 k in length, or 3.6 kb to 4.6 kb in length, or range from about 3.4 kb to about 4.4 kb in length, or range from about 3.2 kb to about 4.2 kb in length, or range from about 3.0 kb to 4.0 kb in length, or range from about 3.5 kb to about 4.0 kb in length, or range from about 3.0 kb to about 3.5 kb in length, or range from about 4 kb to about 4.5 kb in length.

[0044] While AAV particles have been reported in the literature having AAV genomes of > 5.0 kb, in many of these cases the 5' or 3' ends of the encoded genes appear to be truncated (see Hirsch et ah, Molec. Ther. 18:6-8, 2010, and Ghosh et al., Biotech. Genet. Engin. Rev. 24:165- 178, 2007). It has been shown, however, that overlapping homologous recombination occurs in AAV infected cells between nucleic acids having 5' end truncations and 3' end truncations so that a "complete" nucleic acid encoding the large protein is generated, thereby reconstructing a functional, full-length gene.

[0045] Oversized AAV vectors are randomly truncated at the 5' ends and lack a 5' AAV ITR. Because AAV is a single-stranded DNA virus, and packages either the sense or antisense strand, the sense strand in oversized AAV vectors lacks the 5' AAV ITR and possibly portions of the 5' end of the target protein-coding gene, and the antisense strand in oversized AAV vectors lacks the 3' ITR and possibly portions of the 3' end of the target protein-coding gene. A functional transgene is produced in oversized AAV vector infected cells by annealing of the sense and antisense truncated genomes within the target cell. Thus, in certain embodiments, the AAV a- galactosidase A vectors and/or viral particles comprise at least one ITR.

[0046] The term "inverted terminal repeat (ITR)" as used herein refers to the art-recognized regions found at the 5' and 3' termini of the AAV genome which function in cis as origins of DNA replication and as packaging signals for the viral genome. AAV ITRs, together with the AAV rep coding region, provide for efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a host cell genome. Sequences of certain AAV-associated ITRs are disclosed by Yan et al., J. Virol. (2005) vol. 79, pp. 364-379 which is herein incorporated by reference in its entirety. ITR sequences that find use herein may be full length, wild-type AAV ITRs or fragments thereof that retain functional capability, or may be sequence variants of full-length, wild-type AAV ITRs that are capable of functioning in cis as origins of replication. AAV ITRs useful in the recombinant AAV a-galactosidase A vectors of the embodiments provided herein may be derived from any known AAV serotype and, in certain embodiments, derived from the AAV2 or AAV5 serotype.

[0047] The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0048] A "transcription regulatory element" refers to nucleotide sequences of a gene involved in regulation of genetic transcription including a promoter, plus response elements, activator and enhancer sequences for binding of transcription factors to aid RNA polymerase binding and promote expression, and operator or silencer sequences to which repressor proteins bind to block RNA polymerase attachment and prevent expression. The term "liver specific transcription regulatory element" or “liver-specific transcription regulatory region” refers to a regulatory element or region that produces preferred gene expression specifically in the liver tissue. Examples of liver specific regulatory elements include, but are not limited to, the mouse thyretin promoter (mTTR), the endogenous human factor VIII promoter (F8), human apolipoprotein E hepatic control region and active fragments thereof, human alpha- 1 -antitrypsin promoter (hAAT) and active fragments thereof, human alpha- 1 -microglobulin promoter and fragments thereof, human prothrombin promoter and active fragments thereof, human albumin minimal promoter, and mouse albumin promoter. Enhancers derived from liver-specific transcription factor binding sites are also contemplated, such as EBP, DBP, HNF1, HNF3,

HNF4, HNF6, and Enhl.

[0049] As used herein, the term “operably linked” is used to describe the connection between regulatory elements and a gene or its coding region. Typically, gene expression is placed under the control of one or more regulatory elements, for example, without limitation, constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. A gene or coding region is said to be “operably linked to” or “operatively linked to” or “operably associated with” the regulatory elements, meaning that the gene or coding region is controlled or influenced by the regulatory element. For instance, a promoter is operably linked to a coding sequence if the promoter effects transcription or expression of the coding sequence.

[0050] In one embodiment, the vector construct comprises a nucleic acid encoding a functionally active a-galactosidase A protein. The a-galactosidase A encoding sequence may be wild-type, codon optimized, or a variant.

[0051] In one embodiment, the functionally active a-galactosidase A protein is a fusion protein comprising an a-galactosidase A enzyme and a peptide tag that improves lysosomal uptake of the fusion protein, optionally including a spacer sequence between the enzyme and peptide tag. In a further embodiment, the peptide tag is IFG-II or any suitable peptide tag as disclosed in U.S. Patent Pub. No. 2014/0161788, incorporated by reference herein in its entirety. [0052] As used herein, wild-type a-galactosidase A (GLA gene) has the following nucleic acid sequence (GenBank Accession No. NM_000169.3): atgctgtccggtcaccgtgacaatgcagctgaggaacccagaactacatctgggctgcgcgcttgcgctt cgcttcctggccctcgtttcctgggacatccctggggctagagcactggacaatggattggcaaggacgc ctaccatgggctggctgcactgggagcgcttcatgtgcaaccttgactgccaggaagagccagattcctg catcagtgagaagctcttcatggagatggcagagctcatggtctcagaaggctggaaggatgcaggttat gagtacctctgcattgatgactgttggatggctccccaaagagattcagaaggcagacttcaggcagacc ctcagcgctttcctcatgggattcgccagctagctaattatgttcacagcaaaggactgaagctagggat ttatgcagatgttggaaataaaacctgcgcaggcttccctgggagttttggatactacgacattgatgcc cagacctttgctgactggggagtagatctgctaaaatttgatggttgttactgtgacagtttggaaaatt tggcagatggttataagcacatgtccttggccctgaataggactggcagaagcattgtgtactcctgtga gtggcctctttatatgtggccctttcaaaagcccaattatacagaaatccgacagtactgcaatcactgg cgaaattttgctgacattgatgattcctggaaaagtataaagagtatcttggactggacatcttttaacc aggagagaattgttgatgttgctggaccagggggttggaatgacccagatatgttagtgattggcaactt tggcctcagctggaatcagcaagtaactcagatggccctctgggctatcatggctgctcctttattcatg tctaatgacctccgacacatcagccctcaagccaaagctctccttcaggataaggacgtaattgccatca atcaggaccccttgggcaagcaagggtaccagcttagacagggagacaactttgaagtgtgggaacgacc tctctcaggcttagcctgggctgtagctatgataaaccggcaggagattggtggacctcgctcttatacc atcgcagttgcttccctgggtaaaggagtggcctgtaatcctgcctgcttcatcacacagctcctccctg tgaaaaggaagctagggttctatgaatggacttcaaggttaagaagtcacataaatcccacaggcactgt tttgcttcagctagaaaatacaatgcagatgtcattaaaagacttactttaaaatgtt (SEQ ID NO: 1). [0053] As used herein, wild-type a-galactosidase A protein (alpha-Gal A protein) has the following amino acid sequence (GenBank Accession No. NP 000160.1) of which amino acids 32 to 429 of SEQ ID NO: 2 represent the mature sequence:

MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWEREMCNLDCQEEPDSCI SEKLEMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKG LKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKS IKSILDWTSFNQERIVDV AGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLEMSNDLRHISPQAKALLQDKDVIAIN QDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFI TQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO: 2).

[0054] The vector constructs described herein may comprise a nucleotide sequence that differs from wild type nucleotide sequence but still encodes a functional a-galactosidase A amino acid sequence at least 90%, 95% or 98% identical to amino acids 32 to 429 of SEQ ID NO: 2. According to this aspect, the nucleotide sequence may comprise a portion having at least 80%, 85%, 90% or 95% homology to at least 100 consecutive bases of SEQ ID NO: 1 or 3-23, as long as the nucleotide sequence encodes functional human a-galactosidase A protein at least 90%, 95% or 98% identical to amino acids 32 to 429 of SEQ ID NO: 2. In example embodiments, the nucleotide sequence may comprise a portion having at least 90% homology to at least 100, 200, 300, 400, or 500 consecutive bases of SEQ ID NO: 1 or 3-23, as long as the nucleotide sequence encodes functional human a-galactosidase A protein at least 90% identical to amino acids 32 to 429 of SEQ ID NO: 2. In example embodiments, the nucleotide sequence has substantial homology to the nucleotide sequence of SEQ ID NO: 1 or 3-23 and encodes functional a-galactosidase A. The term substantial homology can be further defined with reference to a percent (%) homology, e.g. at least 80%, 85%, 90% or 95% homologous. This is discussed in further detail elsewhere herein.

[0055] The term "isolated" when used in relation to a nucleic acid molecule of the present disclosure typically refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid may be present in a form or setting that is different from that in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells. [0056] As used herein, the term “variant” refers to a polynucleotide (or polypeptide) having a sequence substantially similar to a reference polynucleotide (or polypeptide). Procedures for the introduction of nucleotide and amino acid changes in a polynucleotide, protein or polypeptide are known to the skilled artisan (see, e.g., Sambrook et al. (1989)). In the case of a polynucleotide, a variant can have deletions, substitutions, additions of one or more nucleotides at the 5' end, 3' end, and/or one or more internal sites in comparison to the reference polynucleotide. Similarities and/or differences in sequences between a variant and the reference polynucleotide can be detected using conventional techniques known in the art, for example polymerase chain reaction (PCR) and hybridization techniques. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis. Generally, a variant of a polynucleotide, including, but not limited to, a DNA, can have at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to the reference polynucleotide as determined by sequence alignment programs known by skilled artisans. In the case of a polypeptide, a variant can have deletions, substitutions, additions of one or more amino acids in comparison to the reference polypeptide. Similarities and/or differences in sequences between a variant and the reference polypeptide can be detected using conventional techniques known in the art, for example Western blot. Generally, a variant of a polypeptide, can have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to the reference polypeptide as determined by sequence alignment programs known by skilled artisans.

[0057] The term "identity," "homology" and grammatical variations thereof, mean that two or more referenced entities are the same, when they are "aligned" sequences. Thus, by way of example, when two polypeptide sequences are identical, they have the same amino acid sequence, at least within the referenced region or portion. Where two polynucleotide sequences are identical, they have the same polynucleotide sequence, at least within the referenced region or portion. The identity can be over a defined area (region or domain) of the sequence. An "area" or "region" of identity refers to a portion of two or more referenced entities that are the same. Thus, where two protein or nucleic acid sequences are identical over one or more sequence areas or regions they share identity within that region. An "aligned" sequence refers to multiple polynucleotide or protein (amino acid) sequences, often containing corrections for missing or additional bases or amino acids (gaps) as compared to a reference sequence. "Substantial homology" means that a molecule is structurally or functionally conserved such that it has or is predicted to have at least partial structure or function of one or more of the structures or functions (e.g., a biological function or activity) of the reference molecule, or relevant/corresponding region or portion of the reference molecule to which it shares homology. [0058] “Percent (%) nucleic acid sequence identity or homology” is defined as the percentage of nucleotides in a candidate sequence that are identical with a reference sequence after aligning the respective sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0059] “Percent (%) amino acid sequence identity or homology” with respect to the a- galactosidase A amino acid sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a a- galactosidase A polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0060] “Codon optimization” or “codon optimized” refers to changes made in the nucleotide sequence so that it is more likely to be expressed at a relatively high level compared to the non codon optimized sequence. It does not change the amino acid for which each codon encodes. [0061] As used herein, an “intron” is broadly defined as a sequence of nucleotides that is removable by RNA splicing. “RNA splicing” means the excision of introns from a pre-mRNA to form a mature mRNA. Introns may be upstream, downstream, or within the coding region of a gene. Insertion of an intron into a nucleotide sequence can be accomplished by any method known in the art. The only limitation of where the intron is inserted is in consideration of the packaging limitations of the AAV virus particles (about 5 kb).

[0062] In certain embodiments, the recombinant AAV vector construct comprises (a) a nucleic acid comprising an AAV2 5' inverted terminal repeat (ITR) (which may or may not be modified as known in the art), (b) a liver-specific transcription regulatory region, (c) a functional a-galactosidase A protein coding region, (d) one or more introns, (e) a polyadenylation sequence, and (f) an AAV23' ITR (which may or may not be modified as known in the art).

[0063] Other embodiments provided herein are directed to vector constructs encoding a functional a-galactosidase A polypeptide, wherein the constructs comprise one or more of the individual elements of the above described constructs and combinations thereof, in one or more different orientation(s). Another embodiment provided herein is directed to the above described constructs in an opposite orientation. In another embodiment, provided are recombinant AAV virus particles comprising the herein described AAV vector constructs and their use for the treatment of Fabry Disease or deficiency in functional a-galactosidase A in subjects. In one embodiment the subjects are juvenile subjects.

[0064] An "AAV virion" or "AAV viral particle" or "AAV vector particle" or "AAV virus" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated AAV vector construct as described herein. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as a "recombinant AAV vector particle" or simply an "AAV vector". Production of AAV vector particles necessarily includes production of AAV vector genome, as such a vector genome is contained within an AAV vector particle. It is understood that reference to the polynucleotide AAV vector construct encapsulated within the vector particle, and replication thereof, refers to the AAV vector genome.

[0065] As used herein “therapeutic AAV virus” refers to an AAV virion, AAV viral particle, AAV vector particle, or AAV virus that comprises a heterologous polynucleotide that encodes a therapeutic protein such as the a-galactosidase A described herein. An "AAV vector construct" or “AAV vector genome” as used herein refers to a vector construct comprising one or more polynucleotide encoding a protein of interest (also called transgenes) that are flanked by at least one AAV terminal repeat sequences (ITRs) and operably linked to one or more expression control elements. Such AAV vector constructs can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products.

[0066] As used herein “therapeutic protein” refers to a polypeptide that has a biological activity that replaces or compensates for the loss or reduction of activity of an endogenous protein. For example, a functional a-galactosidase A protein (alpha-Gal A) is a therapeutic protein for Fabry Disease (FD).

[0067] “Fabry Disease (FD)” as used herein refers to an inherited lysosomal storage disorder caused by the deficiency of a-galactosidase A (a-Gal A), that is characterized by symptoms of dizziness, vertigo, and in severe cases, stroke, changes in the appearance of the eyes, such as corneal opacities, heart problems such as an irregular or abnormal heart rhythm or beat, hearing loss, tinnitus, abdominal pain, nausea, vomiting, constipation and diarrhea, kidney impairment including cysts or progressive kidney failure, skin abnormalities such as angiokeratoma (small raised dark red spots), absent sweating (anhidrosis), diminished sweating (hypohidrosis), excessive sweating (hyperhidrosis), temperature sensitivity, recurrent fever, and neuropathic pain (e.g., burning or tingling sensations of the skin). Signs of Fabry disease include reduced a- galactosidase A protein levels in plasma or tissues, reduced a-galactosidase A activity in plasma or tissues, accumulation of Gb-3 or Lyso-Gb-3 in cells, and increased levels of Gb-3 or Lyso- Gb-3 in plasma or tissues.

[0068] “a-galactosidase A deficiency” or a “deficiency in functional a-galactosidase A” as used herein refers to an inherited condition caused by a deficiency of functional a-galactosidase A protein, due to absence of protein, reduced production of protein, production of protein that is inactive or production of protein with reduced activity. This includes Fabry Disease.

[0069] “Therapeutically effective for Fabry Disease” or “Fabry Disease therapy” as used herein refers to any therapeutic intervention of a subject having Fabry Disease that ameliorates the characteristic deficiency in functional a-galactosidase A, increases a-galactosidase A protein levels or a-galactosidase A activity in plasma or tissues, e.g. in plasma, liver, heart, kidney, brain or other tissues, reduces accumulation of Gb-3 or Lyso-Gb-3 in cells, reduces the levels of Gb-3 or Lyso-Gb-3 in plasma or tissues, e.g. in plasma, liver, heart, kidney, brain or other tissues, ameliorates Fabry Disease symptoms, or reduces the frequency, duration or severity of Fabry Disease symptoms. “Fabry Disease gene therapy” as used herein refers to any therapeutic intervention of a subject having Fabry Disease that involves the replacement or restoration or increase of a-galactosidase A activity through the delivery of one or more nucleic acid molecules to the cells of the subject that express functional a-galactosidase A. In certain embodiments,

GLA gene therapy refers to gene therapy involving an adeno associated viral (AAV) particle comprising a vector construct that expresses human a-galactosidase A.

[0070] “Treat” or “treatment” as used herein refers to therapeutic treatment which refers to a treatment administered to a subject who exhibits signs or symptoms of pathology, i.e., Fabry Disease, for the purpose of diminishing or eliminating those signs or symptoms or ameliorating their progression, severity or duration. The signs or symptoms can be biochemical, cellular, histological, functional, subjective or objective.

[0071] “Ameliorate” as used herein refers to the action of lessening the severity of symptoms, progression, or duration of a disease.

[0072] As used herein “stably treating” or “stable treatment” refers to using a therapeutic vector construct, AAV particle or cell administered to a subject where the subject stably expresses a therapeutic protein expressed by the vector construct, AAV particle or cell. Stably expressed therapeutic protein means that the protein is expressed for a clinically significant length of time. “Clinically significant length of time” as used herein means expression at therapeutically effective levels for a length of time that has a meaningful impact on the quality of life of the subject, e.g., demonstrated by reduced signs or symptoms of disease. In certain embodiments clinically, significant length of time is expression for at least six months, for at least eight months, for at least one year, for at least two years, for at least three years, for at least four years, for at least five years, for at least six years, for at least seven years, for at least eight years, for at least nine years, for at least ten years, or for the life of the subject.

[0073] As used herein, the term “effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.

[0074] As used herein, a “subject” refers to an animal that is the object of treatment, observation or experiment. “Animal” includes cold- and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles, and in particular, mammals. The term "avian" as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys and pheasants. “Mammal,” as used herein, refers to an individual belonging to the class Mammalia and includes, but not limited to, humans, domestic and farm animals, zoo animals, sports and pet animals. Non-limiting examples of mammals include mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates, such as monkeys, chimpanzees and apes, and, in particular, humans. In some embodiments, the mammal is a human, including an infant, child or juvenile human, e.g. a human age up to 2, 2-4, 2-6 or 2-12. However, in some embodiments, the mammal is not a human.

[0075] In general, a "pharmaceutically acceptable carrier" is one that is not toxic or unduly detrimental to cells. Exemplary pharmaceutically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline. Pharmaceutically acceptable carriers include physiologically acceptable carriers. The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0076] In another embodiment, provided are methods of producing recombinant adeno- associated virus (AAV) particles comprising any of the AAV vector constructs provided herein. The methods comprise the steps of culturing a cell that has been transfected with any of the AAV vector constructs provided herein (in association with various AAV cap and rep genes) and recovering recombinant therapeutic AAV particles from the transfected cell or supernatant of the transfected cell.

[0077] The cells useful for recombinant AAV production provided herein are any cell type susceptible to baculovirus infection, including insect cells such as High Five, Sf9, Se301, SeIZD2109, SeUCRl, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAml, BM-N, Ha2302, Hz2E5, and Ao38. In another embodiment, mammalian cells such as HEK293, HeLa, CHO, NSO, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5 can be used.

[0078] In another embodiment, provided herein is the use of an effective amount of vector nucleic acid, vector construct, or AAV particle for the preparation of a medicament for the treatment of a subject suffering from Fabry Disease or a-galactosidase A deficiency. In one embodiment, the subject suffering from Fabry Disease is a human. In one embodiment, the medicament is administered by intravenous (IV) administration. In another embodiment, administration of the medicament results in increased levels of functional a-galactosidase A enzyme or activity in the blood, e.g. plasma, or in one or more tissues of the subject, e.g., heart, kidney, liver, to ameliorate Fabry Disease symptoms. In another embodiment, administration of the medicament results in reduced levels of or reduced accumulation of Gb-3 or Lyso-Gb-3 in one or more tissues of the subject, e.g., heart, kidney, liver, to ameliorate Fabry Disease symptoms. In certain embodiments, the medicament is also for co-administration with a prophylactic and/or therapeutic corticosteroid for the prevention and/or treatment of any hepatotoxicity associated with administration of the AAV particle. The prophylactic or therapeutic corticosteroid treatment may comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more mg/day of the corticosteroid. In certain embodiments, the prophylactic or therapeutic corticosteroid may be administered over a continuous period of at least about 3, 4, 5, 6, 7, 8, 9, 10 weeks, or more.

[0079] In another embodiment, the Fabry Disease therapy provided herein optionally further includes administration, e.g. concurrent administration, of other therapies that are used to treat Fabry Disease.

VECTOR CONSTRUCTS AND AAV VECTORS

[0080] The recombinant vector construct of the disclosure may be used itself as gene therapy, or may be used to produce rAAV particles by methods described herein, comprising providing to a suitable host cell the recombinant vector construct, together with Rep and Cap genes. The vector constructs described herein comprise a nucleic acid sequence that encodes a functional a-galactosidase A (alpha-Gal A). The recombinant vector construct may comprise a nucleic acid encoding functional human a-galactosidase A operably linked to a heterologous expression control element, e.g. a promoter and/or enhancer; optionally an intron; and optionally a polyadenylation (poly A) signal. The heterologous expression control element may be a heterologous liver-specific transcription regulatory region, e.g., as described herein.

[0081] When used to produce rAAV particles, the recombinant vector construct may comprise (a) one or both of (i) an AAV 5' inverted terminal repeat (ITR) sequence and (ii) an AAV 3’ ITR, (b) a heterologous liver-specific transcription regulatory region, and (c) a nucleic acid encoding a functional human a-galactosidase A, optionally wherein the AAV ITRs are AAV2 ITRs. Preferably, the nucleic acid encoding the functional a-galactosidase A is operably linked to liver-specific expression control elements. The vector construct may include additional expression control elements, for example: a promoter and/or enhancer; an intron; optionally an exon from the same gene as the intron; and a polyadenylation (poly A) signal. Such elements are further described herein. Preferably, the rAAV particles also comprise an AAV capsid with liver tropism, optionally an AAV5 type capsid.

[0082] In one or more embodiments, the functional a-galactosidase A comprises an amino acid sequence at least 90%, 95% or 98% identical to amino acids 32 to 429 of SEQ ID NO: 2 (a human a-galactosidase A). In example embodiments, the nucleic acid sequence encoding the functional a-galactosidase A is a wild-type GLA sequence, of which SEQ ID NO: 1 is one example, or is codon optimized, or is a variant.

[0083] In one or more embodiments, the nucleic acid sequence encoding a-galactosidase A is operably linked to one or more heterologous expression control elements. Preferably, the expression control element is a liver-specific expression control element. Examples of liver specific control elements include, but are not limited to, the mouse thyretin promoter (mTTR), the endogenous human factor VIII promoter (F8), human apolipoprotein E hepatic control region and active fragments thereof, human alpha- 1 -antitrypsin promoter (hAAT) and active fragments thereof, human alpha- 1 -microglobulin promoter and fragments thereof, human prothrombin promoter and active fragments thereof, human albumin minimal promoter, and mouse albumin promoter. Enhancers derived from liver-specific transcription factor binding sites are also contemplated, such as EBP, DBP, HNF1, HNF3, HNF4, HNF6, and Enhl.

[0084] In some embodiments, in the vector constructs comprise a nucleic acid sequence encoding functional a-galactosidase A that is operably linked to a heterologous liver-specific transcription regulatory region. The vector constructs may comprise other regulatory elements.

In some embodiments, in the vector constructs described herein, the expression control elements include one or more of the following: a promoter and/or enhancer; an intron; and a polyadenylation (poly A) signal.

[0085] The liver-specific transcription regulatory region may comprise one or more liver- specific expression control elements. In one or more embodiments, the liver-specific transcription regulatory region is a synthetic promoter sequence comprising portions of a human alpha- 1 -antitrypsin (hAAT) promoter, a hepatic control region (HCR) enhancer, and/or an apolipoprotein E (ApoE) enhancer.

[0086] In some embodiments, the vector construct comprises at least one or both of a 5' inverted terminal repeat (ITR) of AAV and a 3 ' AAV ITR, a promoter, a nucleic acid encoding functional a-galactosidase A, and optionally a posttranscriptional regulatory element, where the promoter, the nucleic acid encoding a-galactosidase A and the posttranscription regulatory element are located downstream of the 5' AAV ITR and upstream of the 3' AAV ITR. The vector construct can, for example, be used to produce high levels of a-galactosidase A in a subject for therapeutic purposes.

[0087] In certain embodiments, the recombinant AAV vector construct comprises a nucleic acid comprising (a) an AAV2 5' inverted terminal repeat (ITR) (which may or may not be modified as known in the art), (b) a liver-specific transcription regulatory region, a functional a- galactosidase A protein coding region, (c) one or more introns including fragments of longer introns, (d) optionally an exon or fragment thereof, (e) a polyadenylation sequence, and (f) an AAV2 3' ITR (which may or may not be modified as known in the art).

[0088] In some embodiments, the liver-specific transcription regulatory region comprises a shortened ApoE enhancer sequence (SEQ ID NO: 61) or a nucleotide sequence at least 80%, 85%, 90%, 95% or 98% identical thereto; a hAAT proximal promoter sequence (SEQ ID NO:

60) or a nucleotide sequence at least 80%, 85%, 90%, 95% or 98% identical thereto; a 186 base human alpha anti-trypsin (hAAT) proximal promoter, including 42 bases of the 5' untranslated region (UTR) (nucleotides 213-398 of SEQ ID NO: 60) or a nucleotide sequence at least 80%, 85%, 90%, 95% or 98% identical thereto; one or more enhancers selected from the group consisting of (i) a ApoE/HCR enhancer (SEQ ID NO: 61) or a nucleotide sequence at least 80%, 85%, 90%, 95% or 98% identical thereto, (ii) a 32 base human AAT promoter distal X region or a nucleotide sequence at least 80%, 85%, 90%, 95% or 98% identical thereto, and (iii) 80 additional bases of distal element of the human AAT proximal promoter or a nucleotide sequence at least 80%, 85%, 90%, 95% or 98% identical thereto; and a nucleic acid encoding human a-galactosidase A. In another embodiment, the liver-specific transcription regulatory region comprises an ApoE-hAAT promoter sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO: 59. In another embodiment, the liver-specific transcription regulatory region comprises an a-microglobulin enhancer sequence (SEQ ID NO: 62) or a nucleotide sequence at least 80%, 85%, 90%, 95% or 98% identical thereto and the 186 base human alpha anti -trypsin (AAT) proximal promoter (nucleotides 213-398 of SEQ ID NO: 61) or a nucleotide sequence at least 80%, 85%, 90%, 95% or 98% identical thereto.

[0089] Other embodiments provided herein are directed to vector constructs encoding a functional a-galactosidase A polypeptide, wherein the constructs comprise one or more of the individual elements of the above described constructs and combinations thereof, in one or more different orientation(s). Another embodiment provided herein is directed to the above described constructs in an opposite orientation. In another embodiment, provided are recombinant AAV particles comprising the herein described vector constructs and their use for the treatment of Fabry Disease or a-galactosidase A deficiency in subjects. In one embodiment the subjects are juvenile subjects.

[0090] The AAV vector constructs provided herein in single strand form are less than about 7.0 kb in length, or are less than 6.5 kb in length, or are less than 6.4 kb in length, or are less than

3.0 kb in length. The AAV vector constructs provided herein in single strand form range from about 5.0 kb to about 6.5 kb in length, or range from about 4.8 kb to about 5.2 k in length, or 4.8 kb to 5.3 kb in length, or range from about 4.9 kb to about 5.5 kb in length, or about 4.8 kb to about 6.0 kb in length, or about 5.0 kb to 6.2 kb in length or about 5.1 kb to about 6.3 kb in length, or about 5.2 kb to about 6.4 kb in length, or about 5.5 kb to about 6.5 kb in length, or range from about 4.0 kb to about 5.0 kb in length, or range from about 3.8 kb to about 4.8 k in length, or 3.6 kb to 4.6 kb in length, or range from about 3.4 kb to about 4.4 kb in length, or range from about 3.2 kb to about 4.2 kb in length, or range from about 3.0 kb to 4.0 kb in length, or range from about 3.5 kb to about 4.0 kb in length, or range from about 3.0 kb to about 3.5 kb in length, or range from about 4 to about 4.5 kb in length.

[0091] When AAV vectors are produced from oversized recombinant vector constructs, they may lack a portion of the 5' or 3’ ends of the recombinant vector construct. Because AAV is a single-stranded DNA virus, and packages either the sense or antisense strand, the sense strand in oversized AAV vectors lacks the 5' AAV ITR and possibly portions of the 5' end of the target protein-coding gene, and the antisense strand in oversized AAV vectors lacks the 3' ITR and possibly portions of the 3' end of the target protein-coding gene. A functional transgene is produced in oversized AAV vector infected cells by annealing of the sense and antisense truncated genomes within the target cell. Thus, in certain embodiments, the rAAV particles of the invention may comprise recombinant vector constructs that comprise at least one ITR, and a substantial portion of a nucleotide sequence encoding a functional a-galactosidase A, such as a fragment of any of SEQ ID NO: 3-23 that is greater than 50%, 60%, 70%, 80%, or 90% of the length of the nucleotide sequence. For example, the recombinant vector construct may comprise at least one ITR, a liver-specific transcription regulatory region, and a substantial portion of a nucleotide sequence encoding a functional a-galactosidase. The rAAV particles of the invention may also comprise a substantial portion of any of any one of SEQ NOs: 24-58, e.g. a fragment that is greater than 50%, 60%, 70%, 80%, or 90% of the length of the nucleotide sequence set forth in any of SEQ ID NOs: 24-58, including the liver-specific transcription regulatory region. [0092] Generation of the vector constructs can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (1989)).

[0093] The vector constructs can incorporate sequences from the genome of any known organism. The sequences can be incorporated in their native form or can be modified in any way to obtain a desired activity. For example, the sequences can comprise insertions, deletions or substitutions.

[0094] AAV vector constructs can be replicated and packaged into infectious AAV particles, preferably replication deficient AAV particles, when present in a host cell that has been transfected with a polynucleotide encoding and expressing rep and cap gene products. [0095] The vector constructs or AAV particles described herein may also produce beneficial effects in a a-galactosidase A-deficient mouse model that shares characteristics associated with Fabry Disease in humans including increased a-galactosidase A levels or activity in plasma, heart, kidney, liver or brain, or reduced Gb-3 or Lyso-Gb-3 levels in plasma, heart, kidney, liver or brain.

Transcription Regulatory Elements or Region Promoters and Enhancers

[0096] Various promoters can be operably linked with a nucleic acid comprising the coding region of the protein of interest, human a-galactosidase A, in the vector constructs disclosed herein. In some embodiments, the promoter can drive the expression of the protein of interest in a cell infected with a virus derived from the viral vector, such as a target cell. The promoter can be naturally-occurring or non-naturally occurring. In some embodiments the promoter is a synthetic promoter. In one embodiment the synthetic promoter comprises sequences that do not exist in nature and which are designed to regulate the activity of an operably linked gene. In another embodiment the synthetic promoter comprises fragments of natural promoters to form new stretches of DNA sequence that do not exist in nature. Synthetic promoters are typically comprised of regulatory elements, promoters, enhancers, introns, splice donors and acceptors that are designed to produce enhanced tissue specific expression. Examples of promoters, include, but are not limited to, viral promoters, plant promoters and mammalian promoters. In another embodiment the promoter is a liver specific promoter. Specific examples of liver specific promoters include LP1, HLP, HCR-hAAT, ApoE-hAAT, LSP, TBG and TTR. These promoters are described in more detail in the following references: LP1 (human ApoE HCR core sequence (192 bp) with human AAT promoter (255 bp)): Nathwani A. etal. Blood. 2006 April 1; 107(7): 2653-2661; hybrid liver specific promoter (HLP) (human apolipoprotein E (ApoE) hepatic control region (HCR) fragment (34 bp) with modified human a -1 -antitrypsin (aAT) promoter (217 bp)): McIntosh J. etal. Blood. 2013 Apr 25; 121(17): 3335-3344; HCR-hAAT (ApoE-HCR (319 bp) with ApoE enhancer (1-4x154 bp) with human AAT promoter (408 bp) and including an Intron A (1.4 kbp) and 3 ’UTR (1.7 kbp)): Miao CH et al. Mol Ther. 2000; 1 : 522-532; ApoE-hAAT: Okuyama T et al. Human Gene Therapy, 7, 637-645 (1996); LSP: Wang L et al. Proc Natl Acad Sci U S A. 1999 March 30; 96(7): 3906-3910, thyroxine binding globulin (TBG) promoter: Yan et al., Gene 506:289-294 (2012), and transthyretin (TTR) promoter: Costa et al., Mol. Cell. Biol. 8:81-90 (1988)

[0097] For example, De Simone et al. (EMBO Journal vol.6 no.9 pp.2759-2766, 1987) describes a number of promoters derived from human a- 1 -antitrypsin promoter. For example, it characterizes the cis- and trans-acting elements required for liver-specific activity within the human AAT promoter from -1200 to +44. The human A AT promoter in HLP consists of the distal X element (32 bp) and the proximal A and B elements (185 bp). Frain et al. (MOL CELL BIO, Mar. 1990, Vol. 10, No.3, p. 991-999) describes a number of promoters derived from human albumin promoter. For example, it characterizes promoter and enhancer elements within the human albumin gene from -1022 to -1.

[0098] Dang et al. (J BIOL CHEM, Vol. 270, No. 38, Issue of September 22, pp. 22577- 22585, 1995) describes the hepatic control region (HCR) of human apolipoprotein E gene (774 bp). Shachter et al. (J. Lipid Res. 1993. Vol.34: ppl699-1707) characterizes a liver-specific enhancer in the ApoE HCR (154 bp). These HCR fragments can be combined with other transcription regulatory elements such as the human AAT promoter or fragments thereof. Chow et al. (J Biol Chem. 1991 Oct 5;266(28): 18927-33) characterizes the human prothrombin enhancer from -940 to -860 (80 bp). Rouet et al. (Vol. 267, No. 29, Issue of October 15, PP. 20765-20773,1992; Nucleic Acids Res. 1995 Feb 11; 23(3): 395-404; and Biochemical Journal Sep 15, 1998, 334 (3) 577-584) characterize the sequence of the liver-specific human a-1- microglobulin/bikunin enhancer. U.S. Patent No. 7,323,324 also describes human AAT promoter, human a-microglobulin/bikunen enhancers, human albumin promoter, and human prothrombin enhancers.

[0099] In some embodiments, the promoter comprises the human alphal anti-trypsin (hAAT) promoter complex. In some embodiments, the promoter comprises at least a portion of the hAAT promoter. The portion of the hAAT promoter can comprise a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more, sequence identity to SEQ ID NO: 59.

[00100] In some embodiments, the promoter comprises a liver specific enhancer. In some embodiments, the promoter comprises an apolipoprotein E (ApoE) / hepatic control region (HCR) enhancer. In some embodiments, the promoter comprises at least a portion of the ApoE/HCR enhancer. For example, the ApoE/HCR enhancer can comprise a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more, sequence identity to SEQ ID NO: 61.

[00101] In some embodiments, the promoter is a synthetic promoter comprising at least a portion of the hAAT promoter, and at least a portion of the ApoE/HCR enhancer. In some embodiments, the promoter can include a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more, sequence identity to SEQ ID NO: 63.

[00102] In some embodiments, the promoter comprises multiple copies of one or more of the enhancers identified above. In some embodiments, the promoter constructs comprise one or more of the individual enhancer elements described above and combinations thereof, in one or more different orientation(s).

[00103] In some embodiments, the promoter is operably linked with a polynucleotide encoding one or more proteins of interest. In some embodiments, the promoter is operably linked with a polynucleotide encoding the a-galactosidase A protein.

[00104] The size of the promoter can vary. Because of the limited packaging capacity of AAV, it is preferred to use a promoter that is small in size, but at the same time allows high level production of the protein(s) of interest in host cells. For example, in some embodiments the promoter is at most about 1.5 kb, at most about 1.4 kb, at most about 1.35 kb, at most about 1.3 kb, at most about 1.25 kb, at most about 1.2 kb, at most about 1.15 kb, at most about 1.1 kb, at most about 1.05 kb, at most about 1 kb, at most about 800 base pairs, at most about 600 base pairs, at most about 400 base pairs, at most about 200 base pairs, or at most about 100 base pairs.

Other Regulatory Elements

[00105] Various additional regulatory elements can be used in the vector constructs, for example enhancers to further increase expression level of the protein of interest in a host cell, a polyadenylation signal, a ribosome binding sequence, and/or a consensus splice acceptor or splice donor site. In some embodiments, the regulatory element can facilitate maintenance of the recombinant DNA molecule extrachromosomally in a host cell and/or improve vector potency (e.g. scaffold/matrix attachment regions (S/MARs)). Such regulatory elements are well known in the art. [00106] The vectors constructs disclosed herein may include regulatory elements such as a transcription initiation region and/or a transcriptional termination region. Examples of a transcription termination region include, but are not limited to, polyadenylation signal sequences. Examples of polyadenylation signal sequences include, but are not limited to, human growth hormone (hGH) poly(A), bovine growth hormone (bGH) poly(A), SV40 late poly(A), rabbit beta-globin (rBG) poly(A), thymidine kinase (TK) poly(A) sequences, and any variants thereof. In some embodiments, the transcriptional termination region is located downstream of the posttranscriptional regulatory element. In some embodiments, the transcriptional termination region is a polyadenylation signal sequence. In some embodiments, the transcriptional termination region is hGH poly(A) sequence (e.g., SEQ ID NO:73-79).

[00107] In some embodiments, the vector constructs can include additional transcription and translation initiation sequences, and/or additional transcription and translation terminators, which are known in the art.

Protein of Interest and Nucleic Acids Encoding the Protein of Interest

[00108] As used herein, a “protein of interest” is any functional a-galactosidase A protein, including naturally-occurring and non-naturally occurring variants thereof. In some embodiments, a polynucleotide encoding one or more a-galactosidase A proteins of interest can be inserted into the viral vectors disclosed herein, wherein the polynucleotide is operably linked with the promoter. In some instances, the promoter can drive the expression of the protein(s) of interest in a host cell (e.g., a human liver cell).

[00109] In a first aspect, the present disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence which encodes functional wild-type a-galactosidase A protein (e.g., amino acids 32 to 429 of SEQ ID NO: 2). The nucleotide sequence may be homologous to the wild-type nucleotide sequence of SEQ ID NO: 1.

[00110] As described herein, the nucleotide sequence encoding the a-galactosidase A protein can be modified to improve expression efficiency of the protein. The methods that can be used to improve the transcription and/or translation of a gene herein are not particularly limited. For example, the nucleotide sequence can be modified to better reflect host codon usage to increase gene expression (e.g., protein production) in the host (e.g., a mammal). As another non-limiting example for the modification, one or more of the splice donors and/or splice acceptors in the nucleotide sequence of the protein of interest is modified to reduce the potential for extraneous splicing. As another non-limiting example for the modification, one or more introns can be inserted within or adjacent to the nucleotide sequence of the protein of interest to optimize AAV vector packaging and enhance expression.

[00111] The nucleic acid molecule encodes a functional a-galactosidase A protein at least 90% identical to amino acids 32 to 429 of SEQ ID NO: 2, and preferably at least 95% or 98% identical to a wild type amino acid sequence. If the nucleic acid encodes a protein comprising a sequence having changes to any of the wild-type amino acids, the protein should still be a functional protein. A skilled person will appreciate that minor changes can be made to some of the amino acids of the protein without adversely affecting the function of the protein.

[00112] In certain embodiments, the nucleic acid molecule has at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homology, or at least 98% homology to the nucleotide sequence of SEQ ID NO: 1 or 3-23, or at least 100, 200, 300, 400 or 500 consecutive nucleotides of SEQ ID NO: 1 or 3-23. In one embodiment, the nucleic acid molecule encodes for a functional a-galactosidase A protein, that is to say it encodes for a-galactosidase A which, when expressed, has the functionality of wild type a-galactosidase A. In certain embodiments, the nucleic acid molecule, when expressed in a suitable system (e.g. a host cell), produces a functional a-galactosidase A protein and at a relatively high level. Since the a-galactosidase A that is produced is functional, it will have a conformation which is the same as at least a portion of the wild type a-galactosidase A. In certain embodiments, a functional a-galactosidase A protein produced as described herein effectively treats a subject suffering from a-galactosidase A deficiency and/or Fabry Disease.

[00113] In another embodiment, the nucleotide sequence coding for a functional a- galactosidase A has an improved codon usage bias for the human cell as compared to naturally occurring nucleotide sequence coding for the corresponding non-codon optimized sequence. The adaptiveness of a nucleotide sequence encoding a functional a-galactosidase A to the codon usage of human cells may be expressed as codon adaptation index (CAI). A codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed human genes. The relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid. The CAI is defined as the geometric mean of these relative adaptiveness values. Non-synonymous codons and termination codons (dependent on genetic code) are excluded. CAI values range from 0 to 1, with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; also see: Kim et al., Gene. 1997, 199:293-301; zur Megede et al., Journal of Virology, 2000, 74: 2628- 2635). In certain embodiments, a nucleic acid molecule encoding a a-galactosidase A has a CAI of at least 0.75, 0.80, 0.85, 0.90, 0.95, or 0.99.

[00114] The nucleotide sequence of SEQ ID NOs: 3, 4 and 8 are codon optimized human a- galactosidase A nucleic acid sequences which were based on the sequence of the wild-type human a-galactosidase A nucleotide sequence (SEQ ID NO: 1).

[00115] Codon optimization can be performed, for example, using the DNA2.0 codon optimization algorithm, see Villalobos et al., “Gene Designer: a synthetic biology tool for constructing artificial DNA segments,” BMC Bioinformatics, vol. 7, article no: 285 (2006) or Operon/Eurofms Genomics codon optimization software or other codon optimization tools, e.g. Grote et al., “JCat: a novel tool to adapt codon usage of a target gene to its potential expression host,” Nucleic Acids Res. 33:W526-31 (2005).

[00116] This can be done in conjunction with manually reducing CpG di-nucleotide content and removing any extra ORF in the sense and anti-sense direction. CpG di-nucleotide content has been shown to activate TLR9 in dendritic cells leading to potential immune activation and CTL responses. A product in the AAV-vector genome may be delivered as ssDNA, thus reducing the CpG content. Reducing CpG content may reduce liver inflammation and ALT. [00117] Generally, codon optimization does not change the amino acid for which each codon encodes. It simply changes the nucleotide sequence so that it is more likely to be expressed at a relatively high level compared to the non-codon optimized sequence. This means that the nucleotide sequences of the nucleic acid provided herein and, for example, SEQ ID NO: 1 or 3- 23 may be different but when they are translated the amino acid sequence of the protein that is produced is the same.

[00118] In some embodiments, the codon optimized human a-galactosidase A nucleic acid molecule has a CpG di -nucleotide content of less than 25, less than 20, less than 15, or less than 10. In another embodiment, the codon optimized human a-galactosidase A nucleic acid molecule has a GC content of less than 65%, less than 60%, or less than 58%. [00119] It would be well within the capabilities of a skilled person to produce a nucleic acid molecule provided herein. This could be done, for example, using chemical synthesis of a given sequence. Further, suitable methods would be apparent to those skilled in the art for determining whether a nucleic acid described herein expresses a functional protein. For example, one suitable in vitro method involves inserting the nucleic acid into a vector, such as an AAV vector, transducing host cells, such as 293 T or HeLa cells, with the vector, and assaying for a- galactosidase A activity. Alternatively, a suitable in vivo method involves transducing a vector containing the nucleic acid into Fabry mice and assaying for functional a-galactosidase A in the plasma of the mice. Suitable methods are described in more detail below.

[00120] In some embodiments, the vector comprises one or more introns. The introns may facilitate processing of the RNA transcript in mammalian host cells, increase expression of the protein of interest and/or optimize packaging of the vector into AAV particles. Non-limiting examples of such an intron are a hemoglobin (b-globin) intron and/or hAAT intron. In some embodiments, the intron is a synthetic intron.

[00121] In one or more embodiments, the intron comprises a nucleotide sequence at least 80% or 85% or 90% or 95% identical to SEQ ID NO: 63, and the intron may be about 1700 to about 2000 nucleotides in length, or about 1800 to about 1900 nucleotides in length.

[00122] In one or more embodiments, the intron comprises a nucleotide sequence at least 80% or 85% or 90% or 95% identical to SEQ ID NO: 64, and the intron may be about 800 to about 1000 nucleotides in length, or about 850 to about 950 nucleotides in length. In example embodiments, the intron comprises SEQ ID NO: 64 or a fragment thereof that is about 100-900 nucleotides, 200-800 nucleotides, 200-700 nucleotides, 200-600 nucleotides, 200-500 nucleotides, 300-700 nucleotides, 300-600 nucleotides, 300-500 nucleotides, 400-700 nucleotides, 400-600 nucleotides, or 400-500 nucleotides of SEQ ID NO: 64, or a variant of said fragment that is at least 80%, 85%, 90%, or 95% identical to said fragment.

[00123] In one or more embodiments, the intron comprises a nucleotide sequence at least 80% or 85% or 90% or 95% identical to SEQ ID NO: 65, and the intron may be about 300 to about 600 nucleotides in length, or about 400 to about 500 nucleotides in length.

[00124] In one or more embodiments, the intron comprises a nucleotide sequence at least 80% or 85% or 90% or 95% identical to SEQ ID NO: 66. In some embodiments, the intron is missing at least nucleotides 226-900 or 226-800 or 226-700 or 226-600 or 226-500 or 300-900 or 300- 800 or 300-700 or 300-600 or 300-500 or 400-900 or 400-800 or 400-700 or 400-600 or 450- 900 or 500-900 of SEQ ID NO: 64. In one or more embodiments, the intron comprises a nucleotide sequence at least 80% or 85% or 90% or 95% identical to SEQ ID NO: 66, and the intron may be about 150 to about 350 nucleotides in length, or about 200 to about 250 nucleotides in length.

[00125] In one or more embodiments, the intron comprises a nucleotide sequence at least 80% or 85% or 90% or 95% identical to SEQ ID NO: 67, and the intron may be about 100 to about 200 nucleotides in length.

[00126] In one or more embodiments, the intron comprises a nucleotide sequence at least 80% or 85% or 90% or 95% identical to SEQ ID NO: 68, and the intron may be about 50 to about 150 nucleotides in length.

[00127] In one or more embodiments, the intron comprises a nucleotide sequence at least 80% or 85% or 90% or 95% identical to SEQ ID NO: 69, and the intron may be about 100 to about 200 nucleotides in length.

[00128] In some embodiments, the vector constructs may further comprise an exon sequence or fragment thereof; preferably adjacent to an intron sequence. In an example embodiment, the vector construct comprises an hAAT intron adjacent to an exon comprising a nucleotide sequence at least 80% or 85% or 90% or 95% identical to SEQ ID NO: 70. In a further example embodiment, the vector construct comprises a hemoglobin intron adjacent to an exon sequence comprising a nucleotide sequence at least 80% or 85% or 90% or 95% identical to SEQ ID NO: 71. In an example embodiment, the vector comprises both (a) an hAAT intron adjacent to an exon comprising a nucleotide sequence at least 80% or 85% or 90% or 95% identical to SEQ ID NO: 70 and (b) a hemoglobin intron adjacent to an exon sequence comprising a nucleotide sequence at least 80% or 85% or 90% identical to SEQ ID NO: 71. In an example embodiment, the vector construct comprises an hAAT intron and a hemoglobin intron adjacent to a hemoglobin exon sequence comprising a nucleotide sequence at least 80% or 85% or 90% identical to SEQ ID NO: 71.

[00129] In some embodiments, the vector construct comprises a polyadenylation signal, optionally a bovine growth hormone (bGH) polyA signal (e.g., SEQ I D NO: 72) or preferably a human growth hormone (hGH) polyA signal or fragment thereof (e.g., SEQ ID NO: 73-79). [00130] In one or more embodiments, the hGH polyA signal comprises a nucleotide sequence at least 80% or 85% or 90% identical to SEQ ID NO: 73, and the sequence may be about 1900 to about 2400 nucleotides in length, or about 2000 to about 2300 nucleotides in length.

[00131] In one or more embodiments, the hGH polyA signal comprises a nucleotide sequence at least 80% or 85% or 90% identical to SEQ ID NO: 74, and the sequence may be about 1700 to about 2200 nucleotides in length, or about 1800 to about 2100 nucleotides in length.

[00132] In one or more embodiments, the hGH polyA signal comprises a nucleotide sequence at least 80% or 85% or 90% identical to SEQ ID NO: 75, and the sequence may be about 1400 to about 1900 nucleotides in length, or about 1500 to about 1800 nucleotides in length.

[00133] In one or more embodiments, the hGH polyA signal comprises a nucleotide sequence at least 80% or 85% or 90% identical to SEQ ID NO: 76, and the sequence may be about 1300 to about 1800 nucleotides in length, or about 1400 to about 1700 nucleotides in length.

[00134] In one or more embodiments, the hGH polyA signal comprises a nucleotide sequence at least 80% or 85% or 90% identical to SEQ ID NO: 77, and the sequence may be about 1100 to about 1600 nucleotides in length, or about 1200 to about 1500 nucleotides in length.

[00135] In one or more embodiments, the hGH polyA signal comprises a nucleotide sequence at least 80% or 85% or 90% identical to SEQ ID NO: 78, and the sequence may be about 700 to about 1200 nucleotides in length, or about 800 to about 1100 nucleotides in length.

[00136] In one or more embodiments, the hGH polyA signal comprises a nucleotide sequence at least 80% or 85% or 90% identical to SEQ ID NO: 79, and the sequence may be about 400 to about 900 nucleotides in length, or about 500 to about 800 nucleotides in length.

[00137] The location and size of the intron in the vector can vary. In some embodiments, the intron is located between the promoter and the sequence encoding the protein of interest. In some embodiments, the intron is located downstream of the sequence encoding the protein of interest. In some embodiments, the intron is located within the promoter. In some embodiments, the intron includes an enhancer element. In some embodiments, the intron is located within the sequence encoding the protein of interest, preferably between exons of the sequence encoding the protein of interest. In some embodiments, the intron may comprise all or a portion of a naturally occurring intron within the sequence encoding the protein of interest. In some embodiments, the intron is a GLA intron. In other embodiments, the intronic sequence is a hAAT (human alpha- 1 -antitrypsin) intron. In other embodiments, the intronic sequence is a hemoglobin or beta-globin intron. In other embodiments, the intronic sequence is a composite hAAT/beta-globin intron.

[00138] Inclusion of an intron element may enhance expression compared with expression in the absence of the intron element (see e.g. Kurachi et al., 1995, J Biol Chem. 1995 Mar 10; 270(10):5276-81). AAV vectors typically accept inserts of DNA having a defined size range which is generally about 4 kb to about 5.2 kb, or slightly more. However, there is no minimum size for packaging and small vector genomes package very efficiently. Introns and intron fragments fulfill this requirement while also enhancing expression. Thus, the present disclosure is not limited to the inclusion of a-galactosidase A intron sequences in the AAV vector, and include other introns or other DNA sequences in place of portions of a a-galactosidase A intron. Additionally, other 5' and 3' untranslated regions of nucleic acid may be used in place of those recited for human a-galactosidase A.

[00139] Polynucleotides and polypeptides including modified forms can be made using various standard cloning, recombinant DNA technology, via cell expression or in vitro translation and chemical synthesis techniques known to those of skill in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition).

Methods of Gene Delivery

[00140] Also provided is a method of using vector construct or AAV particle as described herein to deliver a gene encoding the protein of interest. In one embodiment, a gene delivery vector may be a viral gene delivery vector, such as a viral particle, or a non-viral gene delivery vector, such as a vector construct or nucleic acid encoding the protein of interest. Viral vectors include lenti-, adeno-, herpes viral vectors. It is preferably a recombinant adeno-associated viral (rAAV) vector. Alternatively, non-viral systems may be used, including using naked DNA (with or without chromatin attachment regions) or conjugated DNA that is introduced into cells by various transfection methods such as lipids or electroporation.

[00141] A non-limiting example of a viral vector construct as described herein are as follows: GLA-co-1: AAV 5 - ApoE-h AAT -h A AT intron 150-GLA-co-JCAT-bGH (2812 bp), SEQ ID NO: 24,

GLA-co-2: AAV 5 - ApoE-h AAT -h A AT intron 1944-GLA-co-JCAT-bGH (4548 bp), SEQ ID NO: 25, GLA-co-3 : AAV 5 - ApoE-h AAT - hAAT intron 900-GLA-co-JCAT-hGH907 (4274 bp) , SEQ ID NO: 26,

GLA-co-4: AAV5- ApoE-h AAT- hAAT intron 450-GLA-co-JCAT-hGH1350 (4274 bp) , SEQ ID NO: 27,

GLA-co-5: AAV 5 - ApoE-h AAT -hAAT intron 225-GLA-co-JCAT-hGH1555 (4274 bp) , SEQ ID NO: 28,

GLA-co-IGF : AAV5-ApoE-hAAT-LGI-900-GLA-co-JCAT-RL-IGF-JCATco-CpG-hGH623 (4260 bp) , SEQ ID NO: 54.

[00142] In some embodiments, the vector construct or AAV vector genome comprises a nucleotide sequence having at least about 80%, 85% 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more, sequence identity to any of SEQ ID NOS: 24-58.

[00143] The present disclosure finds use in both veterinary and medical applications. Suitable subjects for gene delivery methods as described herein include both avians and mammals, with mammals being preferred and humans being most preferred. Human subjects include neonates, infants, juveniles, and adults.

Non- Gene Delivery

[00144] Non-viral gene delivery may be carried out using naked DNA which is the simplest method of non-viral transfection. It may be possible, for example, to administer the vector constructs provided herein using naked plasmid DNA. Alternatively, methods such as electroporation, sonoporation or the use of a "gene gun", which shoots DNA coated gold particles into the cell using, for example, high pressure gas or an inverted .22 calibre gun, may be used (Helios® Gene Gun System (BIO-RAD)).

[00145] To improve the delivery of a vector construct into a cell, it may be necessary to protect it from damage and its entry into the cell may be facilitated. To this end, lipoplexes and polyplexes may be used that have the ability to protect a nucleic acid from undesirable degradation during the transfection process.

[00146] Vector constructs may be coated with lipids in an organized structure such as a micelle or a liposome. When the organized structure is complexed with DNA it is called a lipoplex. Anionic and neutral lipids may be used for the construction of lipoplexes for synthetic vectors. In one embodiment, cationic lipids, due to their positive charge, may be used to condense negatively charged DNA molecules so as to facilitate the encapsulation of DNA into liposomes. If may be necessary to add helper lipids (usually electroneutral lipids, such as DOPE) to cationic lipids so as to form lipoplexes (Dabkowska et ah, JR Soc Interface. 2012 Mar 7; 9(68): 548-561).

[00147] In certain embodiments, complexes of polymers with DNA, called polyplexes, may be used to deliver a vector construct. Most polyplexes consist of cationic polymers and their production is regulated by ionic interactions. Polyplexes typically cannot release their DNA load into the cytoplasm. Thus, co-transfection with endosome-lytic agents (to lyse the endosome that is made during endocytosis, the process by which the polyplex enters the cell), such as inactivated adenovirus, may be necessary (Akinc et ah, The Journal of Gene Medicine. 7 (5): 657-63).

[00148] In certain embodiments, hybrid methods may be used to deliver a vector construct that combines two or more techniques. Virosomes are one example; they combine liposomes with an inactivated HIV or influenza virus. In another embodiment, other methods involve mixing other viral vectors with cationic lipids or hybridizing viruses and may be used to deliver a nucleic acid (Khan, Firdos Alam, Biotechnology Fundamentals, CRC Press, Nov 18, 2015, p. 395).

[00149] In certain embodiments, a dendrimer may be used to deliver a vector construct, in particular, a cationic dendrimer, i.e. one with a positive surface charge. When in the presence of genetic material as DNA or RNA, charge complementarity leads to a temporary association of the nucleic acid with the cationic dendrimer. On reaching its destination the dendrimer-nucleic acid complex is then imported into the cell via endocytosis (Amiji, Mansoor M. ed., Polymeric Gene Delivery: Principles and Applications, CRC Press, Sep 29, 2004, p. 142.)

Viral Particles

[00150] In one embodiment, a suitable viral gene delivery vector such as a viral particle may be used to deliver a nucleic acid. In certain embodiments, viral gene delivery vectors suitable for use herein may be a parvovirus, an adenovirus, a retrovirus, a lentivirus or a herpes simplex virus. The parvovirus may be an adenovirus-associated virus (AAV). [00151] Accordingly, the present disclosure provides viral particles for use as gene delivery vectors (comprising a vector construct provided herein) based on animal parvoviruses, in particular dependoviruses such as infectious human or simian AAV, and the components thereof (e.g., an animal parvovirus genome) for introduction and/or expression of a a-galactosidase A in a mammalian cell. The term "parvoviral" as used herein thus encompasses dependoviruses such as any type of AAV.

[00152] Viruses of the Parvoviridae family are small DNA animal viruses. The family Parvoviridae may be divided between two subfamilies: the Parvovirinae, which infect vertebrates, and the Densovirinae, which infect insects. Members of the subfamily Parvovirinae are herein referred to as the parvoviruses and include the genus Dependovirus. As may be deduced from the name of their genus, members of the Dependovirus are unique in that they usually require coinfection with a helper virus such as adenovirus or herpes virus for productive infection in cell culture. The genus Dependovirus includes AAV, which normally infects humans (e.g., serotypes 1, 2, 3A, 3B, 4, 5, and 6), primates (e.g., serotypes 1 and 4), and related viruses that infect other warm-blooded animals (e.g., bovine, canine, equine, mice, rats, and ovine adeno-associated viruses) in addition to birds and reptiles. Further information on parvoviruses and other members of the Parvoviridae is described in Kenneth I. Bems, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in Fields Virology (3d Ed. 1996). For convenience the present disclosure is further exemplified and described herein by reference to AAV. It is, however, understood that the present disclosure is not limited to AAV but may equally be applied to other parvoviruses.

[00153] Production of AAV particles requires AAV "rep" and "cap" genes, which are genes encoding replication and encapsidation proteins, respectively. AAV rep and cap genes have been found in all AAV serotypes examined to date, and are described herein and in the references cited. In wild-type AAV, the rep and cap genes are generally found adjacent to each other in the viral genome (i.e., they are "coupled" together as adjoining or overlapping transcriptional units), and they are generally conserved among AAV serotypes. AAV rep and cap genes are also individually and collectively referred to as "AAV packaging genes." The AAV cap genes for use herein encode Cap proteins which are capable of packaging AAV vectors in the presence of rep and adeno helper function and are capable of binding target cellular receptors. In some embodiments, the AAV cap gene encodes a capsid protein having an amino acid sequence derived from a particular AAV serotype.

[00154] The AAV sequences employed for the production of AAV can be derived from the genome of any AAV serotype. Generally, the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels, provide a similar set of genetic functions, produce virions which are essentially physically and functionally equivalent, and replicate and assemble by practically identical mechanisms. For the genomic sequence of AAV serotypes and a discussion of the genomic similarities. (See, e.g. , GenBank Accession number U89790; GenBank Accession number JO 1901 ; GenBank Accession number AF043303; GenBank Accession number AF085716; Chiorini et ah, J. Vir. (1997) vol. 71, pp. 6823-6833; Srivastava et ah, J. Vir. (1983) vol. 45, pp. 555-564; Chiorini et ah, J. Vir. (1999) vol. 73, pp. 1309-1319; Rutledge et ah, J. Vir. (1998) vol. 72, pp. 309-319; and Wu et ah, J. Vir. (2000) vol. 74, pp. 8635-8647).

[00155] The genomic organization of all known AAV serotypes is very similar. The genome of AAV is a linear, single-stranded DNA molecule that is less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the non- structural replication (Rep) proteins and the structural (VP) proteins. The VP proteins form the capsid. The assembly-activating protein (AAP) rapidly chaperones capsid assembly and prevents degradation of free capsid proteins (Grosse et ah, J. Virol. 91(20): eOl 198-17, 2017). The terminal 145 nt are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. The Rep genes encode the Rep proteins, Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, and Rep 52 and Rep40 are transcribed from the pl9 promoter. The cap genes encode the VP proteins, VPl, VP2, and VP3. The cap genes are transcribed from the p40 promoter. The ITRs employed in the vectors of the present embodiment may correspond to the same serotype as the associated cap genes, or may differ. In one embodiment, the ITRs employed herein correspond to an AAV2 serotype and the cap genes correspond to an AAV5 serotype.

[00156] The AAV VP proteins are known to determine the cellular tropicity of the AAV virion. The VP protein-encoding sequences are significantly less conserved than Rep proteins and genes among different AAV serotypes. The ability of Rep and ITR sequences to cross complement corresponding sequences of other serotypes allows for the production of pseudotyped AAV particles comprising the capsid proteins of a serotype (e.g., AAV1, 5 or 8) and the Rep and/or ITR sequences of another AAV serotype (e.g., AAV2). Such pseudotyped rAAV particles are a part of the present disclosure.

[00157] The AAV particles described herein (and the encoding AAV vector genomes) may comprise any of the capsid proteins described in WO 2018/022608 or PCT/US19/32097, incorporated by reference herein in its entirety for its disclosure of human and simian AAV capsids and their properties such as transduction efficiency, tissue tropism, glycan-binding, and resistance to neutralization by IVIG, including but not limited to any of the capsids in the sequence listing and variants thereof, e.g. with chimeric swapped variable regions and/or glycan binding sequences and/or GH loop.

[00158] In one embodiment, the AAV ITR sequences for use in the context of the present disclosure are derived from AAV1, AAV2, AAV4 and/or AAV6. Likewise, the Rep (e.g., Rep78 and Rep52) coding sequences are in one embodiment derived from AAV1, AAV2, AAV4 and/or AAV6. The sequences coding for the VP1, VP2, and VP3 capsid proteins for use in the context of the present disclosure may however be taken from any serotype, such as from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV 12, or from simian AAVs, including any of the capsid proteins described in WO 2018/022608 or PCT/US19/32097, or newly developed AAV-like particles obtained by e.g. capsid shuffling techniques and AAV capsid libraries, or any capsid at least 90% identical to any of SEQ ID NOs: 82-98.

[00159] For example, the amino acid sequences of various capsids are published. See, e.g., [00160] AAVRh.1 / hu.14 / AAV9 AAS99264.1 (SEQ ID NO: 82)

[00161] AAVRh.8 SEQ97 of U.S. Pat. Pub. 2013/0045186 (SEQ ID NO: 83)

[00162] AAVRh.10 SEQ81 of U.S. Pat. Pub. 2013/0045186 (SEQ ID NO: 84)

[00163] AAVRh.74 SEQ 1 of IntT. Pat. Pub. WO 2013/123503(SEQ ID NO: 85)

[00164] AAV1 AAB 95452.1 (SEQ ID NO: 86)

[00165] AAV2 YP 680426.1 (SEQ ID NO: 87)

[00166] AAV3 NP 043941.1 (SEQ ID NO: 88)

[00167] AAV3B AAB95452.1 (SEQ ID NO: 89) [00168] AAV4 NP 044927.1 (SEQ ID NO: 90)

[00169] AAV5 YP 068409.1 (SEQ ID NO: 91)

[00170] AAV6 AAB95450.1 (SEQ ID NO: 92)

[00171] AAV7 YP 077178.1 (SEQ ID NO: 93)

[00172] AAV8 YP 077180.1 (SEQ ID NO: 94)

[00173] AAV 10 AAT46337.1 (SEQ ID NO: 95)

[00174] AAV11 AAT46339.1 (SEQ ID NO: 96)

[00175] AAV 12 ABI16639.1 (SEQ ID NO: 97)

[00176] AAV 13 ABZ10812.1 (SEQ ID NO: 98)

[00177] Modified "AAV" sequences also can be used in the context of the present disclosure, e.g. for the production of AAV gene therapy vectors. Such modified sequences e.g. sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more nucleotide and/or amino acid sequence identity (e.g., a sequence having about 75-99% nucleotide sequence identity) to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 ITR, Rep, or VP, can be used in place of wild- type AAV ITR, Rep, or VP sequences.

[00178] In some embodiments, a nucleic acid sequence encoding an AAV capsid protein is operably linked to expression control sequences for expression in a specific cell type, such as Sf9 or HEK cells. Techniques known to one skilled in the art for expressing foreign genes in insect host cells or mammalian host cells can be used to practice the embodiment. Methodology for molecular engineering and expression of polypeptides in insect cells is described, for example, in Summers and Smith (1986) A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex.; Luckow (1991) In Prokop et ah, Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DNA Technology and Applications, 97-152; King, L. A. and R. D. Possee (1992) The baculovirus expression system, Chapman and Hall, United Kingdom; O'Reilly, D. R., L. K. Miller, V. A. Luckow (1992) Baculovirus Expression Vectors:

A Laboratory Manual, New York; W.H. Freeman and Richardson, C. D. (1995) Baculovirus Expression Protocols, Methods in Molecular Biology, volume 39; U.S. Pat. No. 4,745,051; US2003148506; and WO 03/074714, all of which are incorporated by reference in their entireties. A particularly suitable promoter for transcription of a nucleotide sequence encoding an AAV capsid protein is e.g. the polyhedron promoter. However, other promoters that are active in insect cells are known in the art, e.g. the plO, p35 or IE-1 promoters and further promoters described in the above references are also contemplated.

[00179] Use of insect cells for expression of heterologous proteins is well documented, as are methods of introducing nucleic acids, such as vectors, e.g., insect-cell compatible vectors, into such cells and methods of maintaining such cells in culture. (See, e.g., METHODS IN MOLECULAR BIOLOGY, ed. Richard, Humana Press, N J (1995); O'Reilly et al.,

BACULO VIRUS EXPRESSION VECTORS, A LABORATORY MANUAL, Oxford Univ. Press (1994); Samulski et al., J. Vir. (1989) vol. 63, pp.3822-3828; Kajigaya et al., Proc. Nat'l. Acad. Sci. USA (1991) vol. 88, pp. 4646-4650; Ruffing et al., J. Vir. (1992) vol. 66, pp. 6922- 6930; Kirnbauer et al., Vir. (1996) vol. 219, pp. 37-44; Zhao et al., Vir. (2000) vol. 272, pp. 382- 393; and U.S. Pat. No. 6,204,059). In some embodiments, the nucleic acid construct encoding AAV in insect cells is an insect cell-compatible vector. An "insect cell-compatible vector" or "vector" as used herein refers to a nucleic acid molecule capable of productive transformation or transfection of an insect or insect cell. Exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector can be employed as long as it is insect cell-compatible. The vector may integrate into the insect cells genome but the presence of the vector in the insect cell need not be permanent and transient episomal vectors are also included. The vectors can be introduced by any means known, for example by chemical treatment of the cells, electroporation, or infection. In some embodiments, the vector is a baculovirus, a viral vector, or a plasmid. In one embodiment, the vector is a baculovirus, i.e. the construct is a baculoviral vector. Baculoviral vectors and methods for their use are described in the above cited references on molecular engineering of insect cells.

METHODS FOR PRODUCING RECOMBINANT AAV PARTICLES

[00180] The present disclosure provides materials and methods for producing recombinant AAV particles in insect or mammalian cells that comprise any of the vector constructs described herein. In some embodiments, the vector construct further comprises a promoter and a restriction site downstream of the promoter to allow insertion of a polynucleotide encoding one or more proteins of interest, wherein the promoter and the restriction site are located downstream of the 5' AAV ITR and upstream of the 3' AAV ITR. In some embodiments, the vector construct further comprises a posttranscriptional regulatory element downstream of the restriction site and upstream of the 3' AAV ITR. In some embodiments, the vector construct further comprises a polynucleotide inserted at the restriction site and operably linked with the promoter, where the polynucleotide comprises the coding region of a protein of interest. As a skilled artisan will appreciate, any one of the AAV vector constructs disclosed in the present application can be used in methods to produce the recombinant AAV particle.

[00181] In some embodiments, the helper functions are provided by one or more helper plasmids or helper viruses comprising adenoviral or baculoviral helper genes. Non-limiting examples of the adenoviral or baculoviral helper genes include, but are not limited to, El A, E1B, E2A, E4 and VA, which can provide helper functions to AAV packaging.

[00182] Helper viruses of AAV are known in the art and include, for example, viruses from the family Adenoviridae and the family Herpes viridae. Examples of helper viruses of AAV include, but are not limited to, SAdV-13 helper virus and SAdV-13-like helper virus described in US Publication No. 20110201088 (the disclosure of which is incorporated herein by reference), and helper vectors pHELP (Applied Viromics). A skilled artisan will appreciate that any helper virus or helper plasmid of AAV that can provide adequate helper function to AAV can be used herein.

[00183] In some embodiments, the AAV cap genes are present in a plasmid. The plasmid can further comprise an AAV rep gene which may or may not correspond to the same serotype as the cap genes. The cap genes and/or rep gene from any AAV serotype described herein (including, but not limited to, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,

AAV1 1, AAV12, AAV13 and any variants thereof) can be used to produce the recombinant AAV. In some embodiments, the AAV cap genes encode a capsid from serotype 1, serotype 2, serotype 4, serotype 5, serotype 6, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11, serotype 12, serotype 13 or a variant thereof.

[00184] In some embodiments, the insect or mammalian cell can be transfected with the helper plasmid or helper virus, the vector construct and the plasmid encoding the AAV cap genes; and the recombinant AAV virus can be collected at various time points after co transfection. For example, the recombinant AAV virus can be collected at about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, or a time between any of these two time points after the co-transfection. [00185] Recombinant AAV particles can also be produced using any conventional methods known in the art suitable for producing infectious recombinant AAV. In some instances, a recombinant AAV can be produced by using an insect or mammalian cell that stably expresses some of the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) comprising AAV rep and cap genes, and a selectable marker, such as a neomycin resistance gene, can be integrated into the genome of the cell. The insect or mammalian cell can then be co-infected with a helper virus (e.g., adenovirus or baculovirus providing the helper functions) and the viral vector construct comprising the 5' and 3' AAV ITR (and the nucleotide sequence encoding the heterologous protein, if desired). The advantages of this method are that the cells are selectable and are suitable for large-scale production of the recombinant AAV particle. As another non-limiting example, adenovirus or baculovirus rather than plasmids can be used to introduce rep and cap genes into packaging cells. As yet another non-limiting example, both the viral vector construct containing the 5' and 3' AAV ITRs and the rep-cap genes can be stably integrated into the DNA of producer cells, and the helper function can be provided by a wild-type adenovirus to produce the recombinant AAV.

[00186] In one aspect, provided herein are methods for the production of an AAV particle, useful as a gene delivery vector, the method comprising the steps of:

(a) providing a cell permissive for AAV replication (e.g. an insect cell or a mammalian cell) with one or more nucleic acid constructs comprising:

(i) a nucleic acid molecule (e.g. recombinant vector construct) provided herein that is flanked by at least one AAV inverted terminal repeat nucleotide sequence;

(ii) a nucleotide sequence encoding one or more AAV Rep proteins which is operably linked to a promoter that is capable of driving expression of the Rep protein(s) in the cell;

(iii) a nucleotide sequence encoding one or more AAV capsid proteins which is operably linked to a promoter that is capable of driving expression of the capsid protein(s) in the cell;

(iv) and optionally AAP and MAAP contained in the VP2/3 mRNA

(b) culturing the cell defined in (a) under conditions conducive to the expression of the Rep and the capsid proteins; and, optionally, (c) recovering the AAV gene delivery vector, and optionally (d) purifying the AAV particle. For example, the recombinant vector construct of (i) comprises (1) at least one AAV ITR, (2) a heterologous liver-specific transcription regulatory region as described herein, and (3) a nucleic acid encoding a functional a- galactosidase A. Preferably the recombinant vector construct of (i) comprises both a 5’ and 3’ AAV ITR.

[00187] Typically then, a method provided herein for producing a AAV gene delivery vector comprises: providing to a cell permissive for AAV replication (a) a nucleotide sequence encoding a template for producing vector genome, e.g. vector construct of the present disclosure (as described in detail herein); (b) nucleotide sequences sufficient for replication of the template to produce a vector genome (the first expression cassette defined above); (c) nucleotide sequences sufficient to package the vector genome into an AAV capsid (the second expression cassette defined above), under conditions sufficient for replication and packaging of the vector genome into the AAV capsid, whereby AAV particles comprising the vector genome encapsidated within the AAV capsid are produced in the cell.

[00188] Transient transfection of adherent HEK293 cells (Chahal et ah, J. Virol. Meth. 196: 163-73 (2014)) and transfection of Sf9 cells, using the baculovirus expression vector system (BEVS) (Mietzsch et ah, Hum. Gene Ther. 25: 212-22 (2014)), are two of the most commonly used methods to produce AAV vectors.

[00189] In one embodiment, following an expansion of transfected cells in suspension cell culture through a series of increasingly large culture platforms, a suspension of transfected cells is purified through a multi-step process to remove process impurities, including recombinant baculviruses and host cells, and enrich for the virions comprising the recombinant parvoviral (rAAV) vector construct. In another embodiment, method provided herein may comprise the step of affinity-purification of the rAAV vector construct using an anti-AAV antibody, in one embodiment an immobilized antibody. In another embodiment, the anti-AAV antibody is a monoclonal antibody. One antibody for use herein is a single chain camelid antibody or a fragment thereof as e.g. obtainable from camels or llamas (see e.g. Muyldermans, 2001, Biotechnol. 74: 277-302). The antibody for affinity-purification of rAAV is an antibody that specifically binds an epitope on an AAV capsid protein, whereby in one embodiment the epitope is an epitope that is present on capsid protein of more than one AAV serotype. For example, the antibody may be raised or selected on the basis of specific binding to AAV5 capsid but at the same time also it may also specifically bind to AAV1, AAV2, AAV3, AAV6, AAV8 or AAV9 capsids.

[00190] The methods provided herein for producing rAAV particles produce a population of rAAV particles. In some embodiments, the population is enriched for particles comprising full length or nearly full length vector genomes by steps that reduce the number of empty capsids. [00191] The population of rAAV particles produced by the methods provided herein are used, for example, for administration in any of the treatment methods described herein.

CELL TYPES USED IN AAV PARTICLE PRODUCTION

[00192] The viral particles comprising the vector constructs described herein may be produced using any invertebrate cell type which allows for production of AAV or biologic products and which can be maintained in culture. For example, the insect cell line used can be from Spodoptera frugiperda, such as SF9, SF21, SF900+, drosophila cell lines, mosquito cell lines, e.g., Aedes albopictus derived cell lines, domestic silkworm cell lines, e.g. Bombyx mori cell lines, Trichoplusia ni cell lines such as High Five cells or Lepidoptera cell lines such as Ascalapha odorata cell lines. In one embodiment, insect cells are cells from the insect species which are susceptible to baculovirus infection, including High Five, Sf9, Se301, SeIZD2109, SeUCRl, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAml, BM-N, Ha2302, Hz2E5 and Ao38.

[00193] Baculoviruses are enveloped DNA viruses of arthropods, two members of which are well known expression vectors for producing recombinant proteins in cell cultures.

Baculoviruses have circular double-stranded genomes (80-200 kbp) which can be engineered to allow the delivery of large genomic content to specific cells. The viruses used as a vector are generally Autographa califomica multicapsid nucleopolyhedrovirus (AcMNPV) or Bombyx mori nucleopolyhedrovirus (BmNPV) (Kato et ak, (2010), Applied Microbiology and Biotechnology, vol. 85, Issue 3, pp 459-470).

[00194] Baculoviruses are commonly used for the infection of insect cells for the expression of recombinant proteins. In particular, expression of heterologous genes in insects can be accomplished as described in for instance U.S. Pat. No. 4,745,051; EP 127,839; EP 155,476; Vlak et ak, (1988), Journal of General Virology, vol. 68, pp 765-776; Miller et ak, (1988), Annual Review of Microbiology, vol. 42, pp 177-179; Carbonell et al., (1998), Gene, vol. 73, Issue 2, pp 409-418; Maeda et al., (1985), Nature, vol. 315, pp 592-594; Lebacq-Veheyden et al., (1988), Molecular and Cellular Biology, vol. 8, no. 8, pp 3129-3135; Smith et al., (1985), PNAS, vol. 82, pp 8404-8408; and Miyajima et al., (1987), Gene, vol. 58, pp 273-281. Numerous baculovirus strains and variants and corresponding permissive insect host cells that can be used for protein production are described in Luckow et al., (1988), Nature Biotechnology, vol. 6, pp 47-55; Maeda et al., (1985), Nature, vol. 315, pp 592-594; and McKenna et al., (1998), Journal of Invertebrate Pathology, vol. 71, Issue 1, pp 82-90.

[00195] In another embodiment, the methods provided herein are carried out with any mammalian cell type which allows for replication of AAV or production of biologic products, and which can be maintained in culture. In one embodiment, mammalian cells used can be HEK293, HeLa, CHO, NSO, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE- 19, and MRC-5 cells.

Host Organism and/or Cells

[00196] In a further embodiment, a host cell is provided comprising the vector described above. In one embodiment, the vector construct is capable of expressing the nucleic acid molecule provided herein in the host cell. In some embodiments, provided herein are Fabry Disease therapeutics that are host cells comprising a vector construct comprising a nucleic acid encoding a-galactosidase A, for use in Fabry Disease cell therapy.

[00197] As used herein, the term "host" refers to organisms and/or cells which harbour a nucleic acid molecule or a vector construct of the present disclosure, as well as organisms and/or cells that are suitable for use in expressing a recombinant gene or protein. It is not intended that the present disclosure be limited to any particular type of cell or organism. Indeed, it is contemplated that any suitable organism and/or cell will find use herein as a host. A host cell may be in the form of a single cell, a population of similar or different cells, for example in the form of a culture (such as a liquid culture or a culture on a solid substrate), an organism or part thereof. In one embodiment, a host cell may permit the expression of a nucleic acid molecule provided herein. Thus, the host cell may be, for example, a bacterial, a yeast, an insect or a mammalian cell, or a human cell. [00198] In another embodiment, provided is a means for delivering a nucleic acid provided herein into a broad range of cells, including dividing and non-dividing cells. The present disclosure may be employed to deliver a nucleic acid provided herein to a cell in vitro, e. g. to produce a polypeptide encoded by such a nucleic acid molecule in vitro or for ex vivo gene therapy.

[00199] The nucleic acid molecule, vector construct, cells and methods/use of the present disclosure are additionally useful in a method of delivering a nucleic acid provided here into a host, typically a host suffering from Fabry Disease.

PHARMACEUTICAL FORMULATIONS

[00200] In one embodiment, provided is a pharmaceutical composition comprising a nucleic acid or a vector provided herein and a pharmaceutically acceptable diluent, excipient, carrier and/or other medicinal agent, pharmaceutical agent or adjuvant, etc.

[00201] By "pharmaceutically acceptable" it is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects. Thus, such a pharmaceutical composition may be used, for example, in transfection of a cell ex vivo or in administering a viral particle or cell directly to a subject.

[00202] A carrier may be suitable for parenteral administration, which includes intravenous, intraperitoneal or intramuscular administration. Alternatively, the carrier may be suitable for sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions provided herein is contemplated.

[00203] In other embodiments, provided herein are pharmaceutical compositions (i.e. formulations) of AAV particles useful for administration to subjects suffering from a genetic disorder to deliver gene encoding a protein of interest. In certain embodiments, the pharmaceutical formulations provided herein are liquid formulations that comprise recombinant AAV particles comprising any of the vector constructs disclosed herein. The concentration of recombinant AAV virions in the formulation may vary. In certain embodiments, the concentration of recombinant AAV particle in the formulation may range from 1 x 10¹² to about 6 x 10¹⁴ vg/ml.

[00204] In other embodiments, the AAV particle pharmaceutical formulation provided herein comprises one or more sterile pharmaceutically acceptable excipients to provide the formulation with advantageous properties for storage and/or administration to subjects for the treatment of the genetic disorder. In certain embodiments, the pharmaceutical formulations provided herein are capable of being stored at -65°C for a period of at least 2 weeks, in one embodiment at least 4 weeks, in another embodiment at least 6 weeks and yet another embodiment at least about 8 weeks, without detectable change in stability. In this regard, the term "stable" means that the recombinant AAV particle present in the formulation essentially retains its physical stability, chemical stability and/or biological activity during storage. In certain embodiments, the recombinant AAV particle present in the pharmaceutical formulation retains at least about 80% of its biological activity in a human patient during storage for a determined period of time at - 65°C, in other embodiments at least about 85%, 90%, 95%, 98% or 99% of its biological activity in a human subject. In one embodiment the subjects are juvenile human subjects.

[00205] In certain aspects, the formulation comprising recombinant AAV particle further comprises one or more buffering agents.

[00206] In another embodiment, the recombinant AAV particle formulation provided herein may comprise one or more isotonicity agents, such as sodium chloride. Other buffering agents and isotonicity agents known in the art are suitable and may be routinely employed for use in the formulations provided herein.

[00207] In another embodiment, the recombinant AAV particle formulations provided herein may comprise one or more bulking agents. Exemplary bulking agents include without limitation mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24).

[00208] In yet another embodiment, the recombinant AAV particle formulations provided herein may comprise one or more surfactants, which may be non-ionic surfactants. Exemplary surfactants include ionic surfactants, non-ionic surfactants, and combinations thereof. For example, the surfactant can be, without limitation, TWEEN 80 (also known as polysorbate 80, or its chemical name polyoxyethylene sorbitan monooleate), sodium dodecyl sulfate, sodium stearate, ammonium lauryl sulfate, TRITON AG 98 (Rhone-Poulenc), poloxamer 407, poloxamer 188 and the like, and combinations thereof.

[00209] The recombinant AAV particle formulations provided herein are stable and can be stored for extended periods of time without an unacceptable change in quality, potency, or purity. In one aspect, the formulation is stable at a temperature of about 5°C (e.g., 2°C to 8°C) for at least 1 month, for example, at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or more. In another embodiment, the formulation is stable at a temperature of less than or equal to about -20°C for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more. In another embodiment, the formulation is stable at a temperature of less than or equal to about -40°C for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more. In another embodiment, the formulation is stable at a temperature of less than or equal to about -60°C for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more.

[00210] Pharmaceutical compositions are typically sterile and stable under the conditions of manufacture and storage. Pharmaceutical compositions may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to accommodate high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. 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 dispersion and by the use of surfactants. In some embodiments, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride are included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. In certain embodiments, a nucleic acid or vector construct provided herein may be administered in a time or controlled release formulation, for example in a composition which includes a slow release polymer or other carriers that will protect the compound against rapid release, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may for example be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymners (PLG).

[00211] In certain embodiments, the pharmaceutical composition comprising the vector construct or AAV particle provided herein may be of use in transferring genetic material to a cell. Such transfer may take place in vitro, ex vivo or in vivo. Accordingly, one embodiment provides a method for delivering a nucleotide sequence to a cell, which method comprises contacting a nucleic acid, a vector construct, or a pharmaceutical composition as described herein under conditions such the nucleic acid or vector provided herein enters the cell. The cell may be a cell in vitro, ex vivo or in vivo.

METHODS OF TREATMENT

[00212] In certain embodiments, provided herein are methods for treating a subject suffering from a genetic disorder comprising administering to the subject a therapeutically effective amount of a nucleic acid encoding a functionally active a-galactosidase A protein, a vector construct, an AAV particle, or a host cell expressing a functionally active a-galactosidase A protein, or a pharmaceutical composition comprising the same. In this instance, a “therapeutically effective amount” is an amount that after administration results in the expression of the therapeutic protein in a level sufficient to at least partially and preferably fully ameliorate the signs and/or symptoms of the genetic disorder.

[00213] In one embodiment, provided herein is a method of treating a-galactosidase A deficiency comprising administering a therapeutically effective amount of a nucleic acid, a vector construct, an AAV particle, a host cell or a pharmaceutical composition provided herein to a patient suffering from a a-galactosidase A deficiency, for example Fabry Disease. In one embodiment, the patient is human. In one embodiment, the subject patient population is patients with moderate to severe a-galactosidase A deficiency, including those with Fabry Disease, or variant forms of Fabry Disease. In one embodiment, the goal for the treatment is conversion of severe Fabry patients to either moderate or mild Fabry. In one embodiment, the treatment increases functional a-galactosidase A levels or a-galactosidase A activity in blood or tissues, e.g., in plasma, liver, heart, kidney, brain or other tissues, or reduces accumulation of Gb-3 or Lyso-Gb-3 in liver, heart, kidney, brain or other tissues, or reduces any signs and/or symptom described herein or known in the art. [00214] In one embodiment, provided herein are methods for increasing circulating a- galactosidase A protein levels in the blood of a subject in need thereof comprising administering to the subject any of the nucleic acids, vector constructs, AAV particles, host cells, or pharmaceutical compositions provided herein, that express the functionally active a- galactosidase A protein.

[00215] In another embodiment, provided herein is the use of an effective amount of recombinant AAV particle described herein for the preparation of a medicament for the treatment of a subject suffering from deficiency of functional a-galactosidase A or Fabry Disease. In one embodiment, the subject suffering from Fabry Disease is a human. In one embodiment, the medicament is administered by intravenous (IV) administration. In another embodiment, administration of the medicament results in expression of a-galactosidase A protein in the bloodstream of the subject sufficient to increase functional a-galactosidase A levels or a- galactosidase A activity in blood or in tissues in the subject, e.g., in plasma, liver, heart, kidney, brain or other tissues, or reduces accumulation of Gb-3 or Lyso-Gb-3 in liver, heart, kidney, brain or other tissues, or reduces any signs or symptom described herein or known in the art. [00216] In one or more embodiments, the treatment methods provided herein also comprise administration of a prophylactic and/or therapeutic corticosteroid for the prevention and/or treatment of any hepatotoxicity associated with administration of the AAV GLA virus.

[00217] In or more embodiments, the treatment methods provided herein optionally include administration, e.g. concurrent administration, of other therapies that are used to treat Fabry Disease.

[00218] A "therapeutically effective amount" of a nucleic acid, vector construct, AAV particle, host cell, or a pharmaceutical composition comprising the same for purposes of treatment as described herein may be determined empirically and in a routine manner. Example therapeutically effective amounts of recombinant AAV particle range from about 1 x 10¹¹ to about 6 x 10¹⁴ vg/kg.

[00219] In one embodiment, recombinant vector constructs or AAV particles provided herein may be administered to a subject, in one embodiment a mammalian subject, or a human subject, through a variety of known administration techniques. In some embodiments, the vector construct or recombinant AAV particle is administered by intravenous injection either as a single bolus or over a prolonged time period, which may be at least about 1, 5, 10, 15, 30, 45, 60, 75,

90, 120, 150, 180, 210 or 240 minutes, or more.

[00220] In any of the treatment methods described herein, the effectiveness of the treatment can be monitored by measuring levels of or activity of functional a-galactosidase A in the blood of the treated subject. The protocol was adapted from R&D Systems datasheet for Recombinant Human a-Galactosidase A/GLA (R&D Systems, 6146-GH) (Ioannou et al., J Cell Biol. 119(5): 1137-50 (1992)). Precise quantitate assays for quantifying circulating levels of a-galactosidase A are well known in the art and commercially available.

[00221] Administration of an AAV particle of the present disclosure may, in some cases, result in an observable degree of hepatotoxicity. Hepatotoxicity may be measured by a variety of well-known and routinely used techniques for example, measuring concentrations of certain liver-associated enzyme(s) (e.g., alanine transaminase, ALT) in the bloodstream of a subject both prior to AAV administration (i.e., baseline) and after AAV administration. An observable increase in ALT concentration after AAV administration (as compared to prior to administration) is indicative of drug-induced hepatotoxicity. In certain embodiments, in addition to administration of a therapeutically effective amount of AAV virus, the subject may be treated either prophylactically, therapeutically, or both with a corticosteroid to prevent and/or treat any hepatotoxicity associated with administration of the AAV virus.

[00222] "Prophylactic" corticosteroid treatment refers to the administration of a corticosteroid to prevent hepatotoxicity and/or to prevent an increase in measured ALT levels in the subject. "Therapeutic" corticosteroid treatment refers to the administration of a corticosteroid to reduce hepatotoxicity caused by administration of an AVV virus and/or to reduce an elevated ALT concentration in the bloodstream of the subject caused by administration of an AAV virus. In certain embodiments, prophylactic or therapeutic corticosteroid treatment may comprise administration of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more mg/day of the corticosteroid to the subject. In certain embodiments, prophylactic or therapeutic corticosteroid treatment of a subject may occur over a continuous period of at least about 3, 4, 5, 6, 7, 8, 9, 10 weeks, or more. Corticosteroids that find use in the methods described herein include any known or routinely-employed corticosteroid including, for example, dexamethasone, prednisone, fludrocortisone, hydrocortisone, and the like. DETECTION OF ANTI-AAV ANTIBODIES

[00223] To maximize the likelihood of successful liver transduction with systemic AAV- mediated therapeutic gene transfer, prior to administration of an AAV particle in a therapeutic regimen to a human patient as described above, the prospective patient may be assessed for the presence of anti-AAV capsid antibodies or anti-AAV neutralizing antibodies that are capable of blocking cell transduction or otherwise reduce the overall efficiency of the therapeutic regimen. Such antibodies may be present in the serum of the prospective patient and may be directed against an AAV capsid of any serotype. In one embodiment, the serotype against which pre existing antibodies are directed is AAV5.

[00224] Methods to detect pre-existing AAV immunity are well known and routinely employed in the art and include cell-based in vitro transduction inhibition (TI) assays, in vivo (e.g., in mice) TI assays, and ELISA-based detection of total anti-capsid antibodies (TAb) (see, e.g.,Masat et ah, Discov. Med., vol. 15, pp. 379-389 and Boutin et ah, (2010) Hum. Gene Ther., vol. 21, pp. 704-712). TI assays may employ host cells into which an AAV-inducible reporter vector has been previously introduced. The reporter vector may comprise an inducible reporter gene such as GFP, etc. whose expression is induced upon transduction of the host cell by an AAV virus. Anti-AAV capsid antibodies present in human serum that are capable of preventing/reducing host cell transduction would thereby reduce overall expression of the reporter gene in the system. Therefore, such assays may be employed to detect the presence of anti-AAV capsid antibodies in human serum that are capable of preventing/reducing cell transduction by the therapeutic AAV particle.

[00225] The assays to detect anti-AAV capsid antibodies may employ solid-phase-bound AAV capsid as a "capture agent" over which human serum is passed, thereby allowing anti capsid antibodies present in the serum to bind to the solid-phase-bound capsid "capture agent". Once washed to remove non-specific binding, a "detection agent" may be employed to detect the presence of anti-capsid antibodies bound to the capture agent. The detection agent may be an antibody, an AAV capsid, or the like, and may be detectably-labeled to aid in detection and quantitation of bound anti-capsid antibody. In one embodiment, the detection agent is labeled with ruthenium or a ruthenium-complex that may be detected using electrochemiluminescence techniques and equipment. [00226] The same above-described methodology may be employed to assess and detect the generation of an anti-AAV capsid immune response in a patient previously treated with a therapeutic AAV virus of interest. As such, not only may these techniques be employed to assess the presence of anti-AAV capsid antibodies prior to treatment with a therapeutic AAV virus, they may also be employed to assess and measure the induction of an immune response against the administered therapeutic AAV virus after administration. As such, contemplated herein are methods that combine techniques for detecting anti-AAV capsid antibodies in human serum and administration of a therapeutic AAV virus for the treatment of Fabry Disease, wherein the techniques for detecting anti-AAV capsid antibodies in human serum may be performed either prior to or after administration of the therapeutic AAV virus.

[00227] Other aspects and advantages of the present disclosure will be understood upon consideration of the following illustrative examples.

EXAMPLES

EXAMPLE 1: EVALUATION OF DIFFERENT HUMAN A-GALACTOSIDASE A- CODING SEQUENCE OPERABLY LINKED TO A LIVER SPECIFIC PROMOTER IN GLA-KO MICE

[00228] A variety of recombinant AAV gene therapy vector constructs were designed comprising a variety of different GLA cDNAs (wild type, variant or different codon-optimized versions) operably linked to a hybrid human apolipoprotein E (ApoE)/HCR enhancer / human alpha anti-trypsin (AAT) promoter, optionally including an intron and polyadenylation signal, and flanked by AAV2 ITRs. In some cases, the encoded GLA was a fusion protein comprising an IGF tag. The twenty-one coding sequences are set forth in SEQ ID NO: 3-23. The vector constructs were prepared using conventional cloning techniques as described e.g., by Gibson et al. (2009). "Enzymatic assembly of DNA molecules up to several hundred kilobases". Nature Methods. 6 (5): 343-345, and Gibson DG. (2011). "Enzymatic assembly of overlapping DNA fragments". Methods in Enzymology. 498: 349-361, which are incorporated herein by reference. [00229] A preliminary in vitro assay was performed to compare the GLA expression and activity from the four vectors described above. Plasmids of the vector constructs were transiently transfected into the human liver cell line, HepG2. Several codon-optimized cDNAs were demonstrated to be capable of expressing high levels of active a-galactosidase A in transiently transfected HepG2 cells. The two highest expressing constructs, Co-JCAT and CoVWF, produced almost twice as much enzyme as the wild type GLA construct, GLA. See Figure 1.

[00230] AAV virions comprising the top three expressing vector constructs “AAV5-ApoE- hAAT-CoVWF” (SEQ ID NO: 32), “AAV5-ApoE-hAAT-Co-JCAT,” (co-1; SEQ ID NO: 24) and “AAV5-ApoE-hAAT-CoVWF-CpG,” (SEQ ID NO: 42) in AAV5-type capsids were produced in a baculovirus expression system. Baculovirus constructs were generated expressing the three vector constructs, and the AAV Cap and Rep proteins, and then were co-infected into insect Sf9 cells. The resultant AAV virions were purified and analyzed by standard methods known in the art.

[00231] The purified AAV virions were quantified by qPCR and tested for a-galactosidase A activity in GLA knockout (GLAko) mice. GLAko mice have no a-galactosidase A activity and progressively accumulate Gb-3 in tissues and fluids, similarly to Fabry Disease patients. Mice (n=7 each group) were administered via IV 1.6E+14vg/kg AAV virions or vehicle. Plasma was collected pre-dosing and at weeks 3, 5 and 8 post injection. Liver, spleen, heart, kidney and brain tissues were collected at week 8. Levels of functional a-galactosidase A activity were measured by its ability to hydrolyze the synthetic substrate 4-methylumbelliferyl-a-D- galactopyranoside. Gb-3 and Lyso-Gb-3 levels were measured by LC/MS/MS method. Immunohistochemistry for a-galactosidase A was also performed and organ pathology was inspected.

[00232] Results are depicted in Figures 2-6. The single dose of AAV produced rising levels of a-galactosidase A activity in plasma (nmol/hr/ml plasma) over the 8 weeks period, as shown in Figures 2 and 3. Figures 4, 5 and 6 show the levels of a-galactosidase A activity in liver, heart and kidney at 8 weeks post-injection. Activity levels depended on the vector construct and gene variant. Plasma a-galactosidase A activity was correlated to kidney, heart and liver a- galactosidase A activity, with the strength of the correlation depending on the coding sequence variant. The JCAT coding sequence that produced the highest enzyme activity also had the highest correlation. Selection of the JCAT codon optimized sequence improved the plasma a- galactosidase A to 8-fold higher than the plasma a-galactosidase A levels of wild type GLA sequence.

[00233] The combination of the AAV5-type capsid and the hybrid human apolipoprotein E (ApoE)/HCR enhancer / human alpha anti-trypsin (AAT) promoter, operably linked to the optimal coding sequence of a-galactosidase A, in the AAV particles of the invention unexpectedly produced higher levels of plasma and tissue functional a-galactosidase A than other AAV therapy which had different coding sequences, and different promoters or different capsids than the AAV particles of the invention. See Table 1 below, which shows levels of a- galactosidase A in plasma or the specified tissue type, expressed as fold increase over wild type mice. See data in Freeline poster at World 2019; Sangamo poster at World 2018; UniQure 2019 R&D day presentation; Ziegler R. et al. 2007. Molecular Therapy vol. 15 no. 3, p493-500.

Table 1

(10 mg)

[00234] Figures 7A-B, 8A-B and 9A-B show the levels of total Gb-3 and Lyso-Gb-3 (ng Lyso-Gb-3/mg protein) in kidney, heart and liver of the GLAko mice at 8 weeks after injection of AAV particles. Figures 10A-B shows the level of total Gb-3 and Lyso-Gb-3 in plasma of the GLAko mice at 8 weeks. All tested coding sequences were able to reduce levels of Gb-3 and Lyso-Gb-3 to nearly or to wild type levels.

[00235] GLAko mice dosed with 1.6E+14 vg/kg demonstrated plasma a-galactosidase A activities that were orders of magnitude more than wild type mice (AAV5-GLA wild type: ~ 1,400X or AAV5-GLA-CO-JCAT: ~10,500X). All vectors analyzed led to large reductions in Gb3 in kidney, heart, liver, and plasma down to amounts near to or at WT levels. The plasma a- Gal A activity was maintained at ~ 9682 nmol/hr/ml and above (~1,400X wild type) and mice exhibited reduced Gb3 and lysoGb3 levels down to near or at normal levels in kidney, heart, liver and plasma.

[00236] Immunohistochemistry showed that the a-galactosidase A enzyme could be detected throughout the kidney within the tubules and glomeruli. The presence or absence of the IGF tag did not affect enzyme levels within the podocytes. The presence of a-galactosidase A enzyme was detected in all hepatocytes evaluated. The majority of a-galactosidase A immunostaining was detected in cells lining the sinusoids, and enzyme was also detected in endothelial and Kupffer cells in the sinusoidal space. Tissues were evaluated for pathology through H&E staining, TUNEL staining, IBA1 quantitation and LAMP2 quantitation. IBAl is a marker of both resident and infiltrating macrophages, and inclusive of basal and activated states. LAMP2 indicates the size of lysosomes. There were no signs of increased cell death, inflammation, or lysosomal dysfunction or stress. Some groups had an observed increase in vacuoles but there was no significant increase in the number of macrophages, the amount of LAMP2 immunostaining, and no increase in ALT at 8 weeks post-injection. No significant increase in cell death was observed except for a slight increase for the CoVWF construct.

EXAMPLE 2: DOSE RESPONSE STUDY IN RAG 2^{~ ~} MICE

[00237] A dose response study was conducted with a vector construct containing one of the codon optimized sequences encoding a-galactosidase A, “Co-JCAT,” operably linked to a hybrid human apolipoprotein E (ApoE)/HCR enhancer / human alpha anti-trypsin (AAT) promoter, optionally including various introns and polyadenylation signal, and flanked by AAV2 ITRs. AAV virions comprising this vector construct with an AAV5-type capsid were produced in a baculovirus expression vector (BEV) system.

[00238] The purified vectors were quantified by qPCR and dosed at 1 0E+14vg/kg, 6.0E+13vg/kg, 2.0E+13vg/kg, 6.0E+12vg/kg, 2.0E+12vg/kg, 6.0E+llvg/kg and 2.0E+llvg/kg into 8 weeks old Rag2 ^/_ mice alongside a vehicle control group. Plasma was collected pre dosing and at weeks 3, 5 and 8 post injection. Liver, spleen, heart, kidney and brain tissues were collected at week 8. Levels of functional a-galactosidase A activity was determined as described above. [00239] Results are shown in Figures 11-15. All of the groups dosed with 6.0E+1 lvg/kg or above showed increased a-galactosidase A in plasma above vehicle background (Figure 11) that was nearly wild type levels. Plasma a-galactosidase A activity was dose-dependent and was sustained for 8 weeks post-AAV dosing in plasma, kidney heart, liver and spleen. Increased a- galactosidase A activity was detectable in plasma with a dose as low as 6el 1 vg/kg, and the dose of 2el2 vg/kg increased a-galactosidase A activity ~6.7 fold compared to wild type levels from week 3 to week 5 post dose, with a plateau of a-galactosidase A activity between week 5 and week 8 at ~10 fold compared to wild type levels. At doses of 2el3 vg/kg in Rag2 ^/_ mice, plasma a-galactosidase A activity reached the level of- 4,300 nmol/hr/ml (1,000X wild type).

[00240] Increased a-galactosidase A activity was observed in kidney, heart, liver and spleen tissues dosed with 6.0E+12vg/kg or above (Figures 12, 13, 14 and 15, respectively). Plasma a- galactosidase A activity was correlated to kidney a-galactosidase A activity.

[00241] Immunohistochemistry showed that the a-galactosidase A enzyme was detected throughout the liver lobule. The presence of a-galactosidase A enzyme was detected in all hepatocytes evaluated. The majority of a-galactosidase A immunostaining was detected in cells lining the sinusoids and in endothelial and kupffer cells in the sinusoidal space. Tissues were evaluated for pathology through H&E staining, TUNEL staining, P3A1 quantitation and LAMP2 quantitation. No vacuoles were seen for any group. A significant increase in TUNEL (+) cells and P3A1 (+) cells was seen only with the highest dose lel4 vg/kg group. There was no increase in GRP78 (+) signal across groups. Plasma ALT levels were not elevated at any dose, indicating no liver toxicity. The I-Cell disease enzyme markers (a-mannosidase and a-Hex A) were not elevated in mouse plasma.

EXAMPLE 3: TESTING OF FURTHER CONSTRUCTS IN RAG 2-/- MICE

[00242] Additional vector constructs were prepared containing different portions of an hAAT/beta-globin intron sequence, as well as different portions of a bovine or human growth hormone polyadenylation signal (bGH or hGH, respectively). Representative depictions of vector configurations are provided in Figure 16. The vector genomes are flanked by AAV serotype 2 (AAV2) derived inverted terminal repeats (ITRs) and ranged in size from 2834 bp to 4571 in length. Plasmids of the vector constructs were transiently transfected into the human liver cell line, HepG2. The three highest expressing constructs were co-3, co-4 and co-5 in Table 2 below.

Table 2 - AAV-GLA Vector Constructs

[00243] AAV virions comprising these vector construct of Table 2 with an AAV5-type capsid were produced in a baculovirus expression vector (BEV) system. The purified AAV virions were quantified by qPCR and administered at doses of 6el3 vg/kg to Rag2 ^/_ mice. Plasma a- galactosidase A activity was increased compared to vehicle for all AAV virions tested. The single dose of AAV produced rising levels of a-galactosidase A activity in plasma over time, plateauing at weeks 5-8. See Figure 17. Plasma a-Gal A activity was 12-fold higher in Rag2 ^/_ mice (~150,000X wild type) dosed with AAV5-GLA-co-4 compared with AAV5-GLA-co-JCAT (also designated GLA-co-1).

EXAMPLE 4: FURTHER DOSE RESPONSE STUDY IN GLA-KO MICE

[00244] The co-3, co-4 and co-5 constructs were selected for further dose response studies in GLAko mice, which exhibit Gb-3 and lyso-Gb-3 accumulation similar to Fabry disease. Doses of 2el2, 6el2, 2el3, and 6el3 vg/kg of the co-4 construct were administered to GLAko mice (n=8 each group), and levels of a-galactosidase A activity in plasma and various tissues were measured. Doses of 6el2, 2el3, and 6el3 of the co-3 and co-5 constructs and 6el3 vg/kg pf the co-4 construct were administered to GLAko mice (n=5 each group) and levels of a-galactosidase A activity in plasma and various tissues were measured.

[00245] Sample results for one construct, GLA-co-4, are displayed in Figure 18. A dose of 6el3 vg/kg GLA-co-4 in GLAko mice produced plasma a-galactosidase A activity that was ~50,000X wild type levels. A dose of 2el3 vg/kg GLA-co-4 in GLAko mice produced plasma a-galactosidase A activity that was ~1,000X wild type levels. Figure 19 shows that administration of the AAV particles to the GLAko mice produced a decrease in Gb-3 and Lyso- Gb-3 at 8 weeks similar to the normal levels seen in wild type mice (6el3 vg/kg) or to nearly normal levels (2el3 vg/kg), as determined in kidney tissue. The dose-dependent difference in plasma a-galactosidase A activity was much greater for the GLAko mice than for the Rag2 ^/_ mice. For example, the difference in plasma enzyme activity at 8 weeks between the 6el3 and 2el3 vg/kg dose was approximately 50 to 100-fold in the GLAko mice, compared to a difference of approximately 7-fold in Rag2 ^/_ mice (between the 6el3 and 2el3 vg/kg dose). The GLA-co-4 construct achieved superior effects at 8 weeks in the GLAko mice dosed at 6el3 vg/kg, with plasma a-galactosidase A activity levels achieving 3-fold superior activity over GLA-co-JCAT and 21 -fold superior activity over GLA wild type (WT) even when the wild type gene was administered at a much higher 1.6el4 vg/kg dose (Figure 3). A dose-dependent increase a- galactosidase A activity was also observed in kidney, heart and liver tissues at 8 weeks post dosing (Figures 20, 21 and 22, respectively). Dosed at 6el3 vg/kg, the GLA-co-4 construct achieved 1.4 fold higher activity in kidney compared to GLA-co-JCAT (also designated GLA- co-1) and 9 fold superior activity over GLA(WT) wild type at much higher 1.6el4 vg/kg dose (Figure 4).

[00246] All three constructs, GLA-co-3, GLA-co-4, and GLA-co-5, produced significant increases in plasma a-galactosidase A activity, from 5 to 8 weeks post-dosing. Comparative levels of plasma a-galactosidase A activity for the three constructs at the same 6el3 vg/kg dose are shown in Figure 23. A dose-dependent increase a-galactosidase A activity was observed in plasma and kidney tissues at 8 weeks post-dosing (Figures 24 and 25). A dose-dependent decrease in accumulation of Gb-3 and Lyso-Gb-3 was also observed at 8 weeks (Figure 26). [00247] All three constructs, GLA-co-3, GLA-co-4 and GLA-co-5, achieved superior activity in both plasma and kidney tissue compared to a higher dose (compare, for example, 6el3 vg/kg to 1.6el4 vg/kg) of GLA-co-JCAT and GLA WT (Figures 24, 25). At a 6el3 vg/kg dose, GLA- co-3 and GLA-co-5 were equivalent and demonstrated higher plasma and kidney a-galactosidase A activity than GLA-co-4. At a 2el3 vg/kg dose, GLA-co-5 demonstrated the highest plasma and kidney a-galactosidase A activity than any other dosing group. All dosing groups demonstrated reduced Gb3 and Lyso-Gb3 level of the kidney samples from Fabry GLAko mice in a dose-dependent fashion. For all vectors (GLA-co-3, GLA-co-4 and GLA-co-5), the 6el3 vg/kg dose reduced Gb3 and lysoGb3 levels in kidney tissue down to the normal wild type levels. At the 2el3 vg/kg dose, GLA-co-5 reduced Gb3 and lysoGb3 down to nearly normal wild type levels.

[00248] Compared to the Sangamo construct (5el3 vg/kg ST-920PC1), a lower dose of GLA- co-5 (2el3 vg/kg) produced a ~3-fold greater increase in plasma a-galactosidase A activity (1375 vs 408-fold change).

[00249] All three constructs also produced consistent increases in a-galactosidase A DNA copy number, promoter DNA copy number, and a-galactosidase A RNA transcript copy number, in the liver of GLA knockout mice at 8 weeks post-dosing.

EXAMPLE 5: EVALUATION OF AAV VECTORS IN NON-HUMAN PRIMATES.

[00250] A non-human primate study is conducted with cynomolgus monkeys (Macaca fascicularis). Study groups include vehicle and various doses of AAV virions containing a- galactosidase A coding sequence. Efficacy endpoints include a run in of 3-4 weeks of weekly bleeds (plasma) for each animal baseline reads then weekly bleeds for a 13 weeks study. Efficacy is evaluated by plasma and tissue a-galactosidase A activity and protein levels. Clinical pathology and hematology readouts are monitored. Safety endpoints include weekly physical, and body weight measurements, as well as monitoring for anti-AAV5 antibody and anti-a- galactosidase A antibody responses and liver enzyme levels such as ALT. The primates are monitored for adverse clinical signs, and if seen additional analyses are performed. At the time of study termination gross necropsy is performed and all major organs assessed for a-Gal A activity, protein and pathology.

[00251] The embodiments described herein are intended to be merely exemplary, and those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, numerous equivalents of specific compounds, materials, and procedures. All such equivalents are considered to be within the scope of the disclosure.

[00252] All of the patents, patent applications and publications referred to herein are incorporated by reference herein in their entireties. Citation or identification of any reference in this application is not an admission that such reference is available as prior art to this application. The full scope of the disclosure is better understood with reference to the appended claims.

Claims

1. A recombinant vector construct comprising a human a-galactosidase A coding sequence at least 95% identical to any one of SEQ ID NO: 3, 4 or 8.

2. The vector construct of claim 1 further comprising a heterologous liver-specific transcription regulatory region.

3. The vector construct of claim 2 further comprising an intron and a polyadenylation signal.

4. A recombinant vector construct comprising (a) an a-galactosidase A coding sequence that encodes amino acids 32 to 429 of SEQ ID NO: 2, or is at least 85% identical to any one of SEQ ID NO: 3-23, and (b) a heterologous liver-specific transcription regulatory region comprising a fragment of an hAAT promoter and a fragment of an ApoE/HCR enhancer, (c) an intron, (d) a polyadenylation signal, and (e) one or both of 5’ and 3’ AAV inverted terminal repeat (ITR) sequences.

5. The vector construct of any of claims 2, 3 or 4, wherein the liver-specific transcription regulatory region comprises (a) an apoliproprotein E (ApoE)/hepatic control region (HCR) enhancer sequence at least 90% identical to SEQ ID NO: 61, and (b) a human alpha anti -trypsin (hAAT) promoter sequence at least 90% identical to SEQ ID NO: 60 or a fragment thereof.

6. The vector construct of any of claims 2, 3 or 4, wherein the liver-specific transcription regulatory region comprises a nucleotide sequence at least 90% or at least 95% identical to SEQ ID NO: 59.

7. The vector construct of any of claims 3-6 wherein the polyadenylation signal is a growth hormone polyadenylation signal or fragment thereof.

8. The vector construct of any of claims 3-6 wherein the intron comprises a fragment of a hAAT intron, a fragment of a hemoglobin intron or both.

9. The vector construct of claim 8, wherein the intron comprises a nucleotide sequence at least 90% identical to any one of SEQ ID NO: 63-69.

10. The vector construct of claim 8, wherein the intron comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 64 or a fragment thereof that retains expression enhancing activity.

11. The vector construct of claim 10, wherein the intron is about 800 to about 1000 nucleotides in length.

12. The vector construct of claim 10, wherein the intron is about 600-700 nucleotides in length.

13. The vector construct of claim 10, wherein the intron is about 400-500 nucleotides in length.

14. The vector construct of claim 10, wherein the intron is about 200-300 nucleotides in length.

15. The vector construct of claim 10, wherein the intron comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 65.

16. The vector construct of claim 10, wherein the intron comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 66.

17. The vector construct of any of claims 1-16 that is an rAAV vector construct about 4 to about 4.5 kb in size.

18. The vector construct of any of claims 1-17 further comprising an AAV 5' ITR and/or AAV3' ITR from AAV2.

19. The vector construct of any of claims 1-18 that comprises a nucleotide sequence at least 80% identical to any of SEQ ID NO: 24-58.

20. An rAAV particle comprising the vector construct of any of claims 1-19 and an AAV capsid.

21. The rAAV of claim 20 wherein the rAAV particle comprises an AAV capsid with liver tropism.

22. The rAAV particle of claim 20 that comprises an AAV5 type capsid.

23. A method of producing the rAAV particle of any of claims 20-22 comprising the steps of (a) providing an insect cell comprising one or more nucleic acid constructs that comprise (i) the vector construct of any of claims 1-19, (ii) a nucleotide sequence encoding one or more AAV Rep proteins operably linked to a promoter that is capable of driving expression in the insect cell, and (iii) a nucleotide sequence encoding one or more AAV capsid proteins operably linked to a promoter that is capable of driving expression in the insect cell, wherein (ii) and (iii) are in the same nucleic acid construct or in two different nucleic acid constructs, and (b) culturing the insect cell under conditions conducive to the expression of the Rep and capsid proteins, and (c) recovering the rAAV particle.

24. A method of producing an rAAV particle comprising the steps of (a) providing a cell permissive for AAV replication with one or more nucleic acid constructs comprising: (i) a recombinant vector construct comprising (1) at least one AAV ITR, (2) a heterologous liver-specific transcription regulatory region, and (3) a nucleic acid encoding a functional human a-galactosidase A, (ii) a nucleotide sequence encoding one or more AAV Rep proteins which is operably linked to a promoter that is capable of driving expression of the Rep protein(s) in the cell; and (iii) a nucleotide sequence encoding one or more AAV capsid proteins which is operably linked to a promoter that is capable of driving expression of the capsid protein(s) in the cell; (b) culturing the cell under conditions permitting expression of the Rep and the capsid proteins; and optionally (c) recovering the AAV particle.

25. The method of claim 24, wherein the cell is an insect cell.

26. The method of claim 24, wherein the cell is a mammalian cell.

27. The method of claim 24 wherein the cell is provided with a recombinant vector construct of any of claims 1-19.

28. A population of rAAV particles produced by the method of any one of claims 23-27, optionally enriched for particles comprising full length or nearly full-length vector genomes by steps that reduce the number of empty capsids.

29. A pharmaceutical composition comprising the vector construct of any of claims 1-19 or the rAAV particle of any of claims 20-22 or the population of rAAV particles of claim 28 in an aqueous suspension with a sterile pharmaceutically acceptable excipient.

30. A method of delivering a human a-galactosidase A coding sequence, comprising administering to a patient with Fabry Disease the vector construct of any of claims 1-19 or the rAAV particle of any of claims 20-22 or the population of rAAV particles of claim 28, or the pharmaceutical composition of claim 24.

31. A method of treating Fabry Disease comprising administering to a patient with Fabry Disease a therapeutically effective amount of the vector construct of any of claims 1-19 or the rAAV particle of any of claims 20-22 or the population of rAAV particles of claim 28 or the pharmaceutical composition of claim 29.

32. The method of claim 30 or 31 wherein the patient exhibits increased plasma a- galactosidase A activity level, or reduced plasma Lyso-Gb-3 or Gb-3 levels.

33. The method of claim 30 or 31 wherein the patient exhibits reduced accumulation of Lyso- Gb-3 or Gb-3 in one or more tissues, optionally kidney, heart or liver.