Methods and compositions for the treatment of Fabry disease

Abstract

The present disclosure provides expression constructs comprising a GLA transgene encoding at least one α-Gal A protein for use in expressing the α-Gal A protein and preventing, inhibiting, or treating Fabry disease or one or more symptoms associated with Fabry disease. Disclosed herein are methods for expressing at least one α-galactosidase A (α-Gal A) protein in a cell. In some embodiments, the method comprises administering to a cell an expression construct comprising a mutated WPRE sequence, optionally a mut6 mutated WPRE sequence, and a GLA transgene encoding at least one α-Gal A protein, such that the α-Gal A protein is expressed in the cell.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 788,439, filed on Jan. 4, 2019, the entire disclosure of which is incorporated herein by reference. Sequence Listing

[0002] This application includes a sequence listing that was electronically submitted in ASCII format, the entire disclosure of which is incorporated herein by reference. The name of the above ASCII copy created on Dec. 3, 2019 is 8325018840SL.txt, and the size is 10,636 bytes.

[0003] Technical Field The present disclosure pertains to the field of prevention and / or treatment of Fabry disease using gene therapy.

Background Art

[0004] The α - galactosidase A (GLA) gene encodes the lysosomal hydrolase, α - galactosidase A (α - GalA). α - Galactosidase is an enzyme that catalyzes the hydrolysis of the terminal α - galactosyl moieties of oligosaccharides and polysaccharides.

[0005] Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene. Deficiency of α-GalA activity results in the progressive systemic accumulation of its primary substrates, globotriaosylceramide (Gb3) and its deacetylated soluble form, globotriaosylsphingosine (lyso-Gb3). The long-term accumulation of these substrates leads to kidney disease, skin disorders, heart disease, corneal dystrophy (e.g., corneal and lens opacities), and / or cerebrovascular disease, shortening the average life expectancy. Depending on the mutation and residual α-GalA enzyme levels, the disease presents in childhood / adolescence as classical early-onset Fabry disease or later in life as a mild (adult) form. Classical Fabry disease occurs when residual enzyme activity is <5% (Arends et al. 2017) and generally affects males. Initial symptoms can include periodic acroparesthesia, angiokeratomas, corneal and lens opacities, progressive renal insufficiency, heart disease, and cerebrovascular events. The mild or adult form of Fabry disease generally affects only one organ system, usually the heart or kidneys only.

[0006] In both classical and adult forms, current standard treatments are enzyme replacement therapy (ERT) using recombinant α-GalA, FABRAZYME® (agalsidase beta or equivalent), or chaperone therapy, which is only available to patients to whom the mutation applies. Injection of recombinant α-GalA into the bloodstream allows for secondary tissue movement by mannose-6-phosphate receptor-mediated uptake (cross-collection). However, the short half-life of recombinant α-GalA used in ERT (approximately 1 hour in plasma) (Clarke et al. 2007) requires lifelong infusions (Clarke et al. 2007), which are associated with a risk of infusion-related reactions in a large proportion of patients, some of which are severe. Furthermore, a significant proportion of patients eventually develop antibodies against the recombinant enzyme, which can also affect the activity of the ERT enzyme and thus may not allow this enzyme to remove all substrates from organs such as the kidney (Linthorst et al. 2004).

[0007] Recombinant α-GalA products with long half-lives that can be administered at lower frequencies are being developed. However, these are still associated with the risk of infusion-related reactions and / or the need for long-term administration due to inactivation by neutralizing antibodies, and it is expected that α-GalA levels will still vary significantly over time.

[0008] Therefore, alternative therapies are needed to address unmet needs in Fabry disease. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0009] Disclosed herein is a method for expressing at least one α-galactosidase A (α-GalA) protein in a cell. In some embodiments, the method comprises administering to the cell an expression construct comprising a mutant WPRE sequence, optionally a mut6 mutant WPRE sequence, and a GLA transgene encoding at least one α-GalA protein such that the α-GalA protein is expressed in the cell.

[0010] In some embodiments, the expression construct comprises a wild-type GLA sequence or a codon-optimized GLA sequence.

[0011] In some embodiments, the expression construct comprises one or more of a sequence encoding an enhancer, a promoter, an intron, a signal peptide, and / or a polyadenylation signal, and the mutant WPRE sequence, optionally the mut6 mutant WPRE sequence, and the GLA transgene encoding at least one α-GalA protein are located between the signal peptide and the sequence encoding the polyadenylation signal.

[0012] In some embodiments, the expression construct comprises the sequence of SEQ ID NO: 9.

[0013] In some embodiments, the cell is from a subject with Fabry disease.

[0014] In some embodiments, the cell is from a male subject.

[0015] In some embodiments, an expression construct in a pharmaceutically acceptable carrier is administered.

[0016] In some embodiments, the pharmaceutically acceptable carrier includes phosphate buffered saline containing CaCl2, MgCl2, NaCl, sucrose, and Kolliphor (poloxamer) P188.

[0017] In some embodiments, the expression construct sequence includes the sequence shown in Table 1, and the expression construct is delivered to the cell by an AAV viral vector.

[0018] In some embodiments, the AAV viral vector serotype is AAV2 / 6.

[0019] In some embodiments, the expression construct is administered to the subject at a dose between about 5.0E+12 and 1.0E+14 vector genomes (vg / kg) per kilogram.

[0020] In some embodiments, the expression construct is administered to the subject's liver. In other embodiments, the expression vector is administered to the subject by intravenous injection. In yet another embodiment, the expression construct is administered to the subject in a single dose.

[0021] In some embodiments, the subject is administered an immunosuppressant before and / or during administration of the expression construct. In some embodiments, the immunosuppressant includes prednisone.

[0022] In some embodiments, the expression of at least one α-galactosidase A (α-GalA) protein is maintained for at least 3 months, at least 9 months, or at least 12 months.

[0023] In some embodiments, the α-GalA protein expressed from the transgene reduces the amount of sphingolipids in the subject by at least about one-half to about one-ninth compared to an untreated subject.

[0024] In some embodiments, the α-GalA protein expressed from the transgene reduces the amount of sphingolipids in the subject by at least about 80% compared to an untreated subject.

[0025] In some embodiments, the α-GalA protein expressed from the transgene reduces the amount of sphingolipids in one or more of the subject's plasma, liver, heart, kidney, or spleen.

[0026] In some embodiments, an expression construct produced in the HEK293 cell line results in a GLA level in the subject that is about 21-fold higher compared to the GLA level in a subject administered an expression construct produced in the Sf9 cell line.

[0027] In some embodiments, the α-GalA protein activity in the subject is about 100-fold to 1,500-fold higher than physiological normal / wild-type.

[0028] In some embodiments, the α-GalA protein expressed from the transgene is active in the subject's kidney, liver, and heart.

[0029] In some embodiments, the GLA transgene is maintained episomally and not integrated into the cell's genome.

[0030] In some embodiments, one or more nucleases that cleave the endogenous albumin gene are administered to the target liver cells such that the transgene is integrated into the albumin gene and expressed therefrom.

[0031] Genetically modified cells comprising an exogenous GLA transgene, produced by the methods described herein, are presented. In some embodiments, the cells are stem cells or progenitor cells. In some embodiments, the cells are liver or muscle cells. In some embodiments, the GLA transgene is maintained episomally and not integrated into the genome of the cells. In some embodiments, the GLA transgene is integrated into the genome of the cells.

[0032] Methods for preventing, inhibiting, or treating Fabry disease or one or more symptoms associated with Fabry disease are also presented. The method may comprise administering an expression construct to a subject in need thereof, the expression construct comprising a mutant WPRE sequence, optionally a mut6 mutant WPRE sequence, and a GLA transgene encoding at least one α-GalA protein.

[0033] In some embodiments, the symptoms include one or more of elevated Gb3 levels above normal or baseline, elevated lyso-Gb3 levels above normal or baseline, kidney disease, heart disease, acral paresthesia, angiokeratoma, gastrointestinal pain, corneal and lens opacities, or cerebrovascular disease. As described herein, baseline may mean any starting measurement, i.e., a measurement taken prior to the administration of a particular treatment. In some embodiments, the subject is male and has less than about 5% α-GalA enzyme activity. In some embodiments, the expression construct comprises a wild-type GLA sequence or a codon-optimized GLA sequence. In some embodiments, the expression construct further comprises one or more of a sequence encoding an enhancer, a promoter, an intron, a signal peptide, and / or a polyadenylation signal, a variant WPRE sequence, optionally a mut6 variant WPRE sequence, and a GLA transgene encoding at least one α-GalA protein is located between the signal peptide and the sequence encoding the polyadenylation signal. In some embodiments, the expression construct in a pharmaceutically acceptable carrier is administered. In some embodiments, the pharmaceutically acceptable carrier comprises phosphate-buffered saline containing CaCl2, MgCl2, NaCl, sucrose, and Kolliphor (poloxamer) P188.

[0034] In other embodiments, the expression construct sequence comprises the sequence shown in Table 1 and the expression construct is delivered to the cells of the subject by an AAV viral vector. In some embodiments, the AAV viral vector serotype is AAV2 / 6.

[0035] In some embodiments, the expression construct is administered to a subject at a dose between about 5.0E+12 and 1.0E+14 vector genomes (vg / kg) per kilogram. In some embodiments, the expression construct is administered to the liver of the subject. In some embodiments, the expression vector is administered to the subject by intravenous injection. In some embodiments, the expression construct is administered to the subject in a single dose.

[0036] In some embodiments, the subject is administered an immunosuppressant before and / or during administration of the expression construct. In some embodiments, the immunosuppressant comprises prednisone. In some embodiments, the expression of at least one α-galactosidase A (α-GalA) protein is maintained for at least 3 months, at least 9 months, or at least 12 months.

[0037] In other embodiments, the α-GalA protein expressed from the transgene reduces the amount of sphingolipids in the subject to between at least about one-third and about one-ninth compared to an untreated subject.

[0038] In some embodiments, the α-GalA protein expressed from the transgene reduces the amount of sphingolipids in the subject by at least about 80% compared to an untreated subject.

[0039] In some embodiments, the α-GalA protein expressed from the transgene reduces the amount of sphingolipids in one or more of the subject's plasma, liver, heart, kidney, or spleen.

[0040] In some embodiments, the expression construct is produced in the HEK293 cell line, and the GLA level in the subject is 21-fold higher compared to the GLA level in a subject administered an expression construct produced in the Sf9 cell line.

[0041] In some embodiments, the α-GalA protein activity in the subject is about 100-fold to 1,500-fold higher than normal / wild-type.

[0042] In some embodiments, the α-GalA protein expressed from the transgene is active in the subject's kidneys, liver, and heart.

[0043] In some embodiments, the GLA transgene is maintained extrachromosomally and not integrated into the genome of the subject's cells.

[0044] In some embodiments, the method comprises administering one or more nucleases that cleave the endogenous albumin gene in the subject's liver cells such that the transgene is integrated into the albumin gene and expressed therefrom.

[0045] Compositions are described herein that comprise an expression construct, the expression construct comprising a mutant WPRE sequence, optionally a mut6 mutant WPRE sequence, and a GLA transgene encoding at least one α-GalA protein for the treatment of Fabry disease.

[0046] In some embodiments, the composition comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier comprises CaCl2, MgCl2, NaCl, sucrose, and Kolliphor (poloxamer) P188, the composition of claim 56.

[0047] In some embodiments, the composition comprises a wild-type GLA sequence or a codon-optimized GLA sequence.

[0048] In some embodiments, the composition comprises one or more of an enhancer, a promoter, an intron, a signal peptide, and / or a sequence encoding a polyadenylation signal, and the mutated WPRE sequence, optionally the mut6 mutated WPRE sequence, and the GLA transgene encoding at least one α-GalA protein are located between the signal peptide and the sequence encoding the polyadenylation signal.

[0049] In some embodiments, the composition comprises the sequences shown in Table 1, and the expression construct is delivered to cells by an AAV viral vector. In some embodiments, the composition comprises the AAV viral vector serotype, AAV2 / 6.

[0050] In some embodiments, the composition comprises an expression construct comprising between about 5.0E+12 and 1.0E+14 vector genomes (vg / kg) per kilogram of subject.

[0051] In some embodiments, the composition comprises an expression construct comprising the sequence of SEQ ID NO: 9.

[0052] A method for generating an α-GalA protein for the treatment of Fabry disease, comprising expressing the α-GalA protein in isolated cells by the method according to any one of claims 1 to 4, and isolating the α-GalA protein produced by the cells, is also presented.

[0053] A delivery vector comprising a mutated WPRE sequence, optionally a mut6 WPRE sequence, and a GLA transgene for use in the methods described herein is presented.

[0054] In some embodiments, the delivery vector is a viral vector or a lipid nanoparticle (LNP). In some embodiments, the viral vector comprises AAV2 / 6, and the viral vector delivers the expression construct to at least 50%, at least 60%, at least 70%, or at least 80% of the cells.

[0055] The use of the expression construct, AAV vector and / or genetically modified cell according to any one of the preceding claims for the treatment of Fabry disease is also presented herein. In some embodiments, the enhancer comprises SEQ ID NO: 2, the promoter comprises SEQ ID NO: 3, the intron comprises SEQ ID NO: 4, the GLA transgene comprises SEQ ID NO: 5, the mutant WPRE sequence comprises SEQ ID NO: 6, and the polyadenylation signal comprises SEQ ID NO: 7.

[0056] In some embodiments, the composition comprises the enhancer of SEQ ID NO: 2, the promoter of SEQ ID NO: 3, the intron of SEQ ID NO: 4, the GLA transgene of SEQ ID NO: 5, the mutant WPRE sequence of SEQ ID NO: 6, and the polyadenylation signal of SEQ ID NO: 7.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0058] Methods and compositions for treating or preventing Fabry disease are disclosed herein. The present specification provides methods and compositions for the introduction of a GLA transgene encoding a protein that is deficient or inadequately expressed in a subject with Fabry disease such that the gene is expressed in the liver and a therapeutic (replenishing) protein is expressed. The present specification also describes the modification of cells (e.g., progenitor or mature RBCs, iPSCs or liver cells) such that the cells produce high levels of a therapeutic substance and the introduction of a population of these modified cells into a patient will supply the protein for which it is needed. The transgene can encode a desired protein or a structural RNA that is therapeutically beneficial in the patient in need.

[0059] Gene therapy using adeno-associated virus (AAV) vectors has shown great promise in both preclinical and clinical trials for the efficient delivery of a therapeutic transgene to the liver, as reported by stable levels of transgene expression up to 6 years for hemophilia B (Lheriteau E, Davidoff E, Nathwani AC. Haemophilia gene therapy: Progress and challenges. Blood Rev. 2015 Sep;29(5):321-8).

[0060] One particularly promising area is the ability to add to a cell a transgene that causes the cell to express a product that it has not previously produced or has produced suboptimally. Examples of the use of this technology include the insertion of a gene encoding a therapeutic protein, the insertion of a coding sequence encoding a protein that is deficient in a cell or an individual for some reason, and the insertion of a sequence encoding a structural nucleic acid such as a microRNA.

[0061] The introduced gene may be introduced into and retained in cells in various ways. Following the "cDNA" approach, the introduced gene is introduced into cells such that it is retained extrachromosomally rather than by integration of the introduced gene into the chromatin of the cell. The introduced gene may be retained in a circular vector (e.g., a plasmid, or a non-integrating viral vector such as AAV or lentivirus), in which case the vector may include transcriptional control sequences such as a promoter, enhancer, polyA signal sequence, intron, and splicing signal (U.S. Patent No. 10,143,760).

[0062] The introduced gene can be delivered into cells by various methods so that the introduced gene will be integrated into the genome of the cell itself and retained therein. In recent years, strategies for gene integration using cleavage by site-specific nucleases for targeted insertion into selected genomic loci have been developed (see, for example, U.S. Patent No. 7,888,121 owned by the present applicant). Nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or nuclease systems, such as the RNA-guided CRISPR / Cas system (using engineered guide RNAs), are specific for the target gene and can be utilized such that the introduced gene construct is inserted by homologous recombination repair (HDR) or by end capture during a non-homologous end joining (NHEJ)-driven process. See, for example, U.S. Patent Nos. 9,877,988; 9,816,074; 9,616,090; 9,873,894; 9,597,357; 9,567,573; 9,458,205; 9,447,434; 9,394,545; 9,255,250; 9,222,105; 9,206,404; 9,200,266; 9,045,763; 9,005,973; 9,150,847; 8,956,828; 8,945,868; 8,895,264; 8,771,985; 8,703,489; 8,586,526; 8,106,255; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; and U.S. Patent Publications 20030232410 and 20050064474, the entire disclosures of each of which are incorporated herein by reference.

[0063] The transgene can be integrated into a high-expression safe harbor locus, such as the albumin gene (see U.S. Patent No. 9,394,545). This approach has been termed an in vivo protein replacement platform or IVPRP. According to this approach, the transgene is inserted into a safe harbor (e.g., albumin) gene by nuclease-mediated targeted insertion, where expression of the transgene is driven by the albumin promoter. The transgene is engineered to include a signal sequence that aids in the secretion / export of the protein encoded by the transgene.

[0064] Examples of "safe harbor" loci include the AAVS1, HPRT, albumin, and CCR5 genes in human cells, and the Rosa26 locus in mouse cells. See, for example, U.S. Patent Nos. 9,877,988; 9,567,573; 9,447,434; 9,394,545; 9,222,105; 9,206,404; 9,150,847; 8,895,264; 8,771,985; 8,106,255; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; and 8,586,526; and U.S. Patent Publications 20030232410 and 20060063231. Nuclease-mediated integration allows for precise transgene placement to minimize the risk of gene silencing or activation of nearby cancer genes, providing the potential for increased transgene expression, safety, and expression durability compared to standard integration approaches that rely on random transgene integration. Nuclease-mediated transgene insertion of genes encoding therapeutic Fab proteins is described in U.S. Publication No. 20180117181.

[0065] Delivery of the transgene to the target cells is one hurdle that must be overcome to fully establish this technology, but another challenge that must be overcome is to ensure that after the transgene is inserted into the cell and expressed, the gene product thus encoded reaches the necessary location within the organism and is produced at a local concentration sufficient to be effective. For diseases characterized by a deficiency of a protein or the presence of an abnormal non-functional protein, delivery of the transgene-encoded wild-type protein can be extremely useful.

[0066] Lysosomal storage diseases (LSDs) are a group of rare metabolic single-gene disorders typically characterized by the deficiency of individual functional lysosomal proteins involved in the breakdown of unwanted lipids, glycoproteins, and mucopolysaccharides. These diseases are characterized by the accumulation of these compounds in cells due to the malfunction of specific enzymes, which prevents them from being processed for reuse. The most common examples are Gaucher disease (glucocerebrosidase deficiency - gene name: GBA), Fabry disease (α-galactosidase A deficiency - GLA), Hunter disease (iduronate-2-sulfatase deficiency - IDS), Hurler disease (α-L-iduronidase deficiency - IDUA), Pompe disease (α-glucosidase (GAA)), and Niemann-Pick disease (sphingomyelin phosphodiesterase 1 deficiency - SMPD1). Collectively, these account for LSDs occurring at a rate of approximately 1 in 7,000 of the population. See also U.S. Patent Nos. 9,877,988 and 9,956,247 and U.S. Publication No. 20160060656.

[0067] For example, Fabry disease is an X-linked disorder of sphingolipid metabolism caused by a deficiency of the α-galactosidase A enzyme (α-GalA). It is associated with the progressive accumulation of sphingolipids, including globotriaosylceramide (also known as GL-3 and Gb3), globotriaosylsphingosine (lyso-Gb3), galabiogalactosylceramide, and substance B. The symptoms of the disease are diverse and can include burning pain, stabbing pain (acroparesthesia), or severe pain symptoms called "Fabry attacks" that can last from a few minutes to several days. Other symptoms include decreased sweating, low tolerance to exercise, a purplish rash called angiokeratoma, eye abnormalities, gastrointestinal problems, heart problems such as cardiac hypertrophy and heart attacks, kidney problems that can lead to kidney failure, and CNS problems. The average lifespan of Fabry patients is significantly shortened.

[0068] Current treatments for Fabry disease may involve enzyme replacement therapy (ERT) using two different preparations of human α-GalA, agalsidase beta, or agalsidase alpha, which requires costly and time-consuming infusions (generally between about 0.2 and 1 mg / kg) every two weeks. Such treatments only treat the symptoms and do not cure the disease. Therefore, patients must be repeatedly dosed with these proteins until death, and in some cases, neutralizing antibodies to the injected proteins may appear.

[0069] Furthermore, adverse reactions, including immune responses such as the appearance of anti-α-GalA antibodies in subjects treated with α-GalA preparations, are associated with ERT. In fact, α-GalA antibodies appeared in 50% of men treated with agalsidase alpha and 88% of men treated with agalsidase beta. Importantly, a significant proportion of such antibodies are neutralizing antibodies and thus reduce the therapeutic impact of the treatment (Meghdari et al (2015) PLoS One 10(2):e0118341.Doi:10.1371 / journal.pone.0118341). Furthermore, ERT does not stop the progression of the disease in all patients.

[0070] Accordingly, the methods and compositions can be used to express one or more therapeutically beneficial α-GalA proteins from a transgene, for example, delivered by a viral vector or from a cDNA construct inserted into any locus (e.g., the highly expressed albumin locus), to replace the defective and / or missing enzyme in Fabry disease. Furthermore, the present specification provides methods and compositions for the treatment (including reduction of one or more symptoms) of Fabry disease by insertion of a transgene sequence into a highly expressed locus in cells such as liver cells. Methods and compositions for delivery of an α-GalA-encoding transgene to the liver of a subject in need thereof by a viral vector are included in the present disclosure, where the virus may be introduced by injection into the peripheral venous system or by direct injection into a blood vessel leading to the liver (e.g., the portal vein). The methods and compositions can be used to cause insertion of the transgene into a safe harbor locus (e.g., albumin) or to cause episomal maintenance of the viral cDNA construct in liver cells. In either case, the transgene is highly expressed and provided to Fabry patients in need of a therapeutic benefit.

[0071] Furthermore, the transgene can be introduced into patient-derived cells, such as patient-derived induced pluripotent stem cells (iPSCs) or other types of stem cells (embryonic or hematopoietic), for use in the final transplantation. Insertion of the therapeutic transgene into hematopoietic stem cells for transplantation into the patient in need thereof is particularly useful. Stem cells will differentiate into mature cells and will contain high levels of the therapeutic protein for delivery to tissues.

[0072] Summary The practice of the methods disclosed herein, as well as the preparation and use of the compositions, employs conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA, and related fields, within the skill of the art, unless otherwise indicated. These techniques are well explained in the literature. See, for example, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, “Chromatin” (P.M. Wassarman and A.P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, “Chromatin Protocols” (P.B. Becker, ed.) Humana Press, Totowa, 1999.

[0073] Definitions The terms "nucleic acid", "polynucleotide", and "oligonucleotide" are used synonymously and refer to deoxyribonucleotide or ribonucleotide polymers that are in a linear or circular structure and can be in either single-stranded or double-stranded form. For the purposes of the present disclosure, these terms should not be construed as being limited with respect to the length of the polymer. These terms may include known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar, and / or phosphate moieties (e.g., phosphorothioate backbone). Generally, analogs of a particular nucleotide have the same base pairing specificity; that is, an analog of A will base pair with T.

[0074] The terms "polypeptide", "peptide", and "protein" are used synonymously and refer to polymers of amino acid residues. This term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acids.

[0075] "Binding" refers to a sequence-specific non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). As long as the interaction is sequence-specific overall, not all components of the binding interaction need to be sequence-specific (e.g., contacts with phosphate residues in the DNA backbone). Such interactions generally have a dissociation constant (K -6 M -1 as follows. "Affinity" refers to the strength of binding, and increased binding affinity correlates with a lower K d ). d

[0076] A "binding domain" is a molecule that can bind non-covalently to another molecule. The binding molecule can bind, for example, to a DNA molecule (a DNA-binding protein such as a zinc finger protein or a TAL-effector domain protein or a single-guide RNA), an RNA molecule (an RNA-binding protein), and / or a protein molecule (a protein-binding protein). In the case of a protein-binding molecule, it can bind to itself (forming a homodimer, homotrimer, etc.), and / or it can bind to one or more molecules of different proteins or a plurality of proteins. A binding molecule may have two or more types of binding activities. For example, a zinc finger protein has DNA-binding, RNA-binding, and protein-binding activities. Therefore, DNA-binding molecules including the DNA-binding components of artificial nucleases and transcription factors include, but are not limited to, ZFPs, TALEs, and sgRNAs.

[0077] A "zinc finger DNA-binding protein" (or binding domain) is a protein that binds to DNA in a sequence-specific manner by one or more zinc fingers, or a domain within a larger protein, which is a region of the amino acid sequence within the binding domain, and this structure is stabilized by the coordination of zinc ions. The term "zinc finger DNA-binding protein" is often abbreviated as "zinc finger protein" or "ZFP". Artificial nucleases and transcription factors may include a ZFP DNA-binding domain and a functional domain (the nuclease domain of a ZFN or the transcriptional regulatory domain of a ZFP-TF). The term "zinc finger nuclease" includes one ZFN and a pair of ZFNs that dimerize to cleave a target gene.

[0078] A "TALE DNA binding domain" or "TALE" is a polypeptide that contains one or more TALE repeat domains / units. The repeat domains are involved in the binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is generally 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. See, e.g., U.S. Patent No. 8,586,526. Artificial nucleases and transcription factors may include a TALE DNA binding domain and a functional domain (the nuclease domain of a TALEN or the transcriptional regulatory domain of a TALEN-TF). The term "TALEN" includes one TALEN as well as pairs of TALENs that dimerize to cleave a target gene.

[0079] Zinc fingers and TALE binding domains can be "engineered" to bind to a predetermined nucleotide sequence, for example, by engineering the recognition helix region of a naturally occurring zinc finger or TALE protein (modification of one or more amino acids). Thus, an engineered DNA binding protein (zinc finger or TALE) is a protein that does not occur in nature. Non-limiting examples of methods for engineering DNA binding proteins are design and selection. An engineered DNA binding protein is a protein that does not occur in nature whose design / composition is primarily due to rational criteria. Rational criteria for design include substitution rules and the application of computerized algorithms for processing information from databases that store information on existing ZFPs and / or TALE designs and binding data. See, e.g., U.S. Patent Nos. 8,568,526; 6,140,081; 6,453,242; and 6,534,261; also see WO98 / 53058; WO98 / 53059; WO98 / 53060; WO02 / 016536 and WO03 / 016496.

[0080] "Selected" zinc finger proteins or TALEs are proteins not found in nature, and their generation primarily results from experimental processes such as phage display, interaction traps, or hybrid selection. See, e.g., Patent Nos. 8,586,526; 5,789,538; US 5,925,523; US 6,007,988; US 6,013,453; US 6,200,759; WO95 / 19431; WO96 / 06166; WO98 / 53057; WO98 / 54311; WO00 / 27878; WO01 / 60970; WO01 / 88197; WO02 / 099084.

[0081] "Recombination" refers to the process of exchanging genetic information between two polynucleotides. For purposes of this disclosure, "homologous recombination (HR)" refers to a specialized form of such exchange that occurs, for example, by the homology-directed repair mechanism during repair of a double-strand break in a cell. This process is commonly known as "non-crossover gene conversion" or "short-tract gene conversion" because it requires nucleotide sequence homology and involves templated repair of a "target" molecule (i.e., the molecule that has undergone the double-strand break) using a "donor" molecule, resulting in the transfer of genetic information from the donor to the target. While not wishing to be bound by any particular theory, such transfer may involve mismatch correction of heteroduplex DNA that occurs between the cleaved target and the donor, and / or "synthesis-dependent strand annealing," in which the donor is used to resynthesize the genetic information that will become part of the target, and / or related processes. Such specialized HR often results in alteration of the sequence of the target molecule such that some or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

[0082] In the methods of the present disclosure, one or more of the target nucleases described herein introduce double-strand breaks at a predetermined site in a target sequence (e.g., cellular chromatin), and a "donor" polynucleotide having homology to the nucleotide sequence of the region of the break can be introduced into the cell. The presence of the double-strand break has been shown to facilitate the integration of the donor sequence. The donor sequence may be physically integrated, or alternatively, the donor polynucleotide is used as a template for repair of the break by homologous recombination, and as a result, all or part of the nucleotide sequence, like the donor, is introduced into the cellular chromatin. Thus, the first sequence in the cellular chromatin can be modified and, in certain embodiments, converted to the sequence present in the donor polynucleotide. Thus, the use of the term "replace" or "replacement" can be construed to represent the replacement of one nucleotide sequence by another nucleotide sequence (i.e., replacement of the sequence in an informational sense), and does not necessarily require physical or chemical replacement of one polynucleotide by another polynucleotide.

[0083] In any of the methods described herein, additional pairs of zinc finger or TALEN proteins may be used for additional double-strand breaks at additional target sites within the cell.

[0084] In certain embodiments of the methods for targeted recombination and / or replacement and / or modification of sequences within a region of interest in cellular chromatin, the sequence of the chromosome is modified by homologous recombination with an exogenous "donor" nucleotide sequence. Such homologous recombination is stimulated by the presence of a double-strand break in the cellular chromatin when a sequence homologous to the region of the break is present.

[0085] In any of the methods described herein, the first nucleotide sequence (“donor sequence”) may include a sequence that is homologous but not identical to the genomic sequence of the region of interest, thereby stimulating homologous recombination to insert a sequence that is not identical to the region of interest. Thus, in certain embodiments, the portion of the donor sequence that is homologous to the sequence of the region of interest exhibits sequence identity of between about 80 and 99% (or any integer therebetween) with the genomic sequence to be replaced. In other embodiments, the homology between the donor sequence and the genomic sequence is greater than 99%, such as when only one nucleotide differs between the donor sequence and the genomic sequence for 100 or more contiguous base pairs. In certain cases, the non-homologous portion of the donor sequence can include a sequence that is not present in the region of interest such that a new sequence is introduced into the region of interest. In these examples, the non-homologous sequence generally has an adjacent sequence of 50-1,000 base pairs (or any integer value therebetween) or any number of base pairs greater than 1,000, which is homologous or identical to the sequence of the region of interest. In other embodiments, the donor sequence is non-homologous to the first sequence and is inserted into the genome by a non-homologous recombination mechanism.

[0086] Any method described herein can be used for the partial or complete inactivation of one or more target sequences in a cell by targeted integration of a donor sequence that interferes with the expression of the target gene(s). Cell lines having a partially or completely inactivated gene are also provided.

[0087] Furthermore, the targeted integration methods described herein can also be used to integrate one or more exogenous sequences. The exogenous nucleic acid sequence may include, for example, one or more genes or cDNA molecules, or any type of coding or non-coding sequence, as well as one or more regulatory elements (e.g., a promoter). Additionally, the exogenous nucleic acid sequence may in some cases produce one or more RNA molecules (e.g., small hairpin RNA (shRNA), inhibitory RNA (RNAi), microRNA (miRNA), etc.).

[0088] "Cleavage" refers to the cleavage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Either single-strand cleavage or double-strand cleavage is possible, and double-strand cleavage may result from two separate single-strand cleavage events. DNA cleavage may result in the production of either blunt ends or sticky ends. In certain embodiments, a fusion polypeptide is used for targeted double-strand DNA cleavage.

[0089] A "cleavage half-domain" is a polypeptide sequence that forms a complex (either with itself or with a second polypeptide, which may be the same or different) having cleavage activity (preferably, double-strand cleavage activity). The terms "first and second cleavage half-domains", "+ and - cleavage half-domains", and "right and left cleavage half-domains" are used interchangeably and refer to pairs of cleavage half-domains that dimerize.

[0090] An "engineered cleavage half-domain" is a cleavage half-domain that has been modified to form a biased heterodimer with another cleavage half-domain (e.g., another engineered cleavage half-domain). See U.S. Patent Nos. 7,888,121; 7,914,796; 8,034,598; and 8,823,618, which are hereby incorporated by reference in their entirety.

[0091] The term "sequence" refers to a nucleotide sequence of any length, which may be DNA or RNA, and may be linear, circular, or branched, and may be either single-stranded or double-stranded. The term "donor sequence" refers to a nucleotide sequence that is inserted into the genome. Donor sequences can be of any length, for example, between 2 and 10,000 (or any integer value between or exceeding those) nucleotides in length, preferably between about 100 and 1,000 (or any integer between those) nucleotides in length, more preferably between about 200 and 500 nucleotides in length.

[0092] A "disease-related gene" is a gene that has some defect in a single-gene disease. Non-limiting examples of single-gene diseases include severe combined immunodeficiency, cystic fibrosis, hemophilia, lysosomal storage diseases (such as Gaucher disease, Hurler disease, Hunter disease, Fabry disease, Niemann-Pick disease, Tay-Sachs disease, etc.), sickle cell anemia, and thalassemia.

[0093] "Chromatin" is a nuclear protein structure containing the cell genome. Cellular chromatin contains proteins, including nucleic acids, mainly DNA, as well as histones and non-histone chromosomal proteins. Most of the chromatin in eukaryotic cells exists in the form of nucleosomes, and the nucleosome core contains approximately 150 base pairs of DNA bound to an octamer containing two each of histones H2A, H2B, H3, and H4, and linker DNA (of varying lengths depending on the organism) extends between the nucleosome cores. Molecules of histone H1 are generally bound to the linker DNA. For the purposes of the present disclosure, the term "chromatin" is intended to encompass all types of nuclear proteins in both prokaryotes and eukaryotes. Cellular chromatin includes both chromosomal chromatin and episomal chromatin.

[0094] A "chromosome" is a chromatin complex containing all or part of the cell's genome. The cell's genome is often characterized by its karyotype, which is the collection of all the chromosomes containing the cell's genome. The cell's genome may contain one or more chromosomes.

[0095] An "episome" is a replicating nucleic acid, a nuclear protein complex, or other structure containing nucleic acids that is not part of the cell's chromosomal karyotype. Examples of episomes include plasmids and certain viral genomes.

[0096] A "target site" or "target sequence" is a nucleic acid sequence that defines a part of a nucleic acid to which a binding molecule binds if there are sufficient conditions for binding.

[0097] "Exogenous" molecules are typically not present in cells but can be introduced into cells by one or more genetic, biochemical, or other methods. "Normally present in a cell" is determined with respect to a particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during the embryonic development of muscle is an exogenous molecule with respect to mature muscle cells. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to non-heat-shocked cells. Exogenous molecules may include, for example, functional forms of dysfunctional endogenous molecules or dysfunctional forms of normally functioning endogenous molecules.

[0098] Exogenous molecules can be, inter alia, small molecules such as those generated by combinatorial chemistry processes, or macromolecules such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, any modified derivatives of the above molecules, or any complex containing one or more of the above molecules. Nucleic acids include DNA and RNA, can be single-stranded or double-stranded, can be linear, branched, or circular, and can be of any length. Nucleic acids include those capable of forming double-stranded nucleic acids, as well as triple-stranded forming nucleic acids. See, for example, U.S. Patent Nos. 5,176,996 and 5,422,251. Examples of proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA-binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases, and helicases.

[0099] An exogenous molecule may be the same type of molecule as an endogenous molecule, such as an exogenous protein or nucleic acid. For example, an exogenous nucleic acid may include an infectious viral genome, a plasmid, or an episome introduced into a cell, or a chromosome not normally present in the cell. Methods for introducing exogenous molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated introduction (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, biolistics, calcium phosphate coprecipitation, DEAE-dextran-mediated introduction, and viral vector-mediated introduction. An exogenous molecule may be the same type of molecule as an endogenous molecule but derived from a species different from that from which the cell is derived. For example, a human nucleic acid sequence may be introduced into a cell line originally derived from a mouse or hamster.

[0100] In contrast, an "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, endogenous nucleic acids can include chromosomes, the genome of mitochondria, chloroplasts, or other organelles, or naturally occurring episomal nucleic acids. Additional endogenous molecules can include proteins, such as transcription factors and enzymes.

[0101] A "fusion" molecule is a molecule in which two or more subunit molecules are linked, preferably covalently. The subunit molecules may be of the same chemical type or different chemical types. Examples of the first type of fusion molecule include, but are not limited to, fusion proteins (e.g., fusions between a ZFP or TALE DNA binding domain and one or more activation domains) and fusion nucleic acids (e.g., nucleic acids encoding the fusion proteins described above). Examples of the second type of fusion molecule include, but are not limited to, fusions between a triplex-forming nucleic acid and a polypeptide, and fusions between a minor groove binder and a nucleic acid.

[0102] Expression of a fusion protein in a cell can result from delivery of the fusion protein to the cell or from delivery to the cell of a polynucleotide encoding the fusion protein, where the polynucleotide is transcribed and the transcript is translated to yield the fusion protein. Trans-splicing, polypeptide cleavage and polypeptide ligation may also be involved in protein expression in a cell. Methods for polynucleotide and polypeptide delivery to cells are presented elsewhere in this disclosure.

[0103] For the purposes of this disclosure, a "gene" includes a DNA region encoding a gene product (see below), as well as all DNA regions that control production of the gene product, whether or not such regulatory sequences are adjacent to the coding and / or transcribed sequences. Thus, genes include, but are not necessarily limited to, promoter sequences, terminators, translational control sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0104] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. The gene product may be the direct transcriptional product of the gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA), or a protein produced by translation of the mRNA. Gene products include RNAs modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified, for example, by methylation, acetylation, phosphorylation, ubiquitination, ADP ribosylation, myristylation, and glycosylation.

[0105] The "GLA gene" encodes α-galactosidase, an enzyme that degrades globotriaosylceramide. Gene mutations in the GLA gene result in an incomplete enzymatic function of α-galactosidase. The GLA gene is located at Xq22.1, which is position 22.1 on the long arm (q) of the X chromosome. The GLA gene may also be called AGAL_HUMAN, agalsidase alpha, alpha-D-galactosidase A, alpha-D-galactosidase galactohydrolase, alpha-galactosidase, alpha-galactosidase A, ceramidetrihexosidase, GALA, galactosidase, alpha, or melibiase.

[0106] "Regulation" of gene expression refers to a change in the activity of a gene. Regulation of expression can include, but is not limited to, gene activation, gene optimization, and gene repression. Genome editing (e.g., cleavage, modification, inactivation, random mutagenesis) can be used to regulate expression. Gene inactivation refers to any decrease in gene expression compared to cells that do not contain the ZFP, TALE, or CRISPR / Cas systems described herein. Thus, gene inactivation can be partial or complete.

[0107] A "target region" is any region of cellular chromatin, such as, for example, a gene, or a non-coding sequence within a gene or a non-coding sequence adjacent to a gene, in which it is desirable to bind an exogenous molecule. The binding may be for the purpose of targeted DNA cleavage and / or targeted recombination. The target region may be present, for example, in a chromosome, episome, organelle genome (e.g., mitochondrion, chloroplast), or infectious viral genome. The target region may be within the coding region of a gene, within a non-transcribed region such as, for example, a leader sequence, trailer sequence, or intron, or within a non-transcribed region either upstream or downstream of the coding region. The target region may be as small as a single nucleotide pair or up to 2,000 nucleotide pairs in length, or any integer value of nucleotide pairs.

[0108] Examples of "eukaryotic" cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells, and human cells (e.g., liver cells, muscle cells, RBCs, T cells, etc.), including stem cells (pluripotent and multipotent).

[0109] "Red Blood Cell" (RBC) or erythrocyte is a terminally differentiated cell derived from hematopoietic stem cells. Erythrocytes lack nucleases and most organelles. RBCs contain hemoglobin that transports oxygen from the lungs to peripheral tissues. In fact, 33% of individual RBCs is hemoglobin. They also transport CO2 produced by cells during metabolism out of tissues and return it to the lungs for release during exhalation. RBCs are produced in the bone marrow in response to low oxygen levels in the blood, which involves the release of erythropoietin (EPO) by the kidneys. EPO increases the number of proerythroblasts and shortens the time required for complete RBC maturation. After approximately 120 days, RBCs are removed from the bloodstream either by the phagocytic activity of macrophages (about 90%) in the liver, spleen, and lymph nodes or by hemolysis in the plasma (about 10%) because they have no nucleus or any other regenerative capacity. After phagocytosis by macrophages, the chemical components of RBCs are degraded within the macrophage vacuoles due to the action of lysosomal enzymes. RBCs may be derived from the genetically modified stem cells or RBC progenitor cells described herein, either in vitro or in vivo.

[0110] A "secretory tissue" is a tissue in an animal that secretes products from individual cells typically into one of several types of lumens derived from epithelium. Examples of secretory tissues located in the digestive tract include cells lining the intestinal tract, pancreas, and inner surface of the gallbladder. Other secretory tissues include the liver, tissues associated with the eye and mucosa, such as salivary glands, mammary glands, prostate gland, pituitary gland, and other elements of the endocrine system. Furthermore, secretory tissues include individual cells of tissue types that can secrete.

[0111] The terms "operably linked" and "operably connected" (or "connected so as to function") are used synonymously with respect to the juxtaposition of two or more components (such as array elements), and these components are arranged such that both components function properly and at least one component permits the possibility of mediating a function that acts on at least one other component. As an example, a transcriptional control sequence such as a promoter is operably linked to a coding sequence when the transcriptional control sequence controls the transcriptional level of the coding sequence in response to the presence or absence of one or more transcriptional control factors. Transcriptional control sequences are generally operably linked to coding sequences in cis, but need not be directly adjacent thereto. For example, enhancers are transcriptional control sequences that are operably linked to coding sequences even if they are not contiguous.

[0112] With respect to a fusion polypeptide, the term "operably linked" can refer to the fact that each of the components linked to another component performs the same function as it would if it were not so linked. For example, with respect to a fusion polypeptide in which a ZFP, TALE or Cas DNA binding domain is fused to an activation domain, when the ZFP or TALE DNA binding domain portion in the fusion polypeptide can bind to its target site and / or its binding site, and at the same time the activation domain can upregulate gene expression, the ZFP or TALE DNA binding domain and the activation domain are operably linked. When the ZFP or TALE DNA binding domain is a fusion polypeptide fused to a cleavage domain, when the ZFP or TALE DNA binding domain portion in the fusion polypeptide can bind to its target site and / or its binding site, and at the same time the cleavage domain can cleave DNA near the target site, the ZFP or TALE DNA binding domain and the cleavage domain are operably linked.

[0113] A "functional fragment" of a protein, polypeptide, or nucleic acid is a protein, polypeptide, or nucleic acid whose sequence is not identical to the full-length protein, polypeptide, or nucleic acid, but still retains the same function as the full-length protein, polypeptide, or nucleic acid. A functional fragment can have more residues, fewer residues, or the same number of residues as the corresponding native molecule, and / or can include one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well known in the art. Similarly, methods for determining protein function are well known. For example, the DNA-binding function of a polypeptide can be determined, e.g., by filter binding, electrophoretic mobility shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. See Ausubel et al., supra. The ability of a protein to interact with another protein can be determined, e.g., by co-immunoprecipitation, two-hybrid assays, or both genetic and biochemical complementation. See, e.g., Fields et al. (1989) Nature 340:245-246; U.S. Patent No. 5,585,245 and International Patent Publication No. WO98 / 44350.

[0114] A "vector" can introduce a gene sequence into a target cell. Generally, "vector construct", "expression vector", "gene delivery vector", and "expression construct" mean any nucleic acid construct that can induce the expression of a gene of interest and introduce a gene sequence into a target cell. Thus, the term includes cloning, and expression vehicles, as well as integration vectors.

[0115] The term "reporter gene" or "reporter sequence" refers to any sequence that, although not necessarily in a normal assay, preferably results in a protein product that is easily measurable. Suitable reporter genes include sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding colored or fluorescent or luminescent proteins (e.g., green fluorescent protein, hypersensitive green fluorescent protein, red fluorescent protein, luciferase), and proteins that mediate enhanced cell growth and / or gene amplification (e.g., dihydrofolate reductase), but are not limited thereto. Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA, or any detectable amino acid sequence. An "expression tag" includes a sequence encoding a reporter that may be operably linked to a desired gene sequence to monitor the expression of the gene of interest.

[0116] The terms "subject" and "patient" are used synonymously and refer to mammals, such as human patients and non-human primates, as well as laboratory animals, such as rabbits, dogs, cats, rats, mice, and other animals. Thus, the term "subject" or "patient" as used herein, when referring to a mammalian patient or subject to whom the modified cells described herein and / or the proteins produced by the modified cells described herein can be administered. Subjects of the present disclosure include subjects having LSD.

[0117] Methods and compositions for treating and / or preventing Fabry disease are disclosed herein. The disclosure describes methods for inserting a transgene sequence into a suitable target cell (e.g., a cell from a subject with Fabry disease), where the transgene encodes at least one protein that treats the disease (e.g., at least one α-GalA protein). The method may be in vivo (delivery of the transgene sequence to cells of a living subject) or ex vivo (delivery of the modified cells to a living subject). The disclosure also describes methods for transfection and / or transduction of a suitable target cell using an expression system such that the α-GalA-encoding transgene expresses a protein that treats the disease (e.g., alleviates one or more of the symptoms associated with the disease). The α-GalA protein may be secreted (excreted) from the target cell such that it can affect or be taken up (cross-collected) by other cells that do not have the transgene. The disclosure also provides a method for generating cells (e.g., mature or undifferentiated cells) that produce high levels of α-GalA, where introduction of a population of such modified cells into a patient will provide the protein required to treat the disease or condition. Further provided is a method for generating cells (e.g., mature or undifferentiated cells) that produce highly active (therapeutically effective) α-GalA, where introduction or generation of a population of these modified cells in a patient will confer the protein activity required to treat Fabry disease (e.g., reduce or eliminate one or more symptoms). The highly active α-GalA produced as described herein may also be isolated from cells as described herein and administered to patients in need thereof using standard enzyme replacement procedures known to those of skill in the art.

[0118] Methods and compositions for expressing at least one alpha-galactosidase A (α-GalA) protein are described herein. The compositions and methods may be for use in vitro, in vivo, or ex vivo, and comprise administering to a cell a GLA transgene encoding at least one α-GalA protein (e.g., a cDNA having a wild-type or codon-optimized GLA sequence) such that the α-GalA protein is expressed in the cell. In certain embodiments, the cell is in a subject with Fabry disease. In any of the methods described herein, the transgene can be administered to the liver of the subject. Optionally, the method further comprises administering to the liver cells of the subject one or more nucleases that cleave the albumin gene such that the transgene is integrated into the endogenous albumin gene and expressed therefrom. In any of the methods described herein, the α-GalA protein expressed from the transgene can reduce the amount of sphingolipid in the subject by at least about one-half as compared to an untreated subject or a subject treated with a formulation buffer or other carrier. The GLA transgene may further comprise additional elements, such as, for example, a signal peptide and / or one or more regulatory elements. In certain embodiments, the GLA transgene (e.g., a cDNA construct) further comprises a mutated WPRE sequence, such as the WPRE mut6 mutation described in Zanta-Boussif et al. (2009) Gene Therapy 16:605-619 and U.S. Patent No. 10,179,918, which may be a wild-type or engineered WPRE sequence. In some embodiments, the mut6 mutation is created in the J04514 WPRE element, while in other embodiments it is created in the J02442.1 WPRE (Ong et al. (2017) doi.org / 10.1101 / 126904). In certain embodiments, the expressed GLA construct comprises the construct shown in Figure 1B (variant #21).The expression constructs containing WPRE described herein result in improved transgene expression and activity (e.g., 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater increased expression or activity) as compared to expression constructs that do not contain the WPRE array. In certain embodiments, the expression construct is as shown in Table 1.

[0119] In one aspect, the present disclosure describes a method of expressing a transgene encoding one or more modified GLA transgenes in a cell of a subject. The transgene may be inserted into the genome of a target cell (e.g., blood cell, liver cell, brain cell, stem cell, progenitor cell, etc.) suitable such that the α-GalA product encoded by the modified transgene is stably integrated into the genome of the cell (also referred to as the “IVPRP” approach), or the transgene may be maintained extrachromosomally in the cell (also referred to as the “cDNA” approach). In one embodiment, the modified GLA transgene is introduced (stably or extrachromosomally) into cells of a cell line for in vitro production of a replacement protein, and the (optionally, purified and / or isolated) protein is then administered to a subject for treating the subject (e.g., by reducing and / or eliminating one or more symptoms associated with Fabry disease). In certain embodiments, the α-GalA product encoded by the modified transgene increases α-GalA activity in the tissue of the subject by any amount, e.g., 2 to 100-fold (or any value therebetween including 10, 20, 30, 40, 50, 60, 70, 80, 90, 100-fold), 100 to 500-fold (or any value therebetween), 500 to 1000-fold (or any value therebetween), or 1000 to 2000-fold or more, including but not limited to, from about 2 to greater than about 2000-fold (or any value therebetween) as compared to an untreated subject.

[0120] In another aspect, ex vivo or in vivo methods of treating a subject with Fabry disease are described herein, (e.g., by reducing and / or eliminating one or more symptoms associated with Fabry disease), the method comprising inserting a GLA transgene into a cell described herein such that the protein is produced in a subject with Fabry disease (cDNA and / or IVPRP approach). In certain embodiments, the GLA transgene is part of the construct shown in Table 1. In certain embodiments, the isolated cell containing the GLA transgene can be used to treat a patient in need thereof, e.g., by administering the cell to a subject with Fabry disease. In other embodiments, the modified GLA transgene is inserted into a target tissue in the body such that the replacement protein is produced in vivo. In some embodiments, the modified transgene is inserted into the genome of the cells of the target tissue, while in other preferred embodiments, the modified transgene is inserted into the cells of the target tissue and maintained extrachromosomally. In any of the methods described herein, the expressed α-GalA protein may be secreted from the cell and act on or be taken up by a secondary target, including other cells of other tissues lacking the GLA transgene (e.g., by secretion into the blood) (cross collection). In some cases, the primary and / or secondary target tissue is the liver. In other cases, the primary and / or secondary target tissue is the brain. In other cases, the primary and / or secondary target is the blood (e.g., the vasculature). In other cases, the primary and / or secondary target is skeletal muscle.

[0121] In certain embodiments, the methods and compositions described herein are used to reduce the amount of glycosphingolipids, including globotriaosylceramide (also known as GL-3 and Gb3) and globotriaosylsphingosine (lyso-Gb3), galabiocylceramide, deposited in the tissues of a subject with Fabry disease. In certain embodiments, the α-Gal A product encoded by the corrective transgene reduces glycosphingolipids in the tissues of the subject by any amount, for example, from about 2-fold to more than about 100-fold (or any value therebetween), compared to an untreated subject, including, but not limited to, 2- to 100-fold (or any value therebetween, including 10, 20, 30, 40, 50, 60, 70, 80, 90, 100-fold). In certain embodiments, the α-Gal A product encoded by the modified transgene reduces glycosphingolipids in the subject's tissues by any amount, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, compared to an untreated subject.

[0122] In any of the methods described herein, the modified GLA transgene comprises the wild-type sequence of a functional GLA gene; however, in other embodiments, the sequence of the modified GLA transgene is modified in some manner to obtain improved biological activity (e.g., optimized codons to increase biological activity and / or modified transcriptional and translational control sequences to improve gene expression). In some embodiments, the GLA gene is modified to improve expression characteristics. Such modifications can include, but are not limited to, the insertion of a translation start site (e.g., methionine), the addition of an optimized Kozak sequence, the insertion of a signal peptide, and / or codon optimization. In some embodiments, the signal peptide can be selected from an albumin signal peptide, an F.IX signal peptide, an IDS signal peptide, and / or an α-Gal A signal peptide.

[0123] In certain embodiments, the donor is a cDNA donor. A cDNA donor generally includes an enhancer sequence, a promoter sequence, an intron sequence, a signal peptide, a GLA coding sequence, a polyadenylation signal, and optionally, a wild-type or mutant WPRE sequence. Non-limiting examples of cDNA donors are schematically shown in FIGS. 1A and 1B.

[0124] Any promoter, enhancer, intron, signal peptide, GLA coding sequence or polyA sequence and any WPRE sequence can be a sequence usable in a cDNA construct. In some embodiments, the enhancer and / or promoter is liver-specific and is composed of, for example, a human ApoE enhancer and a human α1-antitrypsin (hAAT) promoter (Miao CH et al. (2000) Mol. Ther. 1(6): 522-532 (200)). In some embodiments, the liver-specific promoter includes one or more ApoE enhancer sequences (e.g., 1, 2, 3 and / or 4; see Okuyama et al (1996) Hum Gen Ther 7(5):637-45). In some embodiments, the promoter is ligated to an intron. In some embodiments, the intron is an HBB-IGG chimeric intron that includes the 5' donor site of the first intron of the human β-globin gene as well as the branch and 3' acceptor site of the intron of the immunoglobulin gene heavy chain variable region. In some embodiments, the ApoE / hAAT promoter is specific and highly active in hepatocytes, the intended target tissue, but is inactive in non-liver cells and tissue types, which reduces or prevents expression and activity in non-target tissues. In certain embodiments, the signal peptide includes the GLA signal peptide and the polyadenylation signal includes the SPA51 or bGH polyA sequence. Any WPRE sequence may be any wild-type or mutant WPRE sequence. See, for example, U.S. Patent No. 10,179,918. In certain embodiments, the WPRE sequence includes a mutant WPRE such as the mut6 WPRE sequence.

[0125] The cDNA expression vectors described herein can be delivered by any suitable vector, including viral vectors such as AAV of any serotype (eg, AAV2, AAV6, or AAV2 / 6).

[0126] In a specific embodiment, the expression sequence (i.e., expression vector or expression construct) comprises the elements and sequences of variant #21 depicted in Figure 1B and set forth in Table 1 below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0127] The expression constructs in Table 1 containing the WPRE sequence can be readily produced on a clinical scale and have been shown to exhibit, for example, at least about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold improved GLA activity compared to expression constructs that do not contain the WPRE sequence.

[0128] In another aspect, a nuclease (e.g., ZFN, ZFN pair, TALEN, TALEN pair, and / or CRISPR / Cas system) expression vector is described herein that comprises a polynucleotide encoding one or more of the nucleases described herein operably linked to a promoter. In one embodiment, the expression vector is a viral vector. In another aspect, a GLA expression vector is described herein that comprises a polynucleotide encoding α-GalA described herein operably linked to a promoter. In one embodiment, the expression is a viral vector.

[0129] In another aspect, a host cell is described herein that comprises one or more of the nuclease (e.g., ZFN, ZFN pair, TALEN, TALEN pair, and / or CRISPR / Cas system) expression vectors and / or α-GalA expression vectors described herein. The host cell may be stably transformed with one or more nuclease expression vectors, transiently transfected, or a combination thereof. In some embodiments, the host cell is a liver cell.

[0130] In other embodiments, for example, methods are provided for replacing genomic sequences in any target gene with a therapeutic GLA transgene as described herein using a nuclease (e.g., ZFN, ZFN pair, TALEN, TALEN pair, and / or CRISPR / Cas system) (or one or more vectors encoding said nuclease) as described herein and a “donor” sequence or GLA transgene that is inserted into the gene after targeted cleavage by the nuclease. The GLA sequence may be present in the vector carrying the nuclease (or components thereof), in a separate vector (e.g., an Ad, AAV, or LV vector or mRNA), or may be introduced into the cell using different nucleic acid delivery mechanisms. Such insertion of the donor nucleotide sequence into a target locus (e.g., a highly expressed gene, a disease-related gene, other safe harbor genes, etc.) of the target gene results in the expression of the GLA transgene under the control of the endogenous gene control elements of the target locus (e.g., albumin, globin, etc.). In some embodiments, insertion of the GLA transgene, e.g., into a target gene (e.g., albumin), results in the expression of the complete α-GalA protein sequence and any amino acids encoded by the target (e.g., albumin) are missing. In other embodiments, the expressed exogenous α-GalA protein is a fusion protein and includes amino acids encoded by the GLA transgene and by the endogenous locus into which the GLA transgene is inserted (e.g., from the endogenous target locus or from a sequence on the transgene encoding the sequence of the target locus). The target may be any gene, e.g., a safe harbor gene such as the albumin gene, AAVS1 gene, HPRT gene, etc.; the CCR5 gene; or a highly expressed gene (e.g., beta globin or gamma globin) such as the globin gene in RBC progenitor cells. In some cases, the endogenous sequence will be present in the amino (N)-terminal portion of the exogenous α-GalA protein, while in other cases, the endogenous sequence will be present in the carboxy (C)-terminal portion of the exogenous α-GalA protein. In other cases, the endogenous sequence will be present in both the N and C-terminal portions of the α-GalA exogenous protein.In some embodiments, the endogenous sequence encodes a secretory signal peptide that is removed during the process of secretion of the α-GalA protein from the cell. The endogenous sequence may include the full-length wild-type or mutant endogenous sequence, or may include a partial endogenous amino acid sequence. In some embodiments, the endogenous gene-transgene fusion is located at the endogenous locus within the cell, while in other embodiments, the endogenous sequence-transgene coding sequence is inserted at another locus within the genome (e.g., the GLA-transgene sequence is inserted at the albumin, HPRT, or CCR5 locus). In some embodiments, the GLA transgene is expressed such that the therapeutic α-GalA protein product is retained within the cell (e.g., progenitor or mature cell). In other embodiments, the GLA transgene is fused to the extracellular domain of a membrane protein such that upon expression, the transgene α-GalA fusion results in surface localization of the therapeutic protein. In some aspects, the edited cell further comprises a transmembrane protein that transports the cell to a specific tissue type. In one aspect, the transmembrane protein comprises an antibody, while in other aspects, the transmembrane protein comprises a receptor. In certain embodiments, the cell is a progenitor (e.g., CD34+ or hematopoietic stem cell) or mature RBC (derived from the gene-modified GAL-producing cells described herein). In some aspects, the therapeutic α-GalA protein product encoded by the transgene is excreted from the cell and affects or is taken up by cells lacking the transgene. In certain embodiments, the cell is a liver cell that releases the therapeutic α-GalA protein into the bloodstream and acts on distal tissues (e.g., kidney, spleen, heart, brain, skin, etc.).

[0131] In one embodiment, the GLA transgene is expressed from the albumin promoter after insertion into the albumin locus. The biologic agent encoded by the GLA transgene may then be released into the bloodstream if the transgene is inserted into hepatocytes in vivo. In some embodiments, the GLA transgene in a viral vector is delivered to the liver in vivo by intravenous administration. In some embodiments, the donor GLA transgene contains a Kozak consensus sequence (Kozak (1987) Nucl Acid Res 15(20):8125-48) prior to the α-GalA coding sequence such that the expression product lacks the albumin signal peptide. In some embodiments, the donor α-GalA transgene contains an alternative signal peptide, such as a signal peptide from albumin, IDS, or the F9 gene, in place of the native GLA signal sequence.

[0132] In yet another aspect, methods are provided herein for site-specific integration of a nucleic acid sequence into an endogenous locus of a chromosome (e.g., an albumin locus of hepatocytes or a globin locus of RBC progenitor cells, which are associated with high expression of a disease), e.g., into a chromosome of a non-human embryo. In certain embodiments, the method comprises (a) injecting into a non-human embryo (i) at least one DNA vector comprising an upstream sequence and a downstream sequence adjacent to α-GalA encoding the nucleic acid sequence to be integrated, and (ii) at least one polynucleotide molecule encoding at least one nuclease (zinc finger, ZFN pair, TALE nuclease, TALEN pair, or CRISPR / Cas system) that recognizes the site of integration in the target locus; and (b) culturing the embryo to allow expression of the nuclease (ZFN, TALEN, and / or CRISPR / Cas system), wherein the double-strand break introduced into the site of integration by the nuclease is repaired by homologous recombination with the DNA vector so as to integrate the nucleic acid sequence into the chromosome. In some embodiments, the polynucleotide encoding the nuclease is RNA.

[0133] Nuclease For the implementation of aspects of the methods described herein, systems and / or Ttago guide RNAs comprising at least one ZFN, TALEN, homing endonuclease, and CRISPR / Cas useful for in vivo cleavage of donor molecules carrying the transgene, and any nuclease including but not limited to nucleases for cleavage of the cell's genome such that the transgene is integrated into the genome in a targeted manner can be used. Accordingly, compositions are described herein that comprise one or more nucleases that cleave a selected gene such that the cleavage results in genomic modification of the gene (e.g., insertion and / or removal into the cleaved gene). In certain embodiments, one or more nucleases are naturally occurring. In other embodiments, one or more nucleases are non-naturally occurring, i.e., engineered in the DNA binding molecule (also referred to as the DNA binding domain) and / or cleavage domain. For example, the DNA binding domain of a naturally occurring nuclease may be modified to bind to a selected target site (e.g., ZFP, TALE, and / or sgRNA of CRISPR / Cas engineered to bind to a selected target site). In other embodiments, the nuclease comprises heterologous DNA binding and cleavage domains (e.g., zinc finger nuclease; TAL-effector domain DNA binding protein; meganuclease DNA binding domain with a heterologous cleavage domain). In other embodiments, the nuclease comprises a system such as the Ttago-based CRISPR / Cas.

[0134] DNA binding domain In certain embodiments, the compositions and methods described herein utilize meganuclease (homing endonuclease) DNA binding domains for binding to donor molecules and / or to target regions of the genome of a cell. Naturally occurring meganucleases recognize cleavage sites of 15 to 40 base pairs and are generally classified into the following four families: the LAGLIDADG family (the "LAGLIDADG" disclosed as SEQ ID NO: 10), the GIY-YIG family, the His-Cyst box family, and the HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII. Their recognition sequences are known. See also U.S. Patent No. 5,420,032; U.S. Patent No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalogue. Furthermore, the DNA binding specificities of homing endonucleases and meganucleases can be engineered to bind to non-natural target sites.See, e.g., Chevalier et al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res. 31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al. (2007) Current Gene Therapy 7:49-66; U.S. Patent No. 8,021,867. The DNA-binding domain of homing endonucleases and meganucleases may be modified relative to the entire nuclease (i.e., so that the nuclease contains a homologous cleavage domain) or may be fused to a heterologous cleavage domain.

[0135] In other embodiments, the DNA-binding domain of one or more nucleases used in the methods and compositions described herein comprises a naturally occurring or engineered (non-naturally occurring) TAL effector DNA-binding domain. See, e.g., U.S. Patent No. 8,586,526, incorporated herein by reference in its entirety. Plant pathogens of the genus Xanthomonas are known to cause many diseases in important crop plants. Xanthomonas pathogenicity relies on a conserved type III secretion (T3S) system that injects over 25 different effector proteins into plant cells. Among these injected proteins are transcription activator-like (TAL) effectors, which mimic plant transcription activators and manipulate the plant transcriptome (see Kay et al. (2007) Science 318:648-651). These proteins comprise a DNA-binding domain and a transcription activation domain. One of the best-characterized TAL effectors is AvrBs3 from Xanthomonas campestgris pv. Vesicatoria (see Bonas et al. (1989) Mol Gen Genet 218: 127-136 and WO2010079430). TAL effectors contain a concentrated domain of tandem repeats, each containing approximately 34 amino acids that are important for the DNA-binding specificity of these proteins. In addition, they contain a nuclear localization sequence and an acidic transcriptional activation domain (for review, see Schornack S, et al. (2006) J Plant Physiol 163(3):256-272). Furthermore, in the plant pathogen Ralstonia solanacearum, two genes designated brg11 and hpx17 have been found to be homologous to the AvrBs3 family of Xanthomonas in the R. solanacearum biovar 1 strain GMI1000 and biovar 4 strain RS1000 (see Heuer et al (2007) Appl and Envir Micro 73(13):4379-4384).These genes are 98.9% identical to each other in nucleotide sequence, but differ by a 1,575 bp deletion in the repeat domain of hpx17. However, both gene products share less than 40% sequence identity with Xanthomonas AvrBs3 family proteins. See, e.g., U.S. Patent No. 8,586,526, incorporated herein by reference in its entirety.

[0136] The specificity of these TAL effectors depends on the sequence found in the tandem repeat. The repeat sequence contains approximately 102 bp, and the repeats are generally 91-100% homologous to each other (Bonas et al., ibid.). Polymorphisms in the repeat are usually located at positions 12 and 13, and there appears to be a one-to-one correspondence between the identity of the hypervariable dinucleotides (RVDs) at positions 12 and 13 and the identity of consecutive nucleotides in the target sequence of the TAL-effector (see Moscou and Bogdanove, (2009) Science 326:1501 and Boch et al. (2009) Science 326:1509-1512). Experimentally, the natural codes for DNA recognition of these TAL-effectors were determined: the HD sequence at positions 12 and 13 results in binding to cytosine (C); NG binds to T; NI binds to A, C, G, or T; NN binds to A or G; and ING binds to T. These DNA-binding repeats were assembled into proteins with new combinations and repeat numbers to generate artificial transcription factors that can interact with new sequences and activate expression of non-endogenous reporter genes in plant cells (Boch et al., ibid.). Engineered TAL proteins were linked to a FokI cleavage half-domain to obtain TAL effector domain-nuclease fusions (TALENs) that exhibit activity in yeast reporter assays (plasmid-based targets). See, e.g., U.S. Patent No. 8,586,526; Christian et al. ((2010) Genetics epub 10.1534 / genetics.110.120717).

[0137] In certain embodiments, the DNA binding domain of one or more nucleases used for in vivo cleavage and / or targeted cleavage of a cell's genome comprises a zinc finger protein. Preferably, the zinc finger protein is non-naturally occurring in that it has been engineered to bind to a selected target site. See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct., all of which are incorporated herein by reference in their entirety. Biol. 10:411-416; U.S. Patent Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; 7,888,121; 7,972,854; and U.S. Patent Publication No. 20050267061.

[0138] Engineered zinc finger binding domains may have novel binding specificities compared to naturally occurring zinc finger proteins. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design, for example, involves using a database containing triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, where each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers that bind to that particular triplet or quadruplet sequence. See, for example, commonly owned U.S. Patent Nos. 6,453,242 and 6,534,261, which are incorporated herein by reference in their entireties.

[0139] Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Patent Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as WO 98 / 37186; WO 98 / 53057; WO 00 / 27878; and WO 01 / 88197. Additionally, enhanced binding specificity for zinc finger binding domains is described, for example, in commonly owned WO 02 / 077227.

[0140] Furthermore, as disclosed in these and other references, zinc finger domains and / or multi-fingered zinc finger proteins may be linked together using any suitable linker sequence, including, for example, linkers of five or more amino acids in length. See also U.S. Patent Nos. 8,772,453; 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.

[0141] Methods for target site selection; ZFP selection, and design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and are described in U.S. Patent Nos. 6,140,081; 5,789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; 6, 200,759; WO95 / 19431; WO96 / 06166; WO98 / 53057; WO98 / 54311; WO00 / 27878; WO01 / 60970; WO01 / 88197; WO02 / 099084; WO98 / 53058; WO98 / 53059; WO98 / 53060; WO02 / 016536 and WO03 / 016496.

[0142] Furthermore, as disclosed in these and other references, zinc finger domains and / or multi-fingered zinc finger proteins may be linked together using any suitable linker sequence, including, for example, linkers of 5 amino acids or more in length. See also U.S. Patent Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences of 6 amino acids or more in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.

[0143] In certain embodiments, the DNA binding domain is part of a CRISPR / Cas nuclease system that includes, for example, a single guide RNA (sgRNA). See, e.g., U.S. Patent Nos. 8,697,359 and 9,873,894. The CRISPR (clustered, regularly interspaced short palindromic repeats) locus that encodes the RNA component of the system, and the Cas (CRISPR-associated) locus that encodes the protein (Jansen et al., 2002. Mol. Microbiol. 43: 1565-1575; Makarova et al., 2002. Nucleic Acids Res. 30: 482-496; Makarova et al., 2006. Biol. Direct 1: 7; Haft et al., 2005. PLoS Comput. Biol. 1: e60) constitute the genetic sequences of the CRISPR / Cas nuclease system. The CRISPR locus of a microbial host includes a combination of CRISPR-associated (Cas) genes and non-coding RNA elements that can program the specificity of nucleic acid cleavage to which CRISPR is related.

[0144] Type II CRISPR is one of the most characterized systems and performs targeted DNA double-strand cleavage in four consecutive steps. First, two non-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes with the repeat regions of pre-crRNA and mediates the processing of pre-crRNA into mature crRNA containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex guides Cas9 to the target DNA by Watson-Crick base pairing between the spacer of crRNA and the protospacer of the target DNA adjacent to the protospacer adjacent motif (PAM), which is an additional requirement for target recognition. Finally, Cas9 mediates the cleavage of the target DNA, creating a double-strand break within the protospacer. The activity of the CRISPR / Cas system involves the following three stages: (i) insertion of foreign DNA sequences into the CRISPR array to prevent future attacks in a process called "acquisition", (ii) expression of the associated proteins, and expression and processing of the array, followed by (iii) interference involving RNA with foreign nucleic acids. Thus, in bacterial cells, some of the so-called "Cas" proteins are involved in the original function of the CRISPR / Cas system and play roles in functions such as insertion of foreign DNA and the like.

[0145] In certain embodiments, the Cas protein may be a "functional derivative" of a naturally occurring Cas protein. A "functional derivative" of a polypeptide of a native sequence is a compound that has qualitative biological properties in common with the polypeptide of the native sequence. "Functional derivatives" include, but are not limited to, fragments of the native sequence, as well as derivatives and fragments of the polypeptide of the native sequence, provided that they have a biological activity in common with the corresponding polypeptide of the native sequence. The biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" includes amino acid sequence variants of the polypeptide, covalent modifications thereof, and fusions. Suitable derivatives or fragments of the Cas polypeptide include, but are not limited to, variants, fusions, covalent modifications, or fragments of the Cas protein. The Cas protein, including the Cas protein or a fragment thereof, as well as a derivative or a fragment of the Cas protein, may be obtained from cells, or may be synthesized chemically or by a combination of these two procedures. The cells may be cells that originally produce the Cas protein, or cells that are genetically engineered to produce the Cas protein at a higher expression level and also produce an endogenous Cas protein, or to produce the Cas protein from a nucleic acid encoding an endogenous Cas or a different Cas that has been introduced exogenously. In some cases, the cells are not originally producing the Cas protein and are genetically engineered to produce the Cas protein. Further non-limiting examples of RNA-guided nucleases that may be used in addition to and / or in place of the Cas protein include class 2 CRISPR proteins such as Cpf1. See, for example, Zetsche et al. (2015) Cell 163:1-13.

[0146] The CRISPR-Cpf1 system identified in Francisella species is a class 2 CRISPR-Cas system that mediates robust DNA interference in human cells. Although functionally conserved, Cpf1 and Cas9 differ in many aspects, including guide RNA and substrate specificity (see Fagerlund et al., (2015) Genom Bio 16:251). A key difference between Cas9 and Cpf1 proteins is that Cpf1 does not utilize a tracrRNA; only the crRNA is required. The FnCpf1 crRNA is 42–44 nucleotides long (19-nucleotide repeats and a 23–25-nucleotide spacer) and contains a single stem-loop that tolerates sequence changes to maintain secondary structure. Furthermore, the Cpf1 crRNA is significantly shorter than the approximately 100-nucleotide engineered sgRNA required by Cas9, and the PAM requirement for FnCpf1 is a 5'-TTN-3' and 5'-CTA-3' sequence on the displaced strand. Both Cas9 and Cpf1 create double-stranded breaks in target DNA; however, Cas9 uses the RuvC and HNH-like domains to create a blunt-end cut within the seed sequence of the guide RNA, whereas Cpf1 uses the RuvC-like domain to create a staggered cut outside the seed. Because Cpf1 creates a staggered cut away from the critical seed region, NHEJ does not destroy the target site, thereby ensuring that Cpf1 can continue to cut at the same site until the desired HDR recombination event occurs. Therefore, in the methods and compositions described herein, it is understood that the term "Cas" includes both Cas9 and Cfp1 proteins. Thus, as used herein, a "CRISPR / Cas system" refers to both CRISPR / Cas and / or CRISPR / Cfp1 systems, including both nuclease and / or transcription factor systems.

[0147] In some embodiments, the DNA binding domain is part of the TtAgo system (see Swarts et al, loc. cit.; Sheng et al, loc. cit.). In eukaryotes, gene silencing involves proteins of the Argonaute (Ago) family. In this example, Ago binds to small molecule (19 - 31 nt) RNAs. This protein-RNA silencing complex recognizes the target RNA by Watson-Crick base pairing between the small molecule RNA and the target, and cleaves the target RNA by endonuclease (Vogel (2014) Science 344:972-973). In contrast, prokaryotic Ago proteins may bind to small single-stranded DNA fragments and function to detect and remove foreign (often viral) DNA (Yuan et al., (2005) Mol. Cell 19, 405; Olovnikov, et al.(2013) Mol. Cell 51, 594; Swarts et al., loc. cit.). Examples of prokaryotic Ago proteins include proteins from Aquifex aeolicus, Rhodobacter sphaeroides, and Thermus thermophilus.

[0148] One of the best-characterized prokaryotic Ago proteins is from T. thermophilus (TtAgo; Swarts et al., ibid.). TtAgo binds either 15 nt or 13–25 nt single-stranded DNA fragments bearing a 5′ phosphate group. This TtAgo-bound “guide DNA” serves to guide the protein-DNA complex to bind to complementary Watson-Crick DNA sequences in a third molecule of DNA. Once the sequence information in these guide DNAs allows identification of the target DNA, the TtAgo-guide DNA complex cleaves the target DNA. Such a mechanism is also supported by the structure of the TtAgo-guide DNA complex while bound to the target DNA (G. Sheng et al., ibid.). Ago from Rhodobacter sphaeroides (RsAgo) has similar properties (Olivnikov et al., ibid.).

[0149] Foreign guide DNA of any DNA sequence can be loaded onto the TtAgo protein (Swarts et al., loc. cit.). Since the specificity of TtAgo cleavage is induced by the guide DNA, the TtAgo·DNA complex formed with the exogenous guide DNA defined by the researcher will thus induce TtAgo target DNA cleavage to the complementary target DNA defined by the researcher. In this way, a targeted double-strand break can be created in the DNA. The use of the TtAgo·guide DNA system (or an orthologous Ago·guide DNA system from other microorganisms) enables the targeted cleavage of genomic DNA in cells. Such cleavage can be either single-stranded or double-stranded. For the cleavage of mammalian genomic DNA, it would be preferable to use the type of TtAgo codon optimized for expression in mammalian cells. Furthermore, when the TtAgo protein is fused to a cell-penetrating peptide, it would be preferable to treat the cells with the TtAgo·DNA complex formed in vitro. Furthermore, it would be preferable to use the type of TtAgo protein modified by mutagenesis to have improved activity at 37 °C. The use of DNA cleavage involving TtAgo·RNA can affect numerous outcomes including gene knockout, targeted gene addition, gene modification, and targeted gene removal using standard techniques in the art for the utilization of DNA cleavage.

[0150] Thus, the nuclease comprises a DNA-binding domain that specifically binds to the target site of any gene into which it is desired to insert a donor (transgene).

[0151] Cleavage domain Any suitable cleavage domain can be operably linked to a DNA binding domain to form a nuclease. For example, a ZFP DNA binding domain can be fused to a nuclease domain to create a ZFN. This is a functional element that can recognize its intended nucleic acid target by its engineered (ZFP) DNA binding domain and cleave DNA near the ZFP binding site through nuclease activity. See, for example, Kim et al. (1996) Proc Natl Acad Sci USA 93(3):1156-1160. The term "ZFN" includes a pair of ZFNs that dimerize to cleave a target gene. More recently, ZFNs have been used for genome modification in various organisms. See, for example, U.S. Patent Nos. 7,888,121; 8,409,861; 8,106,255; and 9,447,434. Similarly, a TALE DNA binding domain can be fused to a nuclease to create a TALEN. See, e.g., U.S. Patent No. 8,586,526. CRISPR / Cas nuclease systems have also been demonstrated that include a single guide RNA (sgRNA) that binds to DNA and binds to a cleavage domain (e.g., a Cas domain) to cause targeted cleavage. See, e.g., U.S. Patent Nos. 8,697,359 and 8,932,814, and U.S. Patent No. 9,873,894.

[0152] As described above, the cleavage domain can be heterologous to the DNA-binding domain, for example, between a zinc finger DNA-binding domain and a nuclease cleavage domain or a TALEN DNA-binding domain and a nuclease cleavage domain; between an sgRNA DNA-binding domain and a nuclease (CRISPR / Cas) cleavage domain; and / or between a meganuclease DNA-binding domain and a nuclease cleavage domain different from the meganuclease DNA-binding domain. Heterologous cleavage domains can be derived from any endonuclease or exonuclease. Exemplary endonucleases from which cleavage domains can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, e.g., 2002-2003 Catalogue, New England Biolabs, Beverly, MA; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes that cleave DNA are known (e.g., S1 nuclease, mung bean nuclease, pancreatic DNase I, micrococcal nuclease, yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of the cleavage domain and cleavage half-domains.

[0153] Similarly, the cleavage half-domain may be derived from any nuclease or portion thereof that requires dimerization for cleavage activity, as described above. Generally, when a fusion protein contains a cleavage half-domain, two fusion proteins are required for cleavage. Alternatively, a single protein containing two cleavage half-domains may be used. The two cleavage half-domains may be derived from the same endonuclease (or functional fragments thereof), or each cleavage half-domain may be derived from a different endonuclease (or functional fragments thereof). Furthermore, the target sites of the two fusion proteins are preferably positioned relative to each other such that binding of the two fusion proteins to their respective target sites places the cleavage half-domains in a spatial orientation relative to each other that allows the cleavage half-domains to form a functional cleavage domain, e.g., by dimerization. Thus, in certain embodiments, the proximal ends of the target sites are separated by 5-8 nucleotides or 15-18 nucleotides. However, any integral number of nucleotides or nucleotide pairs may be present between the two target sites (e.g., from 2 to 50 or more nucleotide pairs). Generally, the site of cleavage is located between the target sites.

[0154] Restriction endonucleases (restriction enzymes) exist in many species and can bind to DNA in a sequence-specific manner (at a recognition site) and cleave the DNA at or near the site of binding. Certain restriction enzymes (e.g., type IIS) cleave DNA at a site removed from the recognition site and have separable binding and cleavage domains. For example, the type IIS enzyme FokI catalyzes double-stranded cleavage of DNA at the 9th nucleotide from the recognition site on one strand and the 13th nucleotide from the recognition site on the other strand. See, e.g., U.S. Patent Nos. 5,356,802; 5,436,150 and 5,487,994; and Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment, the fusion protein comprises a cleavage domain (or cleavage half-domain) derived from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered.

[0155] An exemplary Type IIS restriction enzyme in which the cleavage domain is separable from the binding domain is FokI. This particular enzyme is active as a dimer (Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575). Therefore, for purposes of this disclosure, the portion of the FokI enzyme used in the fusion proteins of this disclosure is considered a cleavage half-domain. Thus, for targeted double-strand cleavage and / or targeted replacement of cellular sequences using zinc finger-FokI fusions, two fusion proteins, each containing a FokI cleavage half-domain, can be used to reconstitute a catalytically active cleavage domain. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI cleavage half-domains can be used. Parameters for targeted cleavage and targeted sequence modification using zinc finger-FokI fusions are provided elsewhere in this disclosure.

[0156] A cleavage domain or cleavage half-domain can be any portion of a protein that retains cleavage activity or retains the ability to multimerize (e.g., dimerize) to form a functional cleavage domain.

[0157] Exemplary Type IIS restriction enzymes are described in U.S. Patent No. 7,888,121, which is incorporated herein in its entirety. Additional restriction enzymes also contain separable binding and cleavage domains and are contemplated by the present disclosure. See, e.g., Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

[0158] In certain embodiments, the cleavage domain comprises, for example, one or more engineered cleavage half-domains (also referred to as dimerization domain mutants) that minimize or prevent homodimerization as described in U.S. Patent Nos. 8,772,453; 8,623,618; 8,409,861; 8,034,598; 7,914,796; and 7,888,121, the entire disclosures of which are incorporated herein by reference. All of the amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of FokI are targets that affect the dimerization of the FokI cleavage half-domains.

[0159] Examples of engineered cleavage half-domains of FokI that form biased heterodimers include pairs in which the first cleavage half-domain contains mutations at the amino acid residues at positions 490 and 538 of FokI and the second cleavage half-domain contains mutations at amino acid residue positions 486 and 499.

[0160] Thus, in one embodiment, the mutation at position 490 substitutes Glu(E) with Lys(K), the mutation at position 538 substitutes Iso(I) with Lys(K), the mutation at position 486 substitutes Gln(Q) with Glu(E), and the mutation at position 499 substitutes Iso(I) with Lys(K). Specifically, the engineered cleavage half-domains described herein were generated by mutating positions 490 (E→K) and 538 (I→K) in one cleavage half-domain to generate the engineered cleavage half-domain designated "E490K:I538K" ("KK"), and further by mutating positions 486 (Q→E) and 499 (I→L) in another cleavage half-domain to generate the engineered cleavage half-domain designated "Q486E:I499L" ("EL"). The engineered cleavage half-domains described herein are obligate heterodimer mutants in which aberrant cleavage is minimized or eliminated. U.S. Patent Nos. 7,914,796 and 8,034,598, the disclosures of which are incorporated by reference in their entireties. In certain embodiments, the engineered cleavage half-domains contain mutations at positions 486, 499, and 496 (numbering relative to wild-type FokI), e.g., substitutions of the wild-type Gln (Q) residue at position 486 with a Glu (E) residue, the wild-type Iso (I) residue at position 499 with a Leu (L) residue, and the wild-type Asn (N) residue at position 496 with an Asp (D) or Glu (E) residue (also referred to as "ELD" and "ELE" domains, respectively). In other embodiments, the engineered cleavage half-domain contains mutations at positions 490, 538, and 537 (numbering relative to wild-type FokI), e.g., substitution of the wild-type Glu (E) residue at position 490 with a Lys (K) residue, the wild-type Iso (I) residue at position 538 with a Lys (K) residue, and the wild-type His (H) residue at position 537 with a Lys (K) residue or an Arg (R) residue (also referred to as "KKK" and "KKR" domains, respectively).In other embodiments, the engineered cleavage half-domains contain mutations at positions 490 and 537 (numbering relative to wild-type FokI), e.g., mutations substituting the wild-type Glu (E) residue at position 490 with a Lys (K) residue and the wild-type His (H) residue at position 537 with a Lys (K) residue or an Arg (R) residue (also referred to as "KIK" and "KIR" domains, respectively). See, e.g., U.S. Patent No. 8,772,453. In other embodiments, the engineered cleavage half-domains contain the "Sharkey" mutation (see Guo et al, (2010) J. Mol. Biol. 400(1):96-107).

[0161] The engineered cleavage half-domains described herein can be made by site-directed mutagenesis of a wild-type cleavage half-domain (FokI) using any suitable method, e.g., as described in U.S. Patent Nos. 8,623,618; 8,409,861; 8,034,598; 7,914,796; and 7,888,121.

[0162] The methods and compositions can also be used to increase the specificity of nuclease pairs for their intended target relative to other unintended cleavage sites, known as off-target sites (see U.S. Patent Publication Nos. 20170218349 and 20180087072). Thus, the nucleases described herein can contain mutations in one or more DNA-binding domain backbone regions and / or one or more mutations in the nuclease cleavage domain. These nucleases can also contain mutations to amino acids within the ZFP DNA-binding domain ("ZFP backbone") that can nonspecifically interact with phosphates on the DNA backbone, but do not contain alterations to the DNA recognition helix. Thus, the ZFPs can contain mutations in cationic amino acid residues in the ZFP backbone that are not required for nucleotide target specificity. In some embodiments, these mutations in the ZFP backbone include mutating cationic amino acid residues to neutral or anionic amino acid residues. In some embodiments, these mutations in the ZFP backbone include mutating polar amino acid residues to neutral or nonpolar amino acid residues. In preferred embodiments, mutations are made at positions (-5), (-9), and / or (-14) relative to the DNA-binding helix. In some embodiments, a zinc finger may contain one or more mutations at positions (-5), (-9), and / or (-14). In yet another embodiment, one or more zinc fingers of a multi-fingered zinc finger protein may contain mutations at positions (-5), (-9), and / or (-14). In some embodiments, the amino acid at positions (-5), (-9), and / or (-14) (e.g., arginine (R) or lysine (K)) is mutated to alanine (A), leucine (L), Ser (S), Asp (N), Glu (E), Tyr (Y), and / or glutamine (Q).

[0163] In certain embodiments, the engineered cleavage half-domains are derived from the FokI nuclease domain and contain mutations in one or more of amino acid residues 416, 422, 447, 448, and / or 525, which are numbered relative to wild-type full-length FokI. In some embodiments, the mutations at amino acid residues 416, 422, 447, 448, and / or 525 are introduced into FokI “ELD”, “ELE”, “KKK”, “KKR”, “KK”, “EL”, “KIK”, “KIR” and / or Sharkey as described above.

[0164] Furthermore, methods are described herein for enhancing the specificity of cleavage activity by independent dosing of the engineered cleavage half-domain partners of a nuclease complex. In some embodiments, the ratio of the two partners (half-cleavage domains) is provided at a ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:9, 1:10 or 1:20, or any value in between. In other embodiments, the ratio of the two partners is greater than 1:30. In other embodiments, the two partners are deployed at a ratio selected to be different from 1:1. When used individually or in combination, the methods and compositions disclosed herein result in a surprising and unexpected increase in targeting specificity due to a reduction in off-target cleavage activity. The nucleases used in these embodiments may include ZFNs, paired ZFNs, TALENs, paired TALENs, CRISPR / Cas, CRISPR / dCas and TtAgo, or any combination thereof.

[0165] Alternatively, nucleases can be assembled at nucleic acid target sites in vivo using so-called "split enzyme" technology (see, e.g., U.S. Patent Publication No. 20090068164). The components of such split enzymes can be expressed in separate expression constructs, or the individual components can be linked in a single open reading frame separated, for example, by a self-cleaving 2A peptide or an IRES sequence. The components can be individual zinc finger binding domains or domains of meganuclease nucleic acid binding domains.

[0166] Nucleases can be screened for activity prior to use, for example, in a yeast-based chromosomal system as described in U.S. Patent No. 8,563, 314. Expression of the nuclease can be under the control of a constitutive or inducible promoter, for example, the galactokinase promoter, which is activated (derepressed) in the presence of raffinose and / or galactose and repressed in the presence of glucose.

[0167] The Cas9-associated CRISPR / Cas system comprises two non-coding RNA components: a tracrRNA and a pre-crRNA array containing a nuclease guide sequence (spacer) separated by identical direct repeats (DRs). To achieve genome engineering using the CRISPR / Cas system, both of these RNAs must be functional (see Cong et al., (2013) Sciencexpress 1 / 10.1126 / science 1231143). In some embodiments, the tracrRNA and pre-crRNA are provided by separate expression constructs or as separate RNAs. In other embodiments, chimeric RNAs are constructed in which an engineered mature crRNA (which confers target specificity) is fused to a tracrRNA (which confers interaction with Cas9) to generate a chimeric cr-RNA·tracrRNA hybrid (also called a single-guide RNA). (See Jinek ibid and Cong, ibid.).

[0168] Target site As described in detail above, the DNA domain can be engineered to bind to a locus, e.g., any sequence selected at an albumin or other safe harbor gene. The engineered DNA binding domain may have a new binding specificity compared to a naturally occurring DNA binding domain. Methods of engineering include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using a database that includes triplet (or quadruplet) nucleotide sequences and individual (e.g., zinc finger) amino acid sequences, where each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of a DNA binding domain that binds to a specific triplet or quadruplet sequence. See, e.g., U.S. Pat. Nos. 6,453,242 and 6,534,261, owned by the applicant and incorporated herein by reference in their entirety. Rational design of TAL-effector domains can also be implemented. See, e.g., U.S. Pat. No. 8,586,526.

[0169] Exemplary selection methods applicable to DNA binding domains, including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as WO98 / 37186; WO98 / 53057; WO00 / 27878; WO01 / 88197 and GB2,338,237.

[0170] Methods for selection of target sites, nucleases, and design and construction of fusion proteins (and polynucleotides encoding the same) are known to those of skill in the art and are described in U.S. Pat. Nos. 7,888,121 and 8,409,891, which are incorporated herein by reference in their entirety.

[0171] Furthermore, as disclosed in these and other references, DNA binding domains (e.g., multi-fingered zinc finger proteins) may be linked together using any suitable linker sequence, e.g., including a linker of 5 amino acids or more. For linker sequences that are 6 amino acids or longer, see, for example, U.S. Patent Nos. 9,567,609; 6,479,626; 6,903,185; and 7,153,949. The proteins described herein may include any combination of suitable linkers between the individual DNA binding domains of the protein.

[0172] Donor As described above, methods and compositions are provided for the introduction of an exogenous sequence (also referred to as a "donor construct" or "donor sequence" or "donor") into a subject, e.g., for modifying a mutant gene or enhancing the expression of a gene encoding a protein (e.g., α-GalA) that is deficient or lacking in Fabry disease.

[0173] It will be readily apparent that the donor sequence is generally not identical to the genomic sequence in which it is placed. The donor sequence may include a non-homologous sequence flanked by two regions of homology ("homology arms") to enable efficient HDR at the desired location. Further, the donor sequence may include a vector molecule that includes sequences that are not homologous to the desired region of the cellular chromatin. The donor molecule may include several non-contiguous regions of homology to the cellular chromatin. For example, for targeted insertion of a sequence not normally present in the desired region, the above sequences may be present in the donor nucleic acid molecule and regions of homology to the sequence of the desired region may be adjacent.

[0174] Described herein are methods for targeted insertion of a transgene encoding an α-Gal A protein for insertion at a selected location. The GLA transgene may encode a full-length or truncated α-Gal A protein. The polynucleotide for insertion is sometimes referred to as the "exogenous" polynucleotide, "donor" polynucleotide or molecule, or "transgene." Non-limiting example GLA donor constructs are shown in Figures 1A and 1B.

[0175] The donor polynucleotide may be single-stranded and / or double-stranded DNA or RNA and may be introduced into cells in linear or circular form. See, e.g., U.S. Patent Nos. 8,703,489 and 9,255,259. The donor sequence(s) may also be contained within a DNA MC, which may be introduced into cells in circular or linear form. See, e.g., U.S. Patent Publication No. 20140335063. When introduced in linear form, the ends of the donor sequence may be protected (e.g., from exonuclease degradation) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues may be added to the 3' end of the linear molecule, and / or self-complementary oligonucleotides may be ligated to one or both ends. See, e.g., Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of terminal amino group(s) and the use of modified internucleoside linkages such as, for example, phosphorothioate, phosphoramidate, and O-methylribose or deoxyribose residues.

[0176] A polynucleotide can be introduced into a cell as part of a viral or non-viral vector molecule having additional sequences such as, for example, an origin of replication, a promoter, and a gene encoding antibiotic resistance. Further, the donor polynucleotide can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or a poloxamer, or can be delivered by a virus (e.g., adenovirus, AAV, herpes virus, retrovirus, lentivirus, and integrase-deficient lentivirus (IDLV)).

[0177] The donor may be inserted such that its expression is driven at the integration site by an endogenous promoter, i.e., a promoter that drives the expression of an endogenous gene (e.g., highly expressed albumin, AAVS1, HPRT, etc.) into which the donor is inserted. However, it will be apparent that the donor may include a promoter and / or enhancer, e.g., a constitutive promoter or an inducible or tissue-specific promoter. In some embodiments, the donor is retained in the cell as an expression plasmid such that the gene is expressed extrachromosomally.

[0178] The donor molecule may be inserted into the endogenous gene such that all or part of the endogenous gene is expressed or not expressed at all. For example, the transgene described herein may be inserted into albumin or other loci such that part of the endogenous albumin sequence (N-terminus and / or C-terminus relative to the transgene encoding a lysosomal enzyme) is expressed or not expressed at all, e.g., as a fusion with a transgene encoding an α-GalA protein(s). In other embodiments, the transgene (with or without additional coding sequences related to, e.g., albumin) is integrated into any endogenous locus, e.g., a safe harbor locus.

[0179] When an endogenous sequence (part of the endogenous or transgene) is expressed together with the transgene, the endogenous sequence (e.g., albumin, etc.) may be a full-length sequence (wild-type or mutant) or a partial sequence. Preferably, the endogenous sequence is functional. Non-limiting examples of the functions of these full-length or partial sequences (e.g., albumin) include extending the serum half-life of the polypeptide expressed by the transgene (e.g., therapeutic gene), and / or acting as a carrier.

[0180] Furthermore, although not required for expression, the exogenous sequence may also include sequences encoding transcriptional or translational control sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, 2A peptides, and / or polyadenylation signals.

[0181] The exogenous sequence linked to the transgene may also include a signal peptide that aids in the processing and / or secretion of the encoded protein. Non-limiting examples of these signal peptides include those derived from albumin, IDS, and factor IX.

[0182] In certain embodiments, the exogenous sequence (donor) includes a fusion of the protein of interest with the extracellular domain of a membrane protein as its fusion partner, positioning the fusion protein on the cell surface. This enables the protein encoded by the transgene to act in the serum in some cases. In the case of Fabry disease, the α-GalA enzyme encoded by the transgene acts on the metabolites accumulating in the serum from its position on the surface of cells (e.g., RBCs). Furthermore, when RBCs are phagocytosed by splenic macrophages as in the normal process of degradation, the lysosomes formed when the macrophages phagocytose the cells will expose the membrane-bound fusion protein to a high concentration of metabolites in the lysosome at a pH higher than the pH preferred for the enzyme. Non-limiting examples of possible fusion partners are shown in Table 2 below.

Table 2

[0183] In some cases, the expression construct may contain a modified endogenous GLA gene. For example, the endogenous gene may be codon-optimized. Furthermore, while antibody responses to enzyme replacement therapy vary for the particular therapeutic enzyme in question and for each individual patient, significant immune responses have been observed in many Fabry disease patients treated with enzyme replacement using wild-type α-Gal A. A transgene is considered to produce a therapeutic protein if it increases the amount (and / or activity) of the protein compared to subjects without the transgene. Furthermore, the relevance of these antibodies to the efficacy of treatment varies (see Katherine Ponder, (2008) J Clin Invest 118(8):2686). Thus, the methods and compositions described herein may involve the generation of expression constructs with modified sequences compared to wild-type GLA, including, but not limited to, functionally silent amino acid changes at sites known to be stimulatory epitopes for the endogenous immune response, and / or modifications resulting in truncations that reduce the immunogenicity of polypeptides produced by such sequences.

[0184] Patients with Fabry disease often suffer from neurological sequelae due to a deficiency of the α-Gal A enzyme in the brain. Unfortunately, due to the impermeability of the blood-brain barrier, it is often difficult to deliver therapeutic substances to the brain via the blood. Therefore, methods and compositions may be used in conjunction with methods to increase delivery of therapeutic substances to the brain, including, but not limited to, methods that cause temporary opening of tight junctions between brain capillaries, such as the use of temporary osmotic disruption by intracarotid administration of hypertonic mannitol solution, the use of focused ultrasound, and the administration of bradykinin analogs (Matsukado et al. (1996) Neurosurgery 39:125). Alternatively, therapeutic substances may be designed to utilize receptors or transport mechanisms for specific delivery to the brain. Examples of specific receptors that can be used include the transferrin receptor, insulin receptor, or low-density lipoprotein receptor-related proteins 1 and 2 (LRP-1 and LRP-2). LRP is known to interact with a series of secreted proteins, such as apoE, tPA, and PAI-1, and therefore fusion of the recognition sequence of one of these proteins to LRP can facilitate transport of the enzyme to the brain after expression of the therapeutic protein in the liver and secretion into the bloodstream (see Gabathuler, (2010) ibid.).

[0185] cell Genetically modified cells (e.g., stem cells, progenitor cells, liver cells, muscle cells, etc.) containing an exogenous GLA transgene (either integrated or episomal) are provided, which cells are produced by the methods described herein. These cells can be used, for example, to provide α-GalA protein to a subject having Fabry disease by administering the cell(s) to a subject in need thereof, or by isolating the α-GalA protein produced by the cell and administering the protein to a subject in need thereof (enzyme replacement therapy). Alternatively, the cells may be generated in vivo in a subject by administration of the expression constructs described herein. Accordingly, isolated cells that are genetically modified in vivo are provided. Vectors (e.g., viral vectors such as AAV or Ad, or lipid nanoparticles) containing a GLA transgene for use in any of the methods described herein, including for use in treating Fabry disease, are also provided.

[0186] In any of the methods described herein, the GLA transgene may be inserted into the genome of a target cell using a nuclease. Non-limiting examples of suitable nucleases include zinc finger nucleases (ZFNs), TALENs (transcription activator-like effector nucleases), and / or CRISPR / Cas nuclease systems, which include a DNA-binding molecule that binds to a target site in a region of interest (e.g., a disease-related gene, a highly expressed gene, the albumin gene, or other safe harbor gene) in the genome of the cell and one or more nuclease domains (e.g., a cleavage domain and / or a cleavage half-domain). The cleavage domain and cleavage half-domain can be obtained, for example, from various restriction endonucleases, Cas proteins, and / or homing endonucleases. In certain embodiments, the zinc finger domain recognizes a target site in the albumin gene or the globin gene of a red blood cell progenitor (RBC). See, e.g., U.S. Patent No. 9,877,988, which is incorporated herein by reference in its entirety. In other embodiments, the nuclease (e.g., ZFN, TALEN, and / or CRISPR / Cas system) binds to and / or cleaves a safe harbor gene, such as the CCR5 gene, the PPP1R12C gene (also known as AAVS1), the albumin, HPRT, or Rosa gene. See, e.g., U.S. Patent Nos. 9,877,988; 9,567,573; 9,447,434; 9,394,545; 9,222,105; 9,206,404; 9,150,847; 8,895,264; 8,771,985; 8,106,255; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; and 8,586,526; U.S. Patent Publications 20030232410 and 20060063231. The nuclease (or a component thereof) may be provided as a polynucleotide encoding one or more nucleases described herein (e.g., ZFN, TALEN, and / or CRISPR / Cas system).The polynucleotide may be, for example, mRNA. In some embodiments, the mRNA may be chemically modified (see, e.g., Kormann et al., (2011) Nature Biotechnology 29(2):154-157). In other embodiments, the mRNA may include an ARCA cap (see, U.S. Patent Nos. 7,074,596 and 8,153,773). In yet other embodiments, the mRNA may include a mixture of unmodified and modified nucleotides (see, U.S. Patent Publication No. 20120195936). In yet other embodiments, the mRNA may include a WPRE element (see, U.S. Patent No. 10,179,918).

[0187] In another aspect, genetically modified cells (e.g., stem cells, progenitor cells, liver cells, muscle cells, etc.) bearing a desired GLA transgene (optionally incorporated using a nuclease) are described. In some embodiments, the edited stem or progenitor cells may then be expanded and induced to differentiate ex vivo into mature edited cells, which are then administered to the patient. Thus, cells derived from the genetically edited (modified) GLA-producing stem or progenitor cells described herein may be used. In other embodiments, edited progenitor cells (e.g., CD34+ stem cells) are administered in a bone marrow transplant, which, after successful transplantation, expands to produce edited cells, which then differentiate and mature in vivo and contain the biologic expressed from the GLA transgene. In some embodiments, edited CD34+ stem cells are administered intravenously to a patient, allowing the edited cells to migrate to the bone marrow, differentiate, mature, and produce α-Gal A protein. In other embodiments, the edited stem cells are muscle stem cells, which are then introduced into muscle tissue. In some embodiments, the engineered nuclease is a zinc finger nuclease (ZFN) (the term "ZFN" includes paired ZFNs), in other embodiments, the nuclease is a TALE nuclease (TALEN) (the term "TALEN" includes paired TALENs), and in other embodiments, a CRISPR / Cas system is used. The nuclease may be engineered to have specificity for safe harbor loci, genes associated with disease, or genes highly expressed in cells. By way of non-limiting example only, a safe harbor locus may be the AAVS1 locus, the CCR5 gene, albumin, or HPRT gene, while a disease-associated gene may be the GLA gene, which encodes alpha-galactosidase A.

[0188] The GLA-introduced gene may be full-length or modified, may be expressed extrachromosomally, or may be integrated into the cell's genome in a targeted manner using one or more nucleases. Unlike random integration, nuclease-mediated targeted integration ensures that the introduced gene is integrated into a specific gene. The introduced gene can be integrated anywhere in the target gene. In certain embodiments, the introduced gene is at or near the nuclease binding and / or cleavage site, e.g., within 1 to 300 base pairs (or any number of base pairs in between), more preferably within 1 to 100 base pairs (or any number of base pairs in between) on either side of the cleavage and / or binding site, even more preferably within 1 to 50 base pairs (or any number of base pairs in between) on either side of the cleavage and / or binding site. In certain embodiments, the sequence to be integrated does not include any vector sequences (e.g., viral vector sequences).

[0189] Any cell type, including but not limited to cells or cell lines, may be genetically modified as described herein to contain the introduced gene. Other non-limiting examples of the genetically modified cells described herein include T cells (e.g., CD4+, CD3+, CD8+, etc.); dendritic cells; B cells; autologous (e.g., patient-derived) muscle cells, brain cells, etc. In certain embodiments, the cells are liver cells and are modified in vivo. In certain embodiments, the cells are stem cells, including heterologous pluripotent, totipotent or multipotent stem cells (e.g., CD34+ cells, induced pluripotent stem cells (iPSCs), embryonic stem cells or homologous ones). In certain embodiments, the cells described herein are patient-derived stem cells.

[0190] The cells described herein are useful in treating and / or preventing Fabry disease in a subject having a disorder, for example, by in vivo therapy. For example, ex vivo therapy is also provided if nuclease-modified cells can be expanded and then reintroduced into a patient using standard techniques. See, for example, Tebas et al (2014) New Eng J Med 370(10):901. In the case of stem cells, in vivo differentiation of these progenitor cells into cells that express the functional protein (from the inserted donor) also occurs after injection into the subject.

[0191] Pharmaceutical compositions comprising the cells described herein are also provided. Further, the cells may be cryopreserved prior to administration to a patient.

[0192] Delivery The cDNA expression constructs, nucleases, polynucleotides encoding these nucleases, donor polynucleotides and / or compositions (e.g., cells, proteins, polynucleotides, etc.) described herein may be delivered in vivo or ex vivo by any suitable means.

[0193] Methods for delivering the nucleases described herein are described, for example, in U.S. Patent Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the entire disclosures of all of which are incorporated herein by reference.

[0194] The expression constructs and / or nucleases described herein may also be delivered using a vector comprising a sequence encoding one or more of zinc fingers, TALENs and / or Cas protein(s). Any vector system may be used, including but not limited to plasmid vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, poxviral vectors; herpesviral vectors and adeno-associated viral vectors. See also U.S. Patent Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, which are hereby incorporated by reference in their entirety. Further, it will be apparent that any of these vectors can comprise one or more of the sequences required for treatment. Thus, when one or more nucleases and donor constructs are introduced into a cell, the nuclease and / or donor polynucleotide may be carried on the same vector or a different vector. If multiple vectors are used, each vector may comprise a sequence encoding one or more nucleases and / or donor constructs.

[0195] Conventional viral and non-viral gene transfer methods can be used to introduce cDNA expression constructs or nucleic acids and / or expression constructs encoding nucleases into cells (e.g., mammalian cells) and target tissues. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes or poloxamers. Viral vector delivery systems include DNA and RNA viruses that have either episomal or integrated genomes after delivery to cells. For reviews of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and See Bohm (eds.) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0196] Methods of non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, particle bombardment, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid complexes, naked DNA, artificial virions, and uptake of DNA enhanced with agents. For example, sonoporation using the Sonitron2000 system (Rich-Mar) can also be used for delivery of nucleic acids.

[0197] Further examples of nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Maryland), BTX Molecular Delivery Systems (Holliston, MA), and Copernicus Therapeutics Inc (see, for example, US6008336). Lipofection is described, for example, in US Patent Nos. 5,049,386; 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include those of Felgner, WO91 / 17424, WO91 / 16024.

[0198] The preparation of lipid: nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those skilled in the art (see, for example, Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Patent Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; and 4,946,787).

[0199] The cDNA and / or nuclease compositions described herein can also be delivered using nanoparticles, such as lipid nanoparticles (LNPs). See, for example, Lee et al (2016) Am J Cancer Res 6(5):1118-1134; U.S. Patent No. 10,166,298; and U.S. Publication No. 20180185516.

[0200] As a further method of delivery, the use of packaging of the nucleic acid to be delivered into an EnGeneIC delivery vehicle (EDV) can be mentioned. These EDVs are specifically delivered to the target tissue using a bispecific antibody in which one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV. After the antibody has carried the EDV to the target cell surface, the EDV is carried into the cell by endocytosis. When it enters the cell, the contents are released (see MacDiarmid et al (2009) Nature Biotechnology 27(7):643).

[0201] The use of RNA- or DNA-virus-based systems to deliver nucleic acids encoding engineered ZFPs takes advantage of highly evolved processes to direct the virus to specific cells in the body and transport the viral payload to the nucleus. Viral vectors can be administered directly to the subject (in vivo) or used to treat cells in vitro, with the modified cells then administered to the subject (ex vivo). Conventional virus-based systems for ZFP delivery include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated viral, vaccinia viral, and herpes simplex viral vectors for gene transfer. Retroviral, lentiviral, and adeno-associated viral gene transfer methods allow for integration into the host genome, often resulting in long-term expression of the inserted transgene. Additionally, high transduction efficiencies have been demonstrated in many different cell types and target tissues.

[0202] The tropism of retroviruses can be modified by incorporating exogenous envelope proteins to expand the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and generally produce high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats and have a packaging capacity for up to 6-10 kb of exogenous sequence. Minimal cis-acting LTRs are sufficient for vector replication and packaging, which are then used to integrate therapeutic genes into target cells to result in persistent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991)).

[0203] In applications where transient expression is preferred, an adenovirus-based system can be used. Adenovirus-based vectors enable very high transduction efficiency in many cell types and do not require cell division. Using such vectors, high titers and high levels of expression were obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors are also used, for example, in the in vitro production of nucleic acids and peptides and for transducing cells having a target nucleic acid for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994)). Construction of recombinant AAV vectors has been described in many publications, including U.S. Patent No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).

[0204] At least six viral vector approaches are currently available for gene transfer in clinical trials, and these utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to produce the transduction agent.

[0205] pLASN and MFG-S are examples of retroviral vectors used in clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn et al., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138 (1997)). PA317 / pLASN was the first therapeutic vector used in a gene therapy trial (Blaese et al., Science 270:475-480 (1995)). Transduction efficiencies of over 50% were observed with MFG-S-packaged vectors (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoff et al., Hum. Gene Ther. 1:111-2 (1997)).

[0206] Recombinant adeno-associated virus vectors (rAAV) are a promising alternative gene delivery system based on the defective, nonpathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from plasmids that retain only the 145-bp inverted terminal repeats of AAV flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery via integration into the genome of transduced cells are key features of this vector system. (Wagner et al., Lancet 351:9117 1702-3 (1998); Kearns et al., Gene Ther. 9:748-55 (1996)) Other AAV serotypes can also be used, including, but not limited to, AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV 8.2, AAV9, and AAVrhlO, as well as pseudotyped AAVs such as AAV2 / 8, AAV2 / 5, and AAV2 / 6.

[0207] AAV can be produced on a clinical scale by many different processes. Examples of available systems include (1) plasmid DNA transfection in mammalian cells, (2) Ad infection of stable mammalian cell lines, (3) infection of mammalian cells with recombinant herpes simplex virus (rHSV), and (4) infection of insect cells (Sf9 cells) with recombinant baculovirus (for a review, see Penaud-Budloo et al. (2018) Mol Ther Methods Clin Dev. 8: 166-180).

[0208] Replication-deficient recombinant adenoviral vectors (Ad) can be produced at high titers and readily infect many different cell types. Most adenoviral vectors are engineered so that a transgene replaces the Ad E1a, E1b, and / or E3 genes; the replication-deficient vector is then propagated in human 293 cells, which supply the deleted gene functions in trans. Ad vectors can transduce multiple tissue types in vivo, including non-dividing, differentiated cells such as those found in the liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity. An example of the use of Ad vectors in clinical trials included polynucleotide therapy for anti-tumor immunization via intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). Further examples of the use of adenoviral vectors for gene transfer in clinical trials include Rosenecker et al., Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

[0209] Packaging cells are used to form viral particles capable of infecting host cells. Examples of such cells include 293 cells, which package adenovirus, and ψ2 or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are typically generated by producer cell lines that package nucleic acid vectors into viral particles. The vectors generally contain the minimum viral sequences required for packaging and subsequent integration into the host (if applicable), with other viral sequences replaced by expression cassettes encoding the proteins to be expressed. Missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy generally contain only the inverted terminal repeat (ITR) sequences from the AAV genome required for packaging and integration into the host genome. Viral DNA is packaged in a cell line containing a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking the ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus facilitates AAV vector replication and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to the lack of ITR sequences. Adenovirus contamination can be reduced by, for example, heat treatment, to which adenovirus is more sensitive than AAV.

[0210] In many gene therapy applications, it is desirable for the gene therapy vector to be delivered with a high degree of specificity to a particular tissue type. Thus, viral vectors can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus. The ligand is selected to have an affinity for a receptor known to be present on the target cell type. For example, Han et al., Proc. Natl. Acad. Sci. USA 92:9747-9751 (1995) reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and that the recombinant virus infects specific human breast cancer cells that express the human epidermal growth factor receptor. This principle can be extended to other virus-target cell pairs where the target cell expresses a receptor and the virus expresses a fusion protein containing a ligand for that cell surface receptor. For example, filamentous phage may be engineered to display antibody fragments (e.g., FAB or Fv) with specific binding affinity for substantially any selected cell receptor. The above description applies primarily to viral vectors, but the same principle is applicable to non-viral vectors. Such vectors may be engineered to contain specific uptake sequences that facilitate uptake by specific target cells.

[0211] As described below, gene therapy vectors can generally be delivered in vivo by administration to individual patients by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial injection) or local application. Alternatively, the vector can be delivered ex vivo to cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy samples) or cells such as universal donor hematopoietic stem cells, followed by re-transplantation of the cells into the patient, usually after selection of the cells into which the vector has integrated.

[0212] Vectors containing nucleases (e.g., retroviruses, adenoviruses, liposomes, etc.) and / or donor constructs (expression constructs) can also be administered directly to an organism for in vivo cell transduction. Alternatively, naked DNA may be administered. Administration is by any route commonly used to ultimately bring the molecule into contact with blood or tissue cells and includes, but is not limited to, injection, infusion, topical application, and electroporation. Suitable methods for administering such nucleic acids are available and well known to those of skill in the art, and two or more routes can be used to administer a particular composition, although a particular route may often result in a more rapid and effective response than another route.

[0213] Vectors suitable for introduction of the polynucleotides described herein include non-integrating lentiviral vectors (IDLVs). See, for example, Ory et al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al. (1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol. 72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222.

[0214] The pharmaceutically acceptable carrier is determined in part by the particular composition being administered and by the particular method used to administer the composition. Thus, as shown below, there are a variety of suitable formulations of the available pharmaceutical compositions (see, for example, Remington’s Pharmaceutical Sciences, 17th ed., 1989).

[0215] It will be apparent that the nuclease coding sequence and the donor construct can be delivered using the same or different systems. For example, the donor polynucleotide can be carried by a plasmid, while one or more nucleases can be carried by an AAV vector. Further, different vectors may be administered by the same or different routes (intramuscular injection, tail vein injection, other intravenous injections, intraperitoneal administration and / or intramuscular injection). The vectors may be delivered simultaneously or in any series of orders.

[0216] Formulations for both ex vivo and in vivo administration include suspensions or emulsions. The active ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, physiological saline, dextrose, glycerol, ethanol, etc. and combinations thereof. Further, the composition may contain minor amounts of auxiliary substances such as wetting agents, emulsifying agents, pH buffering agents, stabilizing agents or other reagents that improve the effectiveness of the pharmaceutical composition.

[0217] Use The methods disclosed herein contemplate the treatment and / or prevention of Fabry disease (e.g., lysosomal storage disorders). Treatment may include inserting a modified disease-related GLA transgene into a cellular safe harbor locus (e.g., albumin) for expression and release of the required enzyme into the bloodstream. The modified α-GalA-encoding transgene may encode a wild-type or modified protein; and / or a codon-optimized GLA transgene; and / or a transgene in which epitopes can be removed without functionally altering the protein. In certain cases, the method includes inserting an episome expressing an α-GalA-encoding transgene into cells for expression and release of the required enzyme into the bloodstream. Insertion into secretory cells such as liver cells is particularly useful for release of the product into the bloodstream. The methods and compositions can be used in any situation where it is desirable to supply a GLA transgene encoding one or more therapeutic substances to hematopoietic stem cells such that mature cells (e.g., RBCs) derived from the hematopoietic stem cells contain the therapeutic α-GalA protein. These stem cells can be differentiated in vitro or in vivo and may be derived from a universal donor type of cells that can be used in all patients. Additionally, the cells may contain transmembrane proteins that pass through cells in the body. Treatment may also include use of patient cells containing a therapeutic transgene, in which case the cells are developed ex vivo and then introduced back into the patient. For example, HSCs containing a suitable α-GalA-encoding transgene may be inserted into a patient by bone marrow transplantation. Alternatively, stem cells such as muscle stem cells or iPSCs edited using an α-GalA-encoding transgene can also be injected into muscle tissue.

[0218] Thus, this technique may be used in situations where a patient is lacking some proteins due to problems (e.g., problems with expression levels or proteins that are expressed as non-functional or non-functioning). Expression of a transgene that modifies or restores functionality in subjects with Fabry disease is particularly useful.

[0219] As a non-limiting example, various methods of generating a functional α-GalA protein that replenishes defective or missing α-GalA protein have been achieved and used to treat Fabry disease. The nucleic acid donor encoding the protein may be inserted into a safe harbor locus (e.g., albumin or HPRT) and expressed either using an exogenous promoter or a promoter present in the safe harbor. Insertion of the GLA transgene into the albumin locus of hepatocytes is particularly useful, in which case the GLA transgene further comprises a sequence encoding a signal peptide involved in the secretion of the expressed α-GalA protein from hepatocytes into the bloodstream. Alternatively, the donor can be used to correct the defective gene in situ. The desired α-GalA-encoding transgene may be inserted into CD34+ stem cells and returned to the patient during bone marrow transplantation. Finally, the nucleic acid donor may be inserted at the beta-globin locus in CD34+ stem cells such that mature red blood cells derived from the stem cells have a high concentration of the biologic agent encoded by the nucleic acid donor. RBCs containing the biologic agent can then be directed to the correct tissue by a transmembrane protein (e.g., a receptor or an antibody). Further, RBCs may be sensitized ex vivo by electro-sensitization to be more readily destroyed after exposure to an energy source (see WO2002007752).

[0220] In some applications, the endogenous gene may be knocked out by use of the methods and compositions described herein. Examples of this aspect include knockout of abnormal gene regulators or abnormal disease-related genes. In some applications, the abnormal endogenous gene may be replaced either functionally or in situ by the wild-type type of the gene. The inserted gene may also be modified to enhance the expression of the therapeutic α-GalA protein or to reduce immunogenicity. In some applications, the inserted α-GalA-encoding transgene is a fusion protein that increases transport to a selected tissue such as the brain.

[0221] Of course, suitable GLA donors include any GLA transgene, including but not limited to those exemplified below.

[0222] The present disclosure also provides methods and compositions for the production of cells (e.g., RBCs) carrying the α-Gal A therapeutic protein for the treatment of Fabry disease, which can be used universally in all patients as a homogenous product. This allows for the development of a single product for the treatment of patients with, for example, Fabry disease. Such carriers may include transmembrane proteins that aid in the transport of the cells. In one embodiment, the transmembrane protein includes an antibody, while in other embodiments, the transmembrane protein includes a receptor.

[0223] In some embodiments, the GLA transgene donor is transfected or transduced into cells for episomal or extrachromosomal maintenance of the transgene. In some embodiments, the GLA transgene donor is carried in a vector containing a regulatory domain that controls expression of the transgene donor. In some cases, the regulatory domain that controls transgene expression is endogenous to the expressed transgene, while in other cases, the regulatory domain is heterologous to the transgene. In some embodiments, the GLA transgene is carried in a viral vector, while in other embodiments, it is carried in a plasmid or minicircle. In some embodiments, the viral vector is AAV, Ad, or LV. In another embodiment, the vector containing the transgene donor is delivered to appropriate target cells in vivo so that the α-Gal A therapeutic protein encoded by the transgene donor is released into the bloodstream when the transgene donor vector is delivered to hepatocytes.

[0224] In another embodiment, the present disclosure describes precursor cells (muscle stem cells, progenitor cells or CD34+ hematopoietic stem cell (HSPC) cells) into which a GLA transgene has been inserted such that the mature cells derived from the precursor cells contain high levels of the α-GalA product encoded by the transgene. In some embodiments, these precursor cells are induced pluripotent stem cells (iPSCs).

[0225] In some embodiments, the method may be used in vivo in a transgenic animal system. In some aspects, the transgenic animal may be used for model development in which the transgene encodes a human α-GalA protein. In some cases, the transgenic animal may be knocked out at the corresponding endogenous locus, thereby enabling the development of an in vivo system in which the human protein can be studied in isolation. Such transgenic models can be used for screening purposes to identify small molecules, or large biomolecules or other factors that may interact with or modify the human protein of interest. In some aspects, the GLA transgene is incorporated into a selected (e.g., high-expressing or safe harbor) locus in a stem cell (e.g., embryonic stem cell, induced pluripotent stem cell, liver stem cell, neural stem cell, etc.) or non-human animal embryo obtained by any of the methods described herein and standard methods in the art, and then the embryo is transplanted so that a live animal is born. The animal is then raised until sexually mature and enabled to produce offspring, at least some of which contain the incorporated GLA transgene.

[0226] In any of the foregoing embodiments, the methods and compounds may be combined with other therapeutic agents for the treatment of subjects with Fabry disease. In some embodiments, the methods and compositions include the use of molecular chaperones to ensure the correct folding of the Fabry protein (Hartl et al (2011) Nature 465: 324-332). In some aspects, the chaperone can be selected from well-known chaperone proteins such as AT1001 (Benjamin et al (2012) Mol Ther 20(4):717-726), AT2220 (Khanna et al (2014) PLoS ONE 9(7): e102092, doi:10.1371), and Migalastat (Benjamin et al (2016) Genet Med doi: 10.1038 / gim.2016.122). In some aspects, the methods and compositions are used in combination with methods and compositions that enable passage across the blood-brain barrier. In other aspects, the methods and compositions are used in combination with compounds that have been found to suppress the immune response of the subject.

[0227] Also provided are kits comprising the nuclease systems and / or GLA donors described herein. The kits may include nucleic acids encoding one or more nucleases (ZFNs, ZFN pairs, TALENs, TALEN pairs, and / or CRISPR / Cas systems) (e.g., RNA molecules or ZFN, TALEN, and / or CRISPR / Cas system-encoding genes contained in a suitable expression vector), donor molecules, expression vectors encoding single-guide RNAs suitable for a host cell line, instructions for performing the methods disclosed herein, and the like.

[0228] These and other aspects will be readily apparent to those skilled in the art in view of the entire disclosure.

[0229] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings as commonly understood by one of ordinary skill in the art. Although exemplary methods and materials are set forth below, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. In general, the nomenclature used in connection with cardiology, medicine, pharmaceutical and medicinal chemistry, and cell biology as described herein, as well as the techniques thereof, are well known and commonly employed in the art. Enzyme reactions and purification techniques are performed according to the manufacturer's specifications, generally as accomplished in the art or as described herein. Further, unless the context dictates otherwise, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and the embodiments, variations such as the words "have", "comprise", or "has", "having", "comprises", or "comprising" are meant to include the recited integer or group of integers but not to exclude any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although many documents are cited herein, this citation does not admit that any of these documents form part of the common general knowledge in the art. As used herein, the term "about" or "approximately" when applied to one or more values of interest refers to a value similar to the specified reference value. In certain embodiments, the term refers to a range of values that are within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the specified reference value, unless otherwise specified or otherwise apparent from the context.

[0230] To better understand the present invention, the following examples are described. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

Example

[0231] Example 1 High plasma α-GalA activity lasting for 3 months in GLAKO mice treated with variant #4 expression construct Samples of the variant #4 expression construct as shown in Figure 1A were administered to male GLAKO mice to evaluate the pharmacodynamic activity and biodistribution after a single IV dose.

[0232] Male GLAKO mice were 8 - 12 weeks old at the start of the study. Animals (n = 10 - 20 males / group) were given a formulation buffer containing phosphate-buffered saline (PBS) with CaCl2, MgCl2, NaCl, sucrose, and Kolliphor (poloxamer) P188 (control mice) or one of three dose levels of the variant #4 expression vector (2.0E+12, 5.0E+12, or 5.0E+13 vg / kg, respectively; n = 10 / group) as a single 200 μl IV tail injection on day 1. The mice were observed for 3 months. The results of the pharmacokinetic evaluation (plasma α-GalA activity) are shown for individual mice in Figure 2 and the group mean (mean + SD) in Figure 3. Plasma α-GalA activity was evaluated at the AAV / construct dose. Furthermore, the plasma α-GalA activity exceeded 300-fold that of α-GalA activity in physiologically normal or wild-type (non-mutated) subjects. (In Figure 3, * indicates that one outlier was removed due to excessive performance).

[0233] Single administration of increasing doses of WPRE-deficient AAV hGLA cDNA (variant #4) was administered using a clinical-scale manufacturing process and resulted in physiological expression of plasma α-Gal A (>300-fold higher than WT) by study day 15, was well tolerated, and remained stable for 3 months after injection. Dose-dependent increases in α-Gal A activity were achieved in the liver, heart, and kidney, with corresponding decreases in Gb3 / lyso-Gb3.

[0234] α-Gal A produced in the liver was secreted into the bloodstream and absorbed by secondary tissues. Figure 4A shows tissue α-Gal A activity in liver lysates, Figure 4B shows tissue α-Gal A activity in kidney lysates, and Figure 4C shows tissue α-Gal A activity in heart lysates.

[0235] Example 2 High levels of α-Gal A activity result in degradation of the corresponding Fabry substrate The variant #4 construct formulation was administered IV to GLAKO mice at doses of 0 vg / kg, 2.0E+12 vg / kg, 5.0E+12 vg / kg or 5.0E+13 vg / kg to evaluate the levels of Fabry substrates in mouse plasma and tissues. Tissues were collected at necropsy on day 91 post-administration and assayed for the levels of α-GalA substrate Gb3 (isoforms C22:0 and C24:0) and its deacylated form lyso-Gb3 using LC-MS. Briefly, tissues were weighed and mechanically disrupted in a tissue lysis buffer (5% MeOH, 95% water and 0.1% acetic acid) at a 5 ml ratio per 1 mg of tissue. Subsequently, 10 μl of plasma or tissue slurry was added to 90 μl of a precipitation solvent (MeOH with internal standard N-tricosanoylceramide trihexoside (C23:0, Matreya) added to the solution) in a silicon tube, vortexed and placed on a shaking plate at room temperature for 30 minutes. The samples were then centrifuged and 10 μl of the sample was added to 90 μl of a single blank matrix (DMSO / MeOH 1:1 + 0.1% FA) in a glass LC-MS vial. Samples were analyzed for Gb3 chain length 24:0, the major Gb3 chemical species present in GLAKO mice, and measured against a standard curve (Gb3, Matreya) composed of ceramide trihexoside.

[0236] Globotriaosylsphingosine (lyso-Gb3) was measured in a similar fashion using glucosylsphingosine (Matreya) and lyso-ceramide trihexoside (lyso-Gb3, Matreya) as internal standards to generate a standard curve. Data are presented as mean + SD for 9 to 20 animals / group as shown in the legend. The Fabry substrates, globotriaosylceramide (Gb3), in mouse plasma and tissues were measured by mass spectrometry.

[0237] Certain production of α-GalA should enable reduction, and in some cases, removal of the Fabry disease substrates, Gb3 and lyso-Gb3. As shown in FIGS. 5A and 5B, dose-related reduction in the levels of the Fabry substrates, Gb3 and lyso-Gb3, was seen in plasma, liver, heart, kidney, and spleen. For the majority of samples from animals in the high-dose group, tissue Gb3 levels were reduced by more than 80% compared to samples from animals in the formulation control group, as shown in FIG. 5B.

[0238] At high dose levels, Gb3 levels in the heart and kidney were reduced to about 10% of untreated animals, as shown in FIG. 6A. In the treated subjects, Gb3 levels were below the lower limit of quantification, as shown in FIG. 6B.

[0239] Example 3 The variant #21 expression vector elicits plasma α-GalA activity in vitro and in vivo. The levels and activities of secreted human α-GalA were evaluated in various mice, cynomolgus monkeys, and human primary cells and cell lines after transduction with the variant #4 or variant #21 expression vector. The variant #4 or variant #21 expression vector was generated in 1) HEK293 cells or 2) Sf9 insect cell lines.

[0240] Using standard techniques, transduction was performed into HepG2 cells and iPSC-derived hepatocytes (iCell hepatocytes) as described in US Publication No. 20180117181. Briefly, cells were seeded at various densities per well and transduced with the variant #21 expression construct or variant #4 expression construct at a multiplicity of infection (MOI) in the range of 100,000 to 600,000 vg / cell. Supernatant samples were collected from day 3 to day 7, and α-GalA enzyme activity was evaluated by α-GalA fluorescence quantitative activity assay, and cell pellets were harvested at the end of the test (day 6 or 7).

[0241] The cDNA approach may involve the use of an AAV delivery expression construct comprising an hAAT promoter (Okuyama et al (1996) Hum Gene Ther 7(5):637-45), an HBB-IGG intron (a chimeric intron composed of the 5'-donor site of the first intron of the human beta-globin gene, the branch and 3'-acceptor site of the intron of the immunoglobulin gene heavy chain variable region), a signal peptide, a coding sequence (the coding sequence is optionally codon-optimized), and an APOE enhancer linked to a bovine growth hormone (e.g., bGH or SPA51) polyA signal sequence.

[0242] HepG2 / C3A cells (also referred to as "HepG2" cells) (ATCC, CRL 10741) were maintained in minimum essential medium (MEM) containing Earle's salts and L-glutamine (Corning) supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and 1× penicillin streptomycin glutamine (Life Technologies), and incubated at 37°C and 5% CO2. The cells were passaged every 3 to 4 days.

[0243] For transduction, the cells were rinsed, trypsinized with 0.25% trypsin / 2.21 mM EDTA (Corning), and resuspended in growth medium. A small aliquot was mixed 1:1 with 0.4% (w / v) trypan blue solution in phosphate buffered saline (PBS; Corning) and counted in a TC20 Automated Cell Counter (Bio Rad). The cells were resuspended in growth medium at a density of 2e5 per mL and seeded at 1e5 in 0.5 mL of medium per well in a 24-well plate (Corning). Recombinant AAV2 / 6 particles were mixed with growth medium at an appropriate multiplicity of infection (MOI) and added to the cells. The MOI of the GLA cDNA construct was either 3e4, 1e5, 3e5, or 1e6 vg / cell.

[0244] After transfection, the cells were placed under culture for 6 - 10 days. The supernatant was collected on days 3, 5, 7, and 10 (where appropriate) and replaced with fresh medium. After the final supernatant collection step, the cells were trypsinized as described above, resuspended, centrifuged to create a cell pellet, washed with PBS, and stored at -80 °C.

[0245] α-GalA activity was evaluated in a fluorescence quantitative assay using the synthetic substrate 4-methylumbelliferyl-α-D-galactopyranoside (4MU-α-Gal, Sigma).

[0246] Briefly, 10 microliters of HepG2 cell culture supernatant was mixed with 40 μL of 5 mM 4MU-α-Gal dissolved in phosphate buffer (0.1 M citrate / 0.2 M phosphate buffer, pH 4.6, 1% Triton® X-100). The reaction was incubated at 37 °C and stopped by the addition of 100 μL of 0.5 M glycine buffer, pH 10.3. The release of 4-methylumbelliferone (4MU) was measured by fluorescence measurement (excitation 365 / fluorescence 450) using a SpectraMax Gemini XS fluorescence reader (Molecular Devices, Sunnyvale CA).

[0247] A standard curve was generated using a two-fold dilution series of 4MU. The obtained data was fitted to a double logarithmic curve, and the concentration of 4MU in the test samples was calculated using this best-fit curve. Enzyme activity is expressed as nmol of 4MU released per hour per mL of cell culture supernatant per hour of assay incubation (nmol / hr / mL).

[0248] Turning to Figures 7A and 7B, the variant #21 expression construct has improved α-GalA potency in vitro over the AAV GLA variant #4 expression vector. In HepG2 cells, as shown in Figure 7A, the α-GalA activity in the supernatant increased between approximately 4-fold and approximately 9-fold. In iPSC-derived human hepatocytes, as shown in Figure 7B, the activity in the supernatant increased between approximately 3-fold and approximately 5-fold.

[0249] Episomal AAV (serotype 2 / 6) vectors encoding human GLA cDNA (hGLA) driven by a liver-specific promoter without a mutant WPRE sequence (variant #4) or containing it (variant #21) were administered to animals at various doses. Figure 8 shows the increase in GLA A activity with increasing construct dose in the plasma of wild-type mice treated with a dose of 2.0E+12 vg / kg or 5E+11 vg / kg of the variant #21 construct, or a dose of 2.0E+12 vg / kg or 5E+11 vg / kg of the variant #4 construct, or the formulation buffer. The results show an improvement in plasma activity between about 7-fold and about 9-fold over 28 days in wild-type mice.

[0250] The expression construct variant #4 was compared with a cDNA construct (variant #21) containing the WPRE sequence in a 1-month study in wild-type C57BL / 6 mice using two different AAV doses (AAV carrying the cDNA donor). Table 1 above shows the complete sequences of the constructs used. The construct containing the cDNA with the WPRE sequence resulted in an average 7-fold higher level of plasma α-GalA activity than mice administered the same dose of the first (WPRE-free) cDNA on day 28 of the study.

[0251] A 4- to 9-fold increase in GLA activity was seen in the supernatant of HepG2 cells treated with variant #21 (construct containing WPRE) compared to variant #4 (without WPRE), and a 3- to 5-fold increase in GLA activity was seen in the supernatant of hepatocytes derived from induced pluripotent cells (iCell) treated with variant #21 (construct containing WPRE) compared to variant #4 (without WPRE). Furthermore, a 7- to 9-fold increase in plasma GLA activity was seen in mice treated with variant #21 (construct containing WPRE) compared to variant #4 (without WPRE).

[0252] The high levels of α-GalA activity, with a marked decrease in Gb3 / lyso-Gb3 accumulated in key tissues of the GLAKO mouse model as seen in these tests, are due to a clinical-scale manufacturing process that enables the rapid and efficient production of therapeutic vectors, demonstrating that AAV-mediated hepatocyte targeting results in therapeutic levels of human α-GalA in the subject.

[0253] Therapeutic levels of α-GalA protein for the treatment of Fabry are generated in vivo using a cDNA approach, including after clinical-scale production of the expression vector.

[0254] The results shown in Figure 9 and Table 3 below indicate that the variant #21 expression construct results in plasma α-GalA activity up to 1,500-fold that of physiological normal (wt) in vivo. The variant #4 expression construct was administered to C57BL / 6 mice by tail vein injection at 5.0E+12 and 5.0E+13 vg / kg. The variant #21 expression construct was administered to C57BL / 6 mice by tail vein injection at 5.0E+12, 5.0E+13, and 5.0E+14 (not shown). Plasma samples were collected 1 week prior to dosing and on days 8, 15, 22, and 29, and later evaluated for α-GalA enzyme activity by fluorescence quantitative assay. Data points represent the mean response + / - SD for each dose. The lower limit of quantification (LLOQ) of the assay is 2.5 nmol / hr / mL.

Table 3

[0255] Figure 9 shows α-GalA plasma activity in C57BL / 6 mice over 29 days after treatment with either the variant #21 construct at a dose of 5.0E+13 vg / kg, the variant #21 construct at a dose of 5.0E+12 vg / kg, the variant #4 construct at a dose of 5.0E+13 vg / kg, the variant #4 construct at a dose of 5.0E+12 vg / kg, or the formulation buffer.

[0256] Consistent with the in vitro data, plasma and liver GLA levels are higher (up to 21-fold higher) in animals administered the variant #4 expression construct produced in HEK293 cells versus the Sf9 cell line.

[0257] Example 4 Treatment with variant #4 expression vector results in high levels of hepatocyte transduction in GLAKO mice and non-human primates To assess the level of expression construct copies in hepatocytes after IV administration of the variant #4 expression construct, formalin-fixed, paraffin-embedded (FFPE) liver samples from a subset of animals were evaluated by BASESCOPE™ in situ hybridization (ISH). After ISH staining, quantitative image analysis was performed using HALO™ software. Non-coding sequences were targeted. A housekeeping gene probe, PPIB (cyclophilin B), was used as a positive control marker for sample QC to assess RNA quality in tissue samples. The bacterial gene DapB was used as a negative control. A semiquantitative score (on a scale of 0 to 4) was obtained for all samples to assess sample quality and determine QC pass / fail. Most samples had a PPIB (cyclophilin B; housekeeping gene control) score of 3, indicating excellent quality RNA. Most DapB (bacterial gene control) scores were 0, indicating no or negligible nonspecific background. Specific DNA staining signals are visible as dark (red) punctate dots within the cell nuclei. Samples were counterstained with Gill's hematoxylin and shown in light gray (blue).

[0258] Representative ISH images of the liver of a GLAKO mouse administered 5.0+13vg / kg of the variant #4 expression vector at various magnifications are shown in Figure 10 . Representative ISH staining images of the liver of an NHP administered 6.0+13vg / kg of the variant #4 expression vector at various magnifications are shown in Figure 11 . Specific DNA staining signals are observed as dark gray punctate dots within the cell nuclei. The samples were counterstained with Gill's hematoxylin light gray. As shown, 57.5% of the mouse hepatocytes in the representative sample in Figure 10 were positive for the expression vector with 2.34 points / cell and an H-score of 126.82. Impressively, 72.9% of the NHP hepatocytes in the representative sample in Figure 11 were positive for the expression construct with 3.20 points / cell and an H-score of 175.39.

[0259] Overall, a dose-response relationship in mouse liver cells was confirmed for all parameters evaluated, including % positive cells, mean number of dots / cell, and H-score, which was calculated by dividing cells into five bins based on the number of dots per cell and then summing the percentage of cells in each bin according to a weighted formula.

[0260] Figure 12A shows the percentage of GLAKO mouse hepatocytes containing hGLA cDNA in GLAKO mice treated with doses of 2E+12vg / kg, 5E+12vg / kg, and 5E+13vg / kg of the variant #4 construct, or formulation buffer as a control. Figure 12C shows the percentage of hepatocytes containing hGLA cDNA in individual subjects. Similarly, Figure 12B is a graph showing the percentage of hepatocytes containing hGLA cDNA in cynomolgus monkey NHPs treated with doses of 6E+12vg / kg, 1E+13vg / kg, 3E+13vg / kg, and 6E+13vg / kg of the variant #4 construct, or formulation buffer as a control. Figure 12D shows the percentage of hepatocytes containing hGLA cDNA for individual NHP subjects.

[0261] The insights hybridization test to measure the level of hGLA DNA construct in the liver showed a dose-response relationship in mouse and NHP hepatocytes and confirmed the translocation of DNA into the nucleus. Positive stained cells in the range of 28% to 58% were obtained at the high dose (5.0E+13 vg / kg) in mice, and positive stained cells in the range of 61% to 73% were obtained at the high dose (6.0E+13 vg / kg; with immunosuppression) in the NHP test. 49% of positive stained cells were obtained in another NHP (without immunosuppression).

[0262] Example 5 α-GalA Protein and Enzyme Activity in Cynomolgus NHP after a Single Intravenous Administration of Variant #4 Expression Construct The variant #4 expression construct was evaluated in NHP with respect to pharmacology and toxicology. A single IV dose of the variant #4 expression construct was administered to male cynomolgus monkeys (n = 3 / group) at 0 (n = 2), 6.0E+12, 1.0E+13, 3.0E+13 or 6.0E+13 vg / kg. To reduce the possible immune response against the expression vector and / or human α-GalA, rituximab (10 mg / kg; IV) was given to the animals before the administration of the expression construct, and methylprednisolone (10 mg / kg; intramuscular) was given to the animals daily throughout the study. An additional group was given the variant #4 expression construct at the highest dose (6.0E+13 vg / kg) without immunosuppression. The variant #4 expression construct used was manufactured in a GMP clinical manufacturing process using the baculovirus / Sf9 cell platform.

[0263] Blood was collected before dosing (5 time points), and on days 7, 14, 21, 28, 35, 42, 49, and 56, and processed to plasma. These plasma samples were evaluated for human α-GalA protein levels and α-GalA activity. At necropsy on day 56, 4 areas of the liver (2 areas each of the lateral left and right lobes) and 2 areas of the spleen were collected to evaluate α-GalA activity. The results are shown in Figures 13A to 13F.

[0264] Circulating α-GalA protein levels and plasma α-GalA activity were generally detected by day 7, and protein levels and activity reached maximum between days 7 and 21, with no clear dose response. Animals administered the variant #4 expression construct without immunosuppression generally had lower levels and activity of α-GalA protein than animals administered the variant #4 expression construct with immunosuppression. This lack of high dose response as well as the removal of α-GalA activity and protein levels are consistent with the appearance of an immune response against human α-GalA (human protein administered to the animals), as confirmed by the presence of anti-human α-GalA antibodies. Despite the decreased levels of human α-GalA, some animals maintained high levels of human α-GalA (activity and protein). In one high dose animal (6.0E+13 IS), a level of 193 nmol / hr / mL was measured on day 56, while levels in vehicle-treated animals were not detectable (<10 nmol / hr / mL). The transient nature of this response in some animals was highly likely related to the expected immune response against the human α-GalA enzyme (human protein administered to the animals).

[0265] Furthermore, samples were evaluated by qPCR method in NHP tests for vector excretion analysis. Low levels of hGLA vector were measured in saliva, urine and feces of some variant #4-treated animals up to day 4 (urine) or day 14 (saliva, feces). At day 60, hGLA vector levels were not detected in these biological fluids.

[0266] Example 6 hGLA and corresponding mRNA levels in NHP liver Western blot analysis of hGLA and corresponding mRNA levels in NHP liver samples from individual animals was performed on day 60 after treatment with variant #4 construct or formulation buffer at doses of 6.0E+12 vg / kg, 1.0E+13 vg / kg, 3.0E+13 vg / kg, 6.0E+13 vg / kg, 6.0E+13 vg / kg without immunosuppressant. As shown in Figure 14, the hGLA protein levels increased with construct dose and the protein levels were correlated with the mRNA levels in most samples.

[0267] Example 7 Evaluation of the Safety, Tolerability, and Pharmacodynamics of Variant #21 Expression Construct in Humans A study is conducted to evaluate the safety and tolerability of the variant #21 expression construct in humans. Additionally, the pharmacodynamics of α-GalA and the presence of its substrate in plasma, urine, and tissues are measured over time. The effects of the variant #21 expression construct on ERT, renal function, immune response, and viral vector DNA excretion with respect to ERT administration to the subjects can also be evaluated over time.

[0268] Overall, the variant #21 and variant #4 expression constructs were well tolerated in a mouse model of Fabry disease (GLAKO), wild-type (C56BL / 6) mice, and cynomolgus monkey NHPs. In GLAKO mice, there were no adverse findings associated with a single IV administration of the variant #4 expression construct up to 5.0E+13 vg / kg, the highest dose level tested. In C57BL / 6 mice, preliminary analysis indicated that the variant #21 expression construct was well tolerated up to 1.5E+14 vg / kg, the highest dose tested. In NHPs, findings associated with the variant #21 expression construct were limited to animals that did not receive immunosuppressive treatment (6.0E+13 vg / kg). These findings consisted of an increase in lymphoid cell types in lymphoid tissues and spleens, potentially consistent with an immune response associated with hGLA and / or rAAV2 / 6 administration. In these studies, the no observed adverse effect level (NOAEL) was 6.0E+13 vg / kg, the highest dose level tested, with or without an immunosuppressive regimen.

[0269] This study uses a recombinant (e.g., rAAV2 / 6) vector construct encoding a cDNA for human α-Gal A. The vector construct encodes a liver-specific promoter, and rAAV2 / 6 exhibits liver tropism, offering the potential for long-term, stable hepatic production of α-Gal A in Fabry disease subjects after a single administration. Various AAV serotypes, including AAV2, 5, 6, and 8, can be used. The rAAV2 / 6 serotype was selected for use in this and the previous examples based on previous NHP data showing that AAV2 / 6 was primarily liver-tropic and had a similar biodistribution to AAV2 / 8, and that AAV2 / 6 and AAV2 / 8 vectors achieved similar levels of circulating FIX transgene expression. Preliminary clinical safety data were collected from 13 subjects who received the investigational product in three research trials, suggesting that infusions using this AAV2 / 6 serotype were well tolerated (data not shown).

[0270] Testing in a Fabry disease mouse model administered rAAV2 / 6 encoding hGLA cDNA by IV shows therapeutic levels of α-GalA production (greater than 300-fold wild type). A single treatment with the expression vector minimizes the occurrence of infusion-related reactions. Therapeutic levels of α-GalA production in humans can enable the reduction, and in some cases, the removal of the Fabry disease substrates Gb3 and lyso-Gb3, and because of the constant production of the enzyme rather than the peaks and troughs seen with ERT, there may also be a reduced risk of antibody generation against the produced enzyme. The variant #21 expression construct was designed to result in stable long-term α-GalA production at therapeutic levels in subjects with Fabry disease. Constant production of α-GalA in humans may also enable the reduction and removal of the Fabry disease substrates Gb3 and lyso-Gb3.

[0271] Test evaluation The evaluation may include the incidence of treatment-emergent adverse events (TEAEs), routine hematology, chemistry, and liver function, vital signs, ECG and ECHO, a series of alpha-fetoprotein (AFP) tests, and liver MRI (or equivalent imaging) to monitor any liver tumor formation. Furthermore, changes from baseline can be evaluated at specific time points over 1 year as follows: alpha-GalA activity in plasma; Gb3 levels in plasma; Lyso-Gb3 levels in plasma; frequency of FABRAZYME® (or equivalent ERT) infusion; estimated glomerular filtration rate (eGFR) calculated by serum creatinine levels; left ventricular volume measured by cardiac magnetic resonance imaging (MRI), total protein and albumin / creatinine ratio in urine; alpha-GalA and Gb3 levels measured in tissue; substrate levels measured in tissue and urine; urinary biomarkers of renal function; neuropathic pain evaluated by the Brief Pain Inventory (BPI), frequency of analgesic use; gastrointestinal (GI) symptoms evaluated by the GI Symptom Assessment Scale; Mainz Severity Score Index (MSSI); quality of life (QOL) patient-reported outcomes evaluated by the SF-36 questionnaire; immune responses to rAAV2 / 6 and alpha-GalA; and rAAV vector clearance measured by the levels of vector genome in blood, plasma, saliva, urine, feces, and semen.

[0272] Subject Selection and Exclusion Criteria The test subjects can include male subjects aged 18 years or older with classical Fabry disease. Male subjects with classical Fabry disease must be recruited such that measurement of enzyme levels produced by the cDNA-introduced gene is not hindered by any residual enzyme levels.

[0273] More specifically, the subject selection criteria may include: (1) subjects who have received a documented diagnosis of classical Fabry disease defined by <5% α-GalA activity in either plasma or leukocytes and one or more of the following characteristic features of classical Fabry disease symptoms: i) whorled corneal dystrophy, ii) acroparesthesia, iii) anhidrosis, iv) angiokeratoma (if clustered periumbilical angiokeratomas are documented in the document, this symptom alone is sufficient as it is a characteristic sign of classical Fabry disease); (2) subjects during ERT (14-day [±1 day] dosing schedule); or subjects during ERT with GalA activity >5%; or subjects naive to ERT; or subjects similar to naive to ERT who have not received ERT treatment in the 6 months prior to consent; (3) for subjects receiving ERT, ERT must be administered at a stable dose (defined as not having missed more than 4 doses of ERT in the 6 months prior to consent) and dosing schedule (14 days ±1 day for at least 3 months prior to enrollment); (4) subjects with mutations indicative of classical Fabry (i.e., listed in databases such as www.dbfgp.org); (5) subjects with trough α-GalA activity below the lower limit of the normal range of the assay; (6) male subjects approximately 18 years of age or older; (7) sexually mature subjects must be negative for AAV in at least 3 consecutive semen samples from the time of expression construct administration until after the application of the test procedure, and must agree to use condoms and refrain from sperm donation for at least 90 days after the application of the test procedure; and (8) a signed written informed consent from the subject.

[0274] For subjects without a documented α-GalA activity level in the diagnosis, blood samples must be taken to measure the α-GalA activity level (in plasma and / or white blood cells). For such subjects on ERT, this blood draw must be taken at least 13 days after the last ERT infusion (trough). If the subject's α-GalA activity level is >5% and the subject is on ERT, this enzyme activity level may also be due to the remaining α-GalA activity from the last ERT infusion. In this case, when the following three criteria are met, the diagnosis of classical Fabry disease can be supported.

[0275] a. Two or more of the following documented symptom characteristics of classical Fabry: whorled corneal dystrophy, acroparesthesia, anhidrosis, angiokeratoma. If a clustered periumbilical angiokeratoma is documented in the record, this symptom alone is sufficient as it is a characteristic sign of classical Fabry disease.

[0276] b. A mutation indicative of classical Fabry (i.e., listed in a database such as www.dbfgp.org).

[0277] c. The α-GalA activity at trough is below the lower limit of the normal range of the assay.

[0278] Fabry disease gene sequencing may be performed at the time of screening to confirm that the subject has a mutation in the GLA gene. An assay may be performed on a blood or saliva sample. If available, the results of gene sequencing obtained prior to the test may also be used.

[0279] Tests for HIV, HAV, HBV, HCV, and TB can be performed at the time of screening. Subjects with a diagnosis of HIV or evidence of active HAV, HBV, HCV, or TB infection may not be eligible to participate in this study.

[0280] To evaluate the subject's existing immune response to AAV6, the level of neutralizing antibodies against AAV6 can be measured at the time of screening. Subjects with elevated existing neutralizing antibodies against AAV6 may not be eligible to participate in this study. If dosing is not completed within 3 months of screening, the serum neutralization assay against AAV6 must be repeated.

[0281] If available, the results of the diagnostic α-GalA activity levels in plasma or white blood cells obtained prior to the study can also be used. For subjects without documented diagnostic α-GalA activity levels, blood samples must be collected to measure the α-GalA activity levels (in plasma and / or white blood cells). For such subjects on ERT, this blood draw must be taken at least 13 days after the last ERT infusion.

[0282] A chest X-ray (also known as a PA chest radiograph) may be obtained to evaluate the subject's overall health status and eligibility for the study. Chest X-rays taken within 6 months of enrollment in the study can be used to determine the subject's eligibility, unless medically indicated otherwise. A physical examination must be performed on each subject and must include at a minimum the following: general appearance, head, eyes, ears, nose, and throat (HEENT); as well as the cardiovascular, dermatologic, respiratory, GI, musculoskeletal, and nervous systems.

[0283] Exclusion criteria for subjects may include the following subjects: (1) known to be non-responsive to ERT as determined by the responsible investigator of the institutional clinical trial and the medical monitor (e.g., no decrease in substrate levels during documented ERT); (2) currently being treated with Migalastat (Galafold (trademark)) or having received previous treatment within 3 months of informed consent; (3) having a neutralizing antibody response to a known AAV (e.g., AAV6); (4) having a concurrent disease that is expected to interfere with the assessment of safety or efficacy during the observation period of the trial as determined by the responsible investigator of the institutional clinical trial or the medical monitor; (5) having an eGFR of ≤60 ml / min / 1.73 m2; (6) having a New York Heart Association class of III or higher; (7) having an active infection with hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV) (HCV-DNA negative), or human immunodeficiency virus (HIV) or an active infection with tuberculosis (TB) as determined by quantitative polymerase chain reaction (qPCR); (8) having a history of liver disease, such as secondary fatty liver, non-alcoholic steatohepatitis (NASH), and cirrhosis, cholangitis, biliary tract disease within 6 months of informed consent; excluding Gilbert's syndrome; abnormal circulating AFP; (9) for subjects receiving ERT, having a new or continuing hypersensitivity reaction to ERT treatment as indicated by a significant infusion reaction to ERT within 6 months prior to consent as determined by the responsible investigator of the institutional clinical trial and the medical monitor; (10) a marker of liver inflammation or an overt or covert cause of liver dysfunction confirmed by one or more of the following: (i) albumin ≤3.5 g / dL; (ii) total bilirubin > upper limit of normal (ULN) and direct bilirubin ≥0.5 mg / dL; (iii) alkaline phosphatase (ALP) > 2.0 × ULN; (iv) > 1.5×ULN alanine aminotransferase (ALT); (11) current or past 6-month history of use of systemic (IV or oral) immunomodulators or steroids (local treatments are permitted, e.g., asthma or eczema) (use of systemic steroids may be permitted after consultation with the medical monitor); (12) contraindicated for use of adrenal cortical steroid drugs for immunosuppression; (13) history of malignancy other than non-melanoma skin cancer; (14) history of alcohol or substance abuse; (15) participated in a previous investigational drug or medical device trial (excluding implantable loop recorders such as in the RaILRoAD trial) within the past 3 months prior to consent; (16) received prior treatment with a gene therapy product; (17) known allergy to the components of the ST-920 formulation; (18) any other reason that, in the judgment of the institutional trial responsible physician or medical monitor, would render the subject ineligible to participate in the trial.

[0284] Combined drug All drugs except potentially hepatotoxic drugs can be permitted. Hepatotoxic drugs such as diclofenac, amiodarone, chlorpromazine, fluconazole, isoniazid, rifampicin, valproic acid, high-dose acetaminophen (4 - 8 gm / day), as well as hepatotoxic herbal supplements such as Senecio / Tanukimame, bitter melon of tea, chaparral, Jinbuhuan, Ephedra (traditional Chinese medicine) should not be taken during the test period. For subjects receiving ERT, ERT must be administered at a stable dose (defined as not missing more than 4 doses of ERT during the past 6 months prior to consent) and dosing schedule (14 days ± 1 day, for at least 3 months prior to enrollment). Subjects must continue to receive ERT at a stable dose and dosing schedule (14 days ± 1 day) during the test as per the standard treatment unless they discontinue ERT.

[0285] Dose cohort The starting dose is 5.0E+12 vg / kg, and any dose increase to the next dose level is based on a review of data from the previous cohort and / or other clinical trials using in vivo rAAV2 / 6-based therapy, and, if necessary, on the recommendations of a Safety Monitoring Committee (SMC) that may include external subject matter experts, trial medical monitors, and the facility's principal investigator. As used herein, SMC members have appropriate medical and scientific expertise and oversee the safety of the trial. Further, depending on the observed enzyme activity levels and safety profiles of the subjects administered, the SMC may recommend an increase to an intermediate dose level of 3.0E+13 vg / kg, a three-fold increase from the dose in Cohort 2, instead of a five-fold increase to the 5.0E+13 vg / kg dose in Cohort 3. A dose of approximately 1.0E+14 vg / kg may also be considered. The three dose cohorts are shown in Table 4.

Table 4

[0286] Subjects 18 years of age or older who meet all inclusion / exclusion criteria are enrolled. At least two subjects are assigned to each of the three dose cohorts, and after SMC review, each cohort may be expanded to a total of 18 subjects by adding an additional four adult subjects. The expression vector can be administered by intravenous injection. To stagger the treatments within each cohort, each successive subject cannot be injected until at least about two weeks after the preceding subject was administered. A dose increase to the next dose level cannot occur until at least about four weeks after the last subject in the preceding cohort was administered and until the SMC has reviewed the safety data from the entire previous cohort.

[0287] Subjects who received ERT before trial registration should continue to receive ERT during the trial and must remain on the current dose and dosing schedule (14 days ± 1 day) according to the standard treatment unless ERT is discontinued. Baseline tests for enzyme and substrate levels in subjects on ERT are adjusted so that samples can be taken at two separate times in the morning at trough, defined as 14 days (+ / - 1 day) after the previous ERT injection. Since additional time points are taken in advance during the review period, there are three time points to evaluate the residual level of α-GalA at trough before gene therapy application. These three samples should be taken at trough, preferably at the same time of day (e.g., in the morning) to minimize non-specific factors that may potentially affect enzyme levels.

[0288] To minimize the possible immune response to the rAAV capsid protein, avoid loss of transgene expression in case of liver injury, and protect liver function, prednisone or an equivalent corticosteroid drug can be prophylactically started and administered starting approximately 2 days before the expression vector injection and can be gradually decreased over a period of up to approximately 20 weeks.

[0289] The expression vector can be injected using a syringe pump or an IV infusion pump (see Study Pharmacy Manual). The total volume will be determined by the subject's cohort assignment and baseline body weight (kg). While the subject is in a hospital or emergency treatment facility where they can stay for at least 24 hours of observation after completion of the expression vector injection, the expression vector can be administered at a controlled rate via an IV catheter while monitoring the subject's vital signs (body temperature, heart rate, respiratory rate, and blood pressure). The subject can be discharged when all vital signs are stable and any adverse event (AE) has resolved, or when the subject is considered stable according to the judgment of the principal investigator of the clinical trial.

[0290] After injection of the expression vector, test visits may be conducted on the 8th day; and at the 2nd, 4th, 6th, 8th, 12th, 16th, 20th, 24th, 28th, 32nd, 36th, 40th, 44th, 48th, and 52nd weeks. The test visits at the 28th, 32nd, 40th, 44th, and 48th weeks may be conducted remotely because they are evaluations that do not require assessment at a clinical facility. Evaluations regarding AEs and concomitant medications may also be conducted remotely over the phone.

[0291] To monitor AAV-related immunogenicity, while the subject is receiving prednisone or an equivalent corticosteroid drug, for the first approximately 20 weeks after expression vector injection, liver tests (AST, ALT, GGT, total and direct bilirubin, ALP, LDH, albumin, and total protein levels) may be performed twice a week and may be conducted remotely. Blood samples for liver tests may be collected, if possible, at intervals of 2 to 4 days, except for the first week when they can be collected at the visits on the 2nd and 8th days. Thereafter, liver tests can be performed once a week (weeks 21 - 24) for 4 weeks after discontinuation of immunosuppression, and then once a month (weeks 28 - 52) to coincide with the test visits.

[0292] Despite pretreatment with prednisone or an equivalent corticosteroid drug, if there are signs of elevated ALT, continue the dose of prednisone or an equivalent corticosteroid drug (prednisone 1 mg / kg [maximum 60 mg] or equivalent; oral or intravenous and / or increased individually separately), twice a week until the liver enzymes normalize, and then evaluate the liver enzymes according to the subsequent protocol.

[0293] For the first two subjects in each cohort, to stagger the treatment, each subsequent subject is not injected until the preceding subject has been observed for at least about 2 weeks. An increase in dose to the next dose level cannot be done until at least 4 weeks after the two subjects in the preceding cohort have been dosed and the SMC has reviewed the safety data from the two subjects in the cohort before that.

[0294] If any of the discontinuation rules are met, dosing and dose increases may be interrupted.

[0295] Treatment with an expression vector can also eliminate the need for ERT by using an rAAV vector encoding cDNA for human α-GalA to effect long-term liver-specific expression of α-GalA in Fabry disease subjects. Subjects who have discontinued ERT are closely monitored for any AEs, any changes in vital signs, any changes in safety laboratory evaluations, and levels of α-GalA and substrates compared to baseline. To allow sufficient time for transduction of target liver cells, discontinuation of ERT should be considered after a 4-week period. Subjects who have discontinued ERT are closely monitored for any clinical symptoms, including fatigue and neuropathic pain, any AEs, any changes in vital signs, any changes in safety laboratory evaluations, including liver function tests, and levels of α-GalA and substrates (Gb3 and Lyso-Gb3) compared to baseline. Discontinuation of ERT may be done at the discretion of the site principal investigator after consultation with the sponsor of the clinical trial and should be considered for subjects who consent and meet the following criteria. (1) It is more than 4 weeks after administration of ST-920. (2) Medically stable and able to tolerate temporary discontinuation of ERT as determined by the site principal investigator. (3) Agree to increased safety monitoring and additional laboratory tests until the follow-up visit for discontinuation of ERT. (4) There is no need to resume ERT after the follow-up visit for discontinuation of ERT. However, ERT can be resumed at any time based on the clinical situation or at the discretion of the site principal investigator.

[0296] If discontinuation of ERT has failed previously, it may be done at least 12 weeks after the previous attempt and may be repeated if the subject consents, and may be done at the discretion of the site principal investigator after consultation with the sponsor of the clinical trial.

[0297] The trial participation period is up to 76 weeks for each subject, divided into up to 8 weeks for screening, up to 12 weeks for baseline, and 52 weeks of follow-up after dosing. An increase of 9 to 12 months is planned. Subjects should be encouraged to participate in an additional separate long-term follow-up study for up to 4 years.

[0298] If any of the following criteria are met, the trial registration should be interrupted and an SMC may be convened to provide advice on appropriate procedures: (1) Any grade 3 or higher adverse event with at least a reasonable possibility of causality with the expression vector formulation; (2) A serious adverse event (SAE) with at least a reasonable possibility of causality with the expression vector formulation; (3) Death of a human subject; (4) Onset of a malignant tumor.

[0299] An overview of all AEs and by dose cohort that occurred as a result of the treatment can be presented. For each subject, the highest reported severity of each AE can be used in the overview by severity grade. Additionally, an overview of all SAEs and AEs related to the trial treatment can be presented. For other safety evaluations, an overview of the data at each time point can be presented. Changes from baseline values can be calculated for continuous parameters and presented by time point. A table of changes for selected parameters can also be created.

[0300] Plasma α-GalA activity must be measured to assess whether α-GalA is being produced and is active. Measurement of α-GalA levels may be performed on plasma, serum, whole blood, dried blood spots, white blood cells, or other blood components. Samples for subjects during ERT must be obtained at trough, defined as 14 (±1) days after the previous ERT dose. Additional samples may be obtained throughout the trial to further understand the pharmacokinetics of the enzyme and to ensure that samples obtained prior to ERT are at trough.

[0301] Gb3 is a type of sphingolipid that accumulates in blood vessels, tissues, and organs in Fabry disease due to a deficiency of α-GalA. Gb3 levels in plasma, urine, and other tissues may be measured throughout the study to assess the impact of treatment application and α-GalA levels. Samples regarding subjects during ERT must be obtained at trough, which is defined as 14 days (±1 day) after the previous ERT administration.

[0302] Lyso-Gb3 is a soluble form of the substrate Gb3. Lyso-Gb3 levels in plasma, urine, and other tissues may be measured throughout the study to assess the impact of treatment application and α-GalA levels. Samples regarding subjects during ERT must be obtained at trough, which is defined as 14 days (±1 day) after the previous ERT administration.

[0303] The actual values regarding α-GalA as well as Gb3 and lyso-Gb3 levels at each sample collection time point, and the summary of changes from baseline can be presented using descriptive statistics and plotted over time for each dose cohort. For subjects who have discontinued ERT, changes in the frequency and dose of ERT injections before and after discontinuation can be evaluated using the total annual dose and number of injections, and a summary can be presented. The duration of ERT discontinuation can also be analyzed. AAV clearance measured by the vector genome in various samples (plasma, saliva, urine, feces, and semen) can be plotted over time for each dose cohort.

[0304] As shown in Figure 1A, the rAAV vector contains the variant #21 hGLA expression cassette (3321 bp) that includes a liver-specific regulatory element driving the expression of the hGLA transgene. The hGLA transgene is under the control of the enhancer and liver control region of the human apolipoprotein E (ApoE) gene and the human α-1-antitrypsin (hAAT) promoter. The ApoE enhancer and hAAT promoter are specific to the intended target tissue, the liver, and are highly active in the liver but inactive in non-liver cells and tissue types, thus preventing hGLA expression and activity in non-target tissues. The modified chimeric intron (HBB-IghGLA transgene) contains the codon-optimized hGLA α-GalA enzyme.

[0305] Variant #21 contains a mutated form of the woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (WPREmut6). WPREmut6 is a 592 bp DNA sequence that includes the promoter region of the WHV X protein and the start codon of the X protein open reading frame with a point mutation in the putative promoter region to prevent X protein expression (mut6), followed by a truncated form of the X protein itself. The polyA sequence is a derivative of the bovine growth hormone polyadenylation signal. The addition of the WPREmut6 element resulted in an increase in α-GalA protein production. Indeed, a greater potency was confirmed in the variant #21 expression construct compared to the variant #4 expression construct (without the WPREmut6 element).

[0306] The variant #21 expression construct is formulated in phosphate-buffered saline (PBS) containing CaCl2, MgCl2, NaCl, sucrose, and Kolliphor (poloxamer) P188 at approximately 1.0E+13 vg / mL, filled into vials in volumes such as 2 mL or 5 mL or 10 mL, and can be stored at ≤ -65°C. The vials have an aluminum seal with a flip-top.

[0307] The expression construct rAAV vector may be packaged with capsid serotype AAV2 / 6 using a Sf9 insect cell / recombinant baculovirus (Sf9 / rBV) expression system. Alternatively, the expression construct rAAV vector may be packaged with capsid serotype AAV2 / 6 using a mammalian expression system, such as HEK293.

[0308] Studies in Fabry disease mouse models, wild-type mice, and cynomolgus macaque NHPs demonstrate the feasibility of achieving safe production of persistently, potentially effective levels of α-GalA following treatment with the variant #21 expression vector.

[0309] No adverse effects were observed at the given maximum dose levels of up to 1.5E+14 vg / kg in mice and up to 6.0E+13 vg / kg in NHPs, respectively. Thus, the clinical starting dose of 5.0E+12 vg / kg is supported by a 30-fold safety margin in mice and a 12-fold safety margin in NHPs.

[0310] Moderate levels of α-GalA are expected at a dose of 5.0E+12 vg / kg in human subjects based on the significant pharmacodynamic response observed in Fabry disease mice given 2.0E+12 vg / kg.

[0311] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entirety.

[0312] Although the disclosure has been described in some detail by way of figures and examples for clarity of understanding, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the disclosure. Accordingly, the foregoing description and examples should not be construed as limiting. In one embodiment, for example, the following items are provided. (Item 1) A method for expressing at least one α-galactosidase A (α-GalA) protein in a cell, comprising administering to the cell an expression construct comprising a mutant WPRE sequence, optionally a mut6 mutant WPRE sequence, and a GLA transgene encoding at least one α-GalA protein such that the α-GalA protein is expressed in the cell. (Item 2) The method according to Item 1, wherein the expression construct comprises a wild-type GLA sequence or a codon-optimized GLA sequence. (Item 3) The expression construct further comprises one or more of a sequence encoding an enhancer, a promoter, an intron, a signal peptide, and / or a polyadenylation signal, and the mutant WPRE sequence, optionally the mut6 mutant WPRE sequence, and the GLA transgene encoding at least one α-GalA protein are located between the signal peptide and the sequence encoding the polyadenylation signal. The method according to Item 1 or 2. (Item 4) The method according to Item 3, wherein the expression construct comprises the sequence of SEQ ID NO: 9. (Item 5) The method according to any one of Items 1 to 4, wherein the cell is from a subject with Fabry disease. (Item 6) The method according to any one of Items 1 to 5, wherein the cell is from a male subject. (Item 7) The method according to any one of Items 1 to 6, wherein the expression construct in a pharmaceutically acceptable carrier is administered. (Item 8) The pharmaceutically acceptable carrier comprises phosphate-buffered saline containing CaCl 2 , MgCl 2 , NaCl, sucrose, and Kolliphor (poloxamer) P188. The method according to Item 7. (Item 9) The expression construct sequence comprises the sequence shown in Table 1, and the expression construct is delivered to the cell by an AAV viral vector. The method according to any one of Items 1 to 8. (Item 10) The method according to item 9, wherein the AAV viral vector serotype is AAV2 / 6. (Item 11) The method according to any one of items 5 to 10, wherein the expression construct is administered to the subject at a dose between about 5.0E+12 and 1.0E+14 vector genomes (vg / kg) per kilogram. (Item 12) The method according to any one of items 5 to 11, wherein the expression construct is administered to the liver of the subject. (Item 13) The method according to any one of items 5 to 12, wherein the expression vector is administered to the subject by intravenous injection. (Item 14) The method according to any one of items 5 to 13, wherein the expression construct is administered to the subject in a single dose. (Item 15) The method according to any one of items 5 to 14, wherein the subject is administered an immunosuppressant before and / or during administration of the expression construct. (Item 16) The method according to item 15, wherein the immunosuppressant comprises prednisone. (Item 17) The method according to any one of items 1 to 16, wherein the expression of the at least one α-galactosidase A (α-GalA) protein is maintained for at least 3 months, at least 9 months, or at least 12 months. (Item 18) The method according to any one of items 5 to 17, wherein the α-GalA protein expressed from the transgene reduces the amount of sphingolipids in the subject to between at least about one-half and about one-ninth compared to an untreated subject. (Item 19) The method according to any one of items 5 to 18, wherein the α-GalA protein expressed from the transgene reduces the amount of sphingolipids in the subject by at least about 80%. (Item 20) The method according to any one of items 5 to 19, wherein the α-GalA protein expressed from the transgene reduces the amount of sphingolipids in one or more of the plasma, liver, heart, kidney, or spleen of the subject. (Item 21) The method according to any one of items 5 to 20, wherein the expression construct produced in the HEK293 cell line results in a GLA level that is about 21-fold higher in the subject compared to the GLA level in a subject administered the expression construct produced in the Sf9 cell line. (Item 22) The method according to any one of items 5 to 21, wherein the α-GalA protein activity in the subject is about 100 to 1,500 times higher than that of a physiologically normal / wild type. (Item 23) The method according to any one of items 5 to 22, wherein the α-GalA protein expressed from the transgene is active in the kidneys, liver, and heart of the subject. (Item 24) The method according to any one of items 1 to 23, wherein the GLA transgene is retained extrachromosomally and not integrated into the genome of the cell. (Item 25) The method according to any one of items 1 to 23, further comprising administering to the liver cells of the subject one or more nucleases that cleave the albumin gene such that the transgene is integrated into the endogenous albumin gene and expressed therefrom. (Item 26) A genetically modified cell containing an exogenous GLA transgene, produced by the method according to any one of items 1 to 4. (Item 27) The genetically modified cell according to item 26, wherein the cell is a stem cell or a progenitor cell. (Item 28) The genetically modified cell according to item 27, wherein the cell is a liver cell or a muscle cell. (Item 29) The genetically modified cell according to any one of items 26 to 28, wherein the GLA transgene is retained extrachromosomally and not integrated into the genome of the cell. (Item 30) The genetically modified cell according to any one of items 26 to 28, wherein the GLA transgene is integrated into the genome of the cell. (Item 31) A method for preventing, inhibiting, or treating Fabry disease or one or more symptoms associated with Fabry disease, comprising administering an expression construct to a subject in need thereof, wherein the expression construct comprises a mutant WPRE sequence, optionally a mut6 mutant WPRE sequence, and a GLA transgene encoding at least one α-GalA protein. (Item 32) The method according to item 31, wherein the symptoms include one or more of elevated Gb3 levels above normal, elevated lyso-Gb3 levels above normal, kidney disease, heart disease, acral paresthesia, angiokeratoma corporis diffusum, gastrointestinal pain, corneal and lens opacities, or cerebrovascular disease. (Item 33) The method according to item 31 or 32, wherein the subject is male and the subject has less than about 5% α-GalA enzyme activity. (Item 34) The method according to item 31, wherein the expression construct comprises a wild-type GLA sequence or a codon-optimized GLA sequence. (Item 35) The expression construct further comprises one or more of an enhancer, a promoter, an intron, a signal peptide and / or an array encoding a polyadenylation signal, and the mutant WPRE array, optionally the mut6 mutant WPRE array, and the GLA transgene encoding at least one α-GalA protein are located between the signal peptide and the array encoding the polyadenylation signal, the method according to item 31, 33, or 34. (Item 36) The method according to item 31, wherein the expression construct in a pharmaceutically acceptable carrier is administered. (Item 37) The pharmaceutically acceptable carrier is 2 CaCl 2 The method according to item 36, comprising phosphate buffered saline containing MgCl, NaCl, sucrose and Kolliphor (poloxamer) P188. (Item 38) The expression construct sequence comprises the sequence shown in Table 1, and the expression construct is delivered to the cells of the subject by an AAV viral vector, the method according to item 31. (Item 39) The AAV viral vector serotype is AAV2 / 6, the method according to item 38. (Item 40) The expression construct is administered to the subject at a dose between about 5.0E+12 and 1.0E+14 vector genomes (vg / kg) per kilogram, the method according to any one of items 31 to 39. (Item 41) The expression construct is administered to the liver of the subject, the method according to any one of items 31 to 40. (Item 42) The expression vector is administered to the subject by intravenous injection, the method according to any one of items 31 to 41. (Item 43) The expression construct is administered to the subject in a single dose, the method according to any one of items 31 to 42. (Item 44) The subject is administered an immunosuppressant before and / or during administration of the expression construct, the method according to any one of items 31 to 43. (Item 45) The immunosuppressant comprises prednisone, the method according to item 44. (Item 46) The expression of the at least one α-galactosidase A (α-GalA) protein is maintained for at least 3 months, at least 9 months, or at least 12 months, the method according to any one of items 31 to 45. (Item 47) The method according to any one of items 31 to 46, wherein the α-GalA protein expressed from the introduced gene reduces the amount of sphingolipids in the subject to between at least about one-third and about one-ninth compared to an untreated subject. (Item 48) The method according to any one of items 31 to 46, wherein the α-GalA protein expressed from the introduced gene reduces the amount of sphingolipids in the subject by at least about 80%. (Item 49) The method according to any one of items 31 to 48, wherein the α-GalA protein expressed from the introduced gene reduces the amount of sphingolipids in one or more of the subject's plasma, liver, heart, kidney, or spleen. (Item 50) The method according to any one of items 31 to 49, wherein the expression construct is produced in a HEK293 cell line, and the GLA level in the subject is 21-fold higher compared to the GLA level in a subject administered the expression construct produced in an Sf9 cell line. (Item 51) The method according to any one of items 31 to 50, wherein the α-GalA protein activity in the subject is between about 100-fold and 1,500-fold higher than physiological normal. (Item 52) The method according to any one of items 31 to 51, wherein the α-GalA protein expressed from the introduced gene is active in the subject's kidney, liver, and heart. (Item 53) The method according to any one of items 31 to 52, wherein the GLA-introduced gene is retained extrachromosomally and not integrated into the genome of the subject's cells. (Item 54) The method according to any one of items 31 to 52, further comprising administering to the subject's liver cells one or more nucleases that cleave the albumin gene such that the introduced gene is integrated into the endogenous albumin gene and expressed therefrom. (Item 55) A composition comprising an expression construct, wherein the expression construct comprises a mutant WPRE sequence, optionally a mut6 mutant WPRE sequence, and a GLA-introduced gene encoding at least one α-GalA protein for the treatment of Fabry disease, said composition. (Item 56) The composition according to item 55, further comprising a pharmaceutically acceptable carrier. (Item 57) The pharmaceutically acceptable carrier is CaCl 2 、MgCl 2 The composition according to item 56, comprising NaCl, sucrose, and Kolliphor (poloxamer) P188. (Item 58) The composition according to item 55, wherein the expression construct comprises a wild-type GLA sequence or a codon-optimized GLA sequence. (Item 59) The composition according to any one of items 55 to 58, wherein the expression construct further comprises one or more of a sequence encoding an enhancer, a promoter, an intron, a signal peptide, and / or a polyadenylation signal, and the mutant WPRE sequence, optionally the mut6 mutant WPRE sequence, and the GLA transgene encoding at least one α-GalA protein are located between the signal peptide and the sequence encoding the polyadenylation signal. (Item 60) The composition according to any one of items 55 to 59, wherein the expression construct sequence comprises the sequence shown in Table 1, and the expression construct is delivered to cells by an AAV viral vector. (Item 61) The composition according to any one of items 55 to 60, wherein the AAV viral vector serotype is AAV2 / 6. (Item 62) The composition according to item 60 or 61, wherein the expression construct comprises between about 5.0E+12 and 1.0E+14 vector genomes (vg / kg) per kilogram of the subject. (Item 63) The composition according to item 59, wherein the expression construct comprises the sequence of SEQ ID NO: 9. (Item 64) A method for producing an α-GalA protein for the treatment of Fabry disease, comprising expressing the α-GalA protein in isolated cells by the method according to any one of items 1 to 4, and isolating the α-GalA protein produced by the cells. (Item 65) A delivery vector comprising a mutant WPRE sequence, optionally a mut6 WPRE sequence, and a GLA transgene for use in the method according to item 1. (Item 66) The vector according to item 65, wherein the delivery vector is a viral vector or a lipid nanoparticle (LNP). (Item 67) The vector according to item 66, wherein the viral vector comprises AAV2 / 6, and the viral vector delivers the expression construct to at least 50%, at least 60%, at least 70%, or at least 80% of the cells. (Item 68) Use of an expression construct, AAV vector and / or genetically modified cell according to any one of the preceding items for the treatment of Fabry disease. (Item 69) (Item 70) (Item 70) The composition according to item 59, wherein the enhancer comprises SEQ ID NO: 2, the promoter comprises SEQ ID NO: 3, the intron comprises SEQ ID NO: 4, the GLA transgene comprises SEQ ID NO: 5, the mutant WPRE sequence comprises SEQ ID NO: 6, and the polyadenylation signal comprises SEQ ID NO: 7.

Claims

**Claim 1** An expression construct comprising a mutant woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (WPRE) sequence and an α-galactosidase A (α-GalA) transgene encoding at least one α-GalA protein, wherein the mutant WPRE sequence is a mut6 mutant WPRE sequence comprising a mut6 mutation, and the mut6 mutation is created in the J04514 WPRE element, the expression construct. **Claim 2** The expression construct according to claim 1, wherein the expression construct comprises a wild-type α-GalA sequence or a codon-optimized α-GalA sequence. **Claim 3** The expression construct according to claim 1 or 2, further comprising one or more of the following: an enhancer, a promoter, an intron, a sequence encoding a signal peptide, and / or a polyadenylation (polyA) signal sequence, wherein the mutant WPRE sequence and the α-GalA transgene encoding the at least one α-GalA protein are located between the sequence encoding the signal peptide and the polyA signal sequence. **Claim 4** The expression construct according to any one of claims 1 to 3, comprising an apolipoprotein E (APOE) enhancer operably linked to an α-1-antitrypsin (hAAT) promoter, a human hemoglobin β (HBB)-IgG intron, a sequence encoding an α-GalA signal peptide, the mutant WPRE sequence, and a bovine growth hormone polyA signal sequence. **Claim 5** The expression construct according to claim 3, comprising one or more of the following: the enhancer comprising the nucleotide sequence set forth in SEQ ID NO: 2, the promoter comprising the nucleotide sequence set forth in SEQ ID NO: 3, the intron comprising the nucleotide sequence set forth in SEQ ID NO: 4, the α-GalA transgene comprising the nucleotide sequence set forth in SEQ ID NO: 5, the mutant WPRE sequence comprising the nucleotide sequence set forth in SEQ ID NO: 6, and / or the polyA signal sequence comprising the nucleotide sequence set forth in SEQ ID NO:

7. **Claim 6** The expression construct according to any one of claims 1 to 5, comprising the nucleotide sequence set forth in SEQ ID NO:

9. **Claim 7** The expression construct according to any one of claims 1 to 6, wherein the expression construct is an adeno-associated virus (AAV) expression construct.

8. The expression construct according to claim 7, wherein the AAV expression construct has an AAV2 / 6 serotype.

9. An expression construct comprising an enhancer containing the nucleotide sequence set forth in SEQ ID NO: 2, a promoter containing the nucleotide sequence set forth in SEQ ID NO: 3, an intron containing the nucleotide sequence set forth in SEQ ID NO: 4, an α-GalA transgene containing the nucleotide sequence set forth in SEQ ID NO: 5, a mutant WPRE sequence containing the nucleotide sequence set forth in SEQ ID NO: 6, and a polyA signal sequence containing the nucleotide sequence set forth in SEQ ID NO:

7.

10. A composition comprising the expression construct according to any one of claims 1 to 9.

11. The composition according to claim 10, further comprising a pharmaceutically acceptable carrier.

12. The pharmaceutically acceptable carrier is CaCl ２ , MgCl ２ , NaCl, sucrose, and Kolliphor (poloxamer) P188, the composition according to claim 11.

13. An in vitro method for expressing at least one α-GalA protein in a cell, the method comprising administering to the cell the expression construct according to any one of claims 1 to 9 such that the α-GalA protein is expressed in the cell.

14. A genetically modified cell comprising the expression construct according to any one of claims 1 to 9.

15. The genetically modified cell according to claim 14, wherein the cell is produced by the method according to claim 13.

16. The genetically modified cell according to claim 14 or 15, wherein (a) the cell is a stem cell or a progenitor cell, or (b) the cell is a liver cell or a muscle cell.

17. For the manufacture of a medicament for use in preventing, inhibiting, or treating Fabry disease or one or more symptoms associated with Fabry disease, the expression construct according to any one of claims 1 to 9, the composition according to any one of claims 10 to 12, or the cell according to any one of claims 14 to 16.

18. The use according to claim 17, wherein the symptoms include one or more of elevated levels of Gb3 above normal, elevated levels of lyso-Gb3 above normal, kidney disease, heart disease, acroparesthesia, angiokeratoma corporis diffusum, gastrointestinal pain, corneal and lens opacities, or cerebrovascular disease.

19. The expression construct is administered to a subject by intravenous injection for the use according to claim 17 or 18.

20. The expression construct is administered to the subject in a single dose for the use according to claim 19.

21. The subject is administered an immunosuppressant before and / or during administration of the expression construct for the use according to claim 19 or 20.

22. The immunosuppressant includes prednisone for the use according to claim 21.

23. The expression of the at least one α-GalA protein is maintained for at least three months for the use according to any one of claims 19 to 22.

24. The α-GalA protein expressed from the transgene reduces the amount of sphingolipids in the subject by at least one half for the use according to any one of claims 19 to 23.

25. The α-GalA protein expressed from the transgene reduces the amount of sphingolipids in the subject by at least 80% for the use according to any one of claims 19 to 24.

26. The α-GalA protein expressed from the transgene reduces the amount of sphingolipids in one or more of the subject's plasma, liver, heart, kidney, or spleen for the use according to any one of claims 19 to 25.

27. The expression construct produced in the HEK293 cell line results in an α-GalA protein level that is 21-fold higher in the subject compared to the α-GalA protein level in a subject administered the expression construct produced in the Sf9 cell line for the use according to any one of claims 19 to 26.

28. The α-GalA protein activity in the subject is 100-fold to 1,500-fold higher than physiological normal / wild type for the use according to any one of claims 19 to 27.

29. The α-GalA protein expressed from the transgene is active in the subject's kidney, liver, and / or heart for the use according to any one of claims 19 to 28.

30. An in vitro method for generating α-GalA protein for the treatment of Fabry disease, said method comprising expressing said α-GalA protein in isolated cells by the method according to claim 13, and isolating said α-GalA protein produced by said cells.

31. A composition comprising an expression construct according to any one of claims 1 to 9, a composition according to any one of claims 10 to 12, or a composition comprising a cell according to any one of claims 14 to 16 for use in preventing, inhibiting or treating Fabry disease or one or more symptoms associated with Fabry disease.

32. A method for generating an expression construct, said expression construct comprising a transgene encoding at least one α-GalA protein according to any one of claims 1 to 9.

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