Frataxin gene therapy

Abstract

The present invention provides compositions and methods for stimulating the expression of the human frataxin gene. The compositions described herein can be used, for example, to produce genes and RNA equivalents optimized for expression in specific cell types. Compositions and methods can be used to treat Friedreich's ataxia. The compositions and methods of the present disclosure can be used to administer a plasmid (e.g., a viral vector) containing the human frataxin gene (hFXN) or its RNA equivalent to a patient (e.g., a mammalian patient, such as a human patient) with Friedreich's ataxia.

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy created on September 9, 2022 is named 51037-060WO4_Sequence_Listing_9_8_22_ST26.XML and is 23,124 bytes in size.

[0002] The present invention relates to the field of nucleic acid biotechnology, providing compositions and methods for, for example, the production of codon-optimized nucleic acids for enhancing gene expression in target cells or tissues. [Background technology]

[0003] Friedreich's ataxia is the most common autosomal recessive movement disorder, with 6,000 Americans diagnosed with the disease and a prevalence of approximately 15,000-20,000 patients diagnosed worldwide. The clinical symptoms of Friedreich's ataxia are the result of frataxin protein deficiency, with 95% of cases resulting from mutations that cause a repeat expansion of GAA. Frataxin is a ubiquitously expressed mitochondrial iron-binding protein that is important for the function of the heart, cerebellum, and spinal cord, including dorsal root ganglion neurons. The phenotype of Friedreich's ataxia includes degeneration and demyelination of spinocerebellar dorsal root ganglion neurons, causing progressive weakness, spasticity, and sensory loss, with most Friedreich's patients being wheelchair bound by the age of 20 (Dun and Brice, Curr Opin Neurol (2000) 13:407-413). Additionally, most patients with Friedreich's ataxia develop cardiac abnormalities (e.g., left ventricular hypertrophy) leading to death from heart failure in 60% of patients by approximately age 30-40 years. The development of gene therapy for the treatment of Friedreich's ataxia has been hampered by the difficulties associated with achieving expression of therapeutically effective amounts of frataxin in affected tissues, and currently there are no approved disease-modifying treatments. There remains a need for a set of compositions and methods that address these impediments. Summary of the Invention

[0004] The present disclosure provides compositions and methods that can be used to treat Friedreich's ataxia. The compositions and methods of the present disclosure may be used to administer to a patient (e.g., a mammalian patient, such as a human patient) with Friedreich's ataxia a plasmid (e.g., a viral vector) containing the human frataxin gene (hFXN) or its RNA equivalent.

[0005] In one aspect, the disclosure provides a DNA polynucleotide encoding hFXN, or an RNA equivalent thereof, wherein the polynucleotide has a nucleic acid sequence that is at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:1.

[0006] In another aspect, the present disclosure provides a vector comprising the composition of the above aspect, wherein the vector is a plasmid, a DNA vector, an RNA vector, a virion, or a viral vector.For example, in some embodiments, the vector is a viral vector.In some embodiments, the viral vector is an adeno-associated virus (AAV).

[0007] In some embodiments of the above aspects, the polynucleotide is operably linked to a muscle-specific promoter, optionally, the promoter is positioned 5' to the polynucleotide.

[0008] In some embodiments, the vector further comprises a polyadenylation site (pA), optionally, the pA is positioned 3' to the polynucleotide.

[0009] In some embodiments, the vector further comprises an intron, optionally positioned 3' to the promoter and 5' to the polynucleotide.

[0010] In some embodiments, the AAV further comprises two inverted terminal repeats (ITRs), the two ITRs comprising a first ITR (ITR1) and a second ITR (ITR2), where ITR1 is positioned 5' to the polynucleotide and ITR2 is positioned 3' to the polynucleotide to form a cassette comprising the structure ITR1-hFXN-ITR2.

[0011] In some embodiments, the length of the nucleic acid between ITR1 and ITR2 is about 3.7 Kb to about 4.3 Kb (e.g., about 3.8 Kb to about 4.2 Kb or about 3.9 Kb to about 4.1 Kb). For example, in some embodiments, the length of the nucleic acid between ITR1 and ITR2 is about 3.8 Kb to about 4.2 Kb. In some embodiments, the length of the nucleic acid between ITR1 and ITR2 is about 3.9 Kb to about 4.1 Kb. In some embodiments, the length of the nucleic acid between ITR1 and ITR2 is about 4.0 Kb.

[0012] In another aspect, the present disclosure provides a plasmid encoding the viral vector of the above aspect.

[0013] In another aspect, the disclosure provides a nucleic acid molecule comprising ITR1, hFXN or its RNA equivalent, and ITR2, the components being operably linked to each other in the 5'-3' direction as ITR1-hFXN-ITR2, and the length of the nucleic acid between ITR1 and ITR2 is about 3.7 Kb to about 4.3 Kb (e.g., about 3.8 Kb to about 4.2 Kb or about 3.9 Kb to about 4.1 Kb). For example, in some embodiments, the length of the nucleic acid between ITR1 and ITR2 is about 3.8 Kb to about 4.2 Kb. In some embodiments, the length of the nucleic acid between ITR1 and ITR2 is about 3.9 Kb to about 4.1 Kb. In some embodiments, the length of the nucleic acid between ITR1 and ITR2 is about 4.0 Kb.

[0014] In some embodiments of any of the aforementioned aspects, the length of the nucleic acid between and including ITR1 and ITR2 is about 3.9 Kb to about 4.7 Kb (e.g., about 4.0 Kb to about 4.6 Kb, about 4.1 Kb to about 4.5 Kb, about 4.2 Kb to about 4.4 Kb, or about 4.3 Kb). For example, in some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.0 Kb to about 4.6 Kb. In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.1 Kb to about 4.5 Kb. In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.2 Kb to about 4.4 Kb. In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.3 Kb.

[0015] In some embodiments, the nucleic acid molecule comprises a eukaryotic promoter (P Euk ), and the components further comprise ITR1-P Euk - operably linked as hFXN-ITR2.

[0016] In some embodiments, P Euk is a muscle-specific promoter.

[0017] In some embodiments of any of the above aspects, the muscle-specific promoter is a phosphoglycerate kinase (PGK) promoter, a desmin promoter, a muscle creatine kinase promoter, a myosin light chain promoter, a myosin heavy chain promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, an actin alpha promoter, an actin beta promoter, an actin gamma promoter, or a promoter within eye intron 1 paired like homeodomain 3, a cytomegalovirus promoter, or a chicken-β-actin promoter. For example, in some embodiments, the muscle-specific promoter is a PGK promoter.

[0018] In some embodiments of any of the foregoing aspects, the PGK promoter has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid of sequence SEQ ID NO:2. For example, in some embodiments, the PGK promoter has a nucleic acid sequence that is at least 90% identical to the nucleic acid of sequence SEQ ID NO:2. In some embodiments, the PGK promoter has a nucleic acid sequence that is at least 95% identical to the nucleic acid of sequence SEQ ID NO:2, and optionally, the PGK promoter has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid of sequence SEQ ID NO:2. In some embodiments, the PGK promoter has a nucleic acid of SEQ ID NO:2.

[0019] In some embodiments, the nucleic acid molecule further comprises pA, and the components are arranged in a 5'-3' direction relative to each other as ITR1-P Euk -hFXN-pA-ITR2 is operably linked as

[0020] In some embodiments of any of the above aspects, the pA site comprises a Simian Virus 40 (SV40) late polyadenylation site, an SV40 early polyadenylation site, a human β-globin polyadenylation site, or a bovine growth hormone polyadenylation site. For example, in some embodiments, the pA site comprises an SV40 late polyadenylation site.

[0021] In some embodiments, the nucleic acid molecule further comprises an intron, the components being arranged in a 5'-3' direction relative to each other as ITR1-P Euk -intron-hFXN-pA-ITR2 is operably linked as

[0022] In some embodiments of any of the aforementioned aspects, the intron is an SV40 intron.

[0023] In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO:3. For example, in some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 90% identical to the amino acid sequence of SEQ ID NO:3, and optionally, hFXN, or its RNA equivalent, encodes a protein that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein having the amino acid sequence of SEQ ID NO:3.

[0024] In some embodiments of any of the foregoing aspects, hFXN, or its RNA equivalent, has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:1. For example, in some embodiments, hFXN, or its RNA equivalent, has a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXN, or its RNA equivalent, has a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXN, or its RNA equivalent, has a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXN, or its RNA equivalent, has a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXN, or its RNA equivalent, has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXN, or its RNA equivalent, has a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXN, or its RNA equivalent, has a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXN, or its RNA equivalent, has a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXN, or its RNA equivalent, has a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXN, or its RNA equivalent, has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXN, or its RNA equivalent, has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXN, or its RNA equivalent, has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO:1.In some embodiments, hFXN, or its RNA equivalent, has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, hFXN, or its RNA equivalent, has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, hFXN, or its RNA equivalent, has the nucleic acid sequence of SEQ ID NO: 1.

[0025] In another aspect, the present disclosure provides a vector comprising any of the compositions or nucleic acid molecules of the above aspects, wherein the vector is a plasmid, a DNA vector, an RNA vector, a virion, or a viral vector. For example, in some embodiments, the vector is a viral vector.

[0026] In some embodiments of any of the above aspects, the viral vector is selected from the group consisting of AAV, adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus, and synthetic virus. For example, in some embodiments, the viral vector is AAV. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, or AAVrhl74 serotype. In some embodiments, the viral vector is pseudotyped AAV. In some embodiments, the pseudotyped AAV is AAV2 / 8 or AAV2 / 9, and optionally, the pseudotyped AAV is AAV2 / 8.

[0027] In some embodiments of any of the above aspects, ITR1 and / or ITR2 are parvoviral ITRs. For example, in some embodiments, the parvoviral ITR is an AAV ITR. In some embodiments, the AAV ITR is an AAV serotype 2 ITR.

[0028] In some embodiments of any of the aforementioned aspects, the AAV comprises a recombinant capsid protein.

[0029] In some embodiments of any of the aforementioned aspects, the vector has a nucleic acid sequence that is at least 85%, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO:4. For example, in some embodiments, the vector has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the vector has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:4, and optionally, the vector has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the vector has a nucleic acid sequence of SEQ ID NO:4.

[0030] In another aspect, the present disclosure provides a plasmid encoding the viral vector of any one of the preceding aspects.

[0031] In some embodiments of the above aspects, the plasmid further comprises one or more spacers (SS), where the one or more SS is located 5' to ITR1 and / or 3' to ITR2. For example, in some embodiments, the plasmid comprises two SSs, where the two SSs comprise a first spacer (SS1) and a second spacer (SS2), where SS1 is located 5' to ITR1 and SS2 is located 3' to ITR2.

[0032] In some embodiments, one or more SSs do not contain an open reading frame more than 100 amino acids in length.

[0033] In some embodiments, one or more SSs does not contain a prokaryotic transcription factor binding site.

[0034] In some embodiments of any of the above aspects, SS1 is about 1.0 Kb to about 5.0 Kb (e.g., 1.5 Kb to about 4.5 Kb, about 2.0 Kb to about 4.0 Kb, or about 3.0 Kb) in length. For example, in some embodiments, SS1 is about 2.0 Kb to about 5.0 Kb in length.

[0035] In some embodiments of any of the above aspects, SS2 is about 1.0 Kb to about 5.0 Kb (e.g., 1.5 Kb to about 4.5 Kb, about 2.0 Kb to about 4.0 Kb, or about 3.0 Kb) in length. For example, in some embodiments, SS2 is about 2.0 Kb to about 5.0 Kb in length.

[0036] In some embodiments, the plasmid further comprises a prokaryotic promoter operably linked to a selectable marker gene located 5' to one or more SSs located 5' to ITR1 or located 3' to one or more SSs located 3' to ITR2. For example, in some embodiments, the selectable marker gene is an antibiotic resistance gene.

[0037] In some embodiments, the plasmid further comprises a prokaryotic origin of replication positioned 5' to one or more SSs positioned 5' to ITR1 and / or positioned 3' to one or more SSs positioned 3' to ITR2.

[0038] In another aspect, the disclosure provides a pharmaceutical composition comprising a composition, nucleic acid molecule, vector, or plasmid of any of the preceding aspects and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0039] In another aspect, the disclosure provides a method of treating Friedreich's ataxia in a human patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a composition, nucleic acid molecule, vector, plasmid, or pharmaceutical composition of any of the preceding aspects.

[0040] In another aspect, the disclosure provides a method of increasing frataxin expression in a human patient diagnosed with Friedreich's ataxia, the method comprising administering to the patient a therapeutically effective amount of a composition, nucleic acid molecule, vector, plasmid, or pharmaceutical composition of any of the preceding aspects.

[0041] In some embodiments, the patient is between 3 and 17 years old (e.g., between 4 and 16 years old, between 5 and 15 years old, between 6 and 14 years old, between 7 and 13 years old, between 8 and 12 years old, between 9 and 11 years old, or between 10 years old).

[0042] In some embodiments, upon administration of a composition, nucleic acid molecule, vector, plasmid, or pharmaceutical composition of any of the above aspects to a patient, the patient exhibits a change in whole blood frataxin levels, and optionally, the patient exhibits a change in whole blood frataxin levels by about 12 weeks after administration.

[0043] In some embodiments, upon administration to a patient of a composition, nucleic acid molecule, vector, plasmid, or pharmaceutical composition of any of the above aspects, the patient exhibits a reduction in total Friedreich's Ataxia Rating Scale (FARS) score, and optionally, the patient exhibits a reduction in total FARS score by about 12 weeks after administration.

[0044] In another aspect, the disclosure provides a kit comprising a composition, nucleic acid molecule, vector, plasmid, or pharmaceutical composition of any of the preceding aspects and a package insert, the package insert instructing a user of the kit to administer the composition or vector to a human patient diagnosed with Friedreich's ataxia. [Brief description of the drawings]

[0045] [Figure 1]Schematic diagram showing the probability of nucleic acid variation at residue positions 1-270 of the gene encoding human frataxin (FXN) variant 1 (H.FXN.WT), respectively. Residue optimization was compared across databases and residue positions using Integrated DNA Technologies (IDT) and GENEWIZ (GeneWiz) codon optimization tools. While residues 1-270 are shown herein, codon optimization was performed across all residues of the human FXN variant 1 gene, including residues 1-633. The top sequence shows the most frequent nucleic acid at each residue position across the data set. Arrows indicate residues selected for modification in the codon-optimized human FXN variant 1 construct (abbreviated interchangeably as H.FXN.ATX.Co or hFXNco). For example, residue 12 indicates that the wild type cytosine (C) was changed to a guanine (G). [Diagram 2] 1 is a map of an exemplary pseudotyped adeno-associated virus (AAV) 2 / 8 (AAV2 / 8) viral vector for expression of codon-optimized human FXN variant 1 gene (hFXNco). From left to right, the shaded arrows represent a plasmid containing a nucleic acid molecule comprising, from 5' to 3', a first spacer, a first inverted terminal repeat (ITR1), a human phosphoglycerate kinase (hPGK) promoter, hFXNco, a Simian Virus 40 (SV40) late polyadenylation site (SV4 LpA), a second ITR (ITR2), a prokaryotic origin of replication (ori), and a kanamycin (kan) selection gene, with the length between ITR1 and ITR2 containing the payload (e.g., hPGK and hFXNco) being approximately 4.3 Kb in length. [Figure 3A] 1A and 1B are photographs and a graph, respectively, showing an anti-frataxin immunofluorescence expression-based potency assay.FIG. 1C are a set of photographs of cells derived from mouse skeletal muscle (C2C12) stained for frataxin after transduction with 1×107 (1e7) viral genomes (vg) / cell of an AAV2 / 8 viral vector for expression of hFXNco. [Figure 3B]3A and 3B are photographs and a graph, respectively, showing an anti-frataxin immunofluorescence expression-based potency assay, and a graph showing frataxin immunofluorescence as described in FIG. 3A as a function of multiplicity of infection (MOI) normalized to the intensity of Hoechst counterstaining. [Figure 4] FIG. 3 is an immunoblot showing expression of intermediate and mature isoforms of frataxin in C2C12 cells transduced with an exemplary AAV2 / 8 viral vector for expression of hFXNco at an amount of 1×106 (1e6) vg / cell or 1×107 (1e7) vg / cell, respectively, as described in FIGS. 1 and 2. [Diagram 5] FIG. 1 is a graph showing the probability of survival of FXN knockout (KO) mice over time following intravenous (iv) administration of exemplary AAV2 / 8 viral vectors for expression of hFXNco or mouse FXN variant 1 (mFXN) driven by the hPGK promoter at an amount of 3×10 (3e13) vg / kg or 1×10 (1e14) vg / kg compared to non-transduced controls (vehicle wild type (WT) and vehicle KO). [Figure 6]A-E show survival, body weight, and cardiac parameters of FXN KO mice after iv administration of exemplary AAV2 / 8 viral vectors for expression of hFXNco (hFXN) or mFXN driven by the hPGK promoter at doses of 3×10 vg / kg or 1×10 vg / kg compared to non-transduced controls (WT vehicle and FRDA vehicle). A shows the probability of survival of FXN KO mice over time. Survival data are presented as ages at which mice were euthanized if they showed more than 20% weight loss, signs of respiratory distress, unresponsiveness to meaningful stimuli, and / or poor overall body condition prior to scheduled biopsy. B shows the body weight of male and female FXN KO mice over time. C is a graph showing heart weight by left ventricular mass normalized to body weight in FXN KO mice before (6 weeks of age), after (9-10 weeks of age), and at the end of life (18-19 weeks of age). D is a graph showing ejection fraction in FXN KO mice before (6 weeks of age), after (9-10 weeks of age), and at the end of life (18-19 weeks of age). E is a graph showing myosin light chain 3, a marker of myocardial injury, in FXN KO mice before (6 weeks of age), after (9-10 weeks of age), and at the end of life (18-19 weeks of age). [Figure 7] (A and B) are a set of graphs showing frataxin expression in the heart of FXN KO mice following iv administration of exemplary AAV2 / 8 viral vectors for expression of hFXNco or mFXN at an amount of 3×10 vg / kg or 1×10 vg / kg compared to non-transduced controls (vehicle WT and vehicle KO). (A) is a graph showing dose-dependent frataxin expression in the heart as a function of vector copy number (VCN) / decigram (DG), while (B) shows frataxin expression as nanograms (ng) of protein per mg of biopsied heart tissue sampled. [Figure 8]Graph showing levels of frataxin protein in cardiac tissue as measured by enzyme-linked immunosorbent assay (ELISA) 4 weeks (A) and 12 weeks (B) after administration of an exemplary AAV2 / 8 viral vector for expression of hFXNco or mFXN at an amount of 3×10 vg / kg or 1×10 vg / kg compared to non-transduced controls (WT vehicle and FRDA vehicle). [Figure 9] A and B are graphs showing vector copy number / diploid genome detected in cardiac tissue as measured by qPCR at 4 and 12 weeks after administration of exemplary AAV2 / 8 viral vectors for expression of hFXNco or mFXN at amounts of 3×10 vg / kg or 1×10 vg / kg compared to non-transduced controls (WT vehicle and FRDA vehicle). [Figure 10] (A) Immunohistochemistry of frataxin expression in cardiac tissue. (B) Graph showing percentage of cells showing weak, moderate, or strong frataxin expression 4 weeks after administration of exemplary AAV2 / 8 viral vectors for expression of hFXNco or mFXN at doses of 3×10 vg / kg or 1×10 vg / kg. [Figure 11] A and B are photographs of cells stained for frataxin following transduction with 1 x 107 (1e7) viral genomes (vg) / cell of version 2 of the AAV2 / 8 viral vector for expression of hFXNco (AAV2 / 8-PGK-FXN V2) compared to non-transduced controls (vehicle control) for mouse skeletal muscle (A) and human skeletal muscle (B), and graphs showing frataxin immunofluorescence as a function of multiplicity of infection (MOI), normalized to the intensity of Hoechst counterstain. [Figure 12]Comparison of AAV2 / 8-PGK-FXN version 1 (V1) and version 2 (V2). A, A panel of photographs of mouse skeletal muscle (C2C12) cells stained for frataxin after transduction with 1×107 (1e7) viral genomes (vg) / cell of AAV2 / 8-PGK-FXN V1 or V2 compared to a non-transduced control (vehicle control). B, A graph showing frataxin immunofluorescence depicted as a function of multiplicity of infection (MOI) normalized to the intensity of Hoechst counterstain for AAV2 / 8-PGK-FXN V1 or V2. C, A immunoblot showing expression of the mature isoform of frataxin in C2C12 cells transduced with AAV2 / 8-PGK-FXN V1 or V2 at doses of 1×106 (1e6) vg / cell or 1×107 (1e7) vg / cell. [Figure 13] FIG. 1 is a panel of graphs showing frataxin immunofluorescence depicted as a function of multiplicity of infection (MOI) normalized to the intensity of Hoechst counterstaining for version 2 of the AAV2 / 8 viral vector for expression of FXN (AAV2 / 8-PGK-FXN V2), expressing either wild-type FXN (WT) or codon-optimized FXN (CO). A corresponds to transduced mouse skeletal muscle (C2C12) cells and B corresponds to transduced human cells. [Figure 14] Graph showing survival of FXN KO mice over time following intravenous administration of AAV2 / 8-PGK-FXN version 1 (V1) or version 2 (V2) at various doses of viral genomes (vg) / kilogram (kg) compared to non-transduced controls (vehicle WT and vehicle mutant). Survival data are expressed as the age at which mice were euthanized prior to scheduled biopsy if they exhibited any of the following conditions: weight loss of more than 20% of body weight, signs of respiratory distress, unresponsiveness to meaningful stimuli, and / or poor overall physical condition. [Figure 15]FIG. 11 is a panel of graphs showing cardiac ejection fraction of FXN KO mice following intravenous administration of AAV2 / 8-PGK-FXN V1 or V2 at various doses of viral genomes (vg) / kilogram (kg) compared to non-transduced controls (vehicle WT and vehicle mutant) at 5 weeks of age (WOA), 9-10 WOA, and 18-19 WOA. [Figure 16] 4 is a panel of graphs showing fractional shortening of hearts from FXN KO mice following intravenous administration of AAV2 / 8-PGK-FXN V1 or V2 at various doses of viral genomes (vg) per kilogram (kg) compared to non-transduced controls (vehicle WT and vehicle mutant) at 5 weeks of age (WOA), 9-10 WOA, and 18-19 WOA. [Figure 17] 4 is a panel of graphs showing cardiac left ventricular mass in FXN KO mice following intravenous administration of AAV2 / 8-PGK-FXN V1 or V2 at various doses of viral genomes (vg) / kilogram (kg) compared to non-transduced controls (vehicle WT and vehicle mutant) at 5 weeks of age (WOA), 9-10 WOA, and 18-19 WOA. [Figure 18] FIG. 11 is a panel of graphs showing serum cardiac troponin in FXN KO mice following intravenous administration of AAV2 / 8-PGK-FXN V1 or V2 at various doses of viral genomes (vg) / kilogram (kg) compared to non-transduced controls (vehicle WT and vehicle mutant) at 9-10 weeks of age (WOA) and 18-19 WOA. [Figure 19] FIG. 11 is a panel of graphs showing serum myosin light chain in FXN KO mice following intravenous administration of AAV2 / 8-PGK-FXN V1 or V2 at various doses of viral genomes (vg) / kilogram (kg) compared to non-transduced controls (vehicle WT and vehicle mutant) at 9-10 weeks of age (WOA) and 18-19 WOA. [Figure 20]FIG. 11 is a panel of graphs showing serum aspartate transaminase (AST) in FXN KO mice following intravenous administration of AAV2 / 8-PGK-FXN V1 or V2 at various doses of viral genomes (vg) / kilogram (kg) compared to non-transduced controls (vehicle WT and vehicle mutant) at 9-10 weeks of age (WOA) and 18-19 WOA. [Figure 21] FIG. 11 is a panel of graphs showing serum alanine transaminase (ALT) in FXN KO mice following intravenous administration of AAV2 / 8-PGK-FXN V1 or V2 at various doses of viral genomes (vg) / kilogram (kg) compared to non-transduced controls (vehicle WT and vehicle mutant) at 9-10 weeks of age (WOA) and 18-19 WOA. [Figure 22] FIG. 1 is a panel of graphs showing the vector copy number (VCN) / decigram (DG) of AAV2 / 8-PGK-FXN in the heart and quadriceps of FXN KO mice 4 and 12 weeks after intravenous administration of AAV2 / 8-PGK-FXN V1 or V2 at various doses of viral genomes (vg) / kilogram (kg). [Figure 23] 1 is a series of graphs comparing the vector copy number (VCN) / decigram (DG) of AAV2 / 8-PGK-FXN in the heart and quadriceps of FXN KO mice 4 and 12 weeks after intravenous administration of AAV2 / 8-PGK-FXN V1 or V2 at various doses of viral genomes (vg) / kilogram (kg). [Figure 24] FIG. 1 is a panel of graphs showing the vector copy number (VCN) / decigram (DG) of AAV2 / 8-PGK-FXN in the liver of FXN KO mice 4 and 12 weeks after intravenous administration of AAV2 / 8-PGK-FXN V1 or V2 at various doses of viral genomes (vg) / kilogram (kg). [Diagram 25]FIG. 13 is a graph showing FXN mRNA transcripts as RNA relative quantification (RQ) in heart and quadriceps (Quad) of FXN KO mice following intravenous administration of AAV2 / 8-PGK-FXN V1 or V2 at various doses of viral genomes (vg) / kilogram (kg). [Figure 26] FIG. 13 is a graph showing FXN protein expression in the hearts of FXN KO mice at 4 and 12 weeks after intravenous administration of AAV2 / 8-PGK-FXN V1 or V2 at various doses of viral genomes (vg) per kilogram (kg) compared to non-transduced controls (WT vehicle and KO vehicle). [Figure 27] FIG. 11 is a graph showing FXN protein expression in the liver of FXN KO mice 4 and 12 weeks after intravenous administration of AAV2 / 8-PGK-FXN V1 or V2 at various doses of viral genomes (vg) / kilogram (kg) compared to non-transduced controls (WT vehicle and KO vehicle). [Figure 28] FIG. 13 is a graph showing FXN protein expression in the quadriceps muscle of FXN KO mice 4 and 12 weeks after intravenous administration of AAV2 / 8-PGK-FXN V1 or V2 at various doses of viral genomes (vg) per kilogram (kg) compared to non-transduced controls (WT vehicle and KO vehicle). [Figure 29] Figure 13 is a graph showing the time taken in seconds to fall off the rotarod in FXN KO mice following intracerebroventricular (ICV) administration of AAV8-PGK-FXN V1 or V2 at various doses of viral genome (vg) / animal compared to non-transduced controls (vehicle control and vehicle mutant). Measurements were performed at 6-8 weeks of age (WOA), 11-12 WOA, 13-14 WOA, and 16-18 WOA, administered ICV at 8 WOA. [Diagram 30]

[0023] Figure 1 is a panel of graphs showing vector copy number (VCN) / decigram (DG) of AAV8-PGK-FXN at biopsy in the caudal spinal cord, cerebellum, cortex, cranial spinal cord, sciatic nerve, caudal dorsal root ganglion (DRG), left liver lobe, half of the heart, and cranial DRG of FXN KO mice following intracerebroventricular (ICV) administration of AAV8-PGK-FXN V2 at various doses of viral genome (vg) / animal. ICV administration was performed at 8 weeks of age. [Diagram 31] FIG. 1 is a series of graphs showing vector copy number (VCN) / decigram (DG) of AAV8-PGK-FXN at biopsy in the cerebellum, cortex, heart, and liver of FXN KO mice following intracerebroventricular (ICV) or intraparenchymal (IPC) administration of AAV8-PGK-FXN V2 at various doses of viral genome (vg) / animal. Animals were dosed at 8 weeks of age. Tissues from ICV-treated mice were analyzed 10-11 weeks post-dose, and tissues from IPC-treated mice were analyzed 5 weeks post-dose. [Diagram 32]

[0023] Figure 1 is a panel of graphs showing FXN protein in the cortex and cerebellum at biopsy for wild type (WT), non-transduced (FXNPAV / null), and FXN KO mice transduced via intracerebroventricular (ICV) or intraparenchymal (IPC) administration of AAV8-PGK-FXN V1 or V2 at various doses of viral genome (vg) / animal. Animals were dosed at 8 weeks of age. Tissues from ICV-treated mice were analyzed 10 weeks post-dosing and tissues from IPC-treated mice were analyzed 5 weeks post-dosing. [Diagram 33] Figure 1 shows FXN protein / vector copy number (VCN) in the cortex, cerebellum, heart, and liver of FXN KO mice following intracerebroventricular (ICV) or intraparenchymal (IPC) administration of AAV8-PGK-FXN V2. Tissues were analyzed 5 weeks post injection (wpi). ICV was administered on postnatal day 2 (PND2). [Diagram 34]FIG. 13 is a graph showing neurofilament light chain (NFLC) in picograms (pg) / milliliter (mL) in FXN KO mice following administration of AAV8-PGK-FXN V1 or V2 at various doses of viral genome (vg) / animal compared to non-transduced controls (WT vehicle and KO vehicle). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] definition As used herein, the term "about" refers to a value within 10% above or below the stated value.

[0047] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, and any other AAV now known or later discovered. See, e.g., Fields et al. Virology, 4 thed. Lippincott-Raven Publishers, Philadelphia, 1996. Additional AAV serotypes and clades have been identified in recent years. (See, e.g., Gao et al. J. Virol. 78:6381 (2004); Morris et al. Virol. 33:375 (2004). The genomic sequences of various serotypes of AAV, as well as the sequences of the native ITRs, Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Nos. NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_001601, NC_001610, NC_001621, NC_001631, NC_001641, NC_001642, NC_001651, NC_001661, NC_001671, NC_001681, NC_001691, NC_001692, NC_001693, NC_001694, NC_001695, NC_001696, NC_001697, NC_001698, NC_001699, NC_001698, NC_001699, NC_001691, NC_001696, NC_001697, NC_001698, See, e.g., 006152, NC_006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, AY631966, AX753250, EU285562, NC_001358, NC_001540, AF513851, AF513852, and AY530579, the disclosures of which are incorporated by reference herein for their teaching of AAV nucleic acid and amino acid sequences.Also, for example, Bantel-Schaal et al.J.Virol.73:939(1999), Chiorini et al.J.Virol.71:6823(1997), Chiorini et al.J.Virol.73:1309(1999), Gao et al.Proc.Nat.Acad.Sci.USA 99:11854(2002), Morris et al.Virol.33:375(2004), Muramatsu et al.Virol.221:208(1996), Ruffing et al.J.Gen.Virol.75:3385(1994), Rutledge et al.J.Virol.72:309(1998), Schmidt et al. J. Virol. 82:8911 (2008), Shade et al. See, e.g., Srivastava et al. J. Virol. 58:921 (1986), Srivastava et al. J. Virol. 45:555 (1983), Xiao et al. J. Virol. 73:3994 (1999), WO00 / 28061, WO99 / 61601, WO98 / 11244, and US 6,156,303, the disclosures of which are incorporated by reference herein for their teaching of AAV nucleic acid and amino acid sequences.

[0048] "Capsid protein," as used herein, refers to any of the AAV capsid proteins that are components of the AAV viral particle, including AAV8 and AAV9.

[0049] As used herein, the term "cloning site" refers to a nucleic acid sequence that contains a restriction site for restriction endonuclease-mediated cloning by ligation of nucleic acids containing compatible cohesive or blunt ends, a region of nucleic acid that serves as a priming site for PCR-mediated cloning of insert DNA by homology and extension "overlap PCR stitching", or a recombination site for recombinase-mediated insertion of a target nucleic acid by a recombinational exchange reaction, or a mosaic end for transposon-mediated insertion of a target nucleic acid, as well as other techniques common in the art.

[0050] The term "codon" as used herein refers to any group of three consecutive nucleotide bases in a given messenger RNA molecule or coding strand of DNA that specifies a particular amino acid or a start or stop signal for translation. The term codon also refers to a triplet of bases in a DNA strand.

[0051] As used herein, "codon optimization" refers to the process of modifying a nucleic acid sequence according to the principle that the frequency of occurrence of synonymous codons (e.g., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows the same polypeptide to be encoded by a variety of nucleotide sequences. Such modified sequences are referred to herein as "codon optimized." This process can be performed on any of the sequences described herein to enhance expression or stability. Codon optimization can be performed by methods known in the art, for example, as described in U.S. Pat. Nos. 7,561,972, 7,561,973, and 7,888,112, each of which is incorporated herein by reference in its entirety. For example, sequences surrounding the translation start site can be converted to consensus Kozak sequences according to known methods. See, e.g., Kozak et al, Nucleic Acids Res. 15(20):8125-8148, incorporated herein by reference in its entirety. Multiple stop codons can be incorporated. As used herein, the terms "codon-optimized human frataxin gene" and "hFXNco" refer to a polynucleotide that exhibits at least 95% (e.g., 95%, 97%, 98%, or 99%) sequence identity to an endogenous RNA molecule encoding frataxin protein variant 1 (e.g., SEQ ID NO:5). In some embodiments, hFXNco is identical to SEQ ID NO:1.

[0052] Throughout this specification and the claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0053] As used herein, the terms "conservative mutation," "conservative substitution," and "conservative amino acid substitution" refer to the replacement of one or more amino acids with one or more different amino acids that exhibit similar physicochemical properties (e.g., polarity, electrostatic charge, and steric hindrance volume), which are summarized in Table 1 below for each of the 20 naturally occurring amino acids.

[0054] [Table 1]

[0055] From this table it can be seen that conservative amino acid families include, for example, (i) G, A, V, L, I, P, and M; (ii) D and E; (iii) C, S and T; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W. Thus, a conservative mutation or substitution is one that replaces one amino acid with a member of the same amino acid family (e.g., Thr with Ser, or Arg with Lys).

[0056] By "CpG site" is meant a region of DNA in which a cytosine nucleotide is present next to a guanine nucleotide in a linear nucleic acid sequence of nucleotides along its length, e.g., -C-phosphate-G-, a cytosine and guanine separated by only one phosphate, or a cytosine 5' to a guanine nucleotide.

[0057] As used herein, the term "endogenous" refers to a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is naturally found in a particular organism (e.g., humans) or in a particular location within an organism (e.g., an organ, tissue, or cell, e.g., a human cell).

[0058] The term "frataxin" refers to the frataxin protein, and the term "FXN" refers to the gene encoding the frataxin protein (also referred to in the art as "FA," "X25," "CyaY," "FARR," and "MGC57199"). As used herein, the terms "frataxin" and "FXN" refer interchangeably to polypeptides and nucleic acids, respectively, including the following polymorphic variants, alleles, mutants, and interspecies homologs: (1) FXN nucleic acid (SEQ ID NO:5, e.g., GenBank Accession No. NM - 000144.4 (isoform 1)) or to the amino acid sequence of the frataxin polypeptide (SEQ ID NO:3, e.g., GenBank Accession No. NP - 000135.2 (isoform 1)), preferably over a region of at least about 25, 50, 100, 200, 300, 400, or more amino acids, or over the entire length, having an amino acid sequence that has about 905 amino acid sequence identity, e.g., 96%, 97%, 98%, or 99% or more amino acid sequence identity; to FXN nucleic acids (e.g., frataxin polynucleotides described herein, and FXN polynucleotides encoding frataxin polynucleotides described herein), preferably over a region of at least about 25, 50, 100, 200, 500, 1000, 2000, or more nucleotides, or over the entire length, having a nucleic acid sequence that has greater than about 95% nucleotide sequence identity, e.g., greater than about 96%, 97%, 98%, 99% or more nucleotide sequence identity.

[0059] As used herein, the term "Friedreich's ataxia" refers to an autosomal recessive congenital ataxia caused by mutations in the gene FXN (previously known as X25), which codes for frataxin, located on chromosome 9. The genetic basis of Friedreich's ataxia involves a GAA trinucleotide repeat in an intronic region of the gene that codes for frataxin. This segment is usually repeated 5-33 times within the FXN gene. In people with Friedreich's ataxia, the GAA segment is repeated 66 to over 1,000 times. People with fewer than 300 repeated GAA segments tend to have a later onset of symptoms (after age 25) than those with larger GAA trinucleotide repeats. The presence of these repeats causes a decrease in gene transcription and expression. Frataxin is involved in regulating mitochondrial iron content. Mutations in the FXN gene cause progressive damage to the nervous system, resulting in symptoms ranging from gait disturbances to speech disorders, and may also lead to heart disease and diabetes. The ataxia of Friedreich's ataxia results from the degeneration of nerve tissue in the spinal cord, especially sensory neurons that are essential (through connections with the cerebellum) for directing muscle movements in the arms and legs. The spinal cord thins and nerve cells lose some of their myelin sheath (the insulating covering on some nerve cells that helps transmit nerve impulses). Subjects with Friedreich's ataxia may exhibit one or more of the following symptoms: muscle weakness in the arms and legs, loss of coordination, vision problems, hearing problems, slurred speech, curvature of the spine (scoliosis), high plantar arches (pes cavus deformity), carbohydrate intolerance, diabetes, cardiac disorders (e.g., atrial fibrillation, tachycardia (fast heart rate), and hypertrophic cardiomyopathy). Subjects with Friedreich's ataxia may also exhibit involuntary and / or rapid eye movements, loss of deep tendon reflexes, loss of plantar extensor responses, loss of vibration and proprioception, cardiac hypertrophy, symmetric hypertrophy, heart murmurs, and cardiac conduction defects. Pathologic analysis may reveal sclerosis and degeneration of the dorsal root ganglia, spinocerebellar bundles, lateral corticospinal bundles, and dorsal columns.

[0060] As used herein, the term "GC content" refers to the amount of nucleosides in a particular nucleic acid molecule, such as a DNA or RNA polynucleotide, that are either guanosine (G) or cytidine (C) relative to the total amount of nucleosides present in the nucleic acid molecule. GC content may be expressed as a percentage, for example, according to the following formula: GC content = ((total amount of guanosine nucleosides) + (total amount of cytidine nucleosides) / (total amount of nucleosides)) x 100

[0061] As used herein, the term "gene" refers to a region of DNA that codes for a protein. A gene may include a regulatory region and a protein coding region. In some embodiments, a gene includes two or more introns and three or more exons, with each intron forming an intervening sequence between two exons.

[0062] As used herein, the term "intron" refers to a region in the coding region of a gene, the nucleotide sequence of which is not translated into the amino acid sequence of the corresponding protein. The term intron also refers to the corresponding region of the RNA transcribed from the gene. In some embodiments, a gene may, for example, contain at least two introns, each of which forms an intervening sequence between two exons. Introns are transcribed into pre-mRNA but are removed during processing and are not included in the mature mRNA.

[0063] An "ITR" is a palindromic nucleic acid, e.g., an inverted terminal repeat, about 120 nucleotides to about 250 nucleotides in length, capable of forming a hairpin. The term "ITR" includes a site of viral genome replication that can be recognized and bound by a parvoviral protein (e.g., Rep78 / 68). The ITR may be from any adeno-associated virus (AAV), with serotype 2 being preferred. The ITR includes a replication protein-binding element (RBE) and a terminal resolution sequence (TRS). The term "ITR" does not require a wild-type parvoviral ITR (e.g., the wild-type nucleic acid sequence may be altered by insertion, deletion, truncation, or missense mutation), so long as the ITR functions to mediate viral packaging, replication, integration, and / or proviral rescue, etc. The term "5'ITR" is intended to mean a parvoviral ITR located at the 5' border of a nucleic acid molecule, and the term "3'ITR" is intended to mean a parvoviral ITR located at the 3' border of a nucleic acid molecule.

[0064] As used herein, the term "modified nucleotide" refers to a nucleotide or portion thereof (e.g., adenosine, guanosine, thymidine, cytidine, or uridine) that has been altered by one or more enzymatic or synthetic chemical transformations. Exemplary changes observed in modified nucleotides described herein or known in the art include the introduction of chemical substituents, such as halo, thio, amino, azido, alkyl, acyl, or other functional groups, into one or more positions (e.g., the 2', 3', and / or 5' positions) of a 2-deoxyribonucleotide or ribonucleotide.

[0065] As used herein, the term "mutation" refers to a change in the nucleotide sequence of a gene or a change in the polypeptide sequence of a protein. Mutations of genes or proteins can occur naturally, for example, as a result of errors in DNA replication, DNA repair, irradiation, exposure to carcinogens, or mutations can be induced as a result of administration of a transgene that expresses a mutant gene. Mutations can result from the insertion, deletion, or substitution of single or multiple nucleotides.

[0066] "Nucleic acid" and "polynucleotide", used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA.

[0067] As used herein, the term "operably linked" refers to a first molecule being connected to a second molecule, where the molecules are positioned such that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single contiguous molecule, and may or may not be adjacent. For example, a promoter is operably linked to a transcribable polynucleotide molecule if the promoter regulates the transcription of the transcribable polynucleotide molecule of interest in a cell. In addition, two portions of a transcriptional regulatory element are operably linked to each other if they are connected such that the transcriptional activation function of one portion is not adversely affected by the presence of the other portion. Two transcriptional regulatory elements may be operably linked to each other via a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to each other without the presence of any intervening nucleotides.

[0068] The term "parvovirus" as used herein encompasses the family Parvoviridae, which includes autonomously replicating parvoviruses and dependoviruses. Autonomous parvoviruses include members of the genera Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus. Exemplary autonomous parvoviruses include, but are not limited to, minute virus of mice, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, Muscovy duck parvovirus, snake parvovirus, and B19 virus. Other autonomous parvoviruses are known to those of skill in the art. See, for example, Fields et al. Virology, 4 thed. Lippincott-Raven Publishers, Philadelphia, 1996. The Dependovirus genus includes the adeno-associated viruses (AAV), including, but not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, caprine AAV, snake AAV, equine AAV, and ovine AAV.

[0069] "Percent sequence identity" to a reference polynucleotide sequence or a reference polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide sequence or the reference polypeptide sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent identity of nucleic acid or amino acid sequences can be achieved in a variety of ways that are within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. As an example, the percent sequence identity of a given nucleic acid or amino acid sequence A to, with, a given nucleic acid or amino acid sequence B (which can alternatively be translated as a given nucleic acid or amino acid sequence A having a particular percent sequence identity to, with, a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in a programmatic alignment of A and B, and Y is the total number of nucleic acids in B. It will be understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0070] As will be appreciated by those skilled in the art, for purposes of determining "percent sequence identity" as used herein, uridine nucleosides in an RNA molecule are considered equivalent to thymidine nucleosides in a DNA molecule. Thus, an RNA equivalent may be considered to have 100% sequence identity to a DNA polynucleotide where the two polynucleotides differ from each other only by the substitution of uridine nucleosides in the RNA equivalent for thymidine nucleosides in the DNA polynucleotide.

[0071] The term "polyadenylation signal" or "polyadenylation site" is used herein to mean a nucleic acid sequence sufficient to direct the addition of polyadenosine ribonucleic acid to an RNA molecule expressed in a cell.

[0072] A "promoter" is a nucleic acid that enables the initiation of transcription of a gene into messenger RNA; such transcription is initiated by the binding of RNA polymerase on or near the promoter.

[0073] As used herein, the term "pharmaceutical composition" refers to a mixture containing therapeutic compounds that is administered to a subject, e.g., a mammal, e.g., a human, to prevent, treat, or control a particular disease or condition that affects or may affect the subject.

[0074] As used herein, the term "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without undue toxicity, irritation, allergic response, and other significant complications, and with a reasonable benefit / risk ratio.

[0075] As used herein, the term "RNA equivalent" of a gene refers to an RNA polynucleotide that corresponds to a DNA polynucleotide encoding a gene, e.g., an RNA transcript that can be obtained by transcription of a DNA polynucleotide that comprises a gene. Exemplary RNA equivalents include mRNA transcripts that are synthetically produced, such as by solid phase nucleic acid synthesis techniques, as well as recombinant nucleic acid preparation methods known in the art and / or described herein.

[0076] A "spacer" is any polynucleotide at least 1.0 Kb in length that contains an open reading frame (ORF) of less than 100 amino acids, has a CpG content that is less than 1% of the total nucleic acid sequence, or does not contain transcription factor (TF) binding sites (e.g., sites recognized by prokaryotic or baculovirus transcription factors). The term spacer does not include nucleic acids of prokaryotic or baculovirus origin. Spacers can be isolated from naturally occurring sources or modified to, for example, reduce the size of the ORF, the CpG content, or the number of transcription factor binding sites. Spacers can be selected from naturally occurring nucleic acids that promote expression of polynucleotides, such as introns found adjacent to the ORF, or enhancers found near the transcription start site. The use of a "spacer", as defined herein, results in a reduction of contaminating nucleic acids packaged into viral particles.

[0077] As used herein, the term "transcriptional regulatory element" refers to a nucleic acid that at least partially controls the transcription of a gene of interest. Transcriptional regulatory elements can include promoters, enhancers, and other nucleic acids (e.g., polyadenylation signals) that control or aid in the control of gene transcription. Examples of transcriptional regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, 1990).

[0078] As used herein, the term "treat" or "treatment" refers to therapeutic treatment, the purpose of which is, inter alia, to prevent or slow down (reduce) an undesirable physiological change or disorder, e.g., Friedreich's ataxia. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or alleviation of disease state, and remission (whether partial or complete), whether detectable or undetectable. In the context of Friedreich's ataxia, treatment of a patient may be manifested by one or more detectable changes, such as an increase in the concentration of frataxin or a nucleic acid (e.g., DNA or RNA, e.g., mRNA) encoding frataxin (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 100%, 15 ... , 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 500x, 1,000x, or more). The concentration of frataxin can be determined using protein detection assays known in the art, including the ELISA assays described herein. The concentration of nucleic acid encoding frataxin can be determined using nucleic acid detection assays (e.g., RNA Seq assays) described herein. Exemplary protocols for the detection of frataxin protein and nucleic acid are provided in Example 3 below. In addition, the treatment of patients suffering from Friedreich's ataxia may manifest itself in the improvement of the patient's muscle function (e.g., cardiac or skeletal muscle function), as well as the improvement of muscle coordination.For example, the treatment of patients suffering from Friedreich's ataxia may manifest itself in a decrease in total Friedreich's Ataxia Rating Scale (FARS) score (e.g., by about 12 weeks after treatment).

[0079] As used herein, the term "vector" refers to a nucleic acid, e.g., DNA or RNA, that can function as a vehicle to deliver a gene of interest to a cell (e.g., a mammalian cell, such as a human cell), such as for purposes of replication and / or expression. Exemplary vectors useful in conjunction with the compositions and methods described herein are plasmids, DNA vectors, RNA vectors, virions, or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed to deliver polynucleotides encoding exogenous proteins to prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO1994 / 11026, the disclosure of which is incorporated herein by reference. The expression vectors described herein contain not only polynucleotide sequences but also additional sequence elements that are used, for example, for the expression of proteins and / or the integration of these polynucleotide sequences into the genome of a mammalian cell. Particular vectors that can be used to express the transgenes described herein include plasmids that contain regulatory sequences, such as promoter and enhancer regions that direct gene transcription. Other vectors useful for expressing transgenes contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of the mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector. The expression vectors described herein may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0080] Detailed Description The compositions and methods described herein are useful for stimulating expression of human frataxin protein and for treating disorders associated with mutations in the frataxin gene (FXN), such as Friedreich's ataxia. The compositions described herein include a plasmid (e.g., a viral vector, such as an adeno-associated virus (AAV)) encoding codon-optimized human FXN (hFXNco), or its RNA equivalent, for expression of frataxin protein in cells. The plasmids described herein include AAVs containing two inverted terminal repeats (ITRs, e.g., a first ITR (ITR1) and a second ITR (ITR2)), and the length of the nucleic acid between and including ITR1 and ITR2 is about 3.9 Kb to about 4.7 Kb. Without being limited by mechanism, the compositions described herein may ameliorate pathology associated with Friedreich's ataxia by effectively stimulating expression of human frataxin protein.

[0081] The present invention is based, at least in part, on the discovery that delivery of a nucleic acid molecule comprising ITR1-hFXNco-ITR2, the length of the nucleic acid between and including ITR1 and ITR2 being about 3.9 Kb to about 4.7 Kb, results in a surprisingly superior ability to induce expression of human frataxin protein in cells. This property is particularly beneficial in view of the incidence of mutations in the FXN gene in mammalian genomes, such as those of human patients with Friedreich's ataxia. The compositions and methods described herein can be used to effectively enhance expression of vital healthy FXN or its RNA transcripts, and their encoded frataxin protein products.

[0082] The following sections provide exemplary codon optimizations for producing hFXNco that can be used in combination with vectors encoding such constructs described herein, and methods for producing same, as well as methods that can be used to treat Friedreich's ataxia.

[0083] Therapeutic Proteins Genes that can be incorporated into a plasmid (e.g., a viral vector) according to the methods described herein include genes that encode therapeutic proteins (e.g., frataxin), e.g., genes that can be transferred to a subject (e.g., a human patient) suffering from a disease or condition characterized by a deficiency of a protein (e.g., Friedreich's ataxia). For example, genes that can be delivered to a patient according to the methods described herein include a gene encoding frataxin.

[0084] In one approach, the invention provides human FXN (hFXN), or its RNA equivalent, having a polynucleotide sequence that is at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) identical to an endogenous RNA molecule encoding frataxin protein variant 1 (e.g., SEQ ID NO:5). For example, in some embodiments, the polynucleotide exhibits at least 95% sequence identity to an endogenous RNA molecule encoding frataxin protein variant 1 (e.g., SEQ ID NO:5). In some embodiments, the polynucleotide exhibits at least 96% sequence identity to an endogenous RNA molecule encoding frataxin protein variant 1 (e.g., SEQ ID NO:5). In some embodiments, the polynucleotide exhibits at least 97% sequence identity to an endogenous RNA molecule encoding frataxin protein variant 1 (e.g., SEQ ID NO:5). In some embodiments, the polynucleotide exhibits at least 98% sequence identity to an endogenous RNA molecule encoding frataxin protein variant 1 (e.g., SEQ ID NO:5). In some embodiments, the polynucleotide exhibits at least 99% sequence identity to an endogenous RNA molecule encoding frataxin protein variant 1 (e.g., SEQ ID NO: 5). In some embodiments, the polynucleotide is identical to an endogenous RNA molecule encoding frataxin protein variant 1 (e.g., SEQ ID NO: 5).

[0085] In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO:3. For example, in some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 86% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 87% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 88% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 89% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 90% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 91% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 92% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 93% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 94% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 95% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 96% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 97% identical to the amino acid sequence of SEQ ID NO:3.In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 98% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 99% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is identical to the amino acid sequence of SEQ ID NO: 3.

[0086] Codon Optimization The compositions and methods described herein can be used, for example, to optimize the nucleic acid sequence of a gene encoding a protein of interest (e.g., frataxin) or its RNA equivalent (e.g., FXN) to achieve enhanced expression of the protein in a particular cell type. For example, the compositions and methods described herein can be used to optimize a gene and its RNA equivalent for tissue-specific expression of the encoded protein (e.g., frataxin). Genes and their RNA equivalents optimized using the compositions and methods described herein can be synthesized by chemical synthesis techniques and amplified, for example, using polymerase chain reaction (PCR)-based amplification methods, or by transfecting the gene into cells, such as bacterial or mammalian cells, that are capable of replicating exogenous nucleic acids.

[0087] The genes and RNA equivalents described herein may have important clinical utility. A variety of diseases and conditions, including inherited genetic disorders such as Friedreich's ataxia, are symptoms of a deficiency of a natural protein (e.g., frataxin). With the advent of gene therapy, a wide range of vectors and gene delivery techniques have been developed for the introduction of nucleic acids encoding exogenous proteins into target cells (e.g., human cells). However, there remains a need for optimized variants of the transgenes encoded by exogenous nucleic acids to achieve robust and stable expression of the encoded proteins in cells of interest.

[0088] Reduces CpG and homopolymer content Codon optimization can be performed by techniques known in the art. As a non-limiting example, one skilled in the art can engineer the gene sequence encoding the protein of the target gene by incorporating codon substitutions that reduce the CpG and / or homopolymer content of the gene. For example, one can start with a wild-type gene sequence and introduce substitutions (e.g., single nucleotide substitutions) that reduce the CpG and / or homopolymer content of the gene while preserving the identity of the encoded protein sequence. The sequence identity minimization process described above and in Example 1 can then be followed to obtain a gene sequence that is minimally similar to the gene encoded in the cell type of interest (e.g., FXN). Alternatively, one can start with a sequence that has been codon-optimized according to the sequence identity minimization process described above and then engineered by the introduction of mutations (e.g., single nucleotide substitutions) that reduce the CpG and / or homopolymer content of the gene. CpG sites and homopolymers can promote +1 frameshifts during the mRNA translation process. Alternatively, if a homopolymer encodes an amino acid residue that is not essential for protein function (e.g., if the encoded amino acid is not present within the active site of the encoded enzyme or within a site required for non-covalent binding to another biomolecule), one of skill in the art can incorporate codon substitutions that interrupt the homopolymer and introduce conservative substitutions into the encoded protein at the corresponding amino acid site.

[0089] Preparation of codon-optimized genes Once designed, the final codon-optimized gene can be prepared, for example, by solid-phase nucleic acid procedures known in the art. For example, a solid-phase synthesis process using the phosphoramidite method can be used to perform chemical synthesis of nucleic acid molecules such as DNA, RNA, etc. According to this procedure, nucleic acids are generally synthesized by the following steps:

[0090] First, the 5-OH protected nucleoside occurring at the 3'-end of the nucleic acid to be synthesized is esterified to a solid support via the 3'-OH function by attaching the nucleoside to a cleavable linker.Then, the support for solid phase synthesis on which the nucleoside is immobilized can be placed in a reaction column and then loaded into an automated nucleic acid synthesizer.

[0091] Then, a repetitive synthesis process including the following steps can be carried out in the reaction column according to the synthesis program of the automated nucleic acid synthesizer. (1) a deprotection step of the 5'-OH moiety of the protected and immobilized nucleoside (e.g., removing the acid-labile hydroxyl protecting group using an acid such as trichloroacetic acid in a dichloromethane solution, etc.); (2) coupling a 5-OH protected nucleoside phosphoramidite with the deprotected 5'-OH group of the immobilized nucleoside in the presence of an activating agent (e.g., tetrazole, etc.); (3) capping the unreacted 5'-OH group of the 3'-terminal nucleoside (e.g., with acetic anhydride, etc.); and (4) Oxidizing the immobilized phosphite substituents (eg, with aqueous iodine).

[0092] The above process can be repeated to extend the nucleic acid in the 3'-5' direction as necessary, and the 5' end direction is promoted to synthesize a nucleic acid having a desired sequence.

[0093] Finally, the cleavable linker is hydrolyzed (e.g., with aqueous ammonia, methylamine solution, etc.) to cleave the synthesized nucleic acid from the solid support. Procedures such as those described above for chemical synthesis of nucleic acids are known in the art and are described, for example, in U.S. Patent No. 8,835,656, the disclosure of which is incorporated herein by reference in its entirety with respect to protocols for the synthesis of nucleic acid molecules.

[0094] Additionally, the prepared genes can be amplified, for example, using PCR-based techniques described herein or known in the art, and / or by transforming DH5α E. coli with a plasmid containing the designed gene. The bacteria can then be cultured to amplify the DNA therein, and the gene can be isolated using plasmid purification techniques known in the art, optionally followed by restriction digestion and / or sequencing of the plasmid to verify the identity of the codon-optimized gene.

[0095] Exemplary codon-optimized human FXN In one approach, the invention provides hFXNco, or its RNA equivalent, having a polynucleotide sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:1. For example, in some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO:1.In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 1.

[0096] In some embodiments, hFXNco, or its RNA equivalent, has the nucleic acid sequence of SEQ ID NO:1.

[0097] Methods for delivering exogenous nucleic acid to target cells Transfection technology Techniques that can be used to introduce transgenes, such as transgenes operably linked to the transcriptional regulatory elements described herein, into target cells are known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by applying an electrostatic potential to the cells of interest. Mammalian cells, such as human cells, thus exposed to an external electric field are then susceptible to uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, for example, in Chu et al., Nucleic Acids Res 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection™, utilizes an applied electric field to stimulate uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. Nucleofection™ and protocols useful for carrying out this technique are described in detail in, for example, Distler et al., Exp. Dermatol. 14:315 (2005) and US2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0098] Further techniques useful for transfection of target cells include squeeze-poration. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores that form in response to applied stress. This technique is advantageous in that it does not require a vector to deliver nucleic acid to cells, such as human target cells. Squeeze-poration is described in detail, for example, in Sharei et al., JoVE 81:e50980 (2013), the disclosure of which is incorporated herein by reference.

[0099] Lipofection represents another technique useful for transfection of target cells. This method involves loading nucleic acids into liposomes, often presenting cationic functional groups such as quaternary or protonated amines toward the exterior of the liposome. This promotes electrostatic interactions between the liposome and the cells, since the cell membrane is anionic, and ultimately results in the uptake of the exogenous nucleic acid, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in U.S. Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference. A similar technique that utilizes ionic interactions with the cell membrane to induce the uptake of exogenous nucleic acid includes contacting cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge that favors interaction with cell membranes are activated dendrimers (e.g., as described in Dennig, Top Curr Chem 228:227 (2003), the disclosure of which is incorporated herein by reference) and diethylaminoethyl (DEAE)-dextran, the use of which as a transfection agent is described in detail, for example, in Gulick et al., Curr Protoc Mol Biol 40:I:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner, as this method utilizes an applied magnetic field to cause uptake of nucleic acids. This technology is described in detail, for example, in US2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0100] Another useful tool for inducing the uptake of exogenous nucleic acid by target cells is laserfection, a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize cells and allow polynucleotides to penetrate the cell membrane.This technique is described in detail, for example, in Rhodes et al., Methods Cell Biol.82:309 (2007), the disclosure of which is incorporated herein by reference.

[0101] Microvesicles are another vehicle that can potentially be used to modify the genome of target cells according to the methods described herein. For example, microvesicles derived by simultaneously overexpressing glycoprotein VSV-G and genome modification proteins, such as nucleases, can be used to efficiently deliver proteins to cells, and the proteins can then catalyze the site-specific cleavage of endogenous polynucleotide sequences to prepare the genome of cells for covalently incorporating a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also called Gesicle, for genetically modifying eukaryotic cells is described in detail, for example, in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract], in Methylation changes in early embryonic genes in cancer [abstract], and in Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.

[0102] Targeted gene integration using gene editing technology In addition to the above, various tools have been developed that can be used to integrate a gene of interest into a target cell, such as a human cell. One such method that can be used to integrate a polynucleotide encoding a target gene into a target cell involves the use of a transposon. A transposon is a polynucleotide that contains a polynucleotide sequence or gene of interest that encodes a transposase enzyme and is flanked by 5' and 3' cleavage sites. Once the transposon is delivered to a cell, expression of the transposase gene begins, resulting in an active enzyme that cleaves the gene of interest from the transposon. This activity is mediated by the transposase's site-specific recognition of the transposon cleavage sites. In some cases, these excision sites can be terminal repeats or inverted terminal repeats (ITRs). Once the gene of interest is cleaved from the transposon, it can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of similar cleavage sites present in the nuclear genome of the mammalian cell. This allows the gene of interest to be inserted into the complementary cleavage site of the cleaved nuclear DNA, followed by covalent ligation of phosphodiester bonds connecting the gene of interest to the DNA of the mammalian cell genome, completing the integration process. In certain cases, the transposon may be a retrotransposon, in which the gene encoding the target gene is first transcribed into an RNA product, then reverse transcribed into DNA, and then integrated into the mammalian cell genome. Exemplary transposon systems are the piggybac transposon (described in detail, for example, in WO2010 / 085699) and the sleeping beauty transposon (described in detail, for example, in US2005 / 0112764), the disclosures of each of which are incorporated herein by reference with respect to transposons for use in gene delivery to cells of interest.

[0103] Another tool for integrating target genes into the genome of target cells is the clustered regularly interspaced short palindromic repeat (CRISPR) / Cas system, which originally evolved as an adaptive defense mechanism against bacterial and archaeal viral infections. The CRISPR / Cas system contains palindromic repeats in plasmid DNA and the associated Cas9 nuclease. This DNA-protein complex first integrates foreign DNA into the CRISPR locus, thereby inducing site-specific DNA cleavage of the target sequence. Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus are then transcribed in the host cell to generate guide RNAs that can then anneal to the target sequence and localize the Cas9 nuclease to this site. Thus, the interaction of cas9 with the target DNA molecule in close proximity is governed by RNA:DNA hybridization, allowing cas9-mediated highly site-specific DNA cleavage of the foreign polynucleotide. As a result, the CRISPR / Cas system can be designed to cleave any target DNA molecule of interest. This technology has been utilized to edit eukaryotic genomes (see Hwang et al., Nat. Biotechnol. 31:227 (2013)) and can be used as an efficient means of site-specifically editing target cell genomes to cleave DNA prior to integration of a gene encoding a target gene. The use of CRISPR / Cas to modulate gene expression is described, for example, in U.S. Pat. No. 8,697,359, the disclosure of which is incorporated herein by reference with respect to the use of the CRISPR / Cas system for genome editing. Alternative methods for site-specific cleavage of genomic DNA prior to integration of a gene of interest into a target cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).Unlike CRISPR / Cas systems, these enzymes do not contain guide polynucleotides that localize to specific target sequences. Instead, target specificity is controlled by DNA binding domains within these enzymes. The use of ZFNs and TALENs in genome editing applications is described, for example, in Urnov et al., Nat.Rev.Genet.11:636 (2010) and Joung et al., Nat.Rev.Mol.Cell Biol.14:49 (2013), the disclosures of each of which are incorporated herein by reference with respect to compositions and methods for genome editing.

[0104] Additional genome editing techniques that can be used to integrate a polynucleotide encoding a target gene into the genome of a target cell include the use of ARCUS™ meganucleases, which can be rationally designed to site-specifically cleave genomic DNA. Using these enzymes to integrate genes encoding target genes into the genome of mammalian cells is advantageous in view of the clear structure-activity relationship established for the enzymes. Single-chain meganucleases can be modified at specific amino acid positions to create nucleases that selectively cleave DNA at desired positions, thereby allowing site-specific integration of target genes into the nuclear DNA of target cells. These single-chain nucleases have been extensively described, for example, in U.S. Pat. Nos. 8,021,867 and 8,445,251, the disclosures of each of which are incorporated herein by reference with respect to compositions and methods for genome editing.

[0105] Vectors for delivering exogenous nucleic acids to target cells Viral Vectors for Nucleic Acid Delivery Viral genomes provide a rich source of vectors that can be used to efficiently deliver genes of interest into the genome of target cells (e.g., mammalian cells, such as human cells). Viral genomes are particularly useful vectors for gene delivery, because the polynucleotides contained within their genomes are typically integrated into the genome of target cells by general or specific transduction. These processes occur as part of the natural viral replication cycle, and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors include AAV, retroviruses, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive strand RNA viruses such as picornaviruses and alphaviruses, as well as double-stranded DNA viruses, including adenoviruses, herpes viruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and pox viruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses useful for delivering polynucleotides encoding the antibody light and heavy chains or antibody fragments of the invention include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis sarcoma, mammalian C, B, D viruses, HTLV-BLV complex, lentivirus, and spumavirus (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).Other examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in U.S. Patent No. 5,801,030, the disclosure of which is incorporated herein by reference with respect to viral vectors for use in gene therapy.

[0106] AAV vectors for nucleic acid delivery In some embodiments, the nucleic acids of the compositions and methods described herein are incorporated into recombinant AAV (rAAV) vectors and / or virions to facilitate their introduction into cells. rAAV vectors useful in the present invention are recombinant nucleic acid constructs that include (1) a transgene to be expressed (e.g., a polynucleotide encoding a frataxin protein) and (2) viral nucleic acid that facilitates the integration and expression of a heterologous gene. The viral nucleic acid may include those sequences of AAV required in cis for DNA replication and packaging into virions (e.g., functional ITRs). In a typical application, the transgene encodes frataxin, which is useful for correcting frataxin deficiency in patients suffering from Friedreich's ataxia. Such rAAV vectors may also include a marker or reporter gene. In useful rAAV vectors, one or more AAV WT genes are deleted in whole or in part, but functional flanking ITR sequences are retained. The AAV ITRs may be of any serotype (e.g., from serotype 2) suitable for a particular application. In some embodiments, the AAV ITRs can be AAV serotype 2 ITRs. Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279-291 (2000), and Monahan and Samulski, Gene Delivery 7:24-30 (2000), the disclosures of each of which are incorporated herein by reference with respect to AAV vectors for gene delivery.

[0107] The nucleic acids and vectors described herein can be incorporated into rAAV virions to facilitate the introduction of the nucleic acid or vector into cells. The capsid protein of AAV constitutes the outer non-nucleic acid portion of the virion and is encoded by the AAV cap gene. In some embodiments, the AAV of the present invention comprises recombinant capsid protein. The cap gene encodes three viral coat proteins VP1, VP2, and VP3, which are required for virion assembly. Construction of rAAV virions is described, for example, in U.S. Pat. Nos. 5,173,414, 5,139,941, 5,863,541, 5,869,305, 6,057,152, and 6,376,237, as well as in Rabinowitz et al., J. Virol. 76:791-801 (2002), and Bowles et al., J. Virol. 77:423-432 (2003), the disclosures of each of which are incorporated by reference herein with respect to AAV vectors for gene delivery.

[0108] rAAV virions useful in combination with the compositions and methods described herein include those derived from various AAV serotypes, including AAV1, 2, 3, 4, 5, 6, 7, 8 and 9. For targeting muscle cells, rAAV virions containing at least one serotype 1 capsid protein may be particularly useful. rAAV virions containing at least one serotype 6 capsid protein may also be particularly useful, since serotype 6 capsid protein is structurally similar to serotype 1 capsid protein and is therefore also expected to result in high expression of FXN in muscle cells. rAAV serotype 9 has also been found to be an efficient transducer of muscle cells. The construction and use of AAV vectors of different serotypes and AAV proteins are described, for example, in Chao et al., Mol. Ther. 2:619-623 (2000), Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428-3432 (2000), Xiao et al., J. Virol. 72:2224-2232 (1998), Halbert et al., J. Virol. 74:1524-1532 (2000), Halbert et al., J. Virol. 75:6615-6624 (2001), and Auricchio et al., Hum. Molec. Genet. 10:3075-3081 (2001), the disclosures of each of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0109] Also useful in combination with the compositions and methods described herein are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with a capsid gene from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). For example, a representative pseudotyped vector is an AAV8 or AAV9 vector encoding a therapeutic protein (e.g., frataxin) pseudotyped with a capsid gene from AAV serotype 2. Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662-7671 (2001), Halbert et al., J. Virol. 74:1524-1532 (2000), Zolotukhin et al., Methods, 28:158-167 (2002), and Auricchio et al., Hum. Molec. Genet., 10:3075-3081 (2001).

[0110] AAV virions with mutations in the virion capsid can be used to infect specific cell types more efficiently than capsid virions without the mutations. For example, suitable AAV mutants can have ligand insertion mutations to facilitate targeting of AAV to specific cell types. The construction and characterization of AAV capsid mutants, including insertion mutants, alanine screening mutants, and epitope tag mutants, are described in Wu et al., J. Virol. 74:8635-45 (2000). Other rAAV virions that can be used in the methods of the present invention include capsid hybrids generated by molecular breeding and exon shuffling of viruses. See, for example, Soong et al., Nat. Genet., 25:436-439 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423-428 (2001).

[0111] Exemplary AAV Vectors As described herein, exemplary AAV vector components may include a promoter, an intron, a polynucleotide encoding human FXN, or a polynucleotide encoding hFXNco, and / or a polyadenylation site (pA).

[0112] In some embodiments, the AAV is encoded by a prokaryotic promoter (P Euk ). In some embodiments, the AAV may include a muscle-specific promoter. In some embodiments, the muscle-specific promoter is a phosphoglycerate kinase (PGK) promoter, a desmin promoter, a muscle creatine kinase promoter, a myosin light chain promoter, a myosin heavy chain promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, an actin alpha promoter, an actin beta promoter, an actin gamma promoter, or a promoter in eye intron 1 paired like homeodomain 3, a cytomegalovirus promoter, or a chicken-β-actin promoter. For example, in some embodiments, the muscle-specific promoter is a PGK promoter.

[0113] In some embodiments, the PGK promoter has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:2. For example, in some embodiments, the PGK promoter has a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the PGK promoter has a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the PGK promoter has a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the PGK promoter has a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the PGK promoter has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the PGK promoter has a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the PGK promoter has a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the PGK promoter has a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the PGK promoter has a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the PGK promoter has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the PGK promoter has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the PGK promoter has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the PGK promoter has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the PGK promoter has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO:2.In some embodiments, the PGK promoter has a nucleic acid sequence that is identical to the nucleic acid sequence of SEQ ID NO:2.

[0114] In some embodiments, the AAV comprises an intron. In some embodiments, the intron is an SV40 intron.

[0115] In some embodiments, the intron is positioned 5' to the polynucleotide.

[0116] In some embodiments, hFXN, or its RNA equivalent, has a polynucleotide sequence that is at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) identical to an endogenous RNA molecule encoding frataxin protein variant 1 (e.g., SEQ ID NO:5). For example, in some embodiments, the polynucleotide exhibits at least 95% sequence identity to an endogenous RNA molecule encoding frataxin protein variant 1 (e.g., SEQ ID NO:5). In some embodiments, the polynucleotide exhibits at least 96% sequence identity to an endogenous RNA molecule encoding frataxin protein variant 1 (e.g., SEQ ID NO:5). In some embodiments, the polynucleotide exhibits at least 97% sequence identity to an endogenous RNA molecule encoding frataxin protein variant 1 (e.g., SEQ ID NO:5). In some embodiments, the polynucleotide exhibits at least 98% sequence identity to an endogenous RNA molecule encoding frataxin protein variant 1 (e.g., SEQ ID NO:5). In some embodiments, the polynucleotide exhibits at least 99% sequence identity to an endogenous RNA molecule encoding frataxin protein variant 1 (e.g., SEQ ID NO: 5). In some embodiments, the polynucleotide is identical to an endogenous RNA molecule encoding frataxin protein variant 1 (e.g., SEQ ID NO: 5).

[0117] In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO:3. For example, in some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 86% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 87% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 88% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 89% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 90% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 91% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 92% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 93% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 94% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 95% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 96% identical to the amino acid sequence of SEQ ID NO:3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 97% identical to the amino acid sequence of SEQ ID NO:3.In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 98% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is at least 99% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, hFXN, or its RNA equivalent, encodes a protein that is identical to the amino acid sequence of SEQ ID NO: 3.

[0118] In some embodiments, hFXNco, or its RNA equivalent, has a polynucleotide sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:1. For example, in some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO:1.In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, hFXNco, or its RNA equivalent, has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 1.

[0119] In some embodiments, the polynucleotide has a nucleic acid sequence that is identical to the nucleic acid sequence of SEQ ID NO:1.

[0120] In some embodiments, the AAV comprises a polyadenylation site (pA). For example, the pA site can be selected from a non-limiting list including the SV40 late polyadenylation site, the SV40 early polyadenylation site, the human β-globin polyadenylation site, or the bovine growth hormone polyadenylation site. In some embodiments, the AAV comprises the SV40 late polyadenylation site.

[0121] In some embodiments, pA is positioned 3' to the polynucleotide.

[0122] As described herein, exemplary AAV vector components include the human phosphoglycerate kinase (hPGK) promoter, hFXNco, as described herein, exemplified by the nucleic acid sequences in Table 2 shown below, the Simian Virus 40 (SV40) intron, and the SV40 late polyadenylation site.

[0123] [Table 2]

[0124] In some embodiments, the AAV has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequence of SEQ ID NO:4. For example, in some embodiments, the AAV has a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the AAV has a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the AAV has a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the AAV has a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the AAV has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the AAV has a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the AAV has a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the AAV has a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the AAV has a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the AAV has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the AAV has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the AAV has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the AAV has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the AAV has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the AAV has a nucleic acid sequence identical to the nucleic acid sequence of SEQ ID NO:4.

[0125] As described herein, an exemplary AAV2 / 8 vector having the nucleic acid sequence of SEQ ID NO:4 is shown below.

[0126] In some embodiments, the components are operably linked to each other in the 5'-3' direction as ITR1-FXN-ITR2. In some embodiments, the components are operably linked to each other in the 5'-3' direction as ITR1-P Euk In some embodiments, the components are operably linked to each other in the 5'-3' direction as ITR1-P-ITR2. Euk In some embodiments, the components are operably linked to each other in the 5'-3' direction as ITR1-P Euk -intron-FXN-pA-ITR2 is operably linked thereto.

[0127] In some embodiments, the components are operably linked to each other in the 5'-3' orientation as ITR1-hFXNco-ITR2. In some embodiments, the components are operably linked to each other in the 5'-3' orientation as ITR1-promoter-hFXNco-ITR2. In some embodiments, the components are operably linked to each other in the 5'-3' orientation as ITR1-promoter-hFXNco-pA-ITR2. In some embodiments, the components are operably linked to each other in the 5'-3' orientation as ITR1-promoter-intron-hFXNco-pA-ITR2.

[0128] In some embodiments, ITR1-FXN-ITR2 are, in combination, about 3.7 Kb to about 4.3 Kb (e.g., about 3.8 Kb to about 4.2 Kb or about 3.9 Kb to about 4.1 Kb). For example, in some embodiments, ITR1-FXN-ITR2 are, in combination, about 3.8 Kb to about 4.2 Kb in length. In some embodiments, ITR1-FXN-ITR2 are, in combination, about 3.9 Kb to about 4.1 Kb in length. In some embodiments, ITR1-FXN-ITR2 are, in combination, about 4.0 Kb in length.

[0129] In some embodiments, ITR1-FXN-ITR2, together, are about 3.7 Kb in length. In some embodiments, ITR1-FXN-ITR2, together, are about 3.8 Kb in length. In some embodiments, ITR1-FXN-ITR2, together, are about 3.9 Kb in length. In some embodiments, ITR1-FXN-ITR2, together, are about 4.0 Kb in length. In some embodiments, ITR1-FXN-ITR2, together, are about 4.1 Kb in length. In some embodiments, ITR1-FXN-ITR2, together, are about 4.2 Kb in length. In some embodiments, ITR1-FXN-ITR2, together, are about 4.3 Kb in length.

[0130] In some embodiments, the ITR1-hFXNco-ITR2, combined, are about 3.7 Kb to about 4.3 Kb (e.g., about 3.8 Kb to about 4.2 Kb or about 3.9 Kb to about 4.1 Kb). For example, in some embodiments, the ITR1-hFXNco-ITR2, combined, are about 3.8 Kb to about 4.2 Kb in length. In some embodiments, the ITR1-hFXNco-ITR2, combined, are about 3.9 Kb to about 4.1 Kb in length. In some embodiments, the ITR1-hFXNco-ITR2, combined, are about 4.0 Kb in length.

[0131] In some embodiments, ITR1-hFXNco-ITR2, together, are about 3.7 Kb in length. In some embodiments, ITR1-hFXNco-ITR2, together, are about 3.8 Kb in length. In some embodiments, ITR1-hFXNco-ITR2, together, are about 3.9 Kb in length. In some embodiments, ITR1-hFXNco-ITR2, together, are about 4.0 Kb in length. In some embodiments, ITR1-hFXNco-ITR2, together, are about 4.1 Kb in length. In some embodiments, ITR1-hFXNco-ITR2, together, are about 4.2 Kb in length. In some embodiments, ITR1-hFXNco-ITR2, together, are about 4.3 Kb in length.

[0132] In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 3.9 Kb to about 4.7 Kb (e.g., about 4.0 Kb to about 4.6 Kb, about 4.1 Kb to about 4.5 Kb, about 4.2 Kb to about 4.4 Kb, or about 4.3 Kb). For example, in some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.0 Kb to about 4.7 Kb. In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.1 Kb to about 4.7 Kb. In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.2 Kb to about 4.7 Kb. In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.3 Kb to about 4.7 Kb. In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.4 Kb to about 4.7 Kb. In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.5 Kb to about 4.7 Kb.

[0133] In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 3.9 Kb. In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.0 Kb. In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.1 Kb. In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.2 Kb. In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.3 Kb. In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.4 Kb. In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.5 Kb. In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.6 Kb. In some embodiments, the length of the nucleic acid between and including ITR1 and ITR2 is about 4.7 Kb.

[0134] In some embodiments, the plasmid (e.g., transfer vector) comprises an AAV as described herein. In some embodiments, the plasmid may comprise one or more (e.g., two) spacers. The spacer may comprise a naturally occurring nucleic acid molecule or a synthetic nucleic acid molecule. Naturally occurring spacer molecules can be identified using online web tools, e.g., the UCSC genome browser, and can be selected based on intrinsic features of the nucleic acid molecule, e.g., the natural occurrence of the nucleic acid adjacent to the transcription start site. The spacer may be engineered to remove potentially negative features that may result in toxicity if introduced by the viral particle, or to suppress the function of the viral particle. Exemplary sources of toxicity and suppressive features are found in contaminating nucleic acids that are frequently found adjacent to the nucleic acid encoding the viral genome to be packaged. These contaminating nucleic acids include, but are not limited to, prokaryotic (e.g., bacterial and baculovirus) nucleic acids (e.g., origins of replication, nucleic acids with a CpG content greater than 2%, open reading frames, and transcription factor binding sites). In some embodiments, the spacer is designed to minimize the inclusion of contaminating nucleic acids. In some embodiments, one or more of the spacers does not contain an open reading frame more than 100 amino acids in length. In some embodiments, one or more of the spacers does not contain a prokaryotic transcription factor binding site.

[0135] For example, in some embodiments, the plasmid contains two spacers, the two spacers including a first spacer (SS1) and a second spacer (SS2). In some embodiments, SS1 is located 5' to ITR1, and SS2 is located 3' to ITR2. In some embodiments, SS1 is about 1.0 Kb to about 5.0 Kb (e.g., about 1.5 Kb to about 4.5 Kb, about 2.0 Kb to about 4.0 Kb, or about 3.0 Kb) in length. In some embodiments, SS2 is about 1.0 Kb to about 5.0 Kb (e.g., about 1.5 Kb to about 4.5 Kb, about 2.0 Kb to about 4.0 Kb, or about 3.0 Kb) in length.

[0136] Treatment method Friedreich's ataxia Friedreich's ataxia is a neurological and cardiodegenerative disease caused by genetic alterations in the frataxin gene that reduce frataxin polypeptide expression. Identification of effective agents for the treatment of Friedreich's ataxia has previously been hampered by the difficulties associated with achieving therapeutically effective amounts of frataxin expression in affected tissues.

[0137] The present invention is based in part on the identification of new uses for some existing drugs, namely, for treating Friedreich's ataxia or other neurodegenerative diseases.These drugs include the nucleic acid molecules described herein.These drugs increase the level of frataxin protein, which is a characteristic defect of Friedreich's ataxia, in transgenic mouse models of Friedreich's ataxia.

[0138] Suitable patients for treatment include individuals who are at risk of disease but do not show symptoms, and patients who currently show symptoms.Generally, subjects are homozygous for mutations (GAA expansion or point mutations) that inhibit or reduce the expression level of frataxin.For subjects who are homozygous for mutations in frataxin gene that cause insufficient expression level of frataxin polypeptide, the risk of developing symptoms of Friedreich's ataxia generally increases with age.Therefore, in certain embodiments, for asymptomatic subjects who are homozygous for mutations in frataxin gene that cause insufficient expression level of frataxin polypeptide, prophylactic application is contemplated in subjects who are more than 3 years old, for example, subjects who are more than about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more years old.Subjects who develop late or very late onset of disease as above can also be treated.

[0139] In some embodiments, the subject exhibits symptoms of Friedreich's ataxia, such as weakness of the arms and legs, loss of coordination, loss of deep tendon reflexes, loss of plantar extensor response, loss of vibration and proprioception, impaired vision, involuntary and / or rapid eye movements, impaired hearing, slurred speech, curvature of the spine (scoliosis), high plantar arches (pes cavus deformity), carbohydrate intolerance, diabetes, and cardiac disorders (e.g., atrial fibrillation, tachycardia (fast heart rate), hypertrophic cardiomyopathy, cardiac hypertrophy, symmetric hypertrophy, heart murmurs, and cardiac conduction defects).

[0140] In some embodiments, the disclosure provides a method of treating Friedreich's ataxia in a human patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a polynucleotide described herein.

[0141] In some embodiments, the disclosure provides a method of increasing frataxin expression in a human patient diagnosed with Friedreich's ataxia, the method comprising administering to the patient a therapeutically effective amount of a polynucleotide described herein.

[0142] Efficacy monitoring Clinical efficacy may be monitored using biomarkers, among others. Measurable biomarkers for monitoring efficacy include, but are not limited to, monitoring one or more of the physical symptoms of Friedreich's ataxia, including: muscle weakness in arms and legs, loss of coordination, loss of deep tendon reflexes, loss of plantar extensor response, loss of vibration and proprioception, impaired vision, involuntary and / or rapid eye movements, impaired hearing, slurred speech, curvature of the spine (scoliosis), high plantar arch (pes cavus deformity), carbohydrate intolerance, diabetes, and cardiac disorders (e.g., atrial fibrillation, tachycardia (fast heart rate), hypertrophic cardiomyopathy, cardiac hypertrophy, symmetric hypertrophy, heart murmurs, and cardiac conduction defects). Observation of stabilization, improvement, and / or reversal of one or more symptoms indicates that the treatment or prevention plan is effective. Observation of progression, increase, or worsening of one or more symptoms indicates that the treatment or prevention plan is not effective. A preferred biomarker for evaluating treatment in Friedreich's ataxia is the level of frataxin.This marker is preferably evaluated at the protein level, but the measurement of the mRNA encoding frataxin can also be used as a surrogate measurement of frataxin expression.Such levels can be measured in blood samples.Such levels are reduced in subjects with Friedreich's ataxia compared to a control population of unaffected individuals.Therefore, an increase in level provides an indication of favorable treatment response, while an unchanged or decreased level provides an indication of unfavorable or at least suboptimal treatment response.

[0143] Efficacy can also be determined by determining the level of sclerosis and / or degeneration of dorsal root ganglion, spinocerebellar bundle, lateral corticospinal bundle, and dorsal column.This can be achieved using medical imaging techniques, such as magnetic resonance imaging or tomography techniques, such as computed tomography (CT) scan or computed axial tomography (CAT) scan.Subjects that maintain the same level or maintain reversal of sclerosis and / or degeneration indicate that the treatment or prevention plan is effective.Conversely, subjects that show higher levels or progression of sclerosis and / or degeneration indicate that the treatment or prevention plan is not effective.

[0144] In certain embodiments, monitoring methods may involve determining a baseline value of a measurable biomarker or disease parameter in a subject prior to administration of a dosage of a polynucleotide or plasmid (e.g., a viral vector) described herein and comparing this to the value of the same measurable biomarker or parameter after a course of treatment.

[0145] In other methods, a control value (i.e., mean and standard deviation) of a measurable biomarker or parameter is determined for a control population. In certain embodiments, the individuals in the control population have not been previously treated, do not have Friedreich's ataxia, and are not at risk of developing Friedreich's ataxia. In such cases, if the value of the measurable biomarker or clinical parameter approaches the control value, the treatment is considered to be effective. In other embodiments, the individuals in the control population have not been previously treated and have been diagnosed with Friedreich's ataxia. In such cases, if the value of the measurable biomarker or clinical parameter approaches the control value, the treatment is considered to be ineffective.

[0146] In other methods, subjects who are not currently undergoing treatment but have previously undergone a course of treatment are monitored for one or more of the biomarkers or clinical parameters to determine whether treatment should be resumed. Measurements of one or more biomarkers or clinical parameters in a subject can be compared to values ​​previously achieved in the subject after a previous course of treatment. Alternatively, the values ​​measured in a subject can be compared to control values ​​(mean + standard deviation) determined in a population of subjects after undergoing a course of treatment. Alternatively, measurements in a subject can be compared to control values ​​in a population of prophylactically treated subjects who remain free of disease symptoms, or in a population of therapeutically treated subjects who show improvement in disease characteristics. In such cases, if the value of the measurable biomarker or clinical parameter approaches the control value, the treatment is considered to be effective and does not need to be resumed. In all of these cases, a significant difference from the control level (i.e., more than a standard deviation) is an indication that treatment should be resumed in the subject.

[0147] In some embodiments, upon administration of a nucleic acid molecule described herein, the patient exhibits a change in whole blood frataxin levels, e.g., in some embodiments, the patient exhibits a change in whole blood frataxin levels by about 12 weeks after administration.

[0148] In some embodiments, upon administration of a nucleic acid molecule described herein, the patient exhibits a reduction in total Friedreich's Ataxia Rating Scale (FARS) score. For example, in some embodiments, the patient exhibits a reduction in total FARS score by about 12 weeks after administration.

[0149] Methods for measuring gene expression The expression level of a gene expressed by a single cell or cell type (e.g., a cell belonging to a particular tissue) can be ascertained, for example, by assessing the concentration or relative abundance of an RNA transcript (e.g., mRNA) derived from transcription of the gene of interest. Additionally or alternatively, gene expression can be determined by assessing the concentration or relative abundance of a protein produced by transcription and translation of the gene of interest. Protein concentration can also be assessed using functional assays, such as enzyme assays or gene transcription assays, when the gene of interest encodes an enzyme or a regulator of transcription, respectively. The following sections describe exemplary techniques that can be used to measure and rank the expression levels of genes in a cell, cell type, or cell population of interest, for example, at the level of a single cell or cell population. Expression of genes in a sample can be analyzed by a number of methodologies, many of which are known in the art and understood by those of skill in the art, including, but not limited to, nucleic acid sequencing, microarray analysis, proteomics, in situ hybridization (e.g., fluorescent in situ hybridization (FISH)), amplification-based assays, in situ hybridization, fluorescence activated cell sorting (FACS), Northern analysis of mRNA, and / or PCR analysis.

[0150] (i) Nucleic acid detection A nucleic acid-based dataset suitable for analyzing target cell-specific gene expression can have the form of a gene expression profile that represents the identity of the genes expressed in a cell of interest and the degree to which the genes are expressed, which can be used to determine a ranked order of gene expression levels within a cell, cell type, or population of cells of interest. Such a profile can include whole transcriptome sequencing data (e.g., RNA-Seq data), a panel of mRNA, non-coding RNA, or any other nucleic acid sequence that can be expressed from genomic DNA. Other nucleic acid datasets suitable for use with the methods described herein can include expression data collected by imaging-based techniques (e.g., Northern blotting or Southern blotting, as known in the art). Northern blot analysis is a conventional technique well known in the art and is described, for example, in Molecular Cloning, a Laboratory Manual, second edition, 1989, Sambrook, Fritch, Maniatis, Cold Spring Harbor Press, 10 Skyline Drive, Plainview, NY 11803-2500, the disclosure of which is incorporated herein by reference. Exemplary protocols for assessing the status of genes and gene products can be found, for example, in Ausubel et al., eds., 1995, Curr Protoc Mol Biol, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis), the disclosures of which are incorporated herein by reference.

[0151] Gene expression profiles analyzed in conjunction with the methods described herein may include, for example, microarray data or nucleic acid sequencing data generated by sequencing methods known in the art (e.g., Sanger sequencing and next-generation sequencing methods, also known as high-throughput or deep sequencing). Exemplary next-generation sequencing technologies include, but are not limited to, Illumina sequencing, Ion Torrent sequencing, 454 sequencing, SOLiD sequencing, and nanopore sequencing platforms. Additional sequencing methods known in the art may also be used. For example, mRNA expression levels may be determined using RNA-Seq (e.g., as described in Mortazavi et al., Nat. Methods 5:621-628 (2008), the disclosure of which is incorporated herein by reference in its entirety). RNA-Seq is a robust technique known in the art for monitoring expression by directly sequencing RNA molecules in a sample. Briefly, this methodology may involve fragmenting RNA to an average length of 200 nucleotides, converting it to cDNA by random priming, and synthesizing double-stranded cDNA (e.g., using the Just cDNA DoubleStranded cDNA Synthesis Kit from Agilent Technology). The cDNA is then converted into molecular libraries for sequencing by adding sequence adaptors for each library (e.g., from Illumina® / Solexa), and the resulting 50-100 nucleotide reads are mapped onto the genome.

[0152] Because microarray technology offers high resolution, gene expression levels can be determined using microarray-based platforms (e.g., single nucleotide polymorphism (SNP) arrays). Details of various microarray methods can be found in the literature. See, for example, U.S. Pat. No. 6,232,068 and Pollack et al., Nat. Genet. 23:41-46 (1999), the disclosures of each of which are incorporated herein by reference in their entirety. Using nucleic acid microarrays, mRNA samples are reverse transcribed and labeled to generate cDNA. The probes can then be hybridized to one or more complementary nucleic acids arrayed and immobilized on a solid support. The arrays can be constructed, for example, such that the sequence and position of each member of the array is known. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene. Expression levels can be quantified according to the amount of signal detected from the hybridized probe-sample complex. A typical microarray experiment includes the following steps: The process includes the steps of: 1) preparation of fluorescently labeled targets from RNA isolated from a sample, 2) hybridization of the labeled targets to a microarray, 3) washing, staining, and scanning of the array, 4) analysis of the scanned image, and 5) generation of a gene expression profile. One example of a microarray processor is the commercially available Affymetrix GENECHIP® system, which includes arrays manufactured by direct synthesis of oligonucleotides on a glass surface. Other systems known to those skilled in the art may also be used.

[0153] Amplification-based assays can also be used to measure the expression level of one or more markers (e.g., genes). In such assays, the nucleic acid sequence of the gene serves as a template in an amplification reaction (e.g., PCR, such as qPCR). In quantitative amplification, the amount of amplified product is proportional to the amount of template in the original sample. Comparison with an appropriate control provides a measure of the expression level of the gene corresponding to the specific probe used, according to the principles described herein. Methods of real-time qPCR using TaqMan probes are well known in the art. Detailed protocols for real-time qPCR are provided, for example, in Gibson et al., Genome Res. 6:995-1001 (1996) and Heid et al., Genome Res. 6:986-994 (1996), the disclosures of each of which are incorporated herein by reference. The level of gene expression described herein can be determined by RT-PCR techniques. The probes used in PCR may be labeled with a detectable marker, such as, for example, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme.

[0154] (ii) Protein detection Gene expression can additionally be determined by measuring the concentration or relative abundance of the corresponding protein product encoded by the gene of interest. Protein levels can be assessed using standard detection techniques known in the art. Examples of protein expression analyses that generate data suitable for use with the methods described herein include, but are not limited to, proteomic approaches, immunohistochemistry and / or Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, enzyme-linked immunofiltration assays (ELIFA), mass spectrometry, mass spectrometry immunoassays, and biochemical enzyme activity assays. In particular, proteomic methods can be used to multiplex and generate large protein expression data sets. Proteomic methods may utilize capture reagents (e.g., antibodies) specific for a panel of target proteins to detect and quantify polypeptides (e.g., proteins) and / or peptide microarrays, and utilize mass spectrometry to identify and measure the expression levels of proteins expressed in a sample (e.g., a single cell sample or a multi-cell population).

[0155] An exemplary peptide microarray has multiple polypeptides bound to a substrate, and the binding of an oligonucleotide, peptide, or protein to each of the multiple binding polypeptides is separately detectable. Alternatively, the peptide microarray may include multiple binding agents, including, but not limited to, monoclonal antibodies, polyclonal antibodies, phage display binding agents, yeast two-hybrid binding agents, and aptamers, that can specifically detect the binding of a particular oligonucleotide, peptide, or protein. Examples of peptide arrays can be found in U.S. Patent Nos. 6,268,210, 5,766,960, and 5,143,854, the disclosures of each of which are incorporated herein by reference.

[0156] Mass spectrometry (MS) can be used in conjunction with the methods described herein to identify and characterize gene expression profiles of single cells or multi-cell populations. Any MS method known in the art may be used to determine, detect, and / or measure one or more peptides of interest (e.g., LC-MS, ESI-MS, ESI-MS / MS, MALDI-TOF-MS, MALDI-TOF / TOF-MS, tandem MS, etc.). Mass spectrometers generally include an ion source and optics, a mass analyzer, and data processing electronics. Mass analyzers include scanning and ion beam mass analyzers, such as time-of-flight (TOF) and quadrupole (Q), as well as trapping mass analyzers, such as ion trap (IT), orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR), and can be used in the methods described herein. Details of various MS methods can be found in the literature. See, e.g., Yates et al., Annu. Rev. Biomed. Eng. 11:49-79 (2009), the disclosure of which is incorporated herein by reference.

[0157] Prior to MS analysis, proteins in a sample may first be digested into smaller peptides by chemical (e.g., via cyanogen bromide cleavage) or enzymatic (e.g., trypsin) digestion. Complex peptide samples also benefit from the use of front-end separation techniques, such as 2D-PAGE, HPLC, RPLC, and affinity chromatography. The digested and optionally separated sample is then ionized using an ion source to generate charged molecules for further analysis. Ionization of the sample may be performed, for example, by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB) / liquid secondary ionization (LSIMS), matrix-assisted laser desorption / ionization (MALDI), field ionization, field desorption, thermospray / plasma spray ionization, and particle beam ionization. Additional information related to the selection of an ionization method is known to those skilled in the art.

[0158] After ionization, the digested peptides may then be fragmented to generate a signature MS / MS spectrum. Tandem MS, also known as MS / MS, may be particularly useful for the methods described herein that allow for the ionization and subsequent fragmentation of complex peptide samples, such as samples obtained from multicellular populations described herein. Tandem MS involves multiple steps of MS selection, with some form of ion fragmentation occurring between stages, which may be accomplished using individual mass analyzer elements separated in space, or using a single mass analyzer with MS steps separated in time. In spatially separated tandem MS, the elements are physically separated, with physical connections between the elements to maintain a high vacuum. In time separated tandem MS, separation is achieved with ions trapped in the same location, with multiple separation steps occurring over time. The signature MS / MS spectrum may then be compared to a peptide sequence database (e.g., SEQUEST). Post-translational modifications to the peptide may also be determined, for example, by searching the spectrum against the database, allowing for specific peptide modifications.

[0159] Pharmaceutical Compositions The compositions, nucleic acid molecules, and plasmids described herein can be formulated into pharmaceutical compositions for administration to patients, such as human patients exhibiting or at risk of Friedreich's ataxia, in a biologically compatible form suitable for administration in vivo. For example, a pharmaceutical composition containing a nucleic acid molecule comprising one or more transgenes described herein typically comprises a pharma- ceutically acceptable diluent or carrier. The pharmaceutical composition can comprise (e.g., consist of), for example, sterile saline and nucleic acid. The sterile saline is typically pharmaceutical grade saline. The pharmaceutical composition can comprise (e.g., consist of), for example, sterile water and nucleic acid. The sterile water is typically pharmaceutical grade water. The pharmaceutical composition can comprise (e.g., consist of), for example, phosphate buffered saline (PBS) and nucleic acid. The sterile PBS is typically pharmaceutical grade PBS.

[0160] In certain embodiments, pharmaceutical compositions include one or more compositions or nucleic acid molecules and one or more excipients, hi certain embodiments, the excipients are selected from water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0161] In certain embodiments, the nucleic acid molecules may be mixed with pharma- ceutically acceptable active and / or inactive substances for the preparation of pharmaceutical compositions or formulations. The compositions and methods for formulating pharmaceutical compositions depend on several criteria, including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

[0162] In certain embodiments, the pharmaceutical composition comprising the nucleic acid molecule includes any pharma- ceutically acceptable salt of the inhibitor, an ester of the inhibitor, or a salt of such an ester. In certain embodiments, the pharmaceutical composition comprising the nucleic acid molecule, when administered to a subject (e.g., a human), can result (directly or indirectly) in a biologically active metabolite or residue thereof. Thus, for example, the present disclosure is also drawn to pharma- ceutically acceptable salts of the inhibitor, prodrugs, pharma- ceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharma- ceutically acceptable salts include, but are not limited to, sodium and potassium salts. In certain embodiments, the prodrugs include one or more conjugate groups attached to the nucleic acid molecule, and the conjugate groups are cleaved by endogenous nucleases in the body.

[0163] Lipid moieties have been used in various ways in nucleic acid therapy. In certain such methods, nucleic acids are introduced into preformed liposomes or lipoplexes made from a mixture of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or polycationic lipids are formed in the absence of neutral lipids. In certain embodiments, lipid moieties are selected to increase the distribution of pharmaceuticals to certain cells or tissues. In certain embodiments, lipid moieties are selected to increase the distribution of pharmaceuticals to adipose tissue. In certain embodiments, lipid moieties are selected to increase the distribution of pharmaceuticals to muscle tissue.

[0164] In certain embodiments, the pharmaceutical composition comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions, such as those that include hydrophobic compounds. In certain embodiments, certain organic solvents, such as dimethylsulfoxide, are used.

[0165] In certain embodiments, the pharmaceutical composition comprises one or more tissue-specific delivery molecules designed to deliver one or more pharmaceutical agents of the present invention to a particular tissue or cell type. For example, in certain embodiments, the pharmaceutical composition comprises a liposome coated with a tissue-specific antibody.

[0166] In certain embodiments, the pharmaceutical composition includes a co-solvent system. Certain such co-solvent systems include, for example, benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of 3% (w / v) benzyl alcohol, 8% (w / v) of the non-polar surfactant Polysorbate 80™, and 65% (w / v) of polyethylene glycol 300 in absolute ethanol. The proportions of such a co-solvent system may vary significantly without significantly altering its solubility and toxicity properties. Furthermore, the identity of the co-solvent components may be varied, for example, other surfactants may be used in place of Polysorbate 80™, the fractional amount of polyethylene glycol may be changed, other biocompatible polymers, for example, polyvinylpyrrolidone, may replace polyethylene glycol, and dextrose may be replaced with other sugars or polysaccharides.

[0167] In certain embodiments, the pharmaceutical composition is prepared for oral administration. In certain embodiments, the pharmaceutical composition is prepared for buccal administration. In certain embodiments, the pharmaceutical composition is prepared for administration by injection (e.g., intraocular (e.g., intravitreal), intravenous, subcutaneous, intramuscular, intrathecal, intraventricular, intraparenchymal, etc.). In certain such embodiments, the pharmaceutical composition includes a carrier and is formulated in an aqueous solution such as water or a physiologically compatible buffer, e.g., Hank's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients (e.g., ingredients that aid in solubility or act as preservatives) are included. In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents, and the like. Certain injectable pharmaceutical compositions are presented as unit dosage forms, e.g., in ampoules or in multi-dose containers. Certain injectable pharmaceutical compositions are suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending agents, stabilizing agents, and / or dispersing agents. Particular solvents suitable for use in injectable pharmaceutical compositions include, but are not limited to, lipophilic solvents and fatty oils such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.

[0168] kit The compositions described herein can be provided in a kit for use in treating Friedreich's ataxia. The kit can include one or more compositions, nucleic acid molecules, plasmids, or pharmaceutical compositions described herein. The kit can include a package insert that instructs the user of the kit, e.g., a physician, to perform any one of the methods described herein. The kit can optionally include a syringe or other device for administering the composition. In some embodiments, the kit can include one or more additional therapeutic agents. EXAMPLES

[0169] The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein may be used and evaluated, are intended to be purely illustrative of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0170] Example 1. Materials and Methods Codon optimization of the frataxin gene for efficient protein expression and restoration of the expansion of the intron trinucleotide repeat GAA upon codon optimization The FXN isoform 1 gene sequence, excluding intron DNA, is as follows:

[0171] The frataxin isoform 1 amino acid sequence is as follows: MWTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRKSGTLGHPGSLDETTYERLAEETLDSLAEFFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAELTKALKTKLDLSSLAYSGKDA (SEQ ID NO: 3)

[0172] Analysis of SEQ ID NO:5 reveals specific codon preferences for various amino acids throughout the gene. Examination of codon frequency reveals that for certain amino acids, certain codons are used predominantly, while other codons are used less frequently or not at all. One skilled in the art can rationally design variants of the FXN gene, for example, to increase protein stability, reduce local GC content, and / or reduce mRNA secondary structure and unstable motifs. Codon-optimized engineered variants of the FXN gene that contain reduced CpG content and / or reduced homopolymer content to enhance translation of frataxin. For example, one skilled in the art can engineer the gene sequence encoding frataxin by incorporating codon substitutions that reduce the CpG content and / or homopolymer content of the engineered FXN gene (Figure 1). For example, in the FXN isoform 1 gene sequence above, there is an example of the homopolymer GGGGGG. The homopolymer may be the site of a frameshift mutation in the formation of the mRNA transcript and / or during the translation process. If this homopolymer sequence remains in the codon-optimized FXN gene even after minimizing the sequence identity of the gene to endogenously expressed genes in the target cell, one skilled in the art can incorporate additional mutations that interrupt this homopolymer while maintaining the identity of the encoded protein. Alternatively, if the homopolymer encodes an amino acid residue that is not essential for protein function (e.g., if the encoded amino acid is not present in an active site that mediates the function of frataxin (e.g., the active site that mediates the assembly of the iron-sulfur cluster)), one skilled in the art can incorporate codon substitutions that interrupt the homopolymer and introduce conservative substitutions into the encoded protein at the corresponding amino acid site.

[0173] Furthermore, the final codon-optimized gene may exhibit at least 95% sequence identity to an endogenous RNA molecule encoding frataxin variant 1 (SEQ ID NO:5). For example, the final codon-optimized gene may exhibit at least 96%, 97%, 98%, or 99% sequence identity to an endogenous RNA molecule encoding frataxin variant 1 (SEQ ID NO:5). In another example, the final codon-optimized gene may have a nucleic acid sequence identical to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the codon-optimized human FXN gene is

[0174] For nucleic acids 8 to 15 of SEQ ID NO:5, the nucleic acid sequence:

[0175] It may contain a modified Kozak sequence including TIFF2024534415000004.tif13170.

[0176] Once designed, the final codon-optimized gene can be prepared, for example, by solid-phase nucleic acid procedures known in the art. Techniques for solid-phase synthesis of polynucleotides are known in the art and are described, for example, in U.S. Pat. No. 5,541,307, the disclosure of which is incorporated herein by reference as it relates to solid-phase polynucleotide synthesis and purification. In addition, the prepared gene can be amplified, for example, using PCR-based techniques described herein or known in the art, and / or by transforming DH5α E. coli with a plasmid containing the designed gene. The bacteria can then be cultured to amplify the DNA therein, and the gene can be isolated plasmid purification techniques known in the art, optionally followed by restriction digestion and / or sequencing of the plasmid to verify the identity of the codon-optimized gene.

[0177] Immunofluorescence Biopsied hearts were fixed overnight in 10% formalin and then stored in 70% ethanol. Tissues were embedded in paraffin and cut into 5 μm sections.

[0178] For immunofluorescence analysis, tissues were permeabilized, slides were washed, and blocked with 5% goat serum for 30 minutes. Sections were incubated with primary antibody anti-frataxin (Abcam 175402, 2ug / mL, recognizing human, mouse, and rat) for 1 hour at room temperature. Slides were incubated with secondary goat anti-rabbit antibody (Thermo A11008, AlexaFluor 488 conjugate, 4μg / mL) for 30 minutes. Evaluation included detailed quantification of frataxin expression.

[0179] Codon-optimized human frataxin constructs PCR primers were designed to bind and amplify 633 base pairs of FXN nucleic acid sequence from genomic DNA isolated from HEK293 cells by PCR. Using the codon optimization method described above, the isolated FXN was modified by site-directed mutagenesis (Stratgene) to reduce CpG content. The resulting modified amplification product was gel purified and sequenced by methods known in the art.

[0180] A pseudotyped adeno-associated virus (AAV) 2 / 8 (AAV2 / 8) circular parent vector was used as the destination vector for the codon-optimized human FXN variant 1 (hFXNco). The parent vector contains a first spacer (SS1), a first AAV2 inverted terminal repeat (ITR1), a synthetic DNA stuffer, a human phosphoglycerate kinase (PGK) promoter, a simian virus 40 (SV40) intron, a late SV40 polyadenylation site (pA), a second synthetic DNA stuffer, a second AAV2 ITR (ITR2), a prokaryotic origin of replication, a second spacer (SS2), and a kanamycin antibiotic selection gene (Kan R) (Figure 2). The parent vector also contains a cloning site containing a PmeI restriction endonuclease recognition site 5' to the first AAV2 ITR1 and a SwaI restriction endonuclease recognition site 3' to the second AAV2 ITR2. hFXNco was cloned into the parent vector, and the resulting vector contained the following in the 5'-3' direction: SS1-AAV2 ITR1-PGK-SV40 inron-SV40 LpA-AAV2 ITR2-SS2-oriC-Kan, as mediated by ligation with T4 DNA ligase for 1 hour at 16°C. R The resulting vector contained operably linked nucleic acid components as follows: AAV2 / 8-PGK-FXN, PGK-FXN, FXN- ...

[0181] Proof-of-principle experiments were performed using a cell line (C2C12) derived from mouse skeletal muscle cells, and the vector preparation resulted in robust frataxin expression in transduced C2C12 cells, as shown by immunofluorescence (Figures 3A and 3B).

[0182] Friedreich's ataxia mouse model Mice homozygous for the conditional allele of FXN (FXN L3 / L3 ) was transformed with FXN exon 4 (FXN Δ / + ) deletion to induce the striated muscle-restricted exon 4 deletion (see, e.g., Puccio et al., Nat. Genet. 27(2)(2001):181-186). Typically, mutants begin to lose weight in approximately 7 weeks, gradually develop signs of fatigue, and die in 76±10 days.

[0183] MCK knockout (KO) mice were used as a model of Friedreich's ataxia to demonstrate the safety and efficacy of a codon-optimized human frataxin construct, as described in Example 2.

[0184] Immunoblot analysis Western blot analysis was performed on whole cell extracts prepared in radioimmunoprecipitation assay (RIPA) buffer. Proteins were separated on 4-15% polyacrylamide gradient-sodium dodecyl sulfate (SDS) gels (Bio-Rad) and transferred onto nitrocellulose membranes (Invitrogen). Western blots were visualized by Enhanced Chemiluminescence (Perkin-Elmer). The primary antibody was used against frataxin (Invitrogen #45-6300), and the secondary antibody was Licor IR Dye 800 CW Donkey anti-mouse. As a result, pseudotyped AAV2 / 8 viral vectors encoding hFXNco effectively expressed the mature isoform of FXN (Figure 4).

[0185] Example 2. Efficacy of codon-optimized human FXN on heart phenotype and mortality in a model of Friedreich's ataxia This example describes the safety and efficacy of a codon-optimized FXN gene, including its human and mouse genes, for amelioration of cardiac phenotypes and premature mortality associated with Friedreich's ataxia, e.g., in a mouse model of Friedreich's ataxia.

[0186] Materials and Methods Materials and methods are described in Example 1. This example tested AAV2 / 8-PGK-FXN version 2 (V2).

[0187] result 5 and 6A show the 3×10 13 vg / kg or 1×10 14Figure 6 shows the probability of survival of FRDA MCK KO mice administered intravenously with an exemplary AAV2 / 8 encoding the human PGK promoter driving expression of hFXNco (hFXN) or its mouse equivalent (mFXN) at 1000 ng / kg, or vehicle control. Four weeks (weeks, wks) after administration, vehicle KO mice showed a sharp decline in the probability of survival, while mice administered hFXN or mFXN survived comparably to vehicle WT controls. In addition, significant rescue was observed in female and male body weight (Figure 6B), heart weight normalized by body weight (Figure 6C), and ejection fraction (Figure 6D) in KO mice administered hFXN or mFXN compared to vehicle KO mice. Figures 7A and 7B show the levels of frataxin expression in cardiac biopsies taken from mice from the same experiment, measured by Western blot and quantified by vector copy number (VCN, Figure 7A) or frataxin protein levels (Figure 7B), respectively. In the heart, no differences in VCN were observed between dose levels at 4 weeks after intravenous administration. As shown in FIG. 7B, a 3.7-fold and 13.4-fold increase in cardiac frataxin was observed in mFXN-treated KO mice compared to vehicle-treated WT mice (WT average approx. 136 ng / mg), relative to the readouts shown in FIG. 5. Furthermore, a 3.7-fold and 23-fold increase in cardiac frataxin was observed in hFXN- or mFXN-treated KO mice compared to vehicle-treated WT mice, relative to the subsequent readouts shown in FIG. 6A. FIG. 8 shows the levels of frataxin protein in cardiac tissue as measured by enzyme-linked immunosorbent assay (ELISA) at 4 and 12 weeks after administration of AAV2 / 8-PGK-FXN. As shown in FIG. 8, at 4 weeks post-treatment, a 3-fold and 13-fold increase in cardiac frataxin was observed in mFXN-treated KO mice compared to vehicle-treated WT mice, and a 7.3-fold increase in cardiac frataxin was observed in hFXN-treated KO mice compared to vehicle-treated WT mice.As shown in Figure 8, at 12 weeks post-administration, a 5-fold and 21.2-fold increase in cardiac frataxin was observed in mFXN-treated KO mice compared to vehicle-treated WT mice, and a 17.9-fold increase in cardiac frataxin was observed in hFXN-treated KO mice compared to vehicle-treated WT mice. Figure 9 shows vector copy number / diploid genome detected in cardiac tissue as measured by qPCR at 4 and 12 weeks post-administration. Vector DNA was detected in cardiac tissue of all AAV2 / 8-PGK-FXN-treated KO mice at 4 and 12 weeks post-administration, but not in vehicle-treated mice at either time point. As shown in Figure 9, at 12 weeks post-administration, 3x10 13 vg / kg mFXN-treated mice compared with hFXN or 1 × 10 14 Significantly more vector copies / genome were detected in mFXN-treated mice at 1000 mg / kg.

[0188] Figure 10A shows immunohistochemistry of frataxin expression in cardiac tissue. The percentage of cells showing frataxin expression was 3×10 compared to WT mice (54.4%). 13 The results were similar in KO mice receiving AAV2 / 8 plasmid encoding mFXN at 1 × 10 vg / kg (58.6%), whereas the results were similar in KO mice receiving AAV2 / 8 plasmid encoding mFXN at 1 × 10 14 increased (78–85%) in KO mice administered AAV2 / 8 plasmids encoding hFXN or mFXN at 10 vg / kg ( Fig. 10B ).

[0189] Western blot analysis revealed expression of the mature isoform of frataxin in tissues taken from mice treated with hFXN or mFXN. In addition, myosin light chain, a marker of myocardial injury, was reduced in the serum of mice treated with hFXN or mFXN (Figure 6E), and no obvious toxicity or fibrosis was observed in the hearts of treated mice 4 weeks after administration.

[0190] Taken together, these results show that an exemplary AAV2 / 8 plasmid encoding hFXNco rescued mortality in a mouse model of Friedreich's ataxia and induced significant expression of frataxin in the heart.

[0191] Example 3. Use of the codon-optimized FXN gene for the treatment of Friedreich's ataxia The gene encoding frataxin can be codon-optimized using the procedures described herein (e.g., as described in Example 1 above). The gene may be codon-optimized for the purpose of reducing the CpG and / or homopolymer content in the mRNA transcript or adapting the codon bias, for example, by introducing codon substitutions into the optimized FXN gene sequence, as described in Example 1 above. For example, the final codon-optimized FXN gene may exhibit at least 95% sequence identity to the endogenous RNA molecule encoding frataxin variant 1 (SEQ ID NO:5). For example, the final codon-optimized FXN gene may exhibit at least 96%, 97%, 98%, or 99% sequence identity to the endogenous RNA molecule encoding frataxin variant 1 (SEQ ID NO:5). In another example, the final codon-optimized gene may have a nucleic acid sequence identical to the nucleic acid sequence of SEQ ID NO:1.

[0192] The gene can then be incorporated into a plasmid, such as a viral vector, and administered to a patient suffering from Friedreich's ataxia.For example, a patient suffering from Friedreich's ataxia, a disorder characterized by mutations in the FXN gene, can be administered a viral vector containing a codon-optimized FXN gene under the control of a suitable promoter for expression in human cells, such as human muscle cells.For example, an AAV vector can be generated, such as a pseudotyped AAV2 / 8 vector, that incorporates a codon-optimized FXN gene between the 5' and 3' inverted terminal repeats of the vector, and the gene can be placed under the control of a muscle-specific promoter, such as the PGK promoter.The AAV vector can be administered systemically to the subject.

[0193] Those skilled in the art can monitor the expression of the codon-optimized FXN gene by a variety of methods. For example, those skilled in the art can transfect cultured cells, such as C2C12 cells, with the codon-optimized gene to model the expression of the codon-optimized gene in the muscle of a patient. The expression of the encoded protein can then be monitored using expression assays described herein, such as, for example, qPCR, RNA-Seq, ELISA, or immunoblot procedures. Based on the data obtained from the gene expression assay, further iterations of the codon optimization procedure can be performed, for example, to further reduce the CpG content and homopolymer content in the mRNA transcript. The candidate gene sequence with the optimal expression pattern in vitro can then be prepared for incorporation into a suitable vector and administered to a mammalian subject, for example, an animal model of Friedreich's ataxia, or a human patient.

[0194] Example 4. Treatment of Friedreich's ataxia in human patients by administration of codon-optimized human FXN Using the compositions and methods disclosed herein, a patient (e.g., ages 3 to 17) with Friedreich's ataxia may be administered a pseudotyped AAV2 / 8 vector comprising a nucleic acid sequence encoding a codon-optimized human FXN variant 1 gene operably linked to an AAV vector having a PGK promoter, e.g., the nucleic acid of SEQ ID NO:4.

[0195] When a pseudotyped AAV2 / 8 vector comprising a nucleic acid sequence encoding a codon-optimized human FXN variant 1 gene is administered to a patient, the patient exhibits a change in whole blood frataxin levels. For example, the patient exhibits a change in whole blood frataxin levels up to about 12 weeks after administration of a pseudotyped AAV2 / 8 vector comprising a nucleic acid sequence encoding a codon-optimized human FXN variant 1 to the patient. Additionally or alternatively, when a pseudotyped AAV2 / 8 vector comprising a nucleic acid sequence encoding a codon-optimized human FXN variant 1 gene is administered to a patient, the patient exhibits a decrease in total Friedreich's Ataxia Rating Scale (FARS) score. For example, the patient exhibits a decrease in total FARS score up to about 12 weeks after administration of a pseudotyped AAV2 / 8 vector comprising a nucleic acid sequence encoding a codon-optimized human frataxin variant 1 to the patient.

[0196] Example 5. Use of a nucleic acid molecule having a short length of ITR1 to ITR2 and the FXN gene for the treatment of Friedreich's ataxia The gene encoding frataxin can be cloned into a plasmid (e.g., a viral vector) using procedures described herein (e.g., as described in Example 1 above). The parental plasmid can contain a short length, e.g., ITR1 to ITR2, with a payload of about 3.7 Kb to about 4.3 Kb (e.g., about 3.8 Kb to about 4.2 Kb or about 3.9 Kb to about 4.1 Kb). Additionally or alternatively, the length of nucleic acid between and including ITR1 and ITR2 can be about 3.9 Kb to about 4.7 Kb (e.g., about 4.0 Kb to about 4.6 Kb, about 4.1 Kb to about 4.5 Kb, about 4.2 Kb to about 4.4 Kb, or about 4.3 Kb). The parental plasmid can then be administered to a patient suffering from Friedreich's ataxia. For example, a patient suffering from Friedreich's ataxia, a disorder characterized by mutations in the FXN gene, can be administered a viral vector containing a plasmid comprising a nucleic acid molecule comprising ITR1-FXN-ITR2, which together are about 3.7 Kb to about 4.3 Kb in length. The FXN gene can be under the control of a suitable promoter for expression in human cells, such as human muscle cells. For example, an AAV vector can be generated, such as a pseudotyped AAV2 / 8 vector, that incorporates a codon-optimized FXN gene between the 5' and 3' inverted terminal repeats of the vector, and the gene can be placed under the control of a muscle-specific promoter, such as the PGK promoter. The AAV vector can be administered systemically to the subject. The gene can be codon-optimized, for example, to reduce CpG and / or homopolymer content in the mRNA transcript, or to accommodate codon bias, by introducing codon substitutions into the optimized FXN gene sequence, as described in Example 1 above. In another example, the final codon-optimized gene can have a nucleic acid sequence identical to the nucleic acid sequence of SEQ ID NO:1.

[0197] Those skilled in the art can monitor the expression of the codon-optimized FXN gene by a variety of methods. For example, those skilled in the art can transfect cultured cells, such as C2C12 cells, with the codon-optimized gene to model the expression of the codon-optimized gene in the muscle of a patient. The expression of the encoded protein can then be monitored using expression assays described herein, such as, for example, qPCR, RNA-Seq, ELISA, or immunoblot procedures. Based on the data obtained from the gene expression assay, further iterations of the codon optimization procedure can be performed, for example, to further reduce the CpG content and homopolymer content in the mRNA transcript. The candidate gene sequence with the optimal expression pattern in vitro can then be prepared for incorporation into a suitable vector and administered to a mammalian subject, for example, an animal model of Friedreich's ataxia, or a human patient.

[0198] Example 6. Treatment of Friedreich's ataxia in human patients by administration of a nucleic acid molecule having a short stretch of ITR1 to ITR2 and the FXN gene Using the compositions and methods of the present disclosure, a patient (e.g., age 3-17) with Friedreich's ataxia may be administered a pseudotyped AAV2 / 8 vector comprising a nucleic acid sequence encoding frataxin, where the AAV2 / 8 comprises ITR1 and ITR2 flanking the FXN gene, and the ITR1-FXN-ITR2 together is about 3.7 Kb to about 4.3 Kb (e.g., about 3.8 Kb to about 4.2 Kb or about 3.9 Kb to about 4.1 Kb) in length. Additionally or alternatively, the length of the nucleic acid between and including ITR1 and ITR2 may be about 3.9 Kb to about 4.7 Kb (e.g., about 4.0 Kb to about 4.6 Kb, about 4.1 Kb to about 4.5 Kb, about 4.2 Kb to about 4.4 Kb, or about 4.3 Kb). The FXN gene may be under the control of a suitable promoter for expression in a human cell, such as a human muscle cell. For example, the gene may be placed under the control of a muscle-specific promoter, such as the PGK promoter. The gene may be codon-optimized to reduce the CpG and / or homopolymer content in the mRNA transcript or to accommodate codon bias, for example by introducing codon substitutions into the optimized FXN gene sequence, as described in Example 1 above. In another example, the final codon-optimized gene may have a nucleic acid sequence identical to the nucleic acid sequence of SEQ ID NO:1.

[0199] When a patient is administered a pseudotyped AAV2 / 8 vector comprising an ITR1-FXN-ITR2 length of about 3.7 Kb to about 4.3 Kb, the patient exhibits a change in whole blood frataxin levels. For example, the patient exhibits a change in whole blood frataxin levels up to about 12 weeks after administration of a pseudotyped AAV2 / 8 vector comprising an ITR1-FXN-ITR2 length of about 3.7 Kb to about 4.3 Kb to the patient. Additionally or alternatively, when a patient is administered a pseudotyped AAV2 / 8 vector comprising an ITR1-FXN-ITR2 length of about 3.7 Kb to about 4.3 Kb, the patient exhibits a decrease in total Friedreich's Ataxia Rating Scale (FARS) score. For example, the patient exhibits a decrease in total FARS score up to about 12 weeks after administration of a pseudotyped AAV2 / 8 vector comprising an ITR1-FXN-ITR2 length of about 3.7 Kb to about 4.3 Kb to the patient.

[0200] Example 7. Comparison of expression of codon-optimized human FXN in pseudotyped AAV2 / 8 vectors with ITR1-FXN-ITR2 lengths of 1.5 Kb and 4.3 Kb An exemplary pseudotyped AAV2 / 8 vector original version (V1) was generated that encodes the human PGK promoter to drive expression of hFXNco, as described above in Example 1. The AAV2 / 8 vector contains a first spacer (SS1), a first AAV2 inverted terminal repeat (ITR1), a human phosphoglycerate kinase (PGK) promoter, a Simian Virus 40 (SV40) intron, a codon-optimized human FXN gene (hFXNco) late SV40 polyadenylation site (pA), a second AAV2 ITR (ITR2), a prokaryotic origin of replication, a second spacer (SS2), and a kanamycin antibiotic selection gene (Kan R ) nucleic acid molecules having components such as

[0201] A second version (V2) of an exemplary pseudotyped AAV2 / 8 vector was generated that encodes the human PGK promoter to drive expression of hFXNco, as described above in Example 1. The AAV2 / 8 vector contains a first spacer (SS1), a first AAV2 inverted terminal repeat (ITR1), a synthetic DNA stuffer, a human phosphoglycerate kinase (PGK) promoter, a Simian Virus 40 (SV40) intron, a codon-optimized human FXN gene (hFXNco) late SV40 polyadenylation site (pA), a second synthetic DNA stuffer, a second AAV2 ITR (ITR2), a prokaryotic origin of replication, a second spacer (SS2), and a kanamycin antibiotic selection gene (Kan R ) (Figure 2). This second version (V2) of AAV2 / 8-PGK-FXN differs from V1 by the presence of two synthetic DNA stuffers to achieve an optimal sequence length of 4.3 Kb between ITR1 and ITR2. A comparison of AAV2 / 8-PGK-FXN V1 and V2 is summarized in Table 3 below.

[0202] [Table 3]

[0203] To test the expression of FXN in AAV2 / 8-PGK-FXN V2, mouse and human muscle cell lines were transduced with increasing doses of AAV2 / 8-PGK-FXN V2. Dose-dependent FXN expression was observed after transduction with AAV2 / 8-PGK-FXN V2 in mouse (Figure 11A) and human (Figure 11B) muscle cell lines. When compared to AAV2 / 8-PGK-FXN V1, V2 was expressed at the same level (Figures 12A and 12B) and was correctly processed to yield 14 kDa mature FXN (Figure 12C). Codon-optimized human FXN showed similar expression levels as WT human FXN in AAV2 / 8-PGK-FXN V2 when treating C2C12 or AB1079 cells (Figure 13).

[0204] Example 8. Efficacy of codon-optimized human FXN on cardiac phenotype and mortality in a mouse model of Friedreich's ataxia This example describes the safety and efficacy of a codon-optimized FXN (hFXNco) gene for amelioration of Friedreich's ataxia-associated cardiac phenotype and premature mortality in a mouse model of Friedreich's ataxia.

[0205] Materials and Methods Mice were administered a single dose of version 1 (V1 positive control) or version 2 (V2) of an exemplary AAV8 vector encoding the human PGK promoter to drive expression of hFXNco (AAV8-PGK-FXN) intravenously at 6 weeks of age to evaluate the efficacy and toxicity of the codon-optimized FXN gene in the Friedreich's ataxia mouse model (described in Example 1 above). Table 4 below lists the study parameters for each treatment group.

[0206] [Table 4]

[0207] result Survival studies were performed to evaluate the effect of hFXNco on mortality. Prior to scheduled biopsies, mice were euthanized if the animals showed any of the following conditions: loss of more than 20% of body weight, signs of respiratory distress, unresponsiveness to meaningful stimuli, and / or poor overall body condition. Median age at time of euthanasia was significantly different between vehicle-treated (non-transduced control) and all other treatment groups, with a dose-dependent rescue of mortality observed from AAV8-PGK-FXN V1 and V2 treated animals (Figure 14).

[0208] Cardiac function was assessed using high frequency ultrasound over a series of studies starting at 6 weeks of age (WOA) to either endpoints of 9-10 WOA or 18-19 WOA (as detailed in Table 4). Administration of AAV8-PGK-FXN V1 and V2 rescued cardiac function as demonstrated by increased ejection fraction (Figure 15) and left ventricular fractional shortening (Figure 16), as well as reduced left ventricular mass (Figure 17). AAV8-PGK-FXN V1 and V2 reduced serum levels of cardiac injury markers cardiac troponin (Figure 18) and myosin light chain (Figure 19), but no significant changes in AST and ALT were observed (Figures 20 and 21).

[0209] AAV8-PGK-FXN V2 transduction and expression was assessed by measuring vector copy number (VCN), mRNA, and FXN protein expression in the heart, liver, and quadriceps. Dose-dependent VCN was detected in the heart, liver, and quadriceps (Figures 22-24), with VCN in the quadriceps being approximately 3-fold lower than VCN in the heart in most groups (Figure 23), indicating better transduction in the heart than in the quadriceps. FXN mRNA was detected in a dose-dependent manner in the heart and quadriceps of mutant mice treated with AAV8-PGK-FXN V2 (Figure 25). FXN protein expression was increased in a dose-dependent manner in the heart (Figure 26), liver (Figure 27), and quadriceps (Figure 28), with the protein produced by AAV8-PGK-FXN V2 showing much lower expression in the quadriceps than in the heart.

[0210] conclusion Collectively, these results show that an exemplary AAV8 plasmid, V2, encoding hFXNco, under the control of the human PGK promoter, rescued mortality and cardiac function in a mouse model of Friedreich's ataxia and induced significant expression of frataxin in the heart, liver, and quadriceps.

[0211] Example 9. Efficacy of codon-optimized human FXN on central nervous system phenotypes in a neuron-specific mouse model of Friedreich's ataxia This example describes the efficacy of a codon-optimized human FXN (hFXNco) gene in a neuron-specific Friedreich's ataxia mouse model for amelioration of central nervous system (CNS) phenotypes associated with Friedreich's ataxia.

[0212] Materials and Methods Mice were administered a single intracerebroventricular (ICV) or intraparenchymal (IPC) injection of an exemplary AAV8 vector, version 1 (V1 positive control) or version 2 (V2), encoding the human PGK promoter, resulting in expression of hFXNco (AAV8-PGK-FXN) at 8 weeks of age to evaluate the efficacy and toxicity of the codon-optimized FXN gene in a neurospecific Friedreich's ataxia mouse model.

[0213] Mice homozygous for frataxin floxed exon 2 were crossed with mice heterozygous for both PV-Cre knock-in and frataxin global KO to identify Fxn flox / null A ::PV-Cre genotype was induced, which is compound heterozygous at the frataxin locus (floxed exon 2 and global KO on each homologous chromosome) and heterozygous for the PV-Cre knock-in allele. flox / null The ::PV-Cre mice harbor a Cre-conditional frataxin allele, a global knockout frataxin allele, and a parvalbumin neuronal-specific Cre recombinase knock-in allele, generating an early-onset ataxia mouse model useful for the study of Friedreich's ataxia.

[0214] Fxn as a model for Friedreich's ataxia flox / null The efficacy of a codon-optimized human frataxin construct was demonstrated using ::PV-Cre knockout (KO) mice.

[0215] Table 5 below lists the study parameters for each treatment group.

[0216] [Table 5]

[0217] result The effect of hFXNco on motor coordination was evaluated using the rotarod performance test. Treatment with AAV8-PGK-FXN V2 significantly improved the time taken to fall compared to the vehicle (non-transduced control) mutant (Figure 29).

[0218] AAV8-PGK-FXN V2 transduction and expression was assessed by measuring vector copy number (VCN) and FXN protein expression in CNS tissues. Dose-dependent VCN results were observed in animals administered via ICV at 8 weeks of age, with brain VCN showing high variability (Figure 30). IPC-administered animals showed 100-fold higher VCN in the cerebellum and approximately 10-fold higher VCN in the cortex than animals administered via ICV at 8 weeks of age (Figure 31). FXN protein expression was observed in the cortex and cerebellum of animals administered AAV8-PGK-FXN V2 at 8 weeks of age, with IPC injection improving AAV8-PGK-FXN V2 delivery to the cerebellum (Figure 32).

[0219] The cerebellum is a key manifestation of Friedreich's ataxia because the disease causes damage to the cerebellar portion of the brain. After AAV8-PGK-FXN V2 dosing, the cerebellum produced higher amounts of FXN protein per the same amount of VCN than other tissues (Figure 33), indicating that less AAV8-PGK-FXN V2 is required for therapeutic benefit in the cerebellum.

[0220] Neurofilament light chain (NFLC) is a neuron-specific cytoskeletal protein released into the extracellular fluid after axonal injury and is recognized as an important biomarker in many neurodegenerative diseases. After treatment with AAV8-PGK-FXN V2, mutant mice were found to have reduced levels of the NFLC neuronal injury marker (Figure 34).

[0221] conclusion Taken together, these results show that V2 of an exemplary AAV8 plasmid encoding hFXNco, under the control of the human PGK promoter, rescued the CNS phenotype and induced expression of frataxin in CNS tissue in a neurospecific mouse model of Friedreich's ataxia.

[0222] Other embodiments All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0223] While the invention has been described with reference to specific embodiments, it will be understood that the invention is capable of further modifications, and this application is generally intended to cover any variations, uses, or adaptations of the invention in accordance with the principles of the invention, including departures from the invention which come within known or customary practice in the art to which the invention pertains and which may be applicable to the essential characteristics as hereinbefore described, and which comply with the scope of the appended claims.

[0224] Other embodiments are within the scope of the claims.

Claims

1. A DNA polynucleotide encoding human frataxin (hFXN) or its RNA equivalent, said polynucleotide having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:

1.

2. 2. The polynucleotide of claim 1, wherein the polynucleotide has a nucleic acid sequence that is at least 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:

1.

3. 3. A vector comprising the polynucleotide of claim 1 or 2, optionally a plasmid, a DNA vector, an RNA vector, a virion, or a viral vector. (i) the vector is a viral vector; (ii) the polynucleotide is operably linked to a muscle-specific promoter, optionally positioned 5′ to the polynucleotide; or (iii) the vector further comprises a polyadenylation site (pA), optionally positioned 3′ to the polynucleotide; The vector described in claim 3. (i) the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus, and synthetic virus, and optionally, the viral vector is AAV; (ii) the muscle-specific promoter is a phosphoglycerate kinase (PGK) promoter, a desmin promoter, a muscle creatine kinase promoter, a myosin light chain promoter, a myosin heavy chain promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, an actin alpha promoter, an actin beta promoter, an actin gamma promoter, or a promoter within paired eye intron 1 such as homeodomain 3, and optionally the muscle-specific promoter is a PGK promoter; (iii) the pA site comprises the simian virus 40 (SV40) late polyadenylation site, the SV40 early polyadenylation site, the human β-globin polyadenylation site, or the bovine growth hormone polyadenylation site; or (iv) the vector further comprises an intron, optionally positioned 3' to the promoter and 5' to the polynucleotide; The vector according to claim 4. (i) the AAV comprises capsid proteins from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, and AAVrh74; (ii) the viral vector is a pseudotyped AAV; (iii) the AAV comprises a recombinant capsid protein; (iv) the AAV further comprises two inverted terminal repeats (ITRs), the two ITRs comprising a first ITR (ITR1) and a second ITR (ITR2), wherein ITR1 is positioned 5′ to the polynucleotide and ITR2 is positioned 3′ to the polynucleotide to form a cassette comprising the structure ITR1-hFXN-ITR2; (v) the PGK promoter has a nucleic acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid of the sequence of SEQ ID NO:2; (vi) the pA site comprises the SV40 late polyadenylation site; or (vii) the intron is an SV40 intron; The vector according to claim 5 . (i) the pseudotyped AAV is AAV2 / 8 or AAV2 / 9, and optionally, the pseudotyped AAV is AAV2 / 8; (ii) the length of the nucleic acid between ITR1 and ITR2 is from about 3.7 Kb to about 4.3 Kb, optionally the length of the nucleic acid between ITR1 and ITR2 is from about 3.8 Kb to about 4.2 Kb, optionally the length of the nucleic acid between ITR1 and ITR2 is from about 3.9 Kb to about 4.1 Kb, and optionally the length of the nucleic acid between ITR1 and ITR2 is about 4.0 Kb; (iii) the length of the nucleic acid between and including ITR1 and ITR2 is from about 3.9 Kb to about 4.7 Kb, optionally the length of the nucleic acid between and including ITR1 and ITR2 is from about 4.1 Kb to about 4.5 Kb, and optionally the length of the nucleic acid between and including ITR1 and ITR2 is about 4.3 Kb; or (iv) the two ITRs are AAV serotype 2 ITRs; The vector described in claim 6.

8. A pharmaceutical composition comprising the polynucleotide of claim 1 and a pharmaceutically acceptable carrier, diluent, or excipient.

9. A nucleic acid molecule comprising: (i) ITR1; and (ii) hFXN or its RNA equivalent; (iii) ITR2, The components are aligned in the 5'-3' direction relative to each other. The nucleic acid molecule, wherein ITR1 and ITR2 are operably linked as ITR1-hFXN-ITR2, and the length of the nucleic acid between ITR1 and ITR2 is about 3.7 Kb to about 4.3 Kb. (a) the length of the nucleic acid between ITR1 and ITR2 is from about 3.8 Kb to about 4.2 Kb, optionally, the length of the nucleic acid between ITR1 and ITR2 is from about 3.9 Kb to about 4.1 Kb, and optionally, the length of the nucleic acid between ITR1 and ITR2 is about 4.0 Kb; (b) the length of the nucleic acid between and including ITR1 and ITR2 is from about 3.9 Kb to about 4.7 Kb, optionally the length of the nucleic acid between and including ITR1 and ITR2 is from about 4.1 Kb to about 4.5 Kb, and optionally the length of the nucleic acid between and including ITR1 and ITR2 is about 4.3 Kb; (c) the nucleic acid molecule is (iv) eukaryotic promoter (P Ｅｕｋ ) The components are arranged in a 5'-3' direction relative to each other ITR1-P Ｅｕｋ -operably linked as hFXN-ITR2; (d) the hFXN, or its RNA equivalent, encodes a protein having an amino acid sequence that is at least 85%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:3; or (e) the hFXN, or its RNA equivalent, has a nucleic acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid of the sequence of SEQ ID NO: 1; The nucleic acid molecule of claim 9.

11. The P Ｅｕｋ The nucleic acid molecule of claim 10, wherein the promoter is a PGK promoter.

12. 12. The nucleic acid molecule of claim 11, wherein the PGK promoter has a nucleic acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid of sequence SEQ ID NO:

2.

13. A vector comprising the nucleic acid molecule of any one of claims 9 to 12, which is optionally a plasmid, a DNA vector, an RNA vector, a virion, or a viral vector. (i) the vector is a viral vector; or (ii) ITR1 and / or ITR2 are parvoviral ITRs; The vector of claim 13. (i) the viral vector is selected from the group consisting of AAV, adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus, and synthetic virus, and optionally, the viral vector is AAV; or (ii) the parvovirus ITR is an AAV ITR; The vector of claim 14.

16. 4. The vector of claim 3, wherein the vector has a nucleic acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:

4.

17. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 9 to 12 and a pharmaceutically acceptable carrier, diluent, or excipient.

18. A method comprising: (i) a method of treating Friedreich's ataxia in a human patient in need thereof; or (ii) Methods for increasing frataxin expression in human patients diagnosed with Friedreich's ataxia. A composition comprising the polynucleotide of claim 1 for use in

19. 19. The composition of claim 18, wherein the patient is between 3 and 17 years old.

20. A method comprising: (i) a method of treating Friedreich's ataxia in a human patient in need thereof; or (ii) Methods for increasing frataxin expression in human patients diagnosed with Friedreich's ataxia. A composition comprising the nucleic acid according to any one of claims 9 to 12 for use in

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