WO2022147490A1 - Protéines liées à la fukutine optimisées et procédés d'utilisation - Google Patents

Protéines liées à la fukutine optimisées et procédés d'utilisation Download PDF

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WO2022147490A1
WO2022147490A1 PCT/US2022/011010 US2022011010W WO2022147490A1 WO 2022147490 A1 WO2022147490 A1 WO 2022147490A1 US 2022011010 W US2022011010 W US 2022011010W WO 2022147490 A1 WO2022147490 A1 WO 2022147490A1
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vector
synthetic polynucleotide
nucleotide sequence
seq
aav
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PCT/US2022/011010
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Xiao Xiao
Chunping Qiao
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The University Of North Carolina At Chapel Hill
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • the invention relates to synthetic polynucleotides encoding fukutin related protein (FKRP).
  • FKRP fukutin related protein
  • the invention further relates to nucleic acid constructs comprising the synthetic polynucleotides and methods of using these polynucleotides to treat dystroglycanopathy disorders (e.g., that involve a reduction in glycosylation of alpha-dystroglycan (a-DG).
  • dystroglycanopathy disorders e.g., that involve a reduction in glycosylation of alpha-dystroglycan (a-DG).
  • FKRP fukutin-related protein
  • the present invention overcomes previous shortcomings in the art by providing improved compositions and methods for treating diseases associated with FKRP mutations. SUMMARY OF THE INVENTION
  • the present invention is based in part on the surprising finding that double stranded or self-complementary AAV vectors and/or a novel CpG depleted polynucleotide encoding a fukutin-related protein (FKRP) provide improved therapeutic effects over what has previously been known. Accordingly, in one aspect, the invention provides a synthetic polynucleotide encoding a human FKRP, wherein the synthetic polynucleotide comprises the nucleotide sequence of SEQ ID NO:1; and/or a nucleotide sequence having at least 90% identity to SEQ ID NO:1
  • a synthetic polynucleotide encoding a human fukutin-related protein is provided, wherein the synthetic polynucleotide comprises the nucleotide sequence of SEQ ID NO:6 and/or a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO:6.
  • the invention provides a synthetic polynucleotide encoding a human fukutin-related protein (FKRP), wherein the synthetic polynucleotide comprises the nucleotide sequence of SEQ ID NO: 7 and/or a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO:7.
  • FKRP human fukutin-related protein
  • the invention provides a transformed cell comprising a synthetic polynucleotide of the invention and/or a vector comprising the synthetic polynucleotide of the invention.
  • the invention provides a transgenic animal comprising the synthetic polynucleotide, the vector, and/or the transformed cell of the invention.
  • a method of delivering a nucleic acid to a cell comprising delivering to the cell a synthetic polynucleotide of the invention, and/or a vector and/or a transformed cell comprising the same.
  • the invention provides a method of delivering a nucleic acid to a subject, the method comprising delivering to the subject a synthetic polynucleotide of the invention, and/or a vector and/or a transformed cell comprising the same.
  • the synthetic polynucleotide of the invention, and/or vector and/or transformed cell may be delivered to the subject in a therapeutically effective amount.
  • the invention provides a method of treating a dystroglycanopathy in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a synthetic polynucleotide of the invention, and/or a vector and/or a transformed cell comprising the same, thereby treating dystroglycanopathy in the subject.
  • the dystroglycanopathy comprises a mutation in the nucleic acid encoding FKRP and/or a deficiency in glycosylation of alpha-dystroglycan (a-DG), or any combination thereof.
  • the dystroglycanopathy is limb girdle muscular dystrophy 21, congenital muscular dystrophy, Walker-Warburg syndrome, muscle-eye-brain disease, or any combination thereof.
  • FIG. 1 Short-term and long-term therapeutic effects in reducing serum CK level mediated by AAV9-opti-hu-FKRP vectors driven by different promoters.
  • Panel A shows short term effects (3 months post injection);
  • Panel B shows long term effects (8 months post injection);
  • Panel C shows long term effects (15 months post injection).
  • * p ⁇ 0.05; ** p ⁇ 0.01 as compared to the control group.
  • the long-term effects group there was no statistical difference observed because of the limited animal numbers.
  • FIG. 2 Muscle functional analysis. Left panel, treadmill running distance test. Right panel. Grip force measurement. The muscle force (g) was normalized by body weight (g). * p ⁇ 0.05; ** p ⁇ 0.01 as compared to the control group.
  • FIG. 3 Protein expression of varied hu-FKRP AAV plasmids driven by different promoters revealed via immunofluorescent staining after transfection in 293 cells.
  • FIG. 5 Constructions of ds-AAV plasmids containing FKRP gene.
  • ITR inverted terminal repeat
  • D mu D-sequence mutation
  • synlOO synthetic muscle-specific promoter.
  • FIG. 6 Therapeutic effects mediated by different vectors in reducing serum CK level.
  • Panel A shows male 6weeks post injection;
  • Panel B shows mail 12 weeks post injection;
  • Panel C shows male 7.5 months post injection. **, p ⁇ 0.01 as compared to the untreated group.
  • the CpG(-) performed better than the CpC(+) vectors.
  • the terms “increase,” “increasing,” “enhance,” “enhancing,” “improve” and “improving” (and grammatical variations thereof) as used herein refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold or an increase of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 400%, 500% or more.
  • the terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” and “decrease” describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% as compared to a control.
  • the reduction can result in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.
  • the above terms are relative to a reference or control.
  • a method of this invention for reducing serum creatine kinase levels in a subject comprising delivering to the subject a synthetic polynucleotide of the invention, the reduction is relative to the amount of serum creatine kinase in a subject (e.g., a control subject) in the absence of delivery of the synthetic polynucleotide to that control subject.
  • a “treatment effective” amount or “therapeutically effective” amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject.
  • a “treatment effective” amount is an amount that will provide some alleviation, mitigation, decrease or stabilization in at least one clinical symptom in the subject.
  • Determination of a therapeutically effective amount, as well as other factors related to effective administration of a compound of the present invention to a subject of this invention, including dosage forms, routes of administration, and frequency of dosing, may depend upon the particulars of the condition that is encountered, including the subject and condition being treated or addressed, the severity of the condition in a particular subject, the particular compound being employed, the particular route of administration being employed, the frequency of dosing, and the particular formulation being employed. Determination of a therapeutically effective treatment regimen for a subject of this invention is within the level of ordinary skill in the medical or veterinarian arts. In clinical use, an effective amount may be the amount that is recommended by the U.S. Food and Drug Administration, or an equivalent foreign agency. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the subject being treated and the particular mode of administration.
  • an "treatment effective” amount or “therapeutically effective” amount can refer to an amount of a composition of this invention (e.g., the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:6, and/or SEQ ID NO:7; a nucleotide sequence having at least 90% identity to SEQ ID NO:1; a nucleotide sequence having at least 80% identity to SEQ ID NO:6 or SEQ ID NO:7; a nucleotide sequence encoding a FKRP that is CpG depleted, and/or an expression cassette, vector or transgenic cell comprising one or more of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:6, and/or SEQ ID NO:7; a nucleotide sequence having at least 90% identity to SEQ ID NO:1; a nucleotide sequence having at least 80% identity to SEQ ID NO:6 or SEQ ID NO:7) that is sufficient to produce a desired effect, which can be a therapeutic effect
  • the effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art.
  • an "effective amount" in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation. (See, for example, Remington, The Science And Practice of Pharmacy (20th ed. 2000)).
  • prevention effective amount is an amount that is sufficient to prevent and/or delay the onset of a disease, disorder and/or clinical symptoms in a subject and/or to reduce and/or delay the severity of the onset of a disease, disorder and/or clinical symptoms in a subject relative to what would occur in the absence of the methods of the invention.
  • level of prevention need not be complete, as long as some benefit is provided to the subject.
  • the efficacy of treating a dystroglycanopathy by the methods of the invention can be determined by detecting a clinical improvement as indicated by a change in the subject’s symptoms and/or clinical parameters as would be well known to one of skill in the art.
  • treat By the terms “treat,” “treating,” or “treatment,” it is intended that the severity of the subject's condition is reduced or at least partially improved or modified and that some alleviation, mitigation or decrease in at least one clinical symptom is achieved.
  • prevent refers to a decrease or delay in the extent or severity of a disease, disorder and/or clinical symptom(s) after onset relative to what would occur in the absence of carrying out the methods of the invention prior to the onset of the disease, disorder and/or clinical symptom(s).
  • preventing refers to the occurrence of an increase in glycosylation of alpha-dystroglycan (a-DG) as compared to the amount of glycosylation of alpha-dystroglycan (a-DG) that occurs in the absence of the therapeutic treatment.
  • a subject identified to have one or more mutations that are associated with a dystroglycanopathy characterized by reduced glycosylation of a-DG can be administered the synthetic/optimized polynucleotides of this invention to prevent/delay/alleviate onset of said dystroglycanopathy.
  • Such mutations are well known in the art.
  • Concurrently administering or “concurrently administer” as used herein means that the two or more compounds or compositions are administered closely enough in time to produce a combined effect (that is, concurrently may be simultaneously, or it may be two or more events occurring within a short time period before and/or after each other, e.g., sequentially). Simultaneous concurrent administration may be carried out by mixing the compounds prior to administration, or by administering the compounds at the same point in time but at different anatomic sites and/or by using different routes of administration.
  • “Pharmaceutically acceptable” as used herein means that the compound or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.
  • nucleic acid As used herein, “nucleic acid,” “nucleotide sequence,” and “polynucleotide” are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA and chimeras of RNA and DNA.
  • polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain.
  • the nucleic acid can be double-stranded or singlestranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand.
  • the nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides) or produced by cell biology techniques commonly used for vector production. Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • the present invention further provides a nucleic acid that is the complement (which can be either a full complement or a partial complement) of a nucleic acid, nucleotide sequence, or polynucleotide of this invention.
  • an "isolated polynucleotide” is a nucleotide sequence (e.g., an “isolated DNA” or an “isolated RNA”) that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • an isolated nucleic acid may include some or all of the 5' non-coding (e.g., promoter) sequences that are immediately contiguous to a coding sequence.
  • the term therefore includes, for example, a recombinant DNA/synthetic polynucleotide that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment), independent of other sequences. It also includes a recombinant DNA/synthetic polynucleotide that is part of a hybrid nucleic acid encoding an additional polypeptide or peptide sequence.
  • An isolated polynucleotide that includes a gene is not a fragment of a chromosome that includes such gene, but rather includes the coding region and regulatory regions associated with the gene, but no additional genes naturally found on the chromosome.
  • Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 C.F.R. ⁇ 1.822 and established usage.
  • modified refers to a sequence that differs from a wild-type sequence due to one or more deletions, additions, substitutions, or any combination thereof.
  • a “3’ portion” of a polynucleotide indicates a segment of the polynucleotide that is downstream of another segment.
  • the term “3’ portion” is not intended to indicate that the segment is necessarily at the 3’ end of the polynucleotide, or even that it is necessarily in the 3’ half of the polynucleotide, although it may be.
  • a “5’ portion” of a polynucleotide indicates a segment of the polynucleotide that is upstream of another segment.
  • polypeptide encompasses both peptides and proteins, unless indicated otherwise.
  • exogenous and/or heterologous can include a nucleotide sequence that is not naturally occurring in the nucleic acid construct and/or delivery vector (e.g., virus delivery vector) in which it is contained and can also include a nucleotide sequence that is placed into a non-naturally occurring environment and/or position relative to other nucleotide sequences (e.g., by association with a promoter or coding sequence with which it is not naturally associated).
  • delivery vector e.g., virus delivery vector
  • a heterologous or exogenous nucleotide sequence or amino acid sequence of this invention can be any heterologous nucleotide sequence and/or amino acid sequence that has been introduced into a cell and can include a nucleotide sequence and/or amino acid sequence for which an original version is already present in the cell and/or the heterologous nucleotide sequence or amino acid sequence can be introduced into a cell that does not naturally comprise the same nucleotide sequence and/or amino acid sequence.
  • sequence identity has the standard meaning in the art. As is known in the art, a number of different programs can be used to identify whether a polynucleotide or polypeptide has sequence identity or similarity to a known sequence. Sequence identity or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 45:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351 (1987); the method is similar to that described by Higgins & Sharp, CABIOS 5: 151 (1989).
  • BLAST BLAST algorithm
  • WU-BLAST-2 WU-BLAST-2 uses several search parameters, which are preferably set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • a percentage amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region.
  • the "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
  • percent nucleic acid sequence identity is defined as the percentage of nucleotide residues in the candidate sequence that are identical with the nucleotides in the polynucleotide specifically disclosed herein.
  • the alignment may include the introduction of gaps in the sequences to be aligned.
  • the percentage of sequence identity will be determined based on the number of identical nucleotides in relation to the total number of nucleotides.
  • sequence identity of sequences shorter than a sequence specifically disclosed herein will be determined using the number of nucleotides in the shorter sequence, in one embodiment.
  • percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as insertions, deletions, substitutions, etc.
  • identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of "0," which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations.
  • Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the one having the most actual residues in the aligned region.
  • synthetic polynucleotide refers to a polynucleotide sequence that does not exist in nature but instead is made by the hand of man, either chemically, or biologically (i.e., in vitro modified).
  • a synthetic polynucleotide may be a codon optimized human FKRP (e.g., SEQ ID NO:7) or it may be an optimized and CpG depleted human FKRP (e.g., SEQ ID NO:1; SEQ ID NO:6). Codon optimization may be as compared to wild type human FKRP (e.g., SEQ ID NO: 10).
  • operably linked or “operably associated” as used herein, it is meant that the indicated elements are functionally related to each other, and are also generally physically related.
  • operably linked or “operably associated” as used herein, refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated.
  • a first nucleotide sequence that is operably linked to a second nucleotide sequence means a situation when the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence.
  • a promoter is operably associated with a nucleotide sequence if the promoter effects the transcription or expression of said nucleotide sequence.
  • control sequences e.g., promoter
  • the control sequences need not be contiguous with the nucleotide sequence to which it is operably associated, as long as the control sequences function to direct the expression thereof.
  • intervening untranslated sequences that may be transcribed can be present between a promoter and a nucleotide sequence, and the promoter can still be considered "operably linked" to the nucleotide sequence.
  • a “promoter” is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (i.e., a coding sequence) that is operably associated with the promoter.
  • the coding sequence may encode a polypeptide and/or a functional RNA.
  • a “promoter” refers to a nucleotide sequence that contains a binding site for RNA polymerase II and directs the initiation of transcription.
  • promoters are found 5', or upstream, relative to the start of the coding region of the corresponding coding sequence.
  • the promoter region may comprise other elements that act as regulators of gene expression.
  • fragment as applied to a polynucleotide, will be understood to mean a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of, and/or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence.
  • a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.
  • such fragments can comprise, consist essentially of, and/or consist of oligonucleotides having a length of at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive nucleotides of a nucleic acid or nucleotide sequence of the invention (e.g., the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:6 and/or SEQ ID NO:7, a nucleotide sequence having at least 90% identity to SEQ ID NO:1 and/or a nucleotide sequence having at least 80% identity to SEQ ID NO:6 or SEQ ID NO:7).
  • a nucleic acid or nucleotide sequence of the invention e.g., the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:6 and/or SEQ ID NO:7, a nucleotide sequence having at least 90% identity to SEQ ID NO:1 and/or a nucleotide sequence having
  • isolated can refer to a nucleic acid, nucleotide sequence or polypeptide that is substantially free of cellular material, viral material, and/or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized).
  • an "isolated fragment” is a fragment of a nucleic acid, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be found in the natural state. "Isolated” does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose.
  • fragment as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence and comprising, consisting essentially of, and/or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence.
  • a polypeptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent.
  • such fragments can comprise, consist essentially of, and/or consist of peptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive amino acids of a polypeptide or amino acid sequence according to the invention.
  • the nucleic acid of this invention can be present in a vector and such a vector can be present in a cell.
  • a "vector” is any nucleic acid molecule for the cloning of and/or transfer of a nucleic acid into a cell.
  • a vector may be a replicon to which another nucleotide sequence may be attached to allow for replication of the attached nucleotide sequence.
  • a "replicon” can be any genetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome) that functions as an autonomous unit of nucleic acid replication in vivo, i.e., capable of replication under its own control.
  • any suitable vector is encompassed in the embodiments of this invention, including, but not limited to, nonviral vectors (e.g., plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acid-protein complexes, poloxymers and biopolymers), viral vectors (e.g., retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, and adenovirus vectors) and synthetic biological nanoparticles (BNP) (e.g, synthetically designed from different adeno- associated viruses, as well as other parvoviruses).
  • vector includes both viral and nonviral (e.g, plasmid) nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and/or in vivo.
  • any suitable vector can be used to deliver a heterologous nucleic acid of this invention.
  • the choice of delivery vector can be made based on a number of factors known in the art, including age and species of the target host, in vitro vs. in vivo delivery, level and persistence of expression desired, intended purpose (e.g., for therapy or polypeptide production), the target cell or organ, route of delivery, size of the isolated nucleic acid, safety concerns, and the like.
  • Suitable vectors also include virus vectors (e.g., retrovirus, alphavirus; vaccinia virus; adenovirus, adeno-associated virus, or herpes simplex virus), lipid vectors, poly-lysine vectors, synthetic polyamino polymer vectors that are used with nucleic acid molecules, such as plasmids, and the like.
  • virus vectors e.g., retrovirus, alphavirus; vaccinia virus; adenovirus, adeno-associated virus, or herpes simplex virus
  • lipid vectors e.g., retrovirus, alphavirus; vaccinia virus; adenovirus, adeno-associated virus, or herpes simplex virus
  • poly-lysine vectors e.g., lipid vectors
  • synthetic polyamino polymer vectors that are used with nucleic acid molecules, such as plasmids, and the like.
  • Protocols for producing recombinant viral vectors and for using viral vectors for nucleic acid delivery can be found, e.g., in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989) and other standard laboratory manuals e.g., Vectors for Gene Therapy. In: Current Protocols in Human Genetics. John Wiley and Sons, Inc.: 1997).
  • a large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc.
  • the insertion of the nucleic acid fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate nucleic acid fragments into a chosen vector that has complementary cohesive termini.
  • the ends of the nucleic acid molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) to the nucleic acid termini.
  • Such vectors may be engineered to contain sequences encoding selectable markers that provide for the selection of cells that contain the vector and/or have incorporated the nucleic acid of the vector into the cellular genome.
  • a "recombinant" vector refers to a viral or non-viral vector that comprises one or more heterologous nucleotide sequences (i.e., transgenes), e.g., one, two, three, four, five or more heterologous nucleotide sequences (e.g., a vector comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:6 and/or SEQ ID NO:7)
  • a vector may also comprise one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (delivery to specific tissues, duration of expression, etc.).
  • a vector comprising a synthetic polynucleotide of the invention may be used to infect and thereby delivering said synthetic polynucleotide to the infected cells.
  • the exact method of introducing the synthetic polynucleotide into mammalian cells is, of course, not limited to the use of any particular type of vector. Any vector system now known or later identified may be used with the synthetic polynucleotides of this invention. Techniques are widely available for such procedures including the use of, for example, adenoviral vectors (Mitani et al., Hum. Gene Ther.
  • chimeric viral particles which are well known in the art and which can comprise viral proteins and/or nucleic acids from two or more different viruses in any combination to produce a functional viral vector. Chimeric viral particles of this invention can also comprise amino acid and/or nucleotide sequence of non-viral origin (e.g., to facilitate targeting of vectors to specific cells or tissues and/or to induce a specific immune response).
  • Vectors may be introduced into the desired cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a nucleic acid vector transporter (see, e.g., Wu et al., J. Biol. Chem. 267:963 (1992); Wu et al., J. Biol. Chem. 263: 14621 (1988); and Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).
  • nucleic acid in vivo can be used for facilitating delivery of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., WO95/21931), peptides derived from nucleic acid binding proteins (e.g., WO96/25508), and/or a cationic polymer (e.g., WO95/21931). It is also possible to introduce a vector in vivo as naked nucleic acid (see U.S. Patent Nos. 5,693,622, 5,589,466 and 5,580,859). Receptor-mediated nucleic acid delivery approaches can also be used (Curiel et al., Hum. Gene Ther. 3:147 (1992); Wu et al., J. Biol. Chem. 262:4429 (1987)).
  • a cationic oligopeptide e.g., WO95/21931
  • peptides derived from nucleic acid binding proteins e.g.,
  • a synthetic polynucleotide of this invention can be achieved by any one of numerous, well-known approaches, for example, but not limited to, direct transfer of the nucleic acids, in a plasmid or viral vector, or via transfer in cells or in combination with carriers such as cationic liposomes.
  • Such methods are well known in the art and readily adaptable for use in the methods described herein.
  • these methods can be used to target certain diseases and tissues, organs and/or cell types and/or populations by using the targeting characteristics of the carrier, which would be well known to the skilled artisan.
  • cell and tissue specific promoters can be employed in the nucleic acids of this invention to target specific tissues and cells and/or to treat specific diseases and disorders.
  • protein and “polypeptide” are used interchangeably and encompass both peptides and proteins, unless indicated otherwise.
  • a "fusion protein” is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides not found fused together in nature are fused together in the correct translational reading frame.
  • Illustrative fusion polypeptides include fusions of a polypeptide of the invention (or a fragment thereof) to all or a portion of glutathione-S-transferase, maltose-binding protein, or a reporter protein (e.g., Green Fluorescent Protein, P-glucuronidase, 0-galactosidase, luciferase, etc.), hemagglutinin, c-myc, FLAG epitope, etc.
  • a reporter protein e.g., Green Fluorescent Protein, P-glucuronidase, 0-galactosidase, luciferase, etc.
  • express or "expression” of a polynucleotide coding sequence, it is meant that the sequence is transcribed, and optionally, translated. Typically, according to the present invention, expression of a coding sequence of the invention will result in production of the polypeptide of the invention. The entire expressed polypeptide or fragment can also function in intact cells without purification.
  • AAV adeno-associated virus
  • AAV type 1 AAV type 2, AAV type 3 (including types 3 A 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, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV and any other AAV now known or later discovered.
  • AAV serotypes and clades have been identified (see, e.g., Gao etal., (2004) J. Virol. 78:6381-6388), which are also encompassed by the term "AAV.”
  • genomic sequences of various AAV and autonomous parvoviruses as well as the sequences of the inverted terminal repeats (ITRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as the GenBank® database.
  • a "recombinant AAV vector genome” or “rAAV genome” is an AAV genome (i.e., vDNA) that comprises at least one inverted terminal repeat (e.g, one, two or three inverted terminal repeats) and one or more heterologous nucleotide sequences.
  • rAAV vectors generally retain the 145 base terminal repeat(s) (TR(s)) in cis to generate virus; however, modified AAV TRs and non-AAV TRs including partially or completely synthetic sequences can also serve this purpose. All other viral sequences are dispensable and may be supplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97).
  • the rAAV vector optionally comprises two TRs (e.g., AAV TRs), which generally will be at the 5’ and 3’ ends of the heterologous nucleotide sequence(s), but need not be contiguous thereto.
  • the TRs can be the same or different from each other.
  • the vector genome can also contain a single ITR at its 3' or 5' end.
  • terminal repeat includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates the desired functions such as replication, virus packaging, integration and/or provirus rescue, and the like).
  • the TR can be an AAV TR or a non-AAV TR.
  • a non- AAV TR sequence such as those of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or the SV40 hairpin that serves as the origin of SV40 replication can be used as a TR, which can further be modified by truncation, substitution, deletion, insertion and/or addition.
  • the TR can be partially or completely synthetic, such as the "double-D sequence" as described in United States Patent No. 5,478,745 to Samulski et al.
  • An "AAV terminal repeat” or “AAV TR” may be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or any other AAV now known or later discovered.
  • An AAV terminal repeat need not have the native terminal repeat sequence (e.g, a native AAV TR sequence may be altered by insertion, deletion, truncation and/or missense mutations), as long as the terminal repeat mediates the desired functions, e.g., replication, virus packaging, integration, and/or provirus rescue, and the like.
  • rAAV particle and “rAAV virion” are used interchangeably here.
  • a “rAAV particle” or “rAAV virion” comprises a rAAV vector genome packaged within an AAV capsid.
  • a "transformed” cell is a cell that has been transformed, transduced and/or transfected with a synthetic polynucleotide of this invention encoding an optimized human FKRP (e.g., SEQ ID NO:7) and/or optimized and CpG depleted human FKRP (e.g., SEQ ID NO:1 and/or SEQ ID NO:6)
  • dysstroglycanopathy refers to a subset of muscular dystrophies involving a reduction in glycosylation of alpha-dystroglycan (a-DG). Such disorders include but are not limited to limb girdle muscular dystrophy 21, congenital muscular dystrophy, Walker-Warburg syndrome, or muscle-eye-brain disease.
  • a "subject" of the invention includes any animal having or susceptible to a dystroglycanopathy for which prevention or treatment of said dystroglycanopathy is needed and/or desired, which can be treated, ameliorated or prevented by administration/delivery of a synthetic polynucleotide of the invention encoding FKRP to the subject.
  • a subject is generally a mammalian subject (e.g., a laboratory animal such as a rat, mouse, guinea pig, rabbit, primates, etc.), a farm or commercial animal (e.g., a cow, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, etc.).
  • the subject is a primate subject, a non-human primate subject (e.g., a chimpanzee, baboon, monkey, gorilla, etc.) or a human.
  • Subjects of the invention can be a subject known or believed to be at risk of dystroglycanopathy for which prevention or treatment is needed and/or desired.
  • a subject according to the invention can also include a subject not previously known or suspected to be at risk of dystroglycanopathy for which prevention or treatment is needed or desired.
  • the subject can be a laboratory animal and/or an animal model of disease. Suitable subjects include both males and females and subjects of any age, including embryonic (e.g., in utero or in ovo), infantjuvenile, adolescent, adult and geriatric subjects.
  • a "subject in need thereof in the context of treatment or therapy is a subject known to have, or suspected of having or being at risk of having, a disease or disorder (e.g., dystroglycanopathy), and that is likely to benefit from the treatment or therapy, i.e., is in need thereof.
  • a disease or disorder e.g., dystroglycanopathy
  • One aspect of the present invention relates to a synthetic polynucleotide encoding a codon optimized and CpG(-) depleted human fukutin-related protein (FKRP-CpG(-)).
  • the invention provides a synthetic polynucleotide encoding a human fukutin- related protein (FKRP), wherein the synthetic polynucleotide comprises, consists essentially of, or consists of: the nucleotide sequence of SEQ ID NO:1; and/or a nucleotide sequence having at least 90% sequence identity (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO:1.
  • the present inventors have surprisingly discovered that delivery of a vector comprising a synthetic polynucleotide encoding CpG(-) depleted human fukutin-related protein (FKRP- CpG(-)) provided a safer immune profile when compared to a codon optimized human FKRP (SEQ ID NO: 8) in the same vector.
  • FKRP- CpG(-) CpG(-) depleted human fukutin-related protein
  • a synthetic polynucleotide encoding FKRP may be operably associated with control or regulatory sequences.
  • a synthetic polynucleotide of this invention may be operably associated with expression control elements, such as promoters, transcription/translation control signals, origins of replication, polyadenylation signals, internal ribosome entry sites (IRES), enhancers, and the like.
  • the synthetic polynucleotide encoding FKRP e.g., polynucleotide encoding SEQ ID NO:1 and/or a polynucleotide having at least about 90% identity to SEQ ID NO:1 may be operably associated with for example a poly A tail (see, e.g., SEQ ID NO:6).
  • a synthetic polynucleotide encoding a codon optimized and CpG(-) depleted human fukutin-related protein comprises, consists essentially of, or consists of: the nucleotide sequence of SEQ ID NO:6; and/or a nucleotide sequence having at least 80% sequence identity (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO:6 (e.g., an optimized and CpG depleted FKRP (SEQ ID NO:1) operably linked to a poly A tail having the nucleotide sequence of SEQ ID NO:5)
  • SEQ ID NO:6 e.g., an optimized and CpG depleted FKRP (SEQ ID NO:1) operably linked to a poly A tail having the nucleot
  • the present invention provides a synthetic polynucleotide encoding a human fukutin-related protein (FKRP)that comprises, consists essentially of, or consists of: the nucleotide sequence of SEQ ID NO:7 and/or a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO:7.
  • FKRP human fukutin-related protein
  • promoter/enhancer elements may be used depending on the level and tissue-specific expression desired.
  • the promoter/enhancer may be constitutive or inducible, depending on the pattern of expression desired.
  • the promoter/enhancer may be native/endogenous or foreign/heterologous and can be a natural or a synthetic sequence. By foreign/heterologous, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.
  • Promoter/enhancer elements can be native/endogenous to the target cell or subject to be treated and/or native to the heterologous nucleic acid sequence.
  • the promoter/enhancer element is generally chosen so that it will function in the target cell(s) of interest.
  • the promoter/enhancer element is a mammalian promoter/enhancer element.
  • the promoter/enhance element may be constitutive or inducible.
  • Inducible expression control elements are generally used in those applications in which it is desirable to provide regulation over expression of the heterologous nucleic acid sequence(s).
  • Inducible promoter/enhancer elements include, but are not limited to, hormone- inducible and metal-inducible elements.
  • Promoters/enhancer elements for gene delivery can be tissue-specific or tissuepreferred promoter/enhancer elements, and include muscle specific or preferred (including cardiac, skeletal and/or smooth muscle), neural tissue specific or preferred (including brainspecific), eye (including retina-specific and cornea-specific), bone marrow specific or preferred, pancreatic specific or preferred, spleen specific or preferred, and lung specific or preferred promoter/enhancer elements.
  • any promoter operable in the organism or subject in which expression of a synthetic polynucleotide of the invention encoding a FKRP is desired can be used.
  • Promoters useful with this invention include, but are not limited to, a creatine kinase (CK) promoter, a chicken P-actin promoter (CB), desmine promoter, a actin promoter, or any viral promoter.
  • the promoter can be a CK7 promoter.
  • a promoter can be modified to include other regulatory elements, for example, enhancer sequences. In other embodiments, the promoters are not modified to include other regulatory elements such as enhancers.
  • a promoter operably associated with a synthetic polynucleotide of this invention may be a muscle specific promoter.
  • Muscle specific promoters useful with this invention can include, but are not limited to, a creatine kinase (CK) promoter and/or a synthetic muscle specific promoter.
  • CK creatine kinase
  • a synthetic muscle specific promoter may be a Syn 100 promoter, optionally wherein the SynlOO promoter may comprise a nucleotide sequences having at least 90% identity to SEQ ID NO:2.
  • an enhancer sequence useful with this invention can include a CMV enhancer, SV40 enhancer, a muscle creatine kinase enhancer, and/or a myosin light chain enhancer, troponin promoter, tropomycin enhancer, and/or any other synthetic enhancer with or without modification.
  • an enhancer sequence operably associated with a synthetic polynucleotide of this invention may be an intron, optionally a Vh4-Ig-intron3.
  • a Vh4-Ig-intron3 useful with this invention can comprise the nucleotide sequence of SEQ ID NO:3.
  • an expression system or construct can include a 3' untranslated region downstream of the nucleotide sequence encoding the desired recombinant protein. This region can increase expression of the transgene.
  • a poly A tail may be from bovine growth hormone.
  • the poly A tail from bovine growth hormone may comprise the nucleotide sequence of SEQ ID NO:4.
  • the poly A tail may comprise the nucleotide sequence of SEQ ID NO:5.
  • a synthetic polynucleotide of the present invention may be comprised in a vector or expression cassette wherein the synthetic polynucleotide is operably linked to expression control elements as described herein (see, as an example, SEQ ID NO:7).
  • vector generally refers to a virus particle that functions as a nucleic acid delivery vehicle, and which comprises the viral nucleic acid (i.e., the vector genome) packaged within the virion.
  • virus vectors according to the present invention comprise a chimeric AAV capsid according to the invention and can package an AAV or rAAV genome or any other nucleic acid including viral nucleic acids.
  • vector may be used to refer to the vector genome (e.g., vDNA) in the absence of the virion and/or to a viral capsid that acts as a transporter to deliver molecules tethered to the capsid or packaged within the capsid.
  • vector genome e.g., vDNA
  • delivery vector may be used to refer to the vector genome (e.g., vDNA) in the absence of the virion and/or to a viral capsid that acts as a transporter to deliver molecules tethered to the capsid or packaged within the capsid.
  • the virus vectors of the invention can further be duplexed parvovirus particles (double stranded or self-complementary) as described in international patent publication WO 01/92551 (the disclosure of which is incorporated herein by reference in its entirety), wherein double stranded (duplex) genomes can be packaged.
  • a viral vector e.g., AAV
  • AAV a viral vector
  • a viral vector e.g., AAV
  • a viral vector may be double stranded or self-complementary.
  • a “recombinant AAV vector genome” or “rAAV genome” is an AAV genome (z.e., vDNA) that comprises at least one inverted terminal repeat (e.g., one, two or three inverted terminal repeats) and one or more heterologous nucleotide sequences.
  • rAAV vectors generally retain the 145 base terminal repeat(s) (TR(s)) in cis to generate virus; however, modified AAV TRs and non-AAV TRs including partially or completely synthetic sequences can also serve this purpose. All other viral sequences are dispensable and may be supplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97).
  • the rAAV vector optionally comprises two TRs (e.g., AAV TRs), which generally will be at the 5’ and 3’ ends of the heterologous nucleotide sequence(s), but need not be contiguous thereto.
  • the TRs can be the same or different from each other.
  • the vector genome can also contain a single ITR at its 3' or 5' end.
  • terminal repeat includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates the desired functions such as replication, virus packaging, integration and/or provirus rescue, and the like).
  • the TR can be an AAV TR or a non- AAV TR.
  • a non- AAV TR sequence such as those of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or the SV40 hairpin that serves as the origin of SV40 replication can be used as a TR, which can further be modified by truncation, substitution, deletion, insertion and/or addition.
  • a viral vector useful with this invention may comprise a mutated terminal repeat (ITR).
  • ITR mutated terminal repeat
  • Parvovirus genomes have palindromic sequences at both their 5’ and 3’ ends.
  • the palindromic nature of the sequences leads to the formation of a hairpin structure that is stabilized by the formation of hydrogen bonds between the complementary base pairs.
  • This hairpin structure is believed to adopt a " Y" or a "T” shape. See, e.g., Fields et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers).
  • a viral vector useful with this invention is an AAV vector from any known AAV serotype including without limitation AAV type 1, AAV type 2, AAV type 3 (including types 3 A 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, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV and any other AAV now known or later discovered.
  • the AAV vector is an AAV8 vector.
  • the AAV vector is an AAV9 vector.
  • an AAV capsid protein of a virus vector can comprise a modification in the amino acid sequence in the three-fold axis loop 4 (Opie et al., J. Virol. 77: 6995-7006 (2003)).
  • Such modifications have been shown to confer one or more desirable properties to virus vectors comprising the modified AAV capsid protein including without limitation (i) reduced transduction of liver, (ii) enhanced movement across endothelial cells, (iii) systemic transduction; (iv) enhanced transduction of muscle tissue (e.g., skeletal muscle, cardiac muscle and/or diaphragm muscle), and/or (v) reduced transduction of brain tissues (e.g., neurons).
  • such modifications can reduce delivery of the synthetic polynucleotides of the invention to the liver (See, Asokan et al. Nat Biotechnol. 28(l):79-82 (2010); U.S. Patent No. 8,889,641).
  • the vector may further comprise a nucleic acid element that reduces expression in the liver.
  • a vector may further comprise a mirl22 binding element.
  • the mirl22 sequence and its use to reduce expression in the liver is well known in the art (See, e.g., Qiao et al, Gene Therapy 18, 403-410 (April 2011) doi:10.1038/gt.2010.157).
  • a vector can further encode reporter polypeptides (e.g., an enzyme).
  • Reporter polypeptides are known in the art and include, but are not limited to, a fluorescent protein (e.g., EGFP, GFP, RFP, BFP, YFP, or dsRED2), an enzyme that produces a detectable product, such as luciferase (e.g., from Gaussia, Renilla, or Photinus), 0-galactosidase, 0 -glucuronidase, alkaline phosphatase, and chloramphenicol acetyltransferase gene, or proteins that can be directly detected.
  • luciferase e.g., from Gaussia, Renilla, or Photinus
  • 0-galactosidase 0 -glucuronidase
  • alkaline phosphatase alkaline phosphatase
  • chloramphenicol acetyltransferase gene or
  • a further aspect of the invention relates to a cell comprising at least one synthetic polynucleotide of the invention and/or vector comprising the at least one synthetic polynucleotide of the invention (e.g., an isolated cell, a transformed cell, a recombinant cell, etc.).
  • various embodiments of the invention are directed to recombinant host cells comprising a vector (e.g., expression cassette) including the synthetic polynucleotide of the invention.
  • a cell may be isolated and/or present in an animal, e.g., a transgenic animal. Transformation of cells is described further below.
  • Recombinant virus vectors according to the present invention find use in both veterinary and medical applications. Suitable subjects include both avians and mammals.
  • avian as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, pheasant, parrots, parakeets.
  • mammal as used herein includes, but is not limited to, humans, primates, non-human primates (e.g., monkeys and baboons), rodents (e.g., rats, rabbits, mice, hamsters, guinea pigs and the like), cattle, sheep, goats, pigs, horses, cats, dogs, etc.
  • Human subjects include neonates, infants, juveniles, and adults.
  • the subject is "in need of' the methods of the present invention, e.g., because the subject has or is believed at risk for a disorder including those described herein or that would benefit from the delivery of a polynucleotide including those described herein.
  • the subject may be a laboratory animal and/or an animal model of disease.
  • the invention provides a transgenic animal comprising a synthetic polynucleotide of the invention, and/or an expression cassette, vector, and/or transformed cell comprising the same.
  • the transgenic animal is not a human.
  • a transgenic animal may be produced by introducing into a single cell embryo the synthetic polynucleotide of the invention encoding FKRP (e.g., a nucleotide sequence encoding FKRP that is codon optimized and CpG depleted, the nucleotide sequence of SEQ ID NO:1; and/or a nucleotide sequence having at least 90% identity to SEQ ID NO:1, the nucleotide sequence of SEQ ID NO:6; and/or a nucleotide sequence having at least 80% identity to SEQ ID NO:6, the nucleotide sequence of SEQ ID NO:7; and/or a nucleotide sequence having at least 80% identity to SEQ ID NO:7 in a manner such that the synthetic polynucleotide is stably integrated into the DNA of germ line cells of the mature animal, and is inherited in normal Mendelian fashion.
  • FKRP e.g., a nucleotide sequence encoding FKRP that is cod
  • the transgenic animal of this invention would have a phenotype of producing FKRP in body fluids and/or tissues.
  • the FKRP may be removed from these fluids and/or tissues and processed, for example for therapeutic use.
  • Clark et al. "Expression of human anti-hemophilic factor IX in the milk of transgenic sheep” Bio/Technology 7:487-492 (1989); Van Cott et al. "Haemophilic factors produced by transgenic livestock: abundance can enable alternative therapies worldwide” Haemophilia 10(4):70-77 (2004), the entire contents of which are incorporated by reference herein).
  • DNA molecules can be introduced into embryos by a variety of means including but not limited to microinjection, calcium phosphate mediated precipitation, liposome fusion, or retroviral infection of totipotent or pluripotent stem cells.
  • the transformed cells can then be introduced into embryos and incorporated therein to form transgenic animals. Methods of making transgenic animals are described, for example, in Transgenic Animal Generation and Use by L. M. Houdebine, Harwood Academic Press, 1997.
  • Transgenic animals also can be generated using methods of nuclear transfer or cloning using embryonic or adult cell lines as described for example in Campbell et al., Nature 380:64-66 (1996) and Wilmut et al., Nature 385:810-813 (1997).
  • FKRP-producing transgenic animals can be obtained by introducing a chimeric construct comprising a synthetic polynucleotide of the invention (e.g., a nucleotide sequence encoding FKRP that is codon optimized and CpG depleted, the nucleotide sequence of SEQ ID NO:1; and/or a nucleotide sequence having at least 90% identity to SEQ ID NO:1, the nucleotide sequence of SEQ ID NO:6; and/or a nucleotide sequence having at least 80% identity to SEQ ID NO:6, the nucleotide sequence of SEQ ID NO:7; and/or a nucleotide sequence having at least 80% identity to SEQ ID NO:7).
  • a synthetic polynucleotide of the invention e.g., a nucleotide sequence encoding FKRP that is codon optimized and CpG depleted, the nucleotide sequence of SEQ ID NO:1; and/or a nu
  • a synthetic polynucleotide of this invention encoding FKRP, or vector and/or cell comprising the synthetic polynucleotide can be included in a pharmaceutical composition.
  • Some embodiments are directed to a kit which includes said synthetic polynucleotide, or vector and/or cell comprising said synthetic polynucleotide of the invention and/or reagents and/or instructions for using the kit, e.g., to carry out the methods of this invention.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a synthetic polynucleotide of the invention and/or expression cassette, vector, and/or transformed cell comprising the same in a pharmaceutically acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc.
  • the carrier will typically be a liquid.
  • the carrier may be either solid or liquid.
  • the carrier will be respirable, and will preferably be in solid or liquid particulate form.
  • pharmaceutically acceptable it is meant a material that is not toxic or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects.
  • a further aspect of the invention relates to the use of the synthetic polynucleotides encoding FKRP, or vector, expression cassette, and/or cell comprising one or more synthetic polynucleotides encoding FKRP.
  • one aspect relates to a method of producing a FKRP polypeptide in a cell or in a subject, comprising delivering to the cell or the subject a synthetic polynucleotide of the invention and/or a vector, and/or transformed cell comprising the same, thereby producing the FKRP polypeptide in said cell or said subject.
  • the synthetic polynucleotide, vector, and/or transformed cell are delivered under conditions whereby expression of the synthetic polynucleotide encoding FKRP occurs to produce a FKRP polypeptide. Such conditions are well known in the art.
  • One aspect of the present invention is a method of transferring a synthetic polynucleotide of this invention to a cell in vitro.
  • the synthetic polynucleotide and/or expression cassette and/or vector comprising the same may be introduced to the cells in the appropriate amount.
  • the virus vector may be introduced to the cells at the appropriate multiplicity of infection according to standard transduction methods appropriate for the particular target cells. Titers of the virus vector or capsid to administer can vary, depending upon the target cell type and number, and the particular virus vector or capsid, and can be determined by those of skill in the art without undue experimentation. In particular embodiments, at least about 10 3 infectious units, more preferably at least about 10 3 , 10 4 , 10 5 or 10 6 infectious units are introduced to the cell.
  • the invention provides a method of delivering/administering a nucleic acid to a cell comprising, consisting essentially of, or consisting of: delivering to the cell a synthetic polynucleotide of the invention (e.g., a nucleotide sequence encoding FKRP that is codon optimized and CpG depleted, the nucleotide sequence of SEQ ID NO:1; and/or a nucleotide sequence having at least 90% identity to SEQ ID NO:1, the nucleotide sequence of SEQ ID NO:6; and/or a nucleotide sequence having at least 80% identity to SEQ ID NO:6, the nucleotide sequence of SEQ ID NO:7; and/or a nucleotide sequence having at least 80% identity to SEQ ID NO:7); and/or a vector comprising, consisting essentially of, or consisting of the same.
  • the synthetic polynucleotide of the invention, and/or vector comprising, consisting essentially
  • Another aspect of the invention provides a method of delivering/administering a nucleic acid to a subject in need thereof, the method comprising, consisting essentially of, or consisting of: delivering to the subject a synthetic polynucleotide of the invention (e.g., a nucleotide sequence encoding FKRP that is codon optimized and CpG depleted, the nucleotide sequence of SEQ ID NO:1; and/or a nucleotide sequence having at least 90% identity to SEQ ID NO:1, the nucleotide sequence of SEQ ID NO:6; and/or a nucleotide sequence having at least 80% identity to SEQ ID NO:6, the nucleotide sequence of SEQ ID NO:7; and/or a nucleotide sequence having at least 80% identity to SEQ ID NO:7); and/or a vector and/or a transformed cell comprising, consisting essentially of, or consisting of the same.
  • virus vectors of the present invention administered in an effective dose in a pharmaceutically acceptable carrier.
  • the present invention further provides a method of treating a dystroglycanopathy in a subject in need thereof, the method comprising, consisting essentially of, or consisting of: delivering/administering to said subject a synthetic polynucleotide of the invention (e.g., a nucleotide sequence encoding FKRP that is codon optimized and CpG depleted, the nucleotide sequence of SEQ ID NO:1; and/or a nucleotide sequence having at least 90% identity to SEQ ID NO:1, the nucleotide sequence of SEQ ID NO:6; and/or a nucleotide sequence having at least 80% identity to SEQ ID NO:6, the nucleotide sequence of SEQ ID NO:7; and/or a nucleotide sequence having at least 80% identity to SEQ ID NO:7); and/or a vector and/or a transformed cell comprising the same, wherein the synthetic polynucleotide is expressed in the subject, thereby treating dys
  • the dystroglycanopathy comprises, consists essentially of, or consists of a mutation in the native or endogenous nucleic acid encoding FKRP and/or a deficiency in glycosylation of alpha-dystroglycan (a-DG), or any combination thereof.
  • the dystroglycanopathy can include, but is not limited to, limb girdle muscular dystrophy 21, congenital muscular dystrophy, Walker-Warburg syndrome, muscle-eye-brain disease, and/or any combination thereof.
  • the present invention further provides a method of reducing serum creatine kinase levels in a subject in need thereof, the method comprising delivering to the subject a synthetic polynucleotide of the invention (e.g., a double stranded vector comprising, for example, an optimized human FKRP or an optimized human FKRP that is CpG depleted as described herein; e.g., SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7) and/or a vector and/or a transformed cell comprising, consisting essentially of, or consisting of the same, wherein the synthetic polynucleotide is expressed in the subject, thereby producing human FKRP and reducing serum creatine kinase levels in the subject.
  • a synthetic polynucleotide of the invention e.g., a double stranded vector comprising, for example, an optimized human FKRP or an optimized human FKRP that is CpG depleted as described herein; e.g
  • the present invention providers nucleic acid constructs encoding FKRP that provide a safer immune response when delivered to a subject.
  • a method of reducing an immune response when administering an FKRP expressing vector comprising administering to the subject a synthetic polynucleotide of the invention (e.g., an optimized human FKRP that is CpG depleted; e.g., SEQ ID NO:1, SEQ ID NO:6) and/or a vector and/or a transformed cell comprising the same, wherein the synthetic polynucleotide is expressed in the subject, thereby reducing the immune response when compared to administering a codon optimized human FKRP expressing vector (e.g., as compared to SEQ ID NO:8).
  • a synthetic polynucleotide of the invention e.g., an optimized human FKRP that is CpG depleted; e.g., SEQ ID NO:1, SEQ ID NO:6
  • reducing an immune response may comprise reduced IL17A levels as compared to a subject having been administered a vector expressing an optimized human FKRP having the nucleotide sequence of SEQ ID NO:8 (UNC-0090 opt huFKRP).
  • the dosage may be about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10
  • the dosage may be about IxlO 10 vector particles/per kg body weight to about 1x10 15 vector parti cles/per kg body weight (e.g., 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 vector particles/per kg body weight, and any range or value therein).
  • the dosage can be about IxlO 12 vector particles/per kg body weight to about IxlO 15 vector particles/per kg body weight (e.g., 10 12 , 10 13 , 10 14 , 10 15 vector particles/per kg body weight, and any range or value therein).
  • Doses and virus titer transducing units may be calculated as vector or viral genomes (vg).
  • vg vector or viral genomes
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a virus vector of the invention in a pharmaceutically acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc.
  • the carrier will typically be a liquid.
  • the carrier may be either solid or liquid.
  • the carrier will be respirable, and will preferably be in solid or liquid particulate form.
  • composition a material that is not toxic or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects.
  • a “pharmaceutically acceptable” component such as a salt, carrier, excipient or diluent of a composition according to the present invention is a component that (i) is compatible with the other ingredients of the composition in that it can be combined with the compositions of the present invention without rendering the composition unsuitable for its intended purpose, and (ii) is suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are “undue” when their risk outweighs the benefit provided by the composition.
  • Non-limiting examples of pharmaceutically acceptable components include, without limitation, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsion, microemulsions and various types of wetting agents.
  • the pharmaceutically acceptable carrier is sterile and would be deemed suitable for administration into human subjects according to regulatory guidelines for pharmaceutical compositions comprising the carrier.
  • the virus vector may be introduced to the cells at the appropriate multiplicity of infection according to standard transduction methods appropriate for the particular target cells. Titers of the virus vector to administer can vary, depending upon the target cell type and number, and the particular virus vector can be determined by those of skill in the art without undue experimentation. In particular embodiments, at least about 1 infectious unit, more preferably at least about 2 or more infectious units are introduced to one target cell.
  • Cell(s) into which the polynucleotide, expression cassette, and/or vector of the invention, e.g., virus vector, can be introduced may be of any type, including but not limited to neural cells (including cells of the peripheral and central nervous systems, in particular, brain cells such as neurons, oligodendrocytes, glial cells, astrocytes), lung cells, cells of the eye (including retinal cells, retinal pigment epithelium, and corneal cells), epithelial cells (e.g., gut and respiratory epithelial cells), skeletal muscle cells (including myoblasts, myotubes and myofibers), diaphragm muscle cells, dendritic cells, pancreatic cells (including islet cells), hepatic cells, a cell of the gastrointestinal tract (including smooth muscle cells, epithelial cells), heart cells (including cardiomyocytes), bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes,
  • the cell may be any progenitor cell.
  • the cell can be a stem cell (e.g, neural stem cell).
  • the cell may be a cancer or tumor cell (cancers and tumors are described above).
  • the cells can be from any species of origin, as indicated above.
  • a synthetic polynucleotide of the invention, and/or expression cassettes, and/or vectors, e.g., virus vector, comprising the same may be introduced to cells in vitro for the purpose of administering the modified cell to a subject.
  • the cells have been removed from a subject, and the synthetic polynucleotide, and/or expression cassette and/or vector of the invention (e.g., virus vector) comprising the synthetic polynucleotide are introduced therein, and the cells are then replaced back into the subject.
  • Methods of removing cells from subject for treatment ex vivo, followed by introduction back into the subject are known in the art (see, e.g., U.S. patent No. 5,399,346).
  • the synthetic polynucleotide of the invention, and/or expression cassette, and/or vector may be introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need thereof.
  • vector e.g., virus vector
  • Suitable cells for ex vivo gene therapy are as described above. Dosages of the cells to administer to a subject will vary upon the age, condition and species of the subject, the type of cell, the nucleic acid being expressed by the cell, the mode of administration, and the like. Typically, at least about 10 2 to about 10 8 or about 10 3 to about 10 6 cells will be administered per dose in a pharmaceutically acceptable carrier. In particular embodiments, the cells transduced with the virus vector are administered to the subject in an effective amount in combination with a pharmaceutical carrier.
  • an effective amount of a composition of this invention will vary from composition to composition and subject to subject, and will depend upon a variety of factors such as age, species, gender, weight, overall condition of the subject, and the particular disease or disorder to be treated. An effective amount can be determined in accordance with routine pharmacological procedures know to those of ordinary skill in the art. In some embodiments, a dose ranging from about IxlO 12 vector particles/per kg body weight to about IxlO 15 vector particles/per kg body weight will have therapeutic efficacy. In embodiments employing viral vectors for delivery of the nucleic acid of this invention, viral doses can be measured to include a particular number of virus particles or plaque forming units (pfu) or infectious particles, depending on the virus employed.
  • pfu plaque forming units
  • particular unit doses can include about 10 3 to about 10 16 pfu or infectious particles per kg body weight (e.g., about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 or 10 16 pfu or infectious particles per kg body weight), or any range or value therein.
  • about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 or 10 16 pfu or infectious particles per kg body weight e.g., about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 or 10 16 pfu or infectious particles per kg body weight
  • virus titers can be at least about 10 5 to about 10 15 transducing units per kg body weight (e.g., at least about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , IO 10 , IO 11 , 10 12 , IO 3 , 10 14 , IO 15 transducing units per kg body weight) or any range or value therein.
  • doses for achieving therapeutic effects are virus titers can be at least about 10 8 to about 10 15 transducing units per kg body weight (e.g., about 10 8 , 10 9 , IO 10 , 10 11 , 10 12 , 10 13 or 10 14 , 10 15 transducing units per kg body weight) or any range or value therein.
  • Doses and virus titer transducing units may be calculated as vector or viral genomes (vg). As the skilled artisan would understand, the specific dose would depend on the size of the target/subject and the nature of the target/subject.
  • the frequency of administration of a composition of this invention can be as frequent as necessary to impart the desired therapeutic effect.
  • the composition can be administered one, two, or more times per day, one, two, three, four or more times a week, one, two, three, four or more times a month, one, two, three or four times a year and/or as necessary to control a particular condition and/or to achieve a particular effect and/or benefit.
  • more than one administration e.g., two, three, four or more administrations
  • one, two, three or four doses over the lifetime of a subject can be adequate to achieve the desired therapeutic effect.
  • the amount and frequency of administration of the composition of this invention will vary depending on the particular condition being treated or to be prevented and the desired therapeutic effect.
  • Exemplary modes of administration include oral, rectal, transmucosal, topical, intranasal, inhalation (e.g., via an aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, in utero (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [including administration to skeletal, diaphragm and/or cardiac muscle], intrapleural, intracerebral, and intraarticular), topical (e.g., to both skin and mucosal surfaces, including airway surfaces, and transdermal administration), and the like, as well as direct tissue or organ injection (e.g., to skeletal muscle, cardiac muscle, diaphragm muscle or brain).
  • parenteral e.g., intravenous, subcutaneous, intradermal, intramuscular [including administration to skeletal, diaphragm and/or cardiac muscle], intrapleural, intracerebral, and intraarticular
  • the route of delivery for treatment of dystroglycanopathy is intramuscular, intravenous and intraarterial to a part of or the whole body.
  • Delivery to any of these tissues can also be achieved by delivering a depot comprising the virus vector, which can be implanted into the tissue or the tissue can be contacted with a film or other matrix comprising the virus vector.
  • a depot comprising the virus vector
  • the tissue can be contacted with a film or other matrix comprising the virus vector. Examples of such implantable matrices or substrates are described in U.S. Patent No. 7,201,898.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • the virus vector can be delivered dried to a surgically implantable matrix such as a bone graft substitute, a suture, a stent, and the like (e.g., as described in U.S. Patent 7,201,898).
  • compositions suitable for oral administration can be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the composition of this invention; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion.
  • Oral delivery can be performed by complexing a virus vector of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers include plastic capsules or tablets, as known in the art.
  • Such formulations are prepared by any suitable method of pharmacy, which includes the step of bringing into association the composition and a suitable carrier (which may contain one or more accessory ingredients as noted above).
  • a suitable carrier which may contain one or more accessory ingredients as noted above.
  • the pharmaceutical composition according to embodiments of the present invention are prepared by uniformly and intimately admixing the composition with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture.
  • a tablet can be prepared by compressing or molding a powder or granules containing the composition, optionally with one or more accessory ingredients.
  • Compressed tablets are prepared by compressing, in a suitable machine, the composition in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets are made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.
  • compositions suitable for buccal (sub-lingual) administration include lozenges comprising the composition of this invention in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia.
  • compositions suitable for parenteral administration can comprise sterile aqueous and non-aqueous injection solutions of the composition of this invention, which preparations are optionally isotonic with the blood of the intended recipient. These preparations can contain anti-oxidants, buffers, bacteriostats and solutes, which render the composition isotonic with the blood of the intended recipient.
  • Aqueous and non-aqueous sterile suspensions, solutions and emulsions can include suspending agents and thickening agents.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • compositions can be presented in unit/dose or multi-dose containers, for example, in sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-inj ection immediately prior to use.
  • sterile liquid carrier for example, saline or water-for-inj ection immediately prior to use.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.
  • an injectable, stable, sterile composition of this invention in a unit dosage form in a sealed container can be provided.
  • the composition can be provided in the form of a lyophilizate, which can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject.
  • the unit dosage form can be from about 1 pg to about 10 grams of the composition of this invention.
  • a sufficient amount of emulsifying agent which is physiologically acceptable, can be included in sufficient quantity to emulsify the composition in an aqueous carrier.
  • emulsifying agent is phosphatidyl choline.
  • compositions suitable for rectal administration can be presented as unit dose suppositories. These can be prepared by admixing the composition with one or more conventional solid carriers, such as for example, cocoa butter and then shaping the resulting mixture.
  • compositions of this invention suitable for topical application to the skin can take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.
  • Carriers that can be used include, but are not limited to, petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
  • topical delivery can be performed by mixing a pharmaceutical composition of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.
  • a lipophilic reagent e.g., DMSO
  • compositions suitable for transdermal administration can be in the form of discrete patches adapted to remain in intimate contact with the epidermis of the subject for a prolonged period of time.
  • Compositions suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Pharm. Res. 3:318 (1986)) and typically take the form of an optionally buffered aqueous solution of the composition of this invention.
  • Suitable formulations can comprise citrate or bis ⁇ tris buffer (pH 6) or ethanol/water and can contain from 0.1 to 0.2M active ingredient.
  • the virus vectors disclosed herein may be administered to the lungs of a subject by any suitable means, for example, by administering an aerosol suspension of respirable particles comprised of the virus vectors, which the subject inhales.
  • the respirable particles may be liquid or solid.
  • Aerosols of liquid particles comprising the virus vectors may be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Patent No. 4,501,729. Aerosols of solid particles comprising the virus vectors may likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.
  • Example 1 Double-stranded AAV vector containing optimized hu-FKRP gene codon.
  • the AAV vector is based on a single-stranded (ss) DNA virus. Its transgene expression requires the conversion of ssDNA to double-stranded (ds) genome, a rate limiting step for AAV transduction.
  • ds-AAV vector The limitation of using ds-AAV vector is that it only contains half size of the single-stranded AAV genome.
  • AAV9 vectors containing opti-hu-FKRP gene driven by different promoters were delivered into 2 to 3 months old E310 stop /L276I mice via tail vein injection at the dosage of 4 x 10 A 13 vg/kg.
  • the four groups were: CK group which was ss- AAV containing muscle-specific creatine-kinase based promoter (Uawwoy et al. Mol. Ther. Methods Clin Dev. 11 : 106-120 (2016); Qiao et al. 2014.
  • CK-Mirl22T group which was ss-AAV containing CK promoter and liver de-targeting liver-specific micro- RNA Mirl22T sequences( Qiao et al, Gene Therapy 18, 403-410 (April 2011)); CB group which was ss-AAV containing ubiquitous CB promoter (CMV enhancer/chicken P-actin promoter) (Qiao et al. 2014.
  • the serum CK was measured to make sure onset of the dystrophic phenotype.
  • the average CK level of all mice before treatment was 4688 ⁇ 3718 U/L.
  • the AAV9-ds-synl00 group 214 ⁇ 122 U/L
  • Example 2 Evalutation of ss-AAV containing opti-hu-FKRP-CpG(-) and ds-AAV containing opti-hu-FKRP gene
  • the human codon-optimized and CpG depleted FKRP gene (SEQ ID NO:1) was synthesized from GeneArt. Then the opti-hu-FKRP-CpG(-) gene was cloned into ss-AAV vector plasmids driven by different promoters: muscle specific promoter CK, muscle specific promoter synlOO, and ubiquitous CB promoter (chicken beta actin promoter with a cytomegalovirus early gene enhancer). The expression of FKRP was evaluated in vitro by transfection of the plasmids into HEK (human embryonic kidney) cells 293. As revealed in FIG. 3, the expression level of opti-hu-FKRP-CpG(-) plasmid was similar to the original plasmid (opti-hu-FKRP) which contains CpG motifs.
  • the treated and control mice were subjected to bleeding and the serum were subjected to a set of cytokines testing including IL1, IL4, IL6, IL10, IL17, TNF-alpha, TGF-beta, and IFN-gamma.

Abstract

La présente invention concerne des polynucléotides synthétiques codant pour la protéine liée à la fukutine (FKRP). L'invention concerne également des constructions d'acide nucléique comprenant les polynucléotides synthétiques et des procédés d'utilisation de ces polynucléotides synthétiques pour traiter des dystroglycanopathies.
PCT/US2022/011010 2021-01-04 2022-01-03 Protéines liées à la fukutine optimisées et procédés d'utilisation WO2022147490A1 (fr)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2019008157A1 (fr) * 2017-07-07 2019-01-10 Genethon Nouveaux polynucléotides codant pour une protéine fkrp humaine
US20190054037A1 (en) * 2016-12-16 2019-02-21 The Charlotte Mecklenburg Hospital Authority D/B/A Atrium Health Compositions and methods for treating muscular dystrophy and other disorders
US10350305B2 (en) * 2015-02-27 2019-07-16 The University Of North Carolina At Chapel Hill Compositions for treating dystroglycanopathy disorders

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US10350305B2 (en) * 2015-02-27 2019-07-16 The University Of North Carolina At Chapel Hill Compositions for treating dystroglycanopathy disorders
US20190054037A1 (en) * 2016-12-16 2019-02-21 The Charlotte Mecklenburg Hospital Authority D/B/A Atrium Health Compositions and methods for treating muscular dystrophy and other disorders
WO2019008157A1 (fr) * 2017-07-07 2019-01-10 Genethon Nouveaux polynucléotides codant pour une protéine fkrp humaine

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EVELYNE GICQUEL, NATACHA MAIZONNIER, STEVEN J. FOLTZ, WILLIAM J. MARTIN, NATHALIE BOURG, FEDOR SVINARTCHOUK, KARINE CHARTON, AARON: "AAV-mediated transfer of FKRP shows therapeutic efficacy in a murine model but requires control of gene expression", HUMAN MOLECULAR GENETICS, vol. 26, no. 10, 15 May 2017 (2017-05-15), pages 1952 - 1965, XP002773603, DOI: 10.1093/hmg/ddx066 *
XU LEI, LU PEI JUAN, WANG CHI-HSIEN, KERAMARIS ELIZABETH, QIAO CHUNPING, XIAO BIN, BLAKE DEREK J, XIAO XIAO, LU QI LONG: "Adeno-associated Virus 9 Mediated FKRP Gene Therapy Restores Functional Glycosylation of alpha-Dystroglycan and Improves Muscle Functions", MOLECULAR THERAPY, vol. 21, no. 10, 1 October 2013 (2013-10-01), pages 1832 - 1840, XP002773602 *

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