WO2023212643A1 - Method for expressing a muscle-specific gene and cassettes for same - Google Patents

Method for expressing a muscle-specific gene and cassettes for same Download PDF

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WO2023212643A1
WO2023212643A1 PCT/US2023/066296 US2023066296W WO2023212643A1 WO 2023212643 A1 WO2023212643 A1 WO 2023212643A1 US 2023066296 W US2023066296 W US 2023066296W WO 2023212643 A1 WO2023212643 A1 WO 2023212643A1
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utr
fkrp
msec
orf
nucleic acid
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Jeffrey Scott CHAMBERLAIN
Stephen D. Hauschka
Halli Claire BENASUTTI
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University of Washington
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University of Washington
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    • A—HUMAN NECESSITIES
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    • A61K35/76—Viruses; Subviral particles; Bacteriophages
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—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 delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4707—Muscular dystrophy
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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.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1288—Transferases for other substituted phosphate groups (2.7.8)
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    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
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    • 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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    • 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
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    • C12N2830/00—Vector systems having a special element relevant for transcription
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/08—Transferases for other substituted phosphate groups (2.7.8)

Definitions

  • FIELD OF THE INVENTION The field of invention relates to gene therapy for the treatment of FKRP-mediated diseases.
  • BACKGROUND Neuromuscular disorders can result from genetic mutations in key genes that regulate optimal muscular production.
  • FKRP fukutin- related protein
  • DGC dystrophin-glycoprotein complex
  • the methods and compositions described herein are based, in part, on the discovery that muscle expression of FKRP can be improved by (i) using a muscle-specific expression cassette (MSEC) to deliver FKRP to a cell, and (ii) modifying the nucleic acid encoding FKRP such that the resulting transcript comprises a modification to the 5’ and/or 3’ untranslated region (UTR).
  • MSEC muscle-specific expression cassette
  • UTR untranslated region
  • FKRP limb girdle muscular dystrophy type 2I/R9
  • LGMD2i also known as LGMDR9
  • MED muscle-eye-brain disease
  • One aspect provided herein describes a nucleic acid expression cassette comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a fukutin-related protein (FKRP) RNA transcript that comprises a modified 5’ and/or 3’ untranslated region (UTR).
  • FKRP fukutin-related protein
  • the modified 5’ untranslated region is truncated as compared to the 5’ UTR of wild-type FKRP.
  • the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 5’ UTR region.
  • the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR.
  • the modification comprises a modification to the Kozak consensus sequence.
  • the modified 3’ UTR is truncated compared to the 3’ UTR of wild-type FKRP.
  • the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region.
  • the nucleic acid encoding FKRP comprises a modification in each of the 5’ and 3’ UTRs.
  • the modification in the 5’ and/or 3’ UTR of FKRP causes an increase or a decrease in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity expressed from a similar construct comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a FKRP RNA transcript that comprises a wild-type 5’ and/or 3’ untranslated region (UTR).
  • the transcriptional regulatory region comprises a muscle-specific expression cassette (MSEC).
  • the MSEC is selected from the group consisting of CK8e.
  • expression level of an FKRP mRNA or protein upon administration to a cell, expression level of an FKRP mRNA or protein is higher when operably linked to an MSEC than the expression level of the FKRP mRNA or protein when operably linked to a CK8e transcriptional regulatory region.
  • expression level of an FKRP mRNA or protein upon administration to a cell, expression level of an FKRP mRNA or protein is lower when operably linked to an MSEC than the expression level of the FKRP mRNA or protein when operably linked to a CK8e transcriptional regulatory region.
  • RNA transcript generated by transcription of the nucleic acid expression cassette of any one of the embodiments described herein.
  • AAV adeno-associated viral vector
  • the adeno-associated viral vector is selected from the group consisting of: an AAVRh74 vector, an AAV8 vector, an AAV9 vector, an AAV6 vector, an AAV7 vector, an AAV2i8 vector, a NP vector, a NP 66 vector, a NP 22 vector, an AAVpo.1 vector, a MyoAAV vector, and an AAVMyo vector.
  • the adeno-associated viral vector comprises an internal terminal repeat (ITR), a muscle-specific cassette, a nucleic acid specific to FKRP, a polyadenylation signal (pA+), and/or a second ITR.
  • ITR internal terminal repeat
  • pA+ polyadenylation signal
  • Another aspect provided herein describes an engineered cell comprising or expressing a nucleic acid expression cassette of any one of the embodiments described herein.
  • the modified 5’ untranslated region (UTR) is truncated as compared to the wild-type 5’ UTR of FKRP.
  • the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of or all of the nucleotides in the 5’ UTR region.
  • the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR.
  • the modification comprises a modification to the Kozak consensus sequence.
  • the modified 3’ UTR is truncated compared to the 3’UTR of wild-type FKRP.
  • the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region.
  • the nucleic acid encoding FKRP comprises a modification in each of the 5’and 3’ UTRs.
  • the modification in the 5’ and/or 3’ UTR of FKRP causes an increase or a decrease in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity expressed from a similar construct comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a FKRP RNA transcript that comprises a wild-type 5’ and/or 3’ untranslated region (UTR).
  • Another aspect provided herein relates to a method of expressing an FKRP gene product in a subject comprising administering an adeno-associated viral vector according to any one of the embodiments described herein to a subject in need thereof.
  • the FKRP gene product is a RNA transcript and/or a protein.
  • the subject in need thereof comprises limb girdle muscular dystrophy type 2I/R9 (LGMD2i), Walker-Warburg syndrome, or muscle-eye-brain disease (MED).
  • the modified 5’ untranslated region (UTR) is truncated as compared to the 5’ UTR of wild-type FKRP.
  • the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 5’ UTR region.
  • the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR.
  • the modification comprises a modification to the Kozak consensus sequence.
  • the modified 3’ UTR is truncated compared to the 3’ UTR of a wild-type FKRP.
  • the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region.
  • the nucleic acid encoding FKRP comprises a modification in each of the 5’ and 3’ UTRs.
  • the modification in the 5’ and/or 3’ UTR of FKRP causes an increase or a decrease in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity expressed from a similar construct comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a FKRP RNA transcript that comprises a wild-type 5’ and/or 3’ untranslated region (UTR).
  • the administration of the AAV vector comprises intravenous and/or intramuscular injection.
  • the subject is a human.
  • the FKRP-mediated disease or disorder comprises limb girdle muscular dystrophy type 2I/R9 (LGMD2i), Walker- Warburg syndrome, or muscle-eye-brain disease (MED).
  • LGMD2i limb girdle muscular dystrophy type 2I/R9
  • MED muscle-eye-brain disease
  • the modified 5’ untranslated region is truncated as compared to the wild-type 5’ UTR of FKRP.
  • the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 5’ UTR region.
  • the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR.
  • the modification comprises a modification to the Kozak consensus sequence.
  • the modified 3’ UTR is truncated compared to the 3’ UTR of a wild-type FKRP.
  • the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region.
  • the nucleic acid encoding FKRP comprises a modification in each of the 5’ and 3’ UTRs.
  • the modification in the 5’ and/or 3’ UTR of FKRP causes an increase or a decrease in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity of a construct comprising wild type 5’ and 3’ FKRP UTRs under substantially similar conditions.
  • the administration of the AAV vector comprises intravenous and/or intramuscular injection.
  • the subject is a human.
  • FIGs.1A-1B is a schematic depicting an exemplary adeno-associated viral vector (AAV) comprising a muscle specific expression cassette and a nucleic acid sequence encoding an FKRP transcript (FIG.1A).
  • AAV adeno-associated viral vector
  • FIG. 1B shows initial data relating to the removal of the 5’UTR and the resulting increase in FKRP expression, as well as the 5’UTR truncations made based on structure predictions and splice sites.
  • FIG.2 Schematic of the pre-mRNA and mature mRNA of FKRP, illustrating how the 5’UTR is on multiple exons while the entire coding region of the gene exists on a single exon.
  • FIG.3 Subset of 30 MSECs from a library of 300 showing relative expression levels from 1/100th to 20X higher than the powerful, ubiquitously expressed CMV promoter.
  • FIGs.4A-4C are schematics showing exemplary modifications to human FKRP gene constructs featuring the first 401 bases from FKRP (SEQ ID NO: 33).
  • FIG.4A Shown are four types of DNA/RNA sequence motifs found in the 5’ UTR that affect FKRP RNA and protein expression levels in mammalian cells.
  • the full 5’ UTR contains a G-quadruplex, a pseudo-knot, an IRES and hairpin forming sequences that influence RNA and protein expression.
  • FIG.4B Mutation of the Kozak consensus sequence in the FKRP gene, which alters expression levels by modifying protein translation.
  • FIG.4C Expression of FKRP protein or RNA can be increased by removal of portions of, or all of the 5’ UTR of the FKRP gene. As but one example, a modified Kozak consensus sequence was introduced into the FKRP expression construct and the entire 5’ UTR was removed and in addition, the upstream MSEC was inserted immediately upstream of the Kozak sequence.
  • FIG.5 is a schematic depicting exemplary 5’UTR variations encoded into the transgene expressed in AAV6. The truncations are based on secondary structure predictions, determined via RNAfold Web Server and sequence patterns.
  • FIGs.6A-6E shows removal of the FKRP 5’- and 3’-UTRs increases FKRP expression.
  • FIG.6A examines cell lysates from differentiated C2C12 myotubes transduced with either 1x10 11 or 1x10 10 vector genomes (vg) of A6.C8mF-FLAG to verify vector expression of mouse FKRP (mFKRP). Labels on the left indicate antibody target (FKRP, Ab607; FLAG, FLAG-HRP, Sigma F7425).
  • FIG.6B examines FKRP levels in myotubes transduced with untagged AAV6-Ck8e-mFKRP (+UTR) and AAV6-Ck8e-mFKRP (-UTR).
  • FIG.6C shows FKRP expression in tibialis anterior (TA) muscles of wild-type mice injected IM with 1x10 11 vg AAV6-Ck8e-mFKRP (-UTR) relative to an untreated wild-type mouse and C2C12 myotubes transduced with 1x10 12 vg of same vector;
  • FIG.6C shows immunostaining of wild-type tibialis anterior muscle injected with AAV6-CK8e- mFKRP. FKRP, green. Golgi, red (GMI30).
  • FIG.6D shows a schematic illustration of the AAV-CK8e-humanFKRP (hFKRP)construct used in these studies.
  • FIG.6E analyzes a transgene construct expressed from vectors pseudotyped with capsids from AAV6 (A6.C8hF), AAV9 (A9.C8hF), or AAVMYO1 (AM.C8hF). Wild-type mice were then treated and tested at various timepoints post-injection to identify potential physiological and histological changes. WT, wild-type.
  • FIGS.7A-7I examines whole-body and isolated limb physiology of wild-type mice treated with AAV-hFKRP.
  • FIG.7A shows the distance run by mice on a treadmill by mice injected with either saline (WT) or 6.4x10 13 vg/kg of A9.C8hF or AM.C8hF.
  • FIG.7B shows the distance run normalized to mouse weight.
  • FIG.7C examines the percent distance changed relative to previous absolute distance measured.
  • FIG.7D shows forelimb grip strength.
  • FIG.7E shows forelimb grip strength normalized to mouse weight.
  • FIG.7F shows the percent forelimb grip strength changed relative to absolute grip strength.
  • FIGs.7G-7I analyzes repeated cycles of plantarflexion eccentric contraction induced injury (20x) in the right hind limb of mice at 4-, 8-, and 12-weeks post-injection measured by hindlimb torque. Limb was stimulated with 10mA at a frequency of 100Hz for 0.4 seconds with a 9 second rest interval between contractions. Significant differences are represented by different letters, shared letter indicate no difference. WT, untreated.
  • FIGs.8A-8E examines cardiac hemodynamics and skeletal muscle mechanics following AAV delivery to wild-type mice. Mice were untreated or injected with 6.4x10 13 vg/kg A9.C8hF or AM.C8hF.
  • FIGs.8A-8C shows ultrasound imaging of heart and diaphragm function at 6- and 12-weeks post-injection.
  • FIG.8D & 8E examines the force:length relationship of TA and diaphragm muscles from same mice at 18 weeks post- injection. Muscles are stretched end-to-end and optimal length is determined at maximum isomeric twitch force (see Methods). No differences were detected in these data. WT, untreated.
  • FIGs.9A-9B shows AAV-FKRP injection does not increase gastrocnemius muscle susceptibility to contraction-induced injury.
  • FIG.10 shows a schematic illustrating the dystrophin-associated protein complex and the extracellular effects of mutated FKRP.
  • DETAILED DESCRIPTION OF THE INVENTION Provided herein are methods and compositions useful for the delivery of a nucleic acid to induce expression of an RNA transcript for FKRP that comprises a modified 5’ and/or 3’ UTR region. Such modifications to the 5’ and/or 3’ UTRs in combination with muscle- specific (e.g., skeletal and/or cardiac) expression of FKRP show improved delivery of FKRP as a therapeutic, as well as reduced side effects from off-target expression of FKRP.
  • nucleic acid cassette refers to a nucleic acid sequence comprising a transcriptional regulatory region and a region that encodes an FKRP RNA transcript.
  • transcriptional regulatory region refers to a nucleotide sequence located upstream of the nucleotide sequence encoding FKRP as described herein and that permits the recruitment of transcriptional machinery and initiation of transcription of an FKRP RNA transcript.
  • a transcriptional regulatory region comprises a promoter (e.g., a tissue-specific promoter, a constitutive promoter etc.).
  • the transcriptional regulatory region can also comprise one or more regulatory elements, such as an enhancer or repressor or binding site elements for transcription factors.
  • the transcriptional regulatory region comprises a promoter and at least 1 enhancer region (e.g., at least 2, at least 3, at least 4, at least 5 or more).
  • the promoter, enhancer element(s) or repressor element(s) can be nucleic acid sequences that are naturally occurring or can be synthetic.
  • the transcriptional regulatory region can be modified to tune expression of FKRP to a desired level, for example, by using a strong promoter and enhancer to increase FKRP expression or alternatively a weaker promoter (or a strong promoter and a weak repressor element) can be used to tune expression of FKRP to a lower level when desired. Further tuning as desired can be achieved through combination of a given transcriptional regulatory region with modifications of the 5’ and/or 3’ UTR of the encoded transcript, e.g., as described herein.
  • FKRP RNA transcript refers to a messenger RNA that encodes FKRP and comprises a modified 5’ or 3’ untranslated region (UTR) as compared to the 5’ or 3’ untranslated region of wild-type FKRP.
  • the modifications to the 5’ and/or 3’ UTR comprise truncation of at least 1 nucleotide (e.g., at least 2, at least 5, at least 10, at least 25, at least 100 nucleotides) or a complete truncation of the 5’ and/or 3’ UTR (i.e., removal of all the nucleotides in a given UTR region).
  • modifications to the 5’ UTR can comprise disruption or deletion of one or more sequences that form secondary structures that influence protein expression (e.g., G-quadruplexes, RNA hairpins, pseudoknots and the like); typically, removal of secondary structures in the 5’ UTR will result in enhanced expression of FKRP by removing structures that can impede binding of translational machinery.
  • modifications to the 5’ UTR can comprise inclusion of a new secondary structure (e.g., an RNA hairpin) that partially impedes binding of translational machinery to tune expression of FKRP to a lower level, if desired.
  • Modifications to the 3’ UTR can comprise the addition, removal or modulation of one or more elements, including but not limited to elements affecting transcript stability, addition of or changes to a polyadenylation signal, and truncations of reducing length of the 3’ UTR, etc.
  • the term “disruption of” when used in reference to RNA transcript secondary structures refers to the removal of (or alternatively the addition of) nucleotides that in turn disrupt the secondary structure.
  • disruption of a G-quadruplex can be achieved by removal of one or more G-Cs in the region or mutation of one or more of the guanines (G) or cytosines (C) to an adenine (A) or uracil (U) to disrupt the G-C base pair-mediated formation of the G-quadruplex.
  • disruption of an RNA hairpin can comprise nucleotide mutations or small deletions that remove or modify the self- complementary/palindromic sequence that results in RNA hairpin formation.
  • operably linked refers to the placement of components e.g., in an upstream transcriptional regulatory region such that they work in relationship, thereby permitting them to function in their intended manner.
  • a control sequence such as a promoter or enhancer
  • a control sequence is positioned in such a way that expression of a nucleic acid sequence encoding FKRP is under the control of the promoter and/or enhancer sequences.
  • “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease or lessening of a property, level, or other parameter by a statistically significant amount.
  • “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more.
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for a cell or individual without a given disorder.
  • the terms “increased,” “increase,” “increases,” or “enhance” or “activate” are all used herein to generally mean an increase of a property, level, or other parameter by a statistically significant amount; for the avoidance of any doubt, the terms “increased”, “increase” or “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, at least about a 20-fold increase, at least about a 50-fold increase, at least about a 100-fold
  • compositions, methods, and respective component(s) thereof are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.
  • consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • patient refers to an animal, particularly a human, to whom treatment of an FKRP-mediated disease or disorder, including prophylactic treatment is provided.
  • subject refers to human and non-human animals.
  • non-human animals and “non-human mammals” are used interchangeably herein and includes all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dog, rodent (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, and non-mammals such as chickens, amphibians, reptiles etc.
  • the subject is human. In another embodiment, the subject is an experimental animal or animal substitute as a disease model. In another embodiment, the subject is a domesticated animal including companion animals (e.g., dogs, cats, rats, guinea pigs, hamsters etc.).
  • companion animals e.g., dogs, cats, rats, guinea pigs, hamsters etc.
  • a subject can have previously received a treatment for an FKRP-mediated disease, or has never received treatment for an FKRP-mediated disease.
  • a subject can have previously been diagnosed with having an FKRP-mediated disease, or has never been diagnosed with an FKRP-mediated disease.
  • a “therapeutically effective amount” or a “therapeutically effective dose” refers to an amount of a nucleic acid cassette or AAV vector as described herein that, when administered to a subject, is sufficient to effect partial or complete treatment of an FKRP-mediated disease or condition in the subject.
  • the amount of a nucleic acid cassette or AAV vector that constitutes a “therapeutically effective amount” will vary depending on the nucleic acid cassette or AAV vector, the condition and severity of the disease, the manner of administration, and/or the age of the subject to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his or her own knowledge and to this disclosure.
  • nucleic acid cassette or AAV vector when a nucleic acid cassette or AAV vector is said to possess “therapeutic efficacy,” this is intended to mean that the nucleic acid cassette or AAV vector is capable of effecting treatment of FKRP-mediated disease or condition in a subject, provided a “therapeutically effective amount” of the nucleic acid cassette or AAV vector is administered under appropriate conditions.
  • treating refers to the treatment of an FKRP-mediated disease or condition of interest in a subject (e.g., a human) having the disease or condition of interest, and includes: (i) preventing or inhibiting the disease or condition from occurring in the subject, for example, when the subject is predisposed to the condition, but has not yet been diagnosed as having the condition; (ii) inhibiting the disease or condition, i.e., arresting or slowing its development or progression; (iii) relieving the disease or condition, i.e., causing regression of the disease or condition; and/or (iv) relieving one or more symptoms (e.g., muscle weakness, muscle fatigue, abnormalities in the brain and/or eyes), resulting from the disease or condition or an improvement in the disease, for example, beneficial or desired clinical results.
  • a subject e.g., a human having the disease or condition of interest
  • beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, disease stabilization (e.g., not worsening), delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • treating can refer to prolonging survival as compared to expected survival if not receiving treatment.
  • a treatment can improve the disease condition, but may not be a complete cure for the disease.
  • Successful treatment can also be assessed by a reduction in the need for medical interventions, reduction in hospital or emergency room visits, reduction in fatigue, or other markers of an improved quality of life.
  • the phrase “reducing at least one symptom of an FKRP-mediated disease or disorder” refers to a reduction in the presence of a given symptom, or severity of a given symptom associated with an FKRP-mediated disease or disorder following treatment with the methods and compositions described herein.
  • the reduction of at least one symptom can include the prevention or delay of an expected symptom onset in a subject diagnosed with an FKRP-mediated disease and undergoing treatment as described herein.
  • Exemplary symptoms of an FKRP-mediated disease include muscle atrophy (e.g., a decrease in muscle mass), muscle weakness, weak heart rate, muscle fatigue, muscle pain, inflammation, decrease in average myofiber diameter in skeletal muscle, loss of ambulation, abnormalities in the brain and/or eyes, eye problems, delay in development, intellectual disability, seizures, and mortality.
  • At least one symptom of an FKRP- mediated disease is reduced by at least 10% as assessed using an appropriate standard clinical measures such as MRI, CT scan, X-rays, PET scans, electromyography, muscle biopsy, ECG, and patient self-reporting; in other embodiments, the at least one symptom is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or even 100% (i.e., symptom is resolved or is below detectable parameters using a clinical measure).
  • an appropriate standard clinical measures such as MRI, CT scan, X-rays, PET scans, electromyography, muscle biopsy, ECG, and patient self-reporting
  • the at least one symptom is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or even 100% (
  • therapeutic efficacy can be measured by a reduction in hospital visits, a reduction in the duration of hospital stays, reduction in medications or doses of such medications, increased longevity, improved quality of life and the like.
  • about refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1% to a reference quantity, level, number, frequency, percentage, dimension, size, amount, weight, or length. In any embodiment discussed in the context of a numerical value used in conjunction with the term “about,” it is specifically contemplated that the term “about” can be omitted.
  • FKRP-mediated Neuromuscular Disorders Provided herein are methods and compositions that can be used in the treatment of FKRP-mediated diseases or disorders.
  • Fukutin-Related Protein FKRP
  • FKRP Fukutin-Related Protein
  • DGC dystrophin-glycoprotein complex
  • dystroglycanopathies which are a collection of diseases resulting from dysfunction of ⁇ - dystroglycan.
  • diseases include limb girdle muscular dystrophy type 2I/R9, congenital muscular dystrophy (MDC1C), Walker-Warburg syndrome, and muscle-eye-brain disease (MED).
  • MDC1C congenital muscular dystrophy
  • MED muscle-eye-brain disease
  • treatment utilizes gene replacement therapy, however new strategies are being developed in combination with gene replacement therapy.
  • Such strategies include gene upregulation, gene editing, testing novel vectors and delivery systems for gene delivery, and developing improved “muscle-specific expression cassettes” (MSECs) as described herein, which help to optimize gene expression levels from the vectors for FKRP-centric tissue-specific needs.
  • MSECs muscle-specific expression cassettes
  • FKRP modifications and constructs Provided herein are methods and compositions that comprise expression of an RNA transcript of FKRP that has a modified 5’ and/or 3’ UTR.
  • Methods and compositions provided herein include muscle-specific expression cassettes (MSECs) that encode such RNA transcripts.
  • the nucleic acid cassette comprising a transcriptional regulatory region and a nucleic acid encoding an FKRP RNA transcript are inserted into an AAV plasmid or vector.
  • the methods and compositions described herein comprise a modified 5’ UTR and/or 3’UTR region of the FKRP nucleic sequence.
  • Modifications to the 5’ UTR can include truncation of at least 1 nucleotide from the N-terminal end of the 5’ UTR; in other embodiments the truncation is at least 2 nucleotides, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300, at least 310, at least 320, at least 330, at least 337 nucleotides, (e.g., the entire 5’ UTR) from the FKRP sequence.
  • the truncation is at least 2 nucleo
  • truncation when applied to the 5’ UTR generally refers to the removal of nucleotide(s) from the 5’ terminal end, it is also specifically contemplated that the 5’ UTR can be modified to remove one or more nucleotides from the 3’ terminal end of the 5’ UTR.
  • Modifications to the 3’ UTR can include truncation of at least one nucleotide from the 3’ terminal end of the 3’ UTR; in other embodiments, the truncation can comprise removal of at least 2 nucleotides at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300, at least 310, at least 320, at least 330, at least 340, at least 350, at least 360, at least 370, at least 380, at least 390, at least 400, at least 410, at least 410, at
  • the modification comprises complete deletion of the 5’ UTR and/or the 3’ UTR.
  • the wild-type FKRP RNA transcript comprises several secondary structures (e.g., G- quadruplex, hairpins, pseudoknots and the like) in the 5’ UTR that can be deleted or disrupted to enhance binding of transcriptional machinery, thereby increasing FKRP expression.
  • secondary structures e.g., G- quadruplex, hairpins, pseudoknots and the like
  • disruption or deletion of such secondary structures can be utilized.
  • secondary structures can be inserted to partially impede translational machinery, thereby expressing FKRP at lower levels.
  • modification of the 5’ UTR of the FKRP RNA transcript comprises the removal of a G-quadruplex, an RNA hairpin, a pseudoknot, or any combination thereof, with the goal of increasing expression of FKRP in a cell.
  • modification of the 5’ UTR of the FKRP RNA transcript comprises the insertion of a G-quadruplex, an RNA hairpin, a pseudoknot, or any combination thereof, with the goal of decreasing expression of FKRP in a cell.
  • modifications to, or removal of, a given region in the 5’ UTR can be made to improve translation by removal of RNA structures that can impede the association of translational machinery.
  • thermodynamically stable structures such as G-quadruplexes and RNA hairpins in the 5’ UTR of an RNA transcript can result in reduced expression of a gene, such as FKRP.
  • a modification to the 5’ UTR comprises removal or disruption of a G-quadruplex and/or an RNA hairpin.
  • the G- quadruplex sequence in the 5’ UTR of human FKRP comprises attgctccaagatggcggcggcggcggcagcg (SEQ ID NO.9).
  • the G- quadruplex sequence in the 5’ UTR of murine FKRP comprises tgtacaattgctccaagatggcggcggcggcggcggcggcggcggcggcag (SEQ ID NO.10).
  • disruption of a G-quadruplex can include removal of at least 1 nucleotide, at least 2 nucleotides, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, or at least 32 (e.g., all) nucleotides of the G-quadruplex sequence (SEQ ID NO.9 and SEQ ID NO.10) such that it modulates the expression of FKRP.
  • disruption of a G-quadruplex can be performed by substituting adenosine (A) and thymine(T)/uracil (U) residues for guanine (G) and cytosine (C) residues in the G-quadruplex sequence (SEQ ID NO.9 and SEQ ID NO.10). Disruptions of the G- quadruplex that in turn result in modifications of FKRP expression are preferred.
  • disruption of a G-quadruplex can be made by inserting adenosine (A) and thymine (T)/uracil (U) residues for guanine (G) and cytosine (C) residues in the G- quadruplex sequence (SEQ ID NO.9 and SEQ ID NO.10). Disruptions of the G- quadruplex that in turn result in modifications of FKRP expression are preferred.
  • the activity of G-quadruplexes can be modulated using small molecule inhibitors and/or small molecule ligands such that the expression of a cassette is modified.
  • disruption of a hairpin can be achieved by introducing or increasing the number of intramolecular base pair mismatches through nucleotide substitution, thereby modulating the expression of FKRP.
  • RNA hairpins can tolerate a small number of mismatches and that appropriate disruption of an RNA hairpin can require including at least 3, at least 4, at least 5, or more mismatches.
  • disruption of a hairpin can be achieved by removing or adding at least 1 nucleotide, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least the full nucleotide length of one or both strands of a hairpin, thereby modulating the expression of FKRP.
  • disruption of a hairpin can be achieved by inserting non- complementary nucleotides into the hairpin sequence, such that it disrupts the folding or formation of a base-paired stem in the nucleic acid strand, and in turn modulates the expression of FKRP.
  • modifications to the 5’ UTR can comprise introducing a hairpin that prevents, at least partially, proteins from being recruited to initiate translation (e.g. a splicing hairpin), thereby producing FKRP at a lower level of expression.
  • the methods and compositions provided herein comprise a 5’ UTR with a modification comprising a disrupted or deleted pseudoknot.
  • Exemplary pseudoknots that can be deleted or disrupted include pseudoknots that are classified as either a H-, K-, L-, or M-type of pseudoknot.
  • a pseudoknot can also include long-range pseudoknots.
  • RNA structure Software that allows the user to predict the formation of pseudoknots in an RNA structure include PseudoViewer and CyloFold. (Staple and Butcher, PLoS Biol. 2005 Jun; 3(6): e213; Bindewald et al. Nucleic Acids Res.2010 Jul; 38; W368-72).
  • the pseudoknot sequence in the 5’ UTR of human FKRP comprises (SEQ ID NO.29): AGCTCAGCTGGGCTGGAACTGCCCTCCTGGAACTCCCCCAGCCTACAACCTAGGA GGTGCAGGGACTGAGGCTCAGGCCAAATCGCAACTCAGACCCAGTGAACCCAAG GCCTGAAGAGAATTTGGATTCATT [00105]
  • disruption of a pseudoknot can be achieved by the removal of at least one stem-loop region (at least 2, at least 3, or more stem-loop regions) of the pseudoknot.
  • disruption of a pseudoknot can be achieved by the insertion and/or substitution of nucleotides into the pseudoknot sequence, such that it disrupts the folding of the nucleic acid strand.
  • disruption of a pseudoknot can be achieved by increasing mismatches through nucleotide substitution.
  • disruption of a pseudoknot can be achieved by a point mutation of one or more nucleotides in the pseudoknot sequence that participates in formation of the pseudoknot structure.
  • modifications can include alteration of a Kozak consensus sequence.
  • the Kozak consensus sequence is a nucleotide motif that functions as the protein translation initiation site in many mRNA transcripts.
  • MSECs Muscle-Specific Expression Cassettes
  • an MSEC as described herein can comprise control elements (e.g., MSEC enhancers and promoters) that bind both ubiquitous and/or muscle type-specific transcription factors; and the activity of each MSEC is determined by differences in control element types, sequences, numbers, and linear order within the enhancer and promoter regions.
  • MSECs can be used to avoid toxicity and immune activation that occurs with uncontrolled expression of muscle therapeutic proteins in other sites, as well as to restrict the expression of a desired gene (e.g., FKRP) in muscle (e.g., skeletal and/or cardiac).
  • the muscle-specific transcriptional regulatory cassette is derived from an M-creatine kinase enhancer and/or a M-creatine kinase promoter sequence.
  • the muscle-specific transcriptional regulatory cassette can be derived from an M-creatine kinase enhancer with an M-creatine kinase promoter.
  • the muscle-specific transcriptional regulatory cassette can include one or more enhancers derived from conserved regions of muscle creatine kinase and/or a CK8 transcriptional regulatory cassette (SEQ ID NO.: 8).
  • the muscle-specific transcriptional regulatory cassette can be a muscle-specific CK8 transcriptional regulatory cassette (CK8) or a derivative thereof.
  • CK8 is a non-naturally occurring nucleotide sequence including multiple muscle and non-muscle gene control elements arranged in a miniaturized array. CK8 can provide high or very high transcriptional expression of a predetermined RNA and/or protein in skeletal and cardiac muscle cells.
  • an MSEC useful for the methods and compositions described herein comprises a modified CK8 transcriptional regulatory cassette (e.g., CK8e).
  • CK8e modified CK8 transcriptional regulatory cassette
  • excess FKRP may be differentially toxic in cardiac muscle.
  • MSECs that have at least 3-fold, at least 10-fold, at least 30- fold, at least 100-fold, at least 300-fold, at least 1000-fold less transcriptional activity than CK8e can be used.
  • MSEC would produce low levels of FKRP.
  • Different MSECs, their makeup, and how their activity is measured are described in, e.g., PCT/US2022/023915, which is incorporated herein by reference in its entirety.
  • the muscle-specific transcriptional regulatory cassette can be a CK8e transcriptional regulatory cassette having at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or at least 100% sequence identity, to the nucleotide sequence of SEQ ID NO.: 8.
  • SEQ ID NO.8 (CK8e transcriptional regulatory cassette) TGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTA ACCCAGACATGTGGCTGCCCCCCCCCCCAACACCTGCTGCCTCTAAAAATAAC CCTGCATGCCATGTTCCCGGCGAAGGGCCAGCTGTCCCCCGCCAGCTAGAC TCAGCACTTAGTTTAGGAACCAGTGAGCAAGTCAGCCCTTGGGGCAGCCCATAC AAGGCCATGGGGCTGGGCAAGCTGCACGCCTGGGTCCGGGGTGGGCACGGTGCC CGGGCAACGAGCTGAAAGCTCATCTGCTCTCAGGGGCCCCTCCCTGGGGACAGC CCCTCCTGGCTAGTCACACCCTGTAGGCTCCTCTATATAACCCAGGGGCACAGGG GCTGCCCTCATTCTACCACCACCTCCACAGCACAGACAGACACTCAGGAGCCAG CCAGC [00116]
  • the MSEC comprises CK8e.
  • AAV vectors [00117] AAV is a parvovirus which belongs to the genus Dependoparvovirus that is useful for the delivery of therapeutic nucleic acids (e.g., a nucleic acid encoding FKRP). AAV’s usefulness stems from its ability to infect a wide range of host cells, including non-dividing cells, as well as its ability to infect cells from different species. AAV has not been associated with any human or animal disease and does not appear to alter the biological properties of the host cell upon integration, thus making it an ideal vector for the delivery of therapeutic nucleic acids.
  • An "AAV vector,” as that term is used herein, comprises a vector derived from an adeno-associated virus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.
  • the AAV vector comprises AAV6.
  • AAV vectors can have one or more of the AAV wild-type viral genes deleted in whole or part, e.g., the rep and/or cap genes, but retain functional flanking ITR sequences. Where functional ITR sequences are necessary for the rescue, replication, and packaging of the AAV virion, in some embodiments, the AAV vectors described herein include at least one functional ITR.
  • the ITRs need not be the wild- type nucleotide sequences, and can be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging.
  • the AAV vectors described herein are engineered to produce synthetic, modified or recombinant AAV vectors.
  • a "recombinant AAV vector” or “rAAV vector” comprises an infectious, replication-defective virus composed of an AAV capsid protein shell encapsulating a heterologous nucleotide sequence of interest that is flanked on both sides by AAV ITRs.
  • An rAAV vector is produced in a suitable host cell comprising an AAV vector, AAV helper functions, and accessory functions.
  • the host cell is rendered capable of encoding AAV polypeptides that are required for packaging the AAV vector (containing a recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.
  • Exemplary recombinant AAV (rAAV) vectors include, but are not limited to, an AAVRh74 vector, an AAV8 vector, an AAV9 vector, an AAV6 vector, an AAV7 vector, an AAV2i8 vector, a NP vector, a NP 66 vector, a NP 22 vector, an AAVpo.1 vector, MyoAAV, and/or an AAVMyo vector.
  • the AAV vector comprises a MyoAAV vector (Weinmann, J. et al.2020, Nat Commun 11, 5432).
  • the AAV vector comprises an AAVMyo vector (Tabebordbar, M.
  • the therapeutically effective amount of the pharmaceutical composition can be between about 10 11 and about 10 16 vector genomes (vg)/kilogram (kg) subject weight, between about 10 12 and about 10 15 vg/kg subject weight, between about 10 13 and about 10 14 vg/kg subject weight, between about 10 12 and about 10 14 vg/kg subject weight , between about 10 12 and about10 15 vg/kg subject weight, between about 10 12 and about 10 16 vg/kg subject weight, between about 10 11 and about10 14 vg/kg subject weight, between about 10 10 and about 10 15 vg/kg subject weight , between about 10 13 and about 10 15 vg/kg subject weight, between about 10 14 and about 10 15 vg/kg subject weight, between about 10 13 and about 10 14 vg/kg subject weight, or
  • the pharmaceutical composition can be administered intravascularly, intraperitoneally, subcutaneously, or by intramuscular injection.
  • it can be beneficial to optimize the level of expression of FKRP in muscle tissue (e.g., skeletal and/or cardiac).
  • muscle tissue e.g., skeletal and/or cardiac
  • it is important to prevent unintended effects from producing too much FKRP, however still expressing enough FKRP to receive its benefits.
  • Some AAV vectors, when combined with different MSECs can produce high levels of expression of FKRP. However, other AAV vectors, when combined with different MSECs, can produce much lower levels of expression of FKRP.
  • Titrating the optimal expression of FKRP protein can be achieved by selecting vectors and/or MSECs that produce different levels of protein in particular muscle tissues (e.g., skeletal and/or cardiac). Such titration is well within the skill set of one of skill in the art given the guidance provided herein. As but one example, if a construct comprising a modified 5’-FKRP UTR cDNA is determined to convey high FKRP protein levels, it may well be that the MSEC in combination with the modified UTRs and AAV vector produces too much FKRP and possibly even toxic levels.
  • an MSEC that produces transcripts in a muscle-specific, but lower level can be used, one or more 5’- or 3’-UTR modifications as discussed herein can be introduced, or a combination of MSEC and 5’- or 3’-UTR modifications can be used to titrate FKRP message and/or protein levels as needed.
  • Measuring FKRP expression levels [00123] Methods to measure the level of FKRP expression products in a cell or tissue are known to a skilled artisan.
  • Such methods to measure FKRP expression products include, e.g., protein level, include ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, histology; immunohistological staining; and/or immunofluoresence assay using detection reagents such as an antibody or protein binding agents.
  • an FKRP antibody is used to measure the expression of FKRP from the nucleic acid cassettes or AAV vectors as described herein.
  • Antibodies for FKRP are commercially available and can be used to measure protein expression levels, (e.g. anti- FKRP (Cat. No. sc-374642; Santa Cruz Biotechnologies, Santa Cruz, CA), anti-FKRP (Cat. No.
  • amino acid sequences for the targets described herein are known and publicly available at the NCBI website, one of skill in the art can raise their own antibodies against these polypeptides of interest for the purpose of the methods described herein.
  • the amino acid sequences of the polypeptides described herein have been assigned NCBI accession numbers for different species such as human, mouse and rat.
  • amino acid sequences of human FKRP and murine FKRP are included herein, e.g. SEQ ID NO: 11 and SEQ ID NO.: 12 respectively.
  • immunohistochemistry (“IHC”) and immunocytochemistry (“ICC”) techniques can be used to detect FKRP expression.
  • IHC is the application of immunochemistry to tissue sections
  • ICC is the application of immunochemistry to cells or tissue imprints after they have undergone specific cytological preparations such as, for example, liquid-based preparations.
  • Immunochemistry is a family of techniques based on the use of an antibody, wherein the antibodies are used to specifically target molecules inside or on the surface of cells.
  • the antibody typically contains a marker that will undergo a biochemical reaction, and thereby experience a change of color, upon encountering the targeted molecules.
  • signal amplification can be integrated into the particular protocol, wherein a secondary antibody, that includes the marker stain or marker signal, follows the application of a primary specific antibody.
  • the assay to detect FKRP expression can be a Western blot analysis.
  • proteins can be separated by two-dimensional gel electrophoresis systems. Two-dimensional gel electrophoresis is well known in the art and typically involves iso-electric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. These methods also require a considerable amount of cellular material.
  • the analysis of 2D SDS-PAGE gels can be performed by determining the intensity of protein spots on the gel, or can be performed using immune detection.
  • protein samples are analyzed by mass spectroscopy.
  • An immunoassay is a biochemical test that measures the concentration of a substance in a biological sample, typically a fluid sample such as blood or serum, using the interaction of an antibody or antibodies to its antigen.
  • the assay takes advantage of the highly specific binding of an antibody with its antigen.
  • specific binding of the FKRP-specific polypeptides with respective FKRP-specific proteins or protein fragments, or an isolated peptide, or a fusion protein described herein occurs in the immunoassay to form a target protein/peptide complex. The complex is then detected by a variety of methods known in the art.
  • An immunoassay also often involves the use of a detection antibody.
  • Detectably labeled enzyme-linked secondary or detection antibodies can then be used to detect and assess the amount of polypeptide in the sample tested.
  • a dot blot immobilizes a protein sample on a defined region of a support, which is then probed with antibody and labelled secondary antibody as in Western blotting.
  • the intensity of the signal from the detectable label in either format corresponds to the amount of enzyme present, and therefore the amount of polypeptide.
  • Levels can be quantified, for example by densitometry.
  • the FKRP expression products as described herein can be instead determined by determining the level of FKRP-specific messenger RNA (mRNA).
  • mRNA messenger RNA
  • the level of an FKRP-specific mRNA can be measured by a quantitative sequencing technology, e.g. a quantitative next-generation sequence technology.
  • Exemplary methods of sequencing include, but are not limited to, Sanger sequencing, dideoxy chain termination, high-throughput sequencing, next generation sequencing, 454 sequencing, SOLiD sequencing, polony sequencing, Illumina sequencing, Ion Torrent sequencing, sequencing by hybridization, nanopore sequencing, Helioscope sequencing, single molecule real time sequencing, RNAP sequencing, and the like. Methods and protocols for performing these sequencing methods are known in the art, see, e.g. “Next Generation Genome Sequencing” Ed. Michal Janitz, Wiley-VCH; “High-Throughput Next Generation Sequencing” Eds.
  • RNA molecules can be isolated from a particular biological sample (e.g. a mouse) using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample. (Roiff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)).
  • a particular biological sample e.g. a mouse
  • the particular isolation procedure chosen being appropriate for the particular biological sample. (Roiff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)).
  • one or more of the reagents e.g.
  • an FKRP-specific antibody reagent and/or nucleic acid probe described herein can comprise a detectable label and/or comprise the ability to generate a detectable signal (e.g. by catalyzing reaction converting a compound to a detectable product).
  • Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into reagents (e.g. FKRP- specific antibodies and nucleic acid probes) are well known in the art.
  • detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means.
  • the detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies).
  • the detectable label can be linked by covalent or non-covalent means to the reagent.
  • a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules.
  • Detectable labels can include, but are not limited to radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.
  • the detection reagent is label with a fluorescent compound. When the fluorescently labeled reagent is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence.
  • a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, Cy3 TM , Cy5 TM , allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5 TM , green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green TM , rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes TM , 6- carboxyfhiorescein (commonly known by the abbreviations FAM and F), 6-carboxy- 2',4',
  • fluorescein fluoresc
  • a detectable label can be a radiolabel including, but not limited to 3 H, 125 I, 35 S, 14 C, 32 P, and 33 P.
  • a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase.
  • An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal.
  • Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.
  • detection reagents can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin.
  • a detectable tag such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin.
  • Other detection systems can also be used, for example, a biotin-streptavidin system.
  • the antibodies immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate.
  • streptavidin peroxidase detection kits are commercially available, e. g.
  • compositions [00137] An aspect of the disclosure relates to pharmaceutical or biopharmaceutical compositions comprising the nucleic acid cassettes or AAV vectors described herein.
  • the pharmaceutical composition can include a transcriptionally regulatory cassette (e.g., a muscle-specific regulatory cassette) operably linked to a nucleotide sequence encoding fukutin-related protein (FKRP), wherein an RNA transcript when expressed from the nucleotide sequence encoding FKRP comprises a modified 5’ and/or 3’ untranslated region (UTR).
  • a transcriptionally regulatory cassette e.g., a muscle-specific regulatory cassette
  • FKRP fukutin-related protein
  • UTR untranslated region
  • the pharmaceutical composition can comprise an AAV vector comprising a transcriptional regulatory cassette operably linked to a nucleotide sequence encoding fukutin-related protein (FKRP), wherein the FKRP RNA transcript when expressed from the nucleotide sequence encoding FKRP comprises a modified 5’ and/or 3’ untranslated region (UTR).
  • the composition can be prepared in pharmaceutically acceptable, physiologically acceptable, and/or pharmaceutical-grade solutions for administration to a cell or a subject (e.g., an animal), either alone, or in combination with one or more other modalities of therapy.
  • the formulations may be administered in combination with other agents, such as other proteins, polypeptides, pharmaceutically active agents, etc.
  • the composition can optionally include a carrier, such as a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions.
  • intramuscular injection can be used to directly deliver the compositions as described herein to muscle (e.g., skeletal and/or cardiac muscle). While delivery to skeletal muscle is preferred, some delivery to cardiac muscle can also be useful for the methods described herein.
  • a targeting moiety can be used that will enhance delivery to muscle (e.g., skeletal and/or cardiac muscle).
  • Exemplary carriers can include aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, preservatives, liposomes, microspheres and emulsions.
  • the composition is formulated for intramuscular delivery.
  • the composition is formulated for intracardiac delivery.
  • Therapeutic compositions contain a physiologically tolerable carrier together with the vectors described herein, dissolved or dispersed therein as an active ingredient.
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
  • a pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired.
  • the preparation of a pharmaceutical composition that contains active ingredients dissolved or dispersed therein is understood in the art and need not be limited based on formulation.
  • compositions are prepared as injectable either as liquid solutions or suspensions; however, solid forms suitable for solution, or suspension in liquid prior to use can also be prepared.
  • the preparation can also be emulsified or presented as a liposome composition.
  • the active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.
  • the therapeutic composition for use with the methods described herein can include pharmaceutically acceptable salts of the components therein.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable carriers are well known in the art.
  • Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
  • compositions for use as described herein can be formulated for any appropriate manner of administration, including for example, intravenous or intramuscular administration.
  • the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer.
  • compositions as described herein can be formulated as a lyophilizate.
  • the pharmaceutical composition may reduce a pathological effect or symptom of a neuromuscular disorder associated with FKRP in a subject.
  • Dosage and Administration Essentially any method of administration can be used with the methods and compositions described herein that permit intramuscular delivery and expression of FKRP in muscle (e.g., skeletal or cardiac muscle).
  • the administrative method will comprise delivery of a therapeutically effective amount of a pharmaceutical composition to one or more muscles in a subject to be treated (e.g., intramuscular injection, intracardiac injection, systemic or intravenous injection or infusion).
  • the pharmaceutical composition can include a nucleic acid expression cassette comprising a transcriptional regulatory region operably linked to a nucleotide sequence encoding a fukutin-related protein (FKRP) RNA transcript comprising a modified 5’ and/or 3’ untranslated region (UTR).
  • FKRP fukutin-related protein
  • UTR untranslated region
  • the compositions can be administered via any suitable route that permits delivery to muscle tissue (e.g. skeletal and/or cardiac), including but not limited to, locally, subcutaneously, systemically, intravenously, intravascularly, intramuscularly, intracardiac, or via a bolus.
  • the compositions can be encapsulated in liposomes, exosomes, microparticles, microcapsules, nanoparticles, and the like, if so desired.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes.
  • Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension can also contain suitable stabilizers or agents that increase the solubility of the active ingredients, to allow for the preparation of highly concentrated solutions.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, water-based solution, before use.
  • a therapeutic agent can be delivered in an immediate release form.
  • the therapeutic agent can be delivered in a controlled-release system or sustained-release system. Controlled- or sustained-release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.
  • the compositions described herein can be administered via a schedule including continuous administration or intermittent administration. Accordingly, in addition to these general schedules, in some embodiments, the composition can be administered twice a day, once a day, once every other day, once a week, once a month, or another suitable period of administration.
  • a pump can be used for administration (Langer, Science 249:1527-1533 (1990); Sefton, CRC Crit. Ref Biomed. Eng.14:201 (1987); Buchwald et al., Surgery 88:507 (1980); and Saudek et al., N. Engl. J. Med 321:574 (1989)).
  • polymeric materials can be used (see Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); Levy et al., Science 228:190 (1985); During et al., Ann. Neurol.25:351 (1989); and Howard et al., J. Neurosurg.71:105 (1989)).
  • the compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • Treatment using the methods and compositions described herein includes both prophylaxis/prevention of disease onset and therapy of an active disease.
  • Prophylaxis or treatment can be accomplished by a single direct injection at a single time point or multiple time points. Administration can also be nearly simultaneous to multiple sites.
  • Patients or subjects include mammals, such as human, bovine, equine, canine, feline, porcine, and ovine animals as well as other veterinary subjects. Preferably, the patients or subjects are human.
  • the methods described herein provide a method for treating a disease or disorder in a subject (e.g., a muscle disease or disorder).
  • the subject can be a mammal.
  • the mammal can be a human, although the approach is effective with respect to all mammals.
  • the method comprises administering to the subject an effective amount of a pharmaceutical composition comprising vector as described herein in a pharmaceutically acceptable carrier.
  • the dosage range for the agent depends upon the potency, the expression level of the therapeutic protein and includes amounts large enough to produce the desired effect, e.g., reduction in at least one symptom of the disease to be treated. The dosage should not be so large as to cause unacceptable adverse side effects.
  • the dosage will vary with the therapeutic composition (e.g., AAV vector vs. plasmid delivery), and with the age, condition, and sex of the patient.
  • the dosage can be determined by one of skill in the art and can also be adjusted by the individual physician in the event of any complication.
  • the vectors are administered at a multiplicity of infection (MOI) of at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 500 or more.
  • MOI multiplicity of infection
  • the vectors are administered at a titer of at least 1 x 10 4 , 1x 10 5 , 1 x 10 6, 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 viral particles or more.
  • Repeated administration can be performed as necessary to maintain therapeutic efficacy.
  • the term “therapeutically effective amount” refers to an amount of a vector or expressed FKRP that is sufficient to produce a statistically significant, measurable change in at least one symptom of a disease (see “Efficacy Measurement” below).
  • a therapeutically effective amount is an amount of a vector or expressed FKRP protein that is sufficient to produce a statistically significant, measurable change in the expression level of a biomarker associated with the disease in the subject. Such effective amounts can be gauged in clinical trials as well as animal studies for a given agent.
  • the vector compositions can be administered directly to a particular site (e.g., intramuscular injection). It is also contemplated herein that the agents can also be delivered intravenously (by bolus or continuous infusion), or systemically, if so desired and provided that FKRP can be expressed in one or more muscle sites (e.g., skeletal and/or cardiac).
  • Therapeutic compositions containing at least one agent can be conventionally administered in a unit dose.
  • unit dose when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier, or vehicle.
  • Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are particular to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more intervals by a subsequent injection or other administration.
  • the doses recited above or as employed by a skilled clinician can be repeated for a limited and defined period of time.
  • the doses are given once a day, or multiple times a day, for example, but not limited to three times a day.
  • the dosage regimen is informed by the half-life of the agent as well as the minimum therapeutic concentration of the agent in blood, serum or localized in a given biological tissue.
  • the doses recited above are administered daily for several weeks or months. The duration of treatment depends upon the subject’s clinical progress and continued responsiveness to therapy. Continuous, relatively low maintenance doses are contemplated after an initial higher therapeutic dose.
  • the methods and compositions described herein comprise a step of diagnosing a subject as having an FKRP-mediated disease.
  • FKRP-mediated diseases can be initially diagnosed by a muscle MRI.
  • FKRP-mediated diseases can show damage to the proximal muscles with relative preservation of the muscles of the anterior compartment of the thighs.
  • a follow up with genetic analysis by specific sequencing of the FKRP gene or of a panel grouping together all the genes involved in the glycosylation of a ⁇ -dystroglycan, or a larger panel of genes can be used to confirm the diagnosis.
  • the efficacy of a given treatment for reducing or preventing FKRP-mediated diseases can be determined by the skilled clinician.
  • a treatment is considered “effective treatment,” as the term is used herein, if any one or all of the signs or symptoms of muscle (e.g., muscle weakness, muscular atrophy), brain, and/or eye deterioration is/are altered in a beneficial manner, or other clinically accepted symptoms or markers of disease are improved, or ameliorated, e.g., by at least 10% following treatment with a therapeutic agent that increases expression of FKRP in muscle (e.g., skeletal and/or cardiac). Efficacy can also be measured by failure of an individual to worsen as assessed by stabilization of the disease, or the need for medical interventions (i.e., progression of the disease is halted or at least slowed).
  • Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing progression of muscle, brain, and/or eye deterioration or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of the disease, or preventing secondary diseases/disorders associated with the deterioration of the muscle, brain, and/or eye.
  • the technology may be as described in any one of the following numbered paragraphs: [00166] Paragraph 1.
  • a nucleic acid expression cassette comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a fukutin-related protein (FKRP) RNA transcript that comprises a modified 5’ and/or 3’ untranslated region (UTR).
  • FKRP fukutin-related protein
  • UTR modified 5’ and/or 3’ untranslated region
  • Paragraph 4 The nucleic acid expression cassette of paragraph 3, wherein the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR.
  • Paragraph 5. The nucleic acid expression cassette of paragraph 4, wherein the modification comprises a modification to the Kozak consensus sequence.
  • Paragraph 6. The nucleic acid expression cassette of paragraph 1, wherein the modified 3’ UTR is truncated compared to the 3’ UTR of wild-type FKRP.
  • nucleic acid expression cassette of paragraph 5 wherein the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region.
  • Paragraph 8 The nucleic acid expression cassette of paragraph 1, wherein the nucleic acid encoding FKRP comprises a modification in each of the 5’ and 3’ UTRs.
  • nucleic acid expression cassette of any of paragraphs 1-8 wherein the modification in the 5’ and/or 3’ UTR of FKRP causes an increase or a decrease in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity expressed from a similar construct comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a FKRP RNA transcript that comprises a wild-type 5’ and/or 3’ untranslated region (UTR).
  • UTR wild-type 5’ and/or 3’ untranslated region
  • Paragraph 12 The nucleic acid expression cassette of paragraph 11, wherein upon administration to a cell, expression level of an FKRP mRNA or protein is higher when operably linked to an MSEC than the expression level of the FKRP mRNA or protein when operably linked to a CK8e transcriptional regulatory region.
  • Paragraph 13 The nucleic acid expression cassette of paragraph 11, wherein upon administration to a cell, expression level of an FKRP mRNA or protein is lower when operably linked to an MSEC than the expression level of the FKRP mRNA or protein when operably linked to a CK8e transcriptional regulatory region.
  • Paragraph 14 The nucleic acid expression cassette of paragraph 11, wherein upon administration to a cell, expression level of an FKRP mRNA or protein is lower when operably linked to an MSEC than the expression level of the FKRP mRNA or protein when operably linked to a CK8e transcriptional regulatory region
  • An adeno-associated viral vector comprising the nucleic acid expression cassette of any one of paragraphs 1- 13.
  • Paragraph 16 The AAV vector of paragraph 15, wherein the adeno-associated viral vector is selected from the group consisting of: an AAVRh74 vector, an AAV8 vector, an AAV9 vector, an AAV6 vector, an AAV7 vector, an AAV2i8 vector, a NP vector, a NP 66 vector, a NP 22 vector, an AAVpo.1 vector, a MyoAAV vector, and an AAVMyo vector.
  • Paragraph 17 The AAV vector of paragraph 15, wherein the adeno-associated viral vector is selected from the group consisting of: an AAVRh74 vector, an AAV8 vector, an AAV9 vector, an AAV6 vector, an AAV7 vector, an AAV2i8 vector, a NP vector, a NP 66 vector
  • the AAV vector of paragraph15 wherein the adeno-associated viral vector comprises an internal terminal repeat (ITR), a muscle-specific expression cassette, a nucleic acid encoding FKRP, a polyadenylation signal (pA+), and/or a second ITR.
  • ITR internal terminal repeat
  • pA+ polyadenylation signal
  • Paragraph 20 An engineered cell comprising or expressing a nucleic acid expression cassette of any one of paragraphs 1- 17.
  • Paragraph 19 The engineered cell of paragraph 18, wherein the modified 5’ untranslated region (UTR) is truncated as compared to the wild-type 5’ UTR of FKRP.
  • Paragraph 21 The engineered cell of paragraph 20, wherein the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR.
  • Paragraph 22 The engineered cell of paragraph 20 or 21, wherein the modification comprises a modification to the Kozak consensus sequence.
  • Paragraph 23 The engineered cell of paragraph 18, wherein the modified 3’ UTR is truncated compared to the 3’UTR of wild-type FKRP.
  • Paragraph 24 The engineered cell of paragraph 23, wherein the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region.
  • Paragraph 25 The engineered cell of paragraph 18, wherein the nucleic acid encoding FKRP comprises a modification in each of the 5’and 3’ UTRs.
  • Paragraph 26 The engineered cell of paragraph 23, wherein the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region.
  • the FKRP gene product is a RNA transcript and/or a protein.
  • Paragraph 29 The method of claim 27, wherein the subject in need thereof comprises limb girdle muscular dystrophy type 2I/R9 (LGMD2i), Walker-Warburg syndrome, or muscle-eye-brain disease (MED).
  • Paragraph 30 The method of paragraph 27, wherein the modified 5’ untranslated region (UTR) is truncated as compared to the 5’ UTR of wild-type FKRP.
  • the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 5’ UTR region.
  • Paragraph 32 The method of paragraph 31, wherein the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR.
  • Paragraph 33 The method of paragraph 32, wherein the modification comprises a modification to the Kozak consensus sequence.
  • Paragraph 34 The method of paragraph 27, wherein the modified 3’ UTR is truncated compared to the 3’ UTR of a wild-type FKRP.
  • Paragraph 35 The method of paragraph 27, wherein the modified 3’ UTR is truncated compared to the 3’ UTR of a wild-type FKRP.
  • a method for reducing at least one symptom of an FKRP-mediated disease or disorder comprising administering an AAV vector of any one of paragraphs 15-17 to a subject in need thereof, thereby reducing at least one symptom of an FKRP-mediated disorder.
  • the FKRP-mediated disease or disorder comprises limb girdle muscular dystrophy type 2I/R9 (LGMD2i), Walker-Warburg syndrome, or muscle-eye-brain disease (MED).
  • Paragraph 42 The method of paragraph 40, wherein the modified 5’ untranslated region (UTR) is truncated as compared to the wild-type 5’ UTR of FKRP.
  • the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 5’ UTR region.
  • the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR.
  • Paragraph 45 The method of paragraph 44, wherein the modification comprises a modification to the Kozak consensus sequence.
  • Paragraph 46 The method of paragraph 40, wherein the modified 3’ UTR is truncated compared to the 3’ UTR of a wild-type FKRP.
  • Paragraph 47 The method of paragraph 46, wherein the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region.
  • Paragraph 48 The method of paragraph 40, wherein the nucleic acid encoding FKRP comprises a modification in each of the 5’ and 3’ UTRs.
  • At least one symptom of a FKRP-mediated disease or disorder comprises: muscle pain, muscle weakness, muscle fatigue, muscle atrophy, inflammation, decrease in average myofiber diameter in skeletal muscle, loss of ambulation, abnormalities in the brain and/or eyes, eye problems, delay in development, intellectual disability, and seizures.
  • FKRP fukutin-related protein
  • MDC1C congenital muscular dystrophy
  • WWS Walker-Warburg syndrome
  • MEB muscle-eye-brain disease
  • FKRP is delivered to a cell by way of expression from a nucleic acid expression cassette.
  • nucleic acid expression cassettes can be encoded in an AAV vector.
  • an AAV plasmid used in the methods and compositions described herein comprises ITRs, a muscle-specific expression cassette (e.g., CK8e), the wild-type FKRP cDNA and a polyA signal.
  • This vector can be adapted as desired and tested using initial dose escalation studies, for example, with the AAV6 capsid.
  • the AAV vectors can be assessed and compared at varying intravascular doses between 5x10 ⁇ 12 vector genomes per kg (vg/kg) up to 2x10 ⁇ 14 vg/kg in different models.
  • the low dose of ⁇ 5x10 ⁇ 12 vg/kg may not be particularly useful for systemic delivery but can still be considered for direct administration to muscle.
  • the high dose of ⁇ 2x10 ⁇ 14 vg/kg is the dose that can be used to obtain near saturating levels of gene transfer in several different models of muscular dystrophy, and is the dose being used in essentially all current human gene therapy trials for DMD, LGMD, SMA and MTM1.
  • a dose of 2x10 ⁇ 14 vg/kg of a given AAV vector is used with the methods and compositions described herein.
  • additional studies can be designed to test and optimize the expression cassette needed for clinical gene transfer. Importantly, ongoing human gene therapy trials have highlighted the critical need for refined MSECs to appropriately target gene expression, while avoiding toxicity and immune activation.
  • EXAMPLE 2 FKRP SEQUENCE OPTIMIZATION [00223] Analysis of the untranslated (UTR) regions of the FKRP gene indicates the presence of inhibitory sequences in the 5’ UTR of the FKRP gene. These include high GC content, an RNA G-quadruplex structure and other inverted repeats.
  • EXAMPLE 3 SELECTION OF AAV SEROTYPES
  • Optimal gene therapy for LGMD2I and CMD can benefit from vectors that display enhanced targeting of striated muscles and muscle satellite (stem) cells, while minimizing transduction of the liver and other organs.
  • Ongoing work in the field aims to develop synthetic capsids to either alter tropism or evade pre-existing immunity to natural serotypes.
  • Three types of synthetic capsids have been developed: (a) insertion of short sequences from one serotype to another, (b) synthesis of novel capsids following ancestral AAV sequence reconstruction (c) shuffling sequences randomly and between different natural serotypes.
  • EXAMPLE 6 TESTING GENE DELIVERY SYSTEMS
  • a wide variety of testing systems can be used to evaluate the various vector and other gene delivery systems contemplated herein. These include conventional mouse models of FKRP disorders using dose escalation combined with functional and immunological testing.
  • the inventors can also test expression cassettes and vectors in various in vitro systems, such as myogenic cultures (primary and iPSC-derived), 2D and 3D human myogenic systems as discussed elsewhere herein.
  • the in vitro systems permit analysis of efficiency, expression levels and some functional readouts.
  • the in vivo systems can also add tropism, efficiency, whole body function and safety studies including immune responses, serum chemistries, blood counts and complement activation.
  • the gene therapy vectors can be optimized to express FKRP at varying levels in muscle cells.
  • the therapeutically effective amount of FKRP expression can be affected by the percentage of muscle fiber and cardiomyocyte myonuclei that are transduced, which varies by the use of different therapeutic vectors; and since new vector designs will improve transduction efficiencies, optimal therapeutic protein product levels may decrease as targeted transduction efficiencies improve.
  • MSECs with attributes for treating LGMD2I have been designed and are currently being optimized, and can be tested for efficacy in FKRP mutant mouse and rat models. This work can be done in the following three exemplary phases.
  • Phase 1 testing can entail a series of studies starting with the AAV gene therapy approaches outlined above.
  • AAV-FKRP vectors can be tested using MSECs that are expected to produce low, medium and high levels of expression.
  • a first goal can be to identify the strongest MSECs that generate therapeutic efficacy without toxicity. If toxicity is observed, the focus can be shifted to weaker MSECs.
  • the Phase 2 testing can compare a series of MSEC cassettes in the general range of activity below those associated with adverse effects and focus on obtaining uniform expression levels in as many skeletal muscles and fiber types as possible, while also allowing good expression levels in cardiac muscle. These studies can involve sequential testing initially with reporter genes, followed by confirmatory and functional studies with the FKRP gene.
  • Phase 3 testing can be performed to optimize MSECs for the potential expression of FKRP having modified 5’ and/or 3’ UTRs as described herein for up-regulating the expression of mutant FKRP in genetic situations in which LGMD2I patients produce low- activity FKRP.
  • Each of these strategies can benefit from the use of existing MSECs, or pairing with additional MSECs, with transcriptional activities that are appropriate for the particular therapeutic strategy.
  • EXAMPLE 7 CARDIAC AND SKELETAL MUSCLE PERFORMANCE
  • the inventors assess longitudinal systolic and diastolic performance by M-mode and Tissue Doppler echocardiographic imaging. Endpoint hemodynamics is assessed in situ by Millar catheter and in vitro by Langendorff perfusion.
  • EXAMPLE 8 DEMEMBRANATED MUSCLE AND ISOLATED MYOFIBRIL CONTRACTILE PROPERTIES
  • Isolated myofibrils allow study of the millisecond timescale kinetics of contractile activation and relaxation, with high fidelity and resolution of forces in the piconewton range.
  • EXAMPLE 9 MUSCLE TISSUE, CELL AND MYOFIBRIL STRUCTURE [00242] Tissue-level structural analysis permits the examination of fibrosis, satellite cells and morphological features such as centralized nuclei and muscle damage.
  • the contractile units called sarcomeres align in the direction perpendicular to the long axis of the cell within myofibrils, and the boundaries of these structures (z-disks) align between myofibrils for high-order parallelization of the contractile apparatus. This order is often disrupted in diseased and damaged muscle, contributing to reduction of force production, altering contraction and relaxation kinetics, and furthering downward spiral muscle damage.
  • electron microscopy can be used to examine the detailed structure of sarcomere thin and thick filaments and z-disks, and these approaches can be used to study structural effects of therapeutic interventions.
  • EXAMPLE 10 ISOLATED CONTRACTILE PROTEIN MECHANICS [00243] An advantage in flow cell assays of isolated contractile protein mechanics is that effects on myosin, actin, troponin and tropomyosin can be assessed as the molecular target for therapeutics, and a large number of conditions can be rapidly assessed since proteins can be separately treated once they are all in a flow cell assay.
  • EXAMPLE 11 METABOLIC PROFILING [00244] Targeted aqueous metabolite profiling analysis can be performed using, for example, the Agilent 1260/AB-Sciex 5500 Qtrap LC-MS/MS instrument and HILIC (hydrophilic interaction chromatography) protocols.
  • EXAMPLE 12 MITOCHONDRIAL FUNCTION
  • Isolated mitochondria and permeabilized cardiac tissue can undergo high-resolution respirometry (HRR) via Oxygraph for the measurement of respiration/respiratory complex analysis (oxygen consumption and flux), mitochondrial membrane potential, ROS and ATP production.
  • HRR high-resolution respirometry
  • Mitochondrial content can be assessed by Western blot to assay ETS (Mitosciences Oxphos profile) and VDAC (Santa Cruz Biotech) protein expression, citrate synthase activity and mtDNA/nuclear DNA.
  • Dystroglycanopathies are a family of muscle disorders (> 20) that are caused by altered glycosylation of ⁇ -dystroglycan ( ⁇ -DG), a peripheral membrane protein located on the extracellular side of the sarcolemma that normally binds to laminin.
  • the laminin- ⁇ -DG association is a crucial portion of the dystrophin-glycoprotein complex (DGC), which provides a mechanical link between the intracellular actin cytoskeleton and the extracellular matrix.
  • DGC dystrophin-glycoprotein complex
  • the DGC enables the lateral transmission of forces from within myofibers, allowing the muscle bundle to contract in unison and preventing cellular damage by internally maintained contractile energy.
  • More than 11 glycosyltransferases are known to post- translationally modify ⁇ -DG, working sequentially to build long glycan chains onto the protein.
  • Fukutin-related protein catalyzes the transfer of ribitol 5-phosphate to a phosphorylated O-mannosyl trisaccharide on ⁇ -DG, but only after Fukutin has added a ribitol 5-phosphate to the growing chain.
  • FKRP Fukutin-related protein
  • LGMDR9 Limb-girdle muscular dystrophy type R9
  • MDC1C congenital muscular dystrophy
  • WWS Walker-Warburg syndrome
  • MEB muscle-eye-brain disease
  • LGMDR9 is slowly progressive, but patients still experience symptoms such as muscle weakness, muscle cramps, hypertrophy, joint contractures, and, in some cases, severe cardiomyopathy and respiratory issues.
  • the age of LGMDR9 onset varies, with a spectrum of symptoms presenting in relation to specific mutations in FKRP.
  • affected LGMDR9 patients are often wheelchair-dependent by 25 years after age of onset. Diagnoses are typically made based on elevated serum creatine kinase (CK) levels and proximal muscle weakness followed by genotyping. There is no cure for LGMDR9, and treatments are limited to temporary symptom amelioration.
  • CK serum creatine kinase
  • LGMDR9 is often due to heterozygous and homozygous mutations in the 1.5 kb coding region of the FKRP gene, the most common of which is 826C>A (L276I). There is a strong genotype-phenotype correlation for this mutation with compound heterozygous patients displaying a more severe phenotype than homozygous patients. Another common mutation is 1343C>T (P448L), and both mutations interfere with the transfer of FKRP from the endoplasmic reticulum to the Golgi apparatus.
  • FKRP is a post-translational glycosyltransferase
  • mislocalization of this enzyme leads to decreased glycosylation and half- life, and results in increased targeting of ⁇ -DG by the proteosome.
  • other insertion, deletion, missense, and nonsense mutations have been reported in patients, though less commonly than the L276I and P448L point mutations.
  • FKRP-null mutations are embryonic lethal, which explains why all patients genotyped to date have at least one mutant allele that leads to expression of a presumably partially functional protein.
  • adeno-associated viral vector (AAV) -mediated systemic delivery of FKRP can significantly ameliorate the dystrophic phenotype in a murine disease model, the FKRP P448L mouse, which will reasonably recapitulate LGMDR9.
  • AAV adeno-associated viral vector
  • CMV human cytomegalovirus immediate early enhancer plus promoter
  • CB CMV enhancer/chicken ⁇ -actin promoter
  • Exercise-based assessments mimic those often used to assess dystrophic patients in the clinic (e.g., 6-minute-walk test) and can be used to exacerbate the dystrophic phenotype in preclinical studies.
  • Therapeutic delivery method is yet another component that should be considered in the development of the safest possible therapeutic for LGMDR9 and related CMDs. Increased gene expression is often a critical part of gene therapy, as therapeutics are limited by the immune responses associated with high doses of AAVs in human patients. For example, the issue of systemically administered AAVs and the accompanying liver toxicity seen in clinical trials remain a barrier moving forward. Therefore, an ideal treatment would maximize gene expression while minimizing the AAV dose. The latest advancements in AAV vector designs have led to increased targeting and gene expression in specific tissues.
  • Transgene optimization provides a tool to improve efficacy and lower necessary treatment doses.
  • Evidence of potential secondary structures was discovered in the untranslated regions of FKRP mRNA.
  • One particularly relevant structure is an RNA G-quadruplex (RGQ), a stable secondary mRNA configuration associated with the inhibition of translation.
  • RGQ RNA G-quadruplex
  • This particular therapeutic differed from the vectors previously tested as it used AAV6 instead of AAV9, as well as a miniaturized mouse muscle creatine kinase enhancer/promoter (CK8e) that is uniquely active and specific for striated muscle (AAV6- Ck8e-humanFKRP, A6.C8hF).
  • CK8e mouse muscle creatine kinase enhancer/promoter
  • C2C12 myotubes were transduced with 1x10 12 vg, 1x10 11 vg, or 1x10 10 vg of AAV6-CK8e-mFKRP- FLAG (the murine Fkrp cDNA with a C-terminal FLAG-tag).
  • Cell lysates revealed co- immunoreactivity with the FKRP and FLAG antibodies and provided confirmation of FKRP production by the AAV6-CK8e-FKRP vector (FIG.6A).
  • potential inhibitory sequences were located in the FKRP untranslated regions.
  • This CK8e-hFKRP construct was encapsulated into AAV6 (A6.C8hF), AAV9 (A9.C8hF), or AAVMYO1 (AM.C8hF) vectors and tested via systemic delivery (FIG. 6D and 6E).
  • AAV6 A6.C8hF
  • AAV9 A9.C8hF
  • AAVMYO1 AM.C8hF
  • Wild-type animals treated with AAV-FKRP vectors displayed normal muscle physiology.
  • additional assays were performed in wild-type mice injected with vectors made with AAV6, AAV9 or AAVMYO1 capsids.
  • the first study consisted of wild-type mice injected with A6.C8hF at doses of 4x10 13 , 2x10 14 , or 4x10 14 vg/kg to determine whether this vector caused an increase in susceptibility of muscles to contraction-induced injury.
  • These high doses were a used in this preliminary safety assessment, as doses at or exceeding 1x10 14 vg/kg have often led to serious adverse events in patients with various neuromuscular disorders. This is important because a high dose AAV-mediated FKRP therapeutic, plus endogenous FKRP, provides the ability to maximize potential expression.
  • Some of these mice underwent gastrocnemius muscle physiology assays 5 weeks later and others were tested at 10 weeks.
  • mice were injected into wild-type mice at doses of 6.4x10 12 , 2x10 13 , and 6.4x10 13 vg/kg.
  • the latter two cohort of mice were analyzed for a variety of properties at 4-, 6-, 8-, 10-, 12-, and 14- weeks post-injection to evaluate effects on muscle physiology (FIGs.7A-7I).
  • fatigue assays were performed in which mice ran on a treadmill at a speed of 10 meter/sec, with 1 meter/sec/minute increases (FIG. 7A-7C).
  • mice treated with AAV9 displayed reduced force over time, there were no differences between groups at any time point. In fact, all the mice (treated and untreated) experienced a decrease in strength between 10- and 14-weeks post-injection groups, which typically occurs when mice habituate to the grip-strength assay.
  • ankle plantarflexion assay Another commonly used assay in mouse muscle analysis is the ankle plantarflexion assay, in which ankle torque over 20 eccentric contractions is quantified using a high yet physiologically relevant stimulation frequency that elicits a maximal response.
  • the assay is designed to assess force generation in addition to fatigue caused by repeated eccentric contractions. At all time points, torque declined to 50% of starting baseline measurements, consistent with contraction-induced fatigue (FIG. 7G-7H). However, no differences were detected between any of the three groups at any timepoint. This includes potential differences in torque at a given contraction as well as the relative change from baseline.
  • Ejection fraction measures the percent of blood leaving the left ventricle
  • fractional shortening is a measure of the heart’s contractility; both of which are common indicators of cardiac function.
  • RNA G-quadruplexes are known to regulate gene expression and have been targeted as a strategy to ameliorate or worsen genetic disease pathologies. It was discovered that complete removal of both the 5’ and 3’ UTRs led to increased gene expression (FIG.6A- 6E). These data suggest the presence of inhibitory regulatory sequences within the UTRs of FKRP, which could provide a potential target for fine-tuning gene expression.
  • a potential G-quadruplex in the 5’ UTR may serve to limit expression, although individual bases were dissected in the 5’ or 3’ UTRs of the FKRP mRNA that mediate this effect.
  • the difficulty encountered by many labs in detecting endogenous FKRP expression suggests that extremely low levels of the enzyme is all that is needed for normal glycosylation of ⁇ -DG. It is possible that inhibitory UTR sequences may serve a role in limiting overexpression during normal muscle activity, although further studies would be needed to clarify this issue. Importantly, the safety of this therapeutic observed in the studies supports continued advancement of methods for gene therapy to treat patients carrying mutations in the FKRP gene.
  • EXAMPLE 14 MATERIALS AND METHODS
  • Animals [00272] All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Washington Mouse studies were performed in C57BL6 wild- type male mice aged 6-35 weeks at age of injection.
  • Plasmid construction and vector production [00274] The coding region of mouse Fkrp was PCR amplified (Forward primer: 5’ TTGTTAACATGCGGCTCACCC 3’ (SEQ ID NO: 30); Reverse primer: 5’ TACCGGTTCAACCGCCTGTC 3’ (SEQ ID NO: 31)) from mouse muscle cDNA.
  • the resulting DNA fragment was digested with HpaI and AgeI and ligated into an AAV backbone vector containing a muscle specific CK8e promoter and synthetic poly A tail, as previously described.
  • a custom AAV transfer plasmid containing the previously described muscle-specific CK8e regulator cassette, a 1,482 bp cDNA expression construct for native mouse Fkrp mRNA (NCBI CCDS20853.1), a synthetic polyA signal and flanking AAV serotype 2 inverted terminal repeats was constructed using standard recombinant methodology.
  • AAV9 pA9.C8hF
  • AAVMYO1 AM.C8hF
  • the human FKRP cDNA was PCR amplified and cloned into pAAV-Ck8e-FKRP in place of the mouse cDNA.
  • Antibody production and purification [00276] Rabbit polyclonal antisera was generated against a peptide near the C-terminus of the FKRP sequence that was identical in the mouse and human proteins (as a fusion with KLH: KLH-C-APNNYRRFLELKFGPGVIENPQYPNP (SEQ ID NO: 32)) by Covance (Denver, PA). Affinity purification was performed using a maltose-binding protein fusion of the antigenic peptide on a MBPTrap HP columns (GE Healthcare) and coupled to Ultralink Biosupport polyacrylamide resin (Thermo Scientific) as per manufacturer’s instructions.
  • the FKRP-C antibody (named Ab607) was affinity purified by HPLC through MBP-FKRP coupled beads and stored in BSA and NaN3.
  • Cell culture [00278] Mouse C2C12 cells were plated at ⁇ 80% confluence on gelatin-coated 6-well plates with standard growth media (DMEM, 20% FBS, 1% penicillin-streptomycin (P/S)) overnight, then washed three times with 1x Saline G prior to infection with rAAV6-CK8- mFKRP. Vectors were diluted to the desired concentrations in differentiation media (DMEM, 2% HS, 1% Penicillin-Streptomycin).
  • RO retro-orbitally
  • FKRP P448L mice which contain a mutation in the FKRP ORF at position 448 (P448L) between the ages of 6 months to a year (the preferred age is 10 months) can be injected with different doses of A6.C8hF (saline, at a range between 1x10 12 , and 1x10 15 vg/kg) and monitored alongside age-matched wild-type controls.
  • A6.C8hF saline, at a range between 1x10 12 , and 1x10 15 vg/kg
  • the purpose of using older mice is to address the effect of treatment in older mice with a more advanced phenotype.
  • Forelimb grip strength of age from wild-type, untreated and treated FKRP P448L mice can be measured between 2-weeks and 16-weeks post-injection.
  • EXAMPLE 17 Exercise capacity of mice treated with FKRP viral vectors as described herein. Additional metrics that can be examined in treated mutant mice include measurements of VO 2 max, distance travelled, energy expended, and energy consumption rate. All of these can be measured using a metabolic treadmill and can include VO2max tests to assess changes in maximal O2 consumption, a measure of exercise capacity and cardiorespiratory function, and several intermittent training sessions meant to exacerbate the dystrophic phenotype.
  • pathological markers can be used for assessing exercise impact training for dystrophic mice.
  • these include several exercise-induced markers in FKRP P448L mice such as variability in respiration (VO2cv), the respiratory exchange ratio (RER, VCO2/VO2), energy expended, and the accumulation of motivational shocks.
  • VO2cv variability in respiration
  • RER respiratory exchange ratio
  • VCO2/VO2 the respiratory exchange ratio
  • energy expended the accumulation of motivational shocks.
  • VO2cv values for untreated FKRP P448L mice will be higher than those for wild-type mice, while the treated FKRP P448L animals will have values that will be either significantly lower than the untreated mice or not different from wild-type mice.
  • a similar pattern is expected when comparing the overall differences between groups, which would be highly significant, and when assessing differences in maximal RER values.
  • EXAMPLE 19 Evaluation of muscle degeneration, muscle fiber size distribution and creatine kinase levels. After assessing exercise impact training, all mice can be sacrificed, and different muscles will be collected. Compared to untreated FKRP P448L mice, it is expected that treated FKRP P448L mice will have larger skeletal muscle myofiber sizes and will have fewer centrally-nucleated myofibers and fewer total centrally-located nuclei, which are hallmarks of prior rounds of muscle necrosis and regeneration. It is expected that treatment will also impact serum creatine kinase levels in treated vs. untreated mice, with levels in treated mice being significantly lower than in untreated.
  • FKRP ORF refers to both wild-type FKRP and a mutated form of FKRP.
  • MSEC refers to any MSEC as defined in the specification and referenced in PCT/US22/023915, which is incorporated by reference in its entirety. The following describes various combinations of 5’ UTR and 3’ UTR modifications in combination with MSECs contemplated for FKRP constructs as described herein.
  • Fukutin-related protein is essential for mouse muscle, brain and eye development and mutation recapitulates the wide clinical spectrums of dystroglycanopathies.
  • Qiao C Wang CH, Zhao C, et al.
  • TREAT-NMD Neuromuscular Network 2011(2.0). http://treat-nmd.eu/resources/research-resources/dmd-sops/ 21. Grange RW. Use of treadmill and wheel exercise to assess dystrophic state. TREAT- NMD Activity A07: Accelerate preclinical phase of new therapeutic treatment development. DMD_M.2.1.003. TREAT-NMD Neuromuscular Network; 2011(1.0). http://treat- nmd.eu/resources/research-resources/dmd-sops/ 22.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Blankinship MJ Gregorevic P, Allen JM, et al. Efficient transduction of skeletal muscle using vectors based on adeno-associated virus serotype 6. Mol Ther. Oct 2004;10(4):671-8. doi:10.1016/j.ymthe.2004.07.016 49. Gao G, Vandenberghe LH, Alvira MR, et al. Clades of Adeno-associated viruses are widely disseminated in human tissues. J Virol. Jun 2004;78(12):6381-8. 50. Alhamidi M, Kjeldsen Buvang E, Fagerheim T, et al.
  • Fukutin-related protein resides in the Golgi cisternae of skeletal muscle fibres and forms disulfide-linked homodimers via an N-terminal interaction.
  • Gregorevic P Blankinship MJ, Allen JM, et al. Systemic delivery of genes to striated muscles using adeno-associated viral vectors. Nat Med. Aug 2004;10(8):828-34. 52.
  • Yin FC Spurgeon HA, Rakusan K, Weisfeldt ML, Lakatta EG.
  • SEQ ID NO.1 is a human FKRP cDNA including 5’ and 3’ UTRs: attgctccaagatggcggcggcggcggcagcgggagcgcagctcagctgggctggaactgccctcctggaactcccccagcctac aacctaggaggtgcagggactgaggctcaggccaaatcgcaactcagacccagtgaacccaaggcctgaagagaatttggattcatt tacctgttttgtggggactggagagacaagtaactctcctctgactaccatttctctgtggggactggagagacaagtaactctcctctgactaccatttctctgactaccatttctctgtggggactggagagacaagtaactctcctctgactaccattt
  • SEQ ID NO.7 is a murine FKRP cDNA sequence lacking the 5’ UTR: ...AGACTCAGCACTTAGTTTAGGAACCAGTGAGCAAGTCAGCCCTTGGGGCAGCC CATACAAGGCCATGGGGCTGGGCAAGCTGCACGCCTGGGTCCGGGGTGGGCACG GTGCCCGGGCAACGAGCTGAAAGCTCATCTGCTCTCAGGGGCCCCTCCCTGGGG ACAGCCCCTCCTGGCTAGTCACACCCTGTAGGCTCCTCTATATAACCCAGGGGCA CAGGGGCTGCCCTCATTCTACCACCACCTCCACAGCACAGACAGACACTCAGGA GCCAGCCAGCGTCGAGATGCGGCTCACCCGCTGCCAGGCTGCCCTGGCGGCCGC CATCACCCTCAACCTTCTGGTCCTCTTCTATGTCGTGGCT... [00634] SEQ ID NO.8 is the CK8e promoter: TGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATA

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Abstract

The invention described here contains a nucleic acid expression cassette comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a FKRP, an RNA transcript comprising a modified 5' and/or 3' untranslated region (UTR) that will be utilized to treat a variety of FKRP-mediated diseases.

Description

METHOD FOR EXPRESSING A MUSCLE-SPECIFIC GENE AND CASSETTES FOR SAME CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No.63/336,678 filed April 29, 2022, the contents of which is incorporated herein by reference in its entirety. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing that has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on April 26, 2023, is named “034186-191920WOPT_SL.xml” and is 101,526 bytes in size. FIELD OF THE INVENTION [0003] The field of invention relates to gene therapy for the treatment of FKRP-mediated diseases. BACKGROUND [0004] Neuromuscular disorders can result from genetic mutations in key genes that regulate optimal muscular production. One gene important for optimal muscular production is fukutin- related protein (FKRP). It is a glycosyl-transferase localized within the trans-golgi complex and mediates glycosylation which is required for the maturation and function of α- dystroglycan, an essential component of the dystrophin-glycoprotein complex (DGC). Currently, there are no significant treatment options for patients with FKRP disorders. One type of treatment that is being utilized to combat genetic muscle diseases is gene therapy, which must be adapted for each individual type of dystrophy. SUMMARY OF THE INVENTION [0005] The methods and compositions described herein are based, in part, on the discovery that muscle expression of FKRP can be improved by (i) using a muscle-specific expression cassette (MSEC) to deliver FKRP to a cell, and (ii) modifying the nucleic acid encoding FKRP such that the resulting transcript comprises a modification to the 5’ and/or 3’ untranslated region (UTR). Such modifications to the 5’ and/or 3’ UTR can increase the expression levels of FKRP as compared to a construct comprising a wild-type 5’ and/or 3’ UTR. The methods and compositions provided herein can be used to express FKRP in a cell or subject for the treatment of FKRP-mediated diseases or disorders including, but not limited to, limb girdle muscular dystrophy type 2I/R9 (LGMD2i, also known as LGMDR9), Walker- Warburg syndrome, or muscle-eye-brain disease (MED). [0006] One aspect provided herein describes a nucleic acid expression cassette comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a fukutin-related protein (FKRP) RNA transcript that comprises a modified 5’ and/or 3’ untranslated region (UTR). [0007] In one embodiment of any of the aspects described herein, the modified 5’ untranslated region (UTR) is truncated as compared to the 5’ UTR of wild-type FKRP. [0008] In one embodiment of any of the aspects described herein, the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 5’ UTR region. [0009] In one embodiment of any of the aspects described herein, the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR. [0010] In one embodiment of any of the aspects described herein, the modification comprises a modification to the Kozak consensus sequence. [0011] In one embodiment of any of the aspects described herein, the modified 3’ UTR is truncated compared to the 3’ UTR of wild-type FKRP. [0012] In one embodiment of any of the aspects described herein, the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region. [0013] In one embodiment of any of the aspects described herein, the nucleic acid encoding FKRP comprises a modification in each of the 5’ and 3’ UTRs. [0014] In one embodiment of any of the aspects described herein, the modification in the 5’ and/or 3’ UTR of FKRP causes an increase or a decrease in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity expressed from a similar construct comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a FKRP RNA transcript that comprises a wild-type 5’ and/or 3’ untranslated region (UTR). [0015] In one embodiment of any of the aspects described herein, the transcriptional regulatory region comprises a muscle-specific expression cassette (MSEC). [0016] In one embodiment of any of the aspects described herein, the MSEC is selected from the group consisting of CK8e. [0017] In one embodiment of any of the aspects described herein, upon administration to a cell, expression level of an FKRP mRNA or protein is higher when operably linked to an MSEC than the expression level of the FKRP mRNA or protein when operably linked to a CK8e transcriptional regulatory region. [0018] In one embodiment of any of the aspects described herein, upon administration to a cell, expression level of an FKRP mRNA or protein is lower when operably linked to an MSEC than the expression level of the FKRP mRNA or protein when operably linked to a CK8e transcriptional regulatory region. [0019] Another aspect provided herein relates to an RNA transcript generated by transcription of the nucleic acid expression cassette of any one of the embodiments described herein. [0020] Also provided herein, in another aspect, is an adeno-associated viral vector (AAV) comprising the nucleic acid expression cassette of any one of the embodiments described herein. [0021] In one embodiment of any of the aspects described herein, the adeno-associated viral vector is selected from the group consisting of: an AAVRh74 vector, an AAV8 vector, an AAV9 vector, an AAV6 vector, an AAV7 vector, an AAV2i8 vector, a NP vector, a NP 66 vector, a NP 22 vector, an AAVpo.1 vector, a MyoAAV vector, and an AAVMyo vector. [0022] In one embodiment of any of the aspects described herein, the adeno-associated viral vector comprises an internal terminal repeat (ITR), a muscle-specific cassette, a nucleic acid specific to FKRP, a polyadenylation signal (pA+), and/or a second ITR. [0023] Another aspect provided herein describes an engineered cell comprising or expressing a nucleic acid expression cassette of any one of the embodiments described herein. [0024] In one embodiment of any of the aspects described herein, the modified 5’ untranslated region (UTR) is truncated as compared to the wild-type 5’ UTR of FKRP. [0025] In one embodiment of any of the aspects described herein, the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of or all of the nucleotides in the 5’ UTR region. [0026] In one embodiment of any of the aspects described herein, the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR. [0027] In one embodiment of any of the aspects described herein, the modification comprises a modification to the Kozak consensus sequence. [0028] In one embodiment of any of the aspects described herein, the modified 3’ UTR is truncated compared to the 3’UTR of wild-type FKRP. [0029] In one embodiment of any of the aspects described herein, the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region. [0030] In one embodiment of any of the aspects described herein, the nucleic acid encoding FKRP comprises a modification in each of the 5’and 3’ UTRs. [0031] In one embodiment of any of the aspects described herein, the modification in the 5’ and/or 3’ UTR of FKRP causes an increase or a decrease in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity expressed from a similar construct comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a FKRP RNA transcript that comprises a wild-type 5’ and/or 3’ untranslated region (UTR). [0032] Another aspect provided herein relates to a method of expressing an FKRP gene product in a subject comprising administering an adeno-associated viral vector according to any one of the embodiments described herein to a subject in need thereof. [0033] In one embodiment of any of the aspects described herein, the FKRP gene product is a RNA transcript and/or a protein. [0034] In one embodiment of any of the aspects described herein, the subject in need thereof comprises limb girdle muscular dystrophy type 2I/R9 (LGMD2i), Walker-Warburg syndrome, or muscle-eye-brain disease (MED). [0035] In one embodiment of any of the aspects described herein, the modified 5’ untranslated region (UTR) is truncated as compared to the 5’ UTR of wild-type FKRP. [0036] In one embodiment of any of the aspects described herein, the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 5’ UTR region. [0037] In one embodiment of any of the aspects described herein, the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR. [0038] In one embodiment of any of the aspects described herein, the modification comprises a modification to the Kozak consensus sequence. [0039] In one embodiment of any of the aspects described herein, the modified 3’ UTR is truncated compared to the 3’ UTR of a wild-type FKRP. [0040] In one embodiment of any of the aspects described herein, the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region. [0041] In one embodiment of any of the aspects described herein, the nucleic acid encoding FKRP comprises a modification in each of the 5’ and 3’ UTRs. [0042] In one embodiment of any of the aspects described herein, the modification in the 5’ and/or 3’ UTR of FKRP causes an increase or a decrease in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity expressed from a similar construct comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a FKRP RNA transcript that comprises a wild-type 5’ and/or 3’ untranslated region (UTR). [0043] In one embodiment of any of the aspects described herein, the administration of the AAV vector comprises intravenous and/or intramuscular injection. [0044] In one embodiment of any of the aspects described herein, the subject is a human. [0045] Also provided herein, in another aspect, is a method for reducing at least one symptom of an FKRP-mediated disease or disorder, the method comprising administering an AAV vector of any one the embodiments to a subject in need thereof, thereby reducing at least one symptom of an FKRP-mediated disorder. [0046] In one embodiment of any of the aspects described herein, the FKRP-mediated disease or disorder comprises limb girdle muscular dystrophy type 2I/R9 (LGMD2i), Walker- Warburg syndrome, or muscle-eye-brain disease (MED). [0047] In one embodiment of any of the aspects described herein, the modified 5’ untranslated region (UTR) is truncated as compared to the wild-type 5’ UTR of FKRP. [0048] In one embodiment of any of the aspects described herein, the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 5’ UTR region. [0049] In one embodiment of any of the aspects described herein, the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR. [0050] In one embodiment of any of the aspects described herein, the modification comprises a modification to the Kozak consensus sequence. [0051] In one embodiment of any of the aspects described herein, the modified 3’ UTR is truncated compared to the 3’ UTR of a wild-type FKRP. [0052] In one embodiment of any of the aspects described herein, the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region. [0053] In one embodiment of any of the aspects described herein, the nucleic acid encoding FKRP comprises a modification in each of the 5’ and 3’ UTRs. [0054] In one embodiment of any of the aspects described herein, the modification in the 5’ and/or 3’ UTR of FKRP causes an increase or a decrease in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity of a construct comprising wild type 5’ and 3’ FKRP UTRs under substantially similar conditions. [0055] In one embodiment of any of the aspects described herein, the administration of the AAV vector comprises intravenous and/or intramuscular injection. [0056] In one embodiment of any of the aspects described herein, the subject is a human. [0057] In one embodiment of any of the aspects described herein, at least one symptom of a FKRP-mediated disease or disorder comprises: muscle pain, muscle weakness, muscle fatigue, muscle atrophy, inflammation, decrease in average myofiber diameter in skeletal muscle, loss of ambulation, abnormalities in the brain and/or eyes, eye problems, delay in development, intellectual disability, and seizures. BRIEF DESCRIPTION OF THE DRAWINGS [0058] FIGs.1A-1B is a schematic depicting an exemplary adeno-associated viral vector (AAV) comprising a muscle specific expression cassette and a nucleic acid sequence encoding an FKRP transcript (FIG.1A). Expression levels of FKRP in muscle cells (e.g., C2C12 cells) and in mouse muscle cells following intramuscular injection (FIG.1B). FIG. 1B shows initial data relating to the removal of the 5’UTR and the resulting increase in FKRP expression, as well as the 5’UTR truncations made based on structure predictions and splice sites. [0059] FIG.2 Schematic of the pre-mRNA and mature mRNA of FKRP, illustrating how the 5’UTR is on multiple exons while the entire coding region of the gene exists on a single exon. [0060] FIG.3 Subset of 30 MSECs from a library of 300 showing relative expression levels from 1/100th to 20X higher than the powerful, ubiquitously expressed CMV promoter. [0061] FIGs.4A-4C are schematics showing exemplary modifications to human FKRP gene constructs featuring the first 401 bases from FKRP (SEQ ID NO: 33). (FIG.4A) Shown are four types of DNA/RNA sequence motifs found in the 5’ UTR that affect FKRP RNA and protein expression levels in mammalian cells. The full 5’ UTR contains a G-quadruplex, a pseudo-knot, an IRES and hairpin forming sequences that influence RNA and protein expression. (FIG.4B) Mutation of the Kozak consensus sequence in the FKRP gene, which alters expression levels by modifying protein translation. (FIG.4C) Expression of FKRP protein or RNA can be increased by removal of portions of, or all of the 5’ UTR of the FKRP gene. As but one example, a modified Kozak consensus sequence was introduced into the FKRP expression construct and the entire 5’ UTR was removed and in addition, the upstream MSEC was inserted immediately upstream of the Kozak sequence. [0062] FIG.5 is a schematic depicting exemplary 5’UTR variations encoded into the transgene expressed in AAV6. The truncations are based on secondary structure predictions, determined via RNAfold Web Server and sequence patterns. [0063] FIGs.6A-6E shows removal of the FKRP 5’- and 3’-UTRs increases FKRP expression. FIG.6A examines cell lysates from differentiated C2C12 myotubes transduced with either 1x1011 or 1x1010 vector genomes (vg) of A6.C8mF-FLAG to verify vector expression of mouse FKRP (mFKRP). Labels on the left indicate antibody target (FKRP, Ab607; FLAG, FLAG-HRP, Sigma F7425). FIG.6B examines FKRP levels in myotubes transduced with untagged AAV6-Ck8e-mFKRP (+UTR) and AAV6-Ck8e-mFKRP (-UTR). FIG.6C (upper) shows FKRP expression in tibialis anterior (TA) muscles of wild-type mice injected IM with 1x1011 vg AAV6-Ck8e-mFKRP (-UTR) relative to an untreated wild-type mouse and C2C12 myotubes transduced with 1x1012 vg of same vector; FIG.6C (lower) shows immunostaining of wild-type tibialis anterior muscle injected with AAV6-CK8e- mFKRP. FKRP, green. Golgi, red (GMI30). FIG.6D shows a schematic illustration of the AAV-CK8e-humanFKRP (hFKRP)construct used in these studies. FIG.6E analyzes a transgene construct expressed from vectors pseudotyped with capsids from AAV6 (A6.C8hF), AAV9 (A9.C8hF), or AAVMYO1 (AM.C8hF). Wild-type mice were then treated and tested at various timepoints post-injection to identify potential physiological and histological changes. WT, wild-type. [0064] FIGS.7A-7I examines whole-body and isolated limb physiology of wild-type mice treated with AAV-hFKRP. FIG.7A shows the distance run by mice on a treadmill by mice injected with either saline (WT) or 6.4x1013 vg/kg of A9.C8hF or AM.C8hF. FIG.7B shows the distance run normalized to mouse weight. FIG.7C examines the percent distance changed relative to previous absolute distance measured. FIG.7D shows forelimb grip strength. FIG.7E shows forelimb grip strength normalized to mouse weight. FIG.7F shows the percent forelimb grip strength changed relative to absolute grip strength. FIGs.7G-7I analyzes repeated cycles of plantarflexion eccentric contraction induced injury (20x) in the right hind limb of mice at 4-, 8-, and 12-weeks post-injection measured by hindlimb torque. Limb was stimulated with 10mA at a frequency of 100Hz for 0.4 seconds with a 9 second rest interval between contractions. Significant differences are represented by different letters, shared letter indicate no difference. WT, untreated. (n=3-4) [0065] FIGs.8A-8E examines cardiac hemodynamics and skeletal muscle mechanics following AAV delivery to wild-type mice. Mice were untreated or injected with 6.4x1013 vg/kg A9.C8hF or AM.C8hF. FIGs.8A-8C shows ultrasound imaging of heart and diaphragm function at 6- and 12-weeks post-injection. FIG.8D & 8E examines the force:length relationship of TA and diaphragm muscles from same mice at 18 weeks post- injection. Muscles are stretched end-to-end and optimal length is determined at maximum isomeric twitch force (see Methods). No differences were detected in these data. WT, untreated. (n=3-4) [0066] FIGs.9A-9B shows AAV-FKRP injection does not increase gastrocnemius muscle susceptibility to contraction-induced injury. Isometric force development in wild- type mice treated with A6.C8hF at doses of 4 x 1013 , 2 x 1014, and 4 x 1014 vg/kg. Shown is the percent of maximum isometric force during 8 rounds of gastrocnemius eccentric contraction in situ at 5-7 weeks (FIG.9A) and 10-weeks (FIG.9B) post-injection. No significant differences were observed between any of the groups (shared letter indicate no difference). WT, untreated. (n= 3-5) [0067] FIG.10 shows a schematic illustrating the dystrophin-associated protein complex and the extracellular effects of mutated FKRP. DETAILED DESCRIPTION OF THE INVENTION [0068] Provided herein are methods and compositions useful for the delivery of a nucleic acid to induce expression of an RNA transcript for FKRP that comprises a modified 5’ and/or 3’ UTR region. Such modifications to the 5’ and/or 3’ UTRs in combination with muscle- specific (e.g., skeletal and/or cardiac) expression of FKRP show improved delivery of FKRP as a therapeutic, as well as reduced side effects from off-target expression of FKRP. The methods and compositions described herein can be used for the treatment or prevention of an FKRP-mediated disease or disorder. Definitions [0069] As used herein, the term “nucleic acid cassette” refers to a nucleic acid sequence comprising a transcriptional regulatory region and a region that encodes an FKRP RNA transcript. [0070] As used herein, the term “transcriptional regulatory region” refers to a nucleotide sequence located upstream of the nucleotide sequence encoding FKRP as described herein and that permits the recruitment of transcriptional machinery and initiation of transcription of an FKRP RNA transcript. At a minimum, a transcriptional regulatory region comprises a promoter (e.g., a tissue-specific promoter, a constitutive promoter etc.). The transcriptional regulatory region can also comprise one or more regulatory elements, such as an enhancer or repressor or binding site elements for transcription factors. In one embodiment, the transcriptional regulatory region comprises a promoter and at least 1 enhancer region (e.g., at least 2, at least 3, at least 4, at least 5 or more). The promoter, enhancer element(s) or repressor element(s) can be nucleic acid sequences that are naturally occurring or can be synthetic. The transcriptional regulatory region can be modified to tune expression of FKRP to a desired level, for example, by using a strong promoter and enhancer to increase FKRP expression or alternatively a weaker promoter (or a strong promoter and a weak repressor element) can be used to tune expression of FKRP to a lower level when desired. Further tuning as desired can be achieved through combination of a given transcriptional regulatory region with modifications of the 5’ and/or 3’ UTR of the encoded transcript, e.g., as described herein. [0071] As used herein, the term “FKRP RNA transcript” refers to a messenger RNA that encodes FKRP and comprises a modified 5’ or 3’ untranslated region (UTR) as compared to the 5’ or 3’ untranslated region of wild-type FKRP. In some embodiments, the modifications to the 5’ and/or 3’ UTR comprise truncation of at least 1 nucleotide (e.g., at least 2, at least 5, at least 10, at least 25, at least 100 nucleotides) or a complete truncation of the 5’ and/or 3’ UTR (i.e., removal of all the nucleotides in a given UTR region). Alternatively, modifications to the 5’ UTR can comprise disruption or deletion of one or more sequences that form secondary structures that influence protein expression (e.g., G-quadruplexes, RNA hairpins, pseudoknots and the like); typically, removal of secondary structures in the 5’ UTR will result in enhanced expression of FKRP by removing structures that can impede binding of translational machinery. Conversely, modifications to the 5’ UTR can comprise inclusion of a new secondary structure (e.g., an RNA hairpin) that partially impedes binding of translational machinery to tune expression of FKRP to a lower level, if desired. Modifications to the 3’ UTR can comprise the addition, removal or modulation of one or more elements, including but not limited to elements affecting transcript stability, addition of or changes to a polyadenylation signal, and truncations of reducing length of the 3’ UTR, etc. [0072] As used herein, the term “disruption of” when used in reference to RNA transcript secondary structures (e.g., G-quadruplexes, RNA hairpins, pseudoknots etc), refers to the removal of (or alternatively the addition of) nucleotides that in turn disrupt the secondary structure. For example, disruption of a G-quadruplex (a G-C rich region) can be achieved by removal of one or more G-Cs in the region or mutation of one or more of the guanines (G) or cytosines (C) to an adenine (A) or uracil (U) to disrupt the G-C base pair-mediated formation of the G-quadruplex. As another example, disruption of an RNA hairpin can comprise nucleotide mutations or small deletions that remove or modify the self- complementary/palindromic sequence that results in RNA hairpin formation. One of skill in the art can identify or predict the mutations or deletions necessary for disrupting RNA hairpin formation based on the current understanding of RNA hairpins in the art and knowledge of the ability of RNA hairpins to tolerate small mismatch regions. [0073] As used herein, the term "operably linked" refers to the placement of components e.g., in an upstream transcriptional regulatory region such that they work in relationship, thereby permitting them to function in their intended manner. For example, a control sequence (such as a promoter or enhancer) is positioned in such a way that expression of a nucleic acid sequence encoding FKRP is under the control of the promoter and/or enhancer sequences. [0074] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease or lessening of a property, level, or other parameter by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for a cell or individual without a given disorder. [0075] The terms “increased," “increase," “increases,” or “enhance" or “activate" are all used herein to generally mean an increase of a property, level, or other parameter by a statistically significant amount; for the avoidance of any doubt, the terms “increased", “increase" or “enhance" or “activate" means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, at least about a 20-fold increase, at least about a 50-fold increase, at least about a 100-fold increase, at least about a 1000-fold increase or more as compared to a reference level. [0076] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not. [0077] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. [0078] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment. [0079] The terms “patient”, “subject” and “individual” are used interchangeably herein, and refer to an animal, particularly a human, to whom treatment of an FKRP-mediated disease or disorder, including prophylactic treatment is provided. The term “subject” as used herein refers to human and non-human animals. The term “non-human animals” and “non-human mammals” are used interchangeably herein and includes all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dog, rodent (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, and non-mammals such as chickens, amphibians, reptiles etc. In one embodiment, the subject is human. In another embodiment, the subject is an experimental animal or animal substitute as a disease model. In another embodiment, the subject is a domesticated animal including companion animals (e.g., dogs, cats, rats, guinea pigs, hamsters etc.). A subject can have previously received a treatment for an FKRP-mediated disease, or has never received treatment for an FKRP-mediated disease. A subject can have previously been diagnosed with having an FKRP-mediated disease, or has never been diagnosed with an FKRP-mediated disease. [0080] As used herein, a “therapeutically effective amount” or a “therapeutically effective dose” refers to an amount of a nucleic acid cassette or AAV vector as described herein that, when administered to a subject, is sufficient to effect partial or complete treatment of an FKRP-mediated disease or condition in the subject. The amount of a nucleic acid cassette or AAV vector that constitutes a “therapeutically effective amount” will vary depending on the nucleic acid cassette or AAV vector, the condition and severity of the disease, the manner of administration, and/or the age of the subject to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his or her own knowledge and to this disclosure. Accordingly, when a nucleic acid cassette or AAV vector is said to possess “therapeutic efficacy,” this is intended to mean that the nucleic acid cassette or AAV vector is capable of effecting treatment of FKRP-mediated disease or condition in a subject, provided a “therapeutically effective amount” of the nucleic acid cassette or AAV vector is administered under appropriate conditions. [0081] As used herein, “treating” or “treatment” refers to the treatment of an FKRP-mediated disease or condition of interest in a subject (e.g., a human) having the disease or condition of interest, and includes: (i) preventing or inhibiting the disease or condition from occurring in the subject, for example, when the subject is predisposed to the condition, but has not yet been diagnosed as having the condition; (ii) inhibiting the disease or condition, i.e., arresting or slowing its development or progression; (iii) relieving the disease or condition, i.e., causing regression of the disease or condition; and/or (iv) relieving one or more symptoms (e.g., muscle weakness, muscle fatigue, abnormalities in the brain and/or eyes), resulting from the disease or condition or an improvement in the disease, for example, beneficial or desired clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, disease stabilization (e.g., not worsening), delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. In some embodiments, treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, one of skill in the art realizes that a treatment can improve the disease condition, but may not be a complete cure for the disease. Successful treatment can also be assessed by a reduction in the need for medical interventions, reduction in hospital or emergency room visits, reduction in fatigue, or other markers of an improved quality of life. [0082] As used herein, the phrase “reducing at least one symptom of an FKRP-mediated disease or disorder” refers to a reduction in the presence of a given symptom, or severity of a given symptom associated with an FKRP-mediated disease or disorder following treatment with the methods and compositions described herein. In one embodiment, the reduction of at least one symptom can include the prevention or delay of an expected symptom onset in a subject diagnosed with an FKRP-mediated disease and undergoing treatment as described herein. Exemplary symptoms of an FKRP-mediated disease include muscle atrophy (e.g., a decrease in muscle mass), muscle weakness, weak heart rate, muscle fatigue, muscle pain,  inflammation, decrease in average myofiber diameter in skeletal muscle, loss of ambulation, abnormalities in the brain and/or eyes, eye problems, delay in development, intellectual disability, seizures, and mortality. In one embodiment, at least one symptom of an FKRP- mediated disease is reduced by at least 10% as assessed using an appropriate standard clinical measures such as MRI, CT scan, X-rays, PET scans, electromyography, muscle biopsy, ECG, and patient self-reporting; in other embodiments, the at least one symptom is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or even 100% (i.e., symptom is resolved or is below detectable parameters using a clinical measure). In some embodiments, therapeutic efficacy can be measured by a reduction in hospital visits, a reduction in the duration of hospital stays, reduction in medications or doses of such medications, increased longevity, improved quality of life and the like. [0083] Unless specifically defined otherwise, the technical terms, as used herein, have their normal meaning as understood in the art. The following terms are specifically defined with examples for the sake of clarity. [0084] As used herein, “a” and “an” denote one or more, unless specifically noted. [0085] As used herein “about” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1% to a reference quantity, level, number, frequency, percentage, dimension, size, amount, weight, or length. In any embodiment discussed in the context of a numerical value used in conjunction with the term “about,” it is specifically contemplated that the term “about” can be omitted. FKRP-mediated Neuromuscular Disorders [0086] Provided herein are methods and compositions that can be used in the treatment of FKRP-mediated diseases or disorders. Fukutin-Related Protein (FKRP) is a glycosyl- transferase localized within the trans-golgi complex. It mediates glycosylation and maturation of α-dystroglycan, an essential component of the dystrophin-glycoprotein complex (DGC). Mutations within FKRP result in decreases in α-dystroglycan glycosylation and lead to the disruption of a critical mechanical link between the extracellular matrix (ECM)and the muscle contractile apparatus. [0087] These mutations to FKRP result in the disease progression of e.g., dystroglycanopathies, which are a collection of diseases resulting from dysfunction of α- dystroglycan. Such diseases include limb girdle muscular dystrophy type 2I/R9, congenital muscular dystrophy (MDC1C), Walker-Warburg syndrome, and muscle-eye-brain disease (MED). Traditionally, treatment utilizes gene replacement therapy, however new strategies are being developed in combination with gene replacement therapy. Such strategies include gene upregulation, gene editing, testing novel vectors and delivery systems for gene delivery, and developing improved “muscle-specific expression cassettes” (MSECs) as described herein, which help to optimize gene expression levels from the vectors for FKRP-centric tissue-specific needs. FKRP modifications and constructs [0088] Provided herein are methods and compositions that comprise expression of an RNA transcript of FKRP that has a modified 5’ and/or 3’ UTR. Methods and compositions provided herein include muscle-specific expression cassettes (MSECs) that encode such RNA transcripts. In some embodiments, the nucleic acid cassette comprising a transcriptional regulatory region and a nucleic acid encoding an FKRP RNA transcript are inserted into an AAV plasmid or vector. [0089] In certain embodiments, the methods and compositions described herein comprise a modified 5’ UTR and/or 3’UTR region of the FKRP nucleic sequence. Modifications to the 5’ UTR can include truncation of at least 1 nucleotide from the N-terminal end of the 5’ UTR; in other embodiments the truncation is at least 2 nucleotides, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300, at least 310, at least 320, at least 330, at least 337 nucleotides, (e.g., the entire 5’ UTR) from the FKRP sequence. While one of skill in the art will recognize that the term truncation when applied to the 5’ UTR generally refers to the removal of nucleotide(s) from the 5’ terminal end, it is also specifically contemplated that the 5’ UTR can be modified to remove one or more nucleotides from the 3’ terminal end of the 5’ UTR. [0090] Modifications to the 3’ UTR can include truncation of at least one nucleotide from the 3’ terminal end of the 3’ UTR; in other embodiments, the truncation can comprise removal of at least 2 nucleotides at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300, at least 310, at least 320, at least 330, at least 340, at least 350, at least 360, at least 370, at least 380, at least 390, at least 400, at least 410, at least 420, at least 430, at least 440, at least 450, at least 460, at least 470, at least 480, at least 490, at least 500, at least 510, at least 520, at least 530, at least 540, at least 550, at least 560, at least 570, at least 580, at least 590, at least 600, at least 610, at least 620, at least 630, at least 640, at least 650, at least 660, at least 670, at least 680, at least 690, at least 700, at least 710, at least 720, at least 730, at least 740, at least 750, at least 760, at least 770, at least 780, at least 790, at least 800, at least 810, at least 820, at least 830, at least 840, at least 850, at least 860, at least 870, at least 880, at least 890, at least 900, at least 910, at least 920, at least 930, at least 940, at least 950, at least 960, at least 970, at least 980, at least 990, at least 1000, at least 1010, at least 1020, at least 1030, at least 1040, at least 1050, at least 1060, at least 1070, at least 1080, at least 1090, at least 1100, at least 1110, at least 1120, at least 1130, at least 1140, at least 1150, at least 1160, at least 1170, at least 1180, at least 1190, at least 1200, at least 1210, at least 1220, at least 1230, at least 1240, at least 1250, at least 1260, at least 1270, at least 1280, at least 1290, at least 1300, at least 1310, at least 1320, at least 1330, at least 1340, at least 1350, at least 1360, at least 1370, at least 1380, at least 1390, at least 1400, at least 1410, at least 1420, at least 1430, at least 1440, at least 1450, at least 1460, at least 1470, at least 1480, at least 1490, at least 1500, at least 1510, at least 1520, at least 1530, at least 1540, at least 1550, at least 1560, at least 1570, at least 1580, at least 1590, at least 1600, at least 1610, at least 1620, at least 1630, at least 1637 nucleotides (e.g., all) from the FKRP sequence. [0091] In one embodiment, the modification comprises complete deletion of the 5’ UTR and/or the 3’ UTR. [0092] The wild-type FKRP RNA transcript comprises several secondary structures (e.g., G- quadruplex, hairpins, pseudoknots and the like) in the 5’ UTR that can be deleted or disrupted to enhance binding of transcriptional machinery, thereby increasing FKRP expression. Thus, where increased FKRP expression is desired, disruption or deletion of such secondary structures can be utilized. Conversely, if a lower degree of FKRP expression is desired, such secondary structures can be inserted to partially impede translational machinery, thereby expressing FKRP at lower levels. One of skill in the art can use such secondary structures to modulate and tune the expression of FKRP to a desired level. [0093] In some embodiments, modification of the 5’ UTR of the FKRP RNA transcript comprises the removal of a G-quadruplex, an RNA hairpin, a pseudoknot, or any combination thereof, with the goal of increasing expression of FKRP in a cell. [0094] In other embodiments, modification of the 5’ UTR of the FKRP RNA transcript comprises the insertion of a G-quadruplex, an RNA hairpin, a pseudoknot, or any combination thereof, with the goal of decreasing expression of FKRP in a cell. [0095] In some embodiments, modifications to, or removal of, a given region in the 5’ UTR can be made to improve translation by removal of RNA structures that can impede the association of translational machinery. For example, thermodynamically stable structures such as G-quadruplexes and RNA hairpins in the 5’ UTR of an RNA transcript can result in reduced expression of a gene, such as FKRP. Features found in the 5’ UTR such as pseudoknots, hairpins, and RNA G-quadruplexes, as well as upstream open reading frames (uORFs) and upstream start codons, (uAUGs) mainly inhibit translation. In embodiments where an increase in expression is desired, a modification to the 5’ UTR comprises removal or disruption of a G-quadruplex and/or an RNA hairpin. In one embodiment, the G- quadruplex sequence in the 5’ UTR of human FKRP comprises  attgctccaagatggcggcggcggcggcagcg (SEQ ID NO.9). In another embodiment, the G- quadruplex sequence in the 5’ UTR of murine FKRP comprises tgtacaattgctccaagatggcggcggcggcggcggcggcag (SEQ ID NO.10). [0096] In one embodiment, disruption of a G-quadruplex can include removal of at least 1 nucleotide, at least 2 nucleotides, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, or at least 32 (e.g., all) nucleotides of the G-quadruplex sequence (SEQ ID NO.9 and SEQ ID NO.10) such that it modulates the expression of FKRP. [0097] In one embodiment, disruption of a G-quadruplex can be performed by substituting adenosine (A) and thymine(T)/uracil (U) residues for guanine (G) and cytosine (C) residues in the G-quadruplex sequence (SEQ ID NO.9 and SEQ ID NO.10). Disruptions of the G- quadruplex that in turn result in modifications of FKRP expression are preferred. [0098] In one embodiment, disruption of a G-quadruplex can be made by inserting adenosine (A) and thymine (T)/uracil (U) residues for guanine (G) and cytosine (C) residues in the G- quadruplex sequence (SEQ ID NO.9 and SEQ ID NO.10). Disruptions of the G- quadruplex that in turn result in modifications of FKRP expression are preferred. [0099] It is also contemplated that the activity of G-quadruplexes can be modulated using small molecule inhibitors and/or small molecule ligands such that the expression of a cassette is modified. [00100] In one embodiment, disruption of a hairpin can be achieved by introducing or increasing the number of intramolecular base pair mismatches through nucleotide substitution, thereby modulating the expression of FKRP. One of skill in the art will recognize that RNA hairpins can tolerate a small number of mismatches and that appropriate disruption of an RNA hairpin can require including at least 3, at least 4, at least 5, or more mismatches. [00101] In one embodiment, disruption of a hairpin can be achieved by removing or adding at least 1 nucleotide, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least the full nucleotide length of one or both strands of a hairpin, thereby modulating the expression of FKRP. [00102] In one embodiment, disruption of a hairpin can be achieved by inserting non- complementary nucleotides into the hairpin sequence, such that it disrupts the folding or formation of a base-paired stem in the nucleic acid strand, and in turn modulates the expression of FKRP. [00103] In one embodiment, modifications to the 5’ UTR can comprise introducing a hairpin that prevents, at least partially, proteins from being recruited to initiate translation (e.g. a splicing hairpin), thereby producing FKRP at a lower level of expression. [00104] In one embodiment, the methods and compositions provided herein comprise a 5’ UTR with a modification comprising a disrupted or deleted pseudoknot. Exemplary pseudoknots that can be deleted or disrupted include pseudoknots that are classified as either a H-, K-, L-, or M-type of pseudoknot. A pseudoknot can also include long-range pseudoknots. Software that allows the user to predict the formation of pseudoknots in an RNA structure include PseudoViewer and CyloFold. (Staple and Butcher, PLoS Biol. 2005 Jun; 3(6): e213; Bindewald et al. Nucleic Acids Res.2010 Jul; 38; W368-72). In one embodiment, the pseudoknot sequence in the 5’ UTR of human FKRP comprises (SEQ ID NO.29): AGCTCAGCTGGGCTGGAACTGCCCTCCTGGAACTCCCCCAGCCTACAACCTAGGA GGTGCAGGGACTGAGGCTCAGGCCAAATCGCAACTCAGACCCAGTGAACCCAAG GCCTGAAGAGAATTTGGATTCATT [00105] In one embodiment, disruption of a pseudoknot can be achieved by the removal of at least one stem-loop region (at least 2, at least 3, or more stem-loop regions) of the pseudoknot. [00106] In one embodiment, disruption of a pseudoknot can be achieved by the insertion and/or substitution of nucleotides into the pseudoknot sequence, such that it disrupts the folding of the nucleic acid strand. [00107] In one embodiment, disruption of a pseudoknot can be achieved by increasing mismatches through nucleotide substitution. [00108] In one embodiment, disruption of a pseudoknot can be achieved by a point mutation of one or more nucleotides in the pseudoknot sequence that participates in formation of the pseudoknot structure. [00109] In certain embodiments, modifications can include alteration of a Kozak consensus sequence. The Kozak consensus sequence is a nucleotide motif that functions as the protein translation initiation site in many mRNA transcripts. Varying this region affects the “strength” of the initiation sequence. Alterations (including, but not limited to, substitution, addition or deletion of sequence) of the Kozak consensus sequence in FKRP can result in improved translation and are specifically contemplated for use with the methods and compositions described herein. Muscle-Specific Expression Cassettes (MSECs) [00110] MSECs are engineered transcriptional regulatory elements that each have varying specificity for different types of muscle tissue, such as skeletal and cardiac muscle tissue. In some embodiments, an MSEC as described herein can comprise control elements (e.g., MSEC enhancers and promoters) that bind both ubiquitous and/or muscle type-specific transcription factors; and the activity of each MSEC is determined by differences in control element types, sequences, numbers, and linear order within the enhancer and promoter regions. MSECs can be used to avoid toxicity and immune activation that occurs with uncontrolled expression of muscle therapeutic proteins in other sites, as well as to restrict the expression of a desired gene (e.g., FKRP) in muscle (e.g., skeletal and/or cardiac). [00111] In some embodiments, the muscle-specific transcriptional regulatory cassette is derived from an M-creatine kinase enhancer and/or a M-creatine kinase promoter sequence. For example, the muscle-specific transcriptional regulatory cassette can be derived from an M-creatine kinase enhancer with an M-creatine kinase promoter. Furthermore, the muscle- specific transcriptional regulatory cassette can include one or more enhancers derived from conserved regions of muscle creatine kinase and/or a CK8 transcriptional regulatory cassette (SEQ ID NO.: 8). [00112] The muscle-specific transcriptional regulatory cassette can be a muscle-specific CK8 transcriptional regulatory cassette (CK8) or a derivative thereof. CK8 is a non-naturally occurring nucleotide sequence including multiple muscle and non-muscle gene control elements arranged in a miniaturized array. CK8 can provide high or very high transcriptional expression of a predetermined RNA and/or protein in skeletal and cardiac muscle cells. In one embodiment, an MSEC useful for the methods and compositions described herein comprises a modified CK8 transcriptional regulatory cassette (e.g., CK8e). [00113] It is contemplated that excess FKRP may be differentially toxic in cardiac muscle. In some preferred embodiments, MSECs that have at least 3-fold, at least 10-fold, at least 30- fold, at least 100-fold, at least 300-fold, at least 1000-fold less transcriptional activity than CK8e can be used. One who is skilled in the art can determine what MSEC would produce low levels of FKRP. Different MSECs, their makeup, and how their activity is measured are described in, e.g., PCT/US2022/023915, which is incorporated herein by reference in its entirety. [00114] In certain embodiments, the muscle-specific transcriptional regulatory cassette can be a CK8e transcriptional regulatory cassette having at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or at least 100% sequence identity, to the nucleotide sequence of SEQ ID NO.: 8. [00115] SEQ ID NO.8: (CK8e transcriptional regulatory cassette) TGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTA ACCCAGACATGTGGCTGCCCCCCCCCCCCCAACACCTGCTGCCTCTAAAAATAAC CCTGCATGCCATGTTCCCGGCGAAGGGCCAGCTGTCCCCCGCCAGCTAGAC TCAGCACTTAGTTTAGGAACCAGTGAGCAAGTCAGCCCTTGGGGCAGCCCATAC AAGGCCATGGGGCTGGGCAAGCTGCACGCCTGGGTCCGGGGTGGGCACGGTGCC CGGGCAACGAGCTGAAAGCTCATCTGCTCTCAGGGGCCCCTCCCTGGGGACAGC CCCTCCTGGCTAGTCACACCCTGTAGGCTCCTCTATATAACCCAGGGGCACAGGG GCTGCCCTCATTCTACCACCACCTCCACAGCACAGACAGACACTCAGGAGCCAG CCAGC [00116] In one embodiment, the MSEC comprises CK8e. Alternative MSECs that exhibit either lower overall transcriptional activities, or lower cardiac muscle activities, can be used and are found at PCT/US22/023915, which is incorporated by reference herein in its entirety. AAV vectors [00117] AAV is a parvovirus which belongs to the genus Dependoparvovirus that is useful for the delivery of therapeutic nucleic acids (e.g., a nucleic acid encoding FKRP). AAV’s usefulness stems from its ability to infect a wide range of host cells, including non-dividing cells, as well as its ability to infect cells from different species. AAV has not been associated with any human or animal disease and does not appear to alter the biological properties of the host cell upon integration, thus making it an ideal vector for the delivery of therapeutic nucleic acids. [00118] An "AAV vector," as that term is used herein, comprises a vector derived from an adeno-associated virus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In one embodiment, the AAV vector comprises AAV6. As will be appreciated by those of skill in the art, AAV vectors can have one or more of the AAV wild-type viral genes deleted in whole or part, e.g., the rep and/or cap genes, but retain functional flanking ITR sequences. Where functional ITR sequences are necessary for the rescue, replication, and packaging of the AAV virion, in some embodiments, the AAV vectors described herein include at least one functional ITR. The ITRs need not be the wild- type nucleotide sequences, and can be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging. [00119] In some embodiments, the AAV vectors described herein are engineered to produce synthetic, modified or recombinant AAV vectors. A "recombinant AAV vector" or "rAAV vector" comprises an infectious, replication-defective virus composed of an AAV capsid protein shell encapsulating a heterologous nucleotide sequence of interest that is flanked on both sides by AAV ITRs. An rAAV vector is produced in a suitable host cell comprising an AAV vector, AAV helper functions, and accessory functions. In this manner, the host cell is rendered capable of encoding AAV polypeptides that are required for packaging the AAV vector (containing a recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery. Exemplary recombinant AAV (rAAV) vectors include, but are not limited to, an AAVRh74 vector, an AAV8 vector, an AAV9 vector, an AAV6 vector, an AAV7 vector, an AAV2i8 vector, a NP vector, a NP 66 vector, a NP 22 vector, an AAVpo.1 vector, MyoAAV, and/or an AAVMyo vector. In one embodiment, the AAV vector comprises a MyoAAV vector (Weinmann, J. et al.2020, Nat Commun 11, 5432). In one embodiment, the AAV vector comprises an AAVMyo vector (Tabebordbar, M. et al.2021, Cell 184, 4919-4938 e4922; Chamberlain, J. S.2022, N. Engl. J. Med.386, 1184-1186). [00120] In various embodiments, the therapeutically effective amount of the pharmaceutical composition can be between about 1011 and about 1016 vector genomes (vg)/kilogram (kg) subject weight, between about 1012 and about 1015 vg/kg subject weight, between about 1013 and about 1014 vg/kg subject weight, between about 1012 and about 1014 vg/kg subject weight , between about 1012 and about1015  vg/kg subject weight, between about 1012 and about 1016 vg/kg subject weight, between about 1011 and about1014 vg/kg subject weight, between about 1010 and about 1015 vg/kg subject weight , between about 1013 and about 1015 vg/kg subject weight, between about 1014 and about 1015 vg/kg subject weight, between about 1013 and about 1014 vg/kg subject weight, or between about 1014 and about 1015 vg/kg subject weight. [00121] In some embodiments, the pharmaceutical composition can be administered intravascularly, intraperitoneally, subcutaneously, or by intramuscular injection. [00122] In some embodiments, it can be beneficial to optimize the level of expression of FKRP in muscle tissue (e.g., skeletal and/or cardiac). In some embodiments, it is important to prevent unintended effects from producing too much FKRP, however still expressing enough FKRP to receive its benefits. Some AAV vectors, when combined with different MSECs, can produce high levels of expression of FKRP. However, other AAV vectors, when combined with different MSECs, can produce much lower levels of expression of FKRP. Titrating the optimal expression of FKRP protein can be achieved by selecting vectors and/or MSECs that produce different levels of protein in particular muscle tissues (e.g., skeletal and/or cardiac). Such titration is well within the skill set of one of skill in the art given the guidance provided herein. As but one example, if a construct comprising a modified 5’-FKRP UTR cDNA is determined to convey high FKRP protein levels, it may well be that the MSEC in combination with the modified UTRs and AAV vector produces too much FKRP and possibly even toxic levels. In order to produce a vector that provides lower levels, an MSEC that produces transcripts in a muscle-specific, but lower level can be used, one or more 5’- or 3’-UTR modifications as discussed herein can be introduced, or a combination of MSEC and 5’- or 3’-UTR modifications can be used to titrate FKRP message and/or protein levels as needed. Measuring FKRP expression levels [00123] Methods to measure the level of FKRP expression products in a cell or tissue are known to a skilled artisan. Such methods to measure FKRP expression products include, e.g., protein level, include ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, histology; immunohistological staining; and/or immunofluoresence assay using detection reagents such as an antibody or protein binding agents. [00124] In one embodiment, an FKRP antibody is used to measure the expression of FKRP from the nucleic acid cassettes or AAV vectors as described herein. Antibodies for FKRP are commercially available and can be used to measure protein expression levels, (e.g. anti- FKRP (Cat. No. sc-374642; Santa Cruz Biotechnologies, Santa Cruz, CA), anti-FKRP (Cat. No. DCABY-1120; Creative Diagnostics, Shirley, NY). Alternatively, since the amino acid sequences for the targets described herein are known and publicly available at the NCBI website, one of skill in the art can raise their own antibodies against these polypeptides of interest for the purpose of the methods described herein. The amino acid sequences of the polypeptides described herein have been assigned NCBI accession numbers for different species such as human, mouse and rat. In particular, the amino acid sequences of human FKRP and murine FKRP are included herein, e.g. SEQ ID NO: 11 and SEQ ID NO.: 12 respectively. [00125] In some embodiments of any of the aspects, immunohistochemistry (“IHC”) and immunocytochemistry (“ICC”) techniques can be used to detect FKRP expression. IHC is the application of immunochemistry to tissue sections, whereas ICC is the application of immunochemistry to cells or tissue imprints after they have undergone specific cytological preparations such as, for example, liquid-based preparations. Immunochemistry is a family of techniques based on the use of an antibody, wherein the antibodies are used to specifically target molecules inside or on the surface of cells. The antibody typically contains a marker that will undergo a biochemical reaction, and thereby experience a change of color, upon encountering the targeted molecules. In some instances, signal amplification can be integrated into the particular protocol, wherein a secondary antibody, that includes the marker stain or marker signal, follows the application of a primary specific antibody. [00126] In some embodiments of any of the aspects, the assay to detect FKRP expression can be a Western blot analysis. Alternatively, proteins can be separated by two-dimensional gel electrophoresis systems. Two-dimensional gel electrophoresis is well known in the art and typically involves iso-electric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. These methods also require a considerable amount of cellular material. The analysis of 2D SDS-PAGE gels can be performed by determining the intensity of protein spots on the gel, or can be performed using immune detection. In other embodiments, protein samples are analyzed by mass spectroscopy. [00127] An immunoassay is a biochemical test that measures the concentration of a substance in a biological sample, typically a fluid sample such as blood or serum, using the interaction of an antibody or antibodies to its antigen. The assay takes advantage of the highly specific binding of an antibody with its antigen. For the methods and assays described herein, specific binding of the FKRP-specific polypeptides with respective FKRP-specific proteins or protein fragments, or an isolated peptide, or a fusion protein described herein occurs in the immunoassay to form a target protein/peptide complex. The complex is then detected by a variety of methods known in the art. An immunoassay also often involves the use of a detection antibody. [00128] Other techniques can be used to detect the level of a FKRP-specific polypeptide in a sample. One such technique is the dot blot, an adaptation of Western blotting (Towbin et at., Proc. Nat. Acad. Sci.76:4350 (1979)). In a Western blot, the polypeptide or fragment thereof can be dissociated with detergents and heat, and separated on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose or PVDF membrane. The membrane is incubated with an antibody reagent specific for the target polypeptide or a fragment thereof. The membrane is then washed to remove unbound proteins and proteins with non-specific binding. Detectably labeled enzyme-linked secondary or detection antibodies can then be used to detect and assess the amount of polypeptide in the sample tested. A dot blot immobilizes a protein sample on a defined region of a support, which is then probed with antibody and labelled secondary antibody as in Western blotting. The intensity of the signal from the detectable label in either format corresponds to the amount of enzyme present, and therefore the amount of polypeptide. Levels can be quantified, for example by densitometry. [00129] In certain embodiments, the FKRP expression products as described herein can be instead determined by determining the level of FKRP-specific messenger RNA (mRNA). Such molecules can be isolated, derived, or amplified from a biological sample, such as a blood sample. Techniques for the detection of mRNA expression is known by persons skilled in the art, and can include, but are not limited to, PCR procedures, reverse-transcription (RT) PCR, quantitative RT-PCR (QRT-PCR), real-time PCR (RT-PCR) methods, quantitative RT- PCR Northern blot analysis, differential gene expression, RNAse protection assay, microarray based analysis, next-generation sequencing; hybridization methods, etc. Such methods are known to those of skill in the art and are not described in detail herein. [00130] In some embodiments of any of the aspects, the level of an FKRP-specific mRNA can be measured by a quantitative sequencing technology, e.g. a quantitative next-generation sequence technology. Exemplary methods of sequencing include, but are not limited to, Sanger sequencing, dideoxy chain termination, high-throughput sequencing, next generation sequencing, 454 sequencing, SOLiD sequencing, polony sequencing, Illumina sequencing, Ion Torrent sequencing, sequencing by hybridization, nanopore sequencing, Helioscope sequencing, single molecule real time sequencing, RNAP sequencing, and the like. Methods and protocols for performing these sequencing methods are known in the art, see, e.g. “Next Generation Genome Sequencing” Ed. Michal Janitz, Wiley-VCH; “High-Throughput Next Generation Sequencing” Eds. Kwon and Ricke, Humanna Press, 2011; and Sambrook et al., Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); which are incorporated by reference herein in their entireties. Methods of sequencing a nucleic acid sequence are known to those of skill in the art and are not described in detail herein. [00131] The nucleic acid sequences of the genes described herein have been assigned NCBI accession numbers for different species such as human, mouse and rat. For example, the human FKRP mRNA (e.g. SEQ ID NO: 11) is known. Accordingly, a skilled artisan can design an appropriate primer based on the known sequence for determining the mRNA level of the respective gene. [00132] Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from a particular biological sample (e.g. a mouse) using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample. (Roiff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)). [00133] In some embodiments of any of the aspects, one or more of the reagents (e.g. an FKRP-specific antibody reagent and/or nucleic acid probe) described herein can comprise a detectable label and/or comprise the ability to generate a detectable signal (e.g. by catalyzing reaction converting a compound to a detectable product). Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into reagents (e.g. FKRP- specific antibodies and nucleic acid probes) are well known in the art. [00134] In some embodiments of any of the aspects, detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means. The detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies). The detectable label can be linked by covalent or non-covalent means to the reagent. Alternatively, a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules. Detectable labels can include, but are not limited to radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes. [00135] In other embodiments, the detection reagent is label with a fluorescent compound. When the fluorescently labeled reagent is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. In some embodiments of any of the aspects, a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, Cy3TM, Cy5TM, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5TM, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon GreenTM, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyesTM, 6- carboxyfhiorescein (commonly known by the abbreviations FAM and F), 6-carboxy- 2',4',7',4,7-hexachlorofiuorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfiuorescein (JOE or J), N,N,N',N'-tetramethyl-6carboxyrhodamine (TAMRA or T), 6-carboxy-X- rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyes and quinoline dyes. In some embodiments of any of the aspects, a detectable label can be a radiolabel including, but not limited to 3H, 125I, 35S, 14C, 32P, and 33P. In some embodiments of any of the aspects, a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase. An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In some embodiments of any of the aspects, a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. In some embodiments of any of the aspects, a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads. [00136] In some embodiments of any of the aspects, detection reagents can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems can also be used, for example, a biotin-streptavidin system. In this system, the antibodies immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate. Such streptavidin peroxidase detection kits are commercially available, e. g. from DAKO; Carpinteria, CA. A reagent can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Pharmaceutical Compositions [00137] An aspect of the disclosure relates to pharmaceutical or biopharmaceutical compositions comprising the nucleic acid cassettes or AAV vectors described herein. The pharmaceutical composition can include a transcriptionally regulatory cassette (e.g., a muscle-specific regulatory cassette) operably linked to a nucleotide sequence encoding fukutin-related protein (FKRP), wherein an RNA transcript when expressed from the nucleotide sequence encoding FKRP comprises a modified 5’ and/or 3’ untranslated region (UTR). In other embodiments, the pharmaceutical composition can comprise an AAV vector comprising a transcriptional regulatory cassette operably linked to a nucleotide sequence encoding fukutin-related protein (FKRP), wherein the FKRP RNA transcript when expressed from the nucleotide sequence encoding FKRP comprises a modified 5’ and/or 3’ untranslated region (UTR). [00138] The composition can be prepared in pharmaceutically acceptable, physiologically acceptable, and/or pharmaceutical-grade solutions for administration to a cell or a subject (e.g., an animal), either alone, or in combination with one or more other modalities of therapy. The formulations may be administered in combination with other agents, such as other proteins, polypeptides, pharmaceutically active agents, etc. [00139] The composition can optionally include a carrier, such as a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions. Where the methods and compositions are directed to the treatment of muscular diseases or disorders, it will be appreciated that methods that permit delivery, at least in part, to muscle are preferred. For example, intramuscular injection can be used to directly deliver the compositions as described herein to muscle (e.g., skeletal and/or cardiac muscle). While delivery to skeletal muscle is preferred, some delivery to cardiac muscle can also be useful for the methods described herein. Alternatively, one can administer the compositions through intravenous injection, as long as sufficient amounts are localized to muscles to produce a desired effect. In order to increase the amount of a composition (e.g., an AAV vector) to a given site, a targeting moiety can be used that will enhance delivery to muscle (e.g., skeletal and/or cardiac muscle). [00140] Exemplary carriers can include aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, preservatives, liposomes, microspheres and emulsions. [00141] In one embodiment, the composition is formulated for intramuscular delivery. In one embodiment, the composition is formulated for intracardiac delivery. [00142] Therapeutic compositions contain a physiologically tolerable carrier together with the vectors described herein, dissolved or dispersed therein as an active ingredient. As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like. A pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired. The preparation of a pharmaceutical composition that contains active ingredients dissolved or dispersed therein is understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable either as liquid solutions or suspensions; however, solid forms suitable for solution, or suspension in liquid prior to use can also be prepared. The preparation can also be emulsified or presented as a liposome composition. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient. The therapeutic composition for use with the methods described herein can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of a vector to be administered herein that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, the expression of the therapeutic agent, and can be determined by standard clinical techniques. [00143] While any suitable carrier known to those of ordinary skill in the art can be employed in the pharmaceutical composition, the type of carrier will vary depending on the mode of administration. Compositions for use as described herein can be formulated for any appropriate manner of administration, including for example, intravenous or intramuscular administration. For parenteral administration, such as intramuscular administration, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. Alternatively, compositions as described herein can be formulated as a lyophilizate. [00144] The pharmaceutical composition may reduce a pathological effect or symptom of a neuromuscular disorder associated with FKRP in a subject. Dosage and Administration [00145] Essentially any method of administration can be used with the methods and compositions described herein that permit intramuscular delivery and expression of FKRP in muscle (e.g., skeletal or cardiac muscle). Typically, the administrative method will comprise delivery of a therapeutically effective amount of a pharmaceutical composition to one or more muscles in a subject to be treated (e.g., intramuscular injection, intracardiac injection, systemic or intravenous injection or infusion). The pharmaceutical composition can include a nucleic acid expression cassette comprising a transcriptional regulatory region operably linked to a nucleotide sequence encoding a fukutin-related protein (FKRP) RNA transcript comprising a modified 5’ and/or 3’ untranslated region (UTR). [00146] The compositions can be administered via any suitable route that permits delivery to muscle tissue (e.g. skeletal and/or cardiac), including but not limited to, locally, subcutaneously, systemically, intravenously, intravascularly, intramuscularly, intracardiac, or via a bolus. The compositions can be encapsulated in liposomes, exosomes, microparticles, microcapsules, nanoparticles, and the like, if so desired. Techniques for formulating and administering therapeutically useful polypeptides are also disclosed in Remington: The Science and Practice of Pharmacy (Alfonso R. Gennaro, et al. eds. Philadelphia College of Pharmacy and Science 2000), which is incorporated herein in its entirety. [00147] Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. [00148] Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the active ingredients, to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, water-based solution, before use. [00149] In some embodiments, a therapeutic agent can be delivered in an immediate release form. In other embodiments, the therapeutic agent can be delivered in a controlled-release system or sustained-release system. Controlled- or sustained-release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds. [00150] In some embodiments, the compositions described herein can be administered via a schedule including continuous administration or intermittent administration. Accordingly, in addition to these general schedules, in some embodiments, the composition can be administered twice a day, once a day, once every other day, once a week, once a month, or another suitable period of administration. [00151] In one embodiment, a pump can be used for administration (Langer, Science 249:1527-1533 (1990); Sefton, CRC Crit. Ref Biomed. Eng.14:201 (1987); Buchwald et al., Surgery 88:507 (1980); and Saudek et al., N. Engl. J. Med 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); Levy et al., Science 228:190 (1985); During et al., Ann. Neurol.25:351 (1989); and Howard et al., J. Neurosurg.71:105 (1989)). [00152] The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered and timing depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. An agent can be targeted by means of a targeting moiety, such as e.g., an antibody or targeted liposome technology. [00153] Treatment using the methods and compositions described herein includes both prophylaxis/prevention of disease onset and therapy of an active disease. Prophylaxis or treatment can be accomplished by a single direct injection at a single time point or multiple time points. Administration can also be nearly simultaneous to multiple sites. Patients or subjects include mammals, such as human, bovine, equine, canine, feline, porcine, and ovine animals as well as other veterinary subjects. Preferably, the patients or subjects are human. [00154] In one aspect, the methods described herein provide a method for treating a disease or disorder in a subject (e.g., a muscle disease or disorder). In one embodiment, the subject can be a mammal. In another embodiment, the mammal can be a human, although the approach is effective with respect to all mammals. The method comprises administering to the subject an effective amount of a pharmaceutical composition comprising vector as described herein in a pharmaceutically acceptable carrier. [00155] The dosage range for the agent depends upon the potency, the expression level of the therapeutic protein and includes amounts large enough to produce the desired effect, e.g., reduction in at least one symptom of the disease to be treated. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage will vary with the therapeutic composition (e.g., AAV vector vs. plasmid delivery), and with the age, condition, and sex of the patient. The dosage can be determined by one of skill in the art and can also be adjusted by the individual physician in the event of any complication. [00156] In some embodiments, the vectors are administered at a multiplicity of infection (MOI) of at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 500 or more. [00157] In certain embodiments, the vectors are administered at a titer of at least 1 x 104, 1x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x 1010, 1 x 1011, 1 x 1012, 1 x 1013, 1 x 1014, 1 x 1015 viral particles or more. [00158] Repeated administration can be performed as necessary to maintain therapeutic efficacy. As used herein, the term “therapeutically effective amount” refers to an amount of a vector or expressed FKRP that is sufficient to produce a statistically significant, measurable change in at least one symptom of a disease (see “Efficacy Measurement" below). Alternatively, a therapeutically effective amount is an amount of a vector or expressed FKRP protein that is sufficient to produce a statistically significant, measurable change in the expression level of a biomarker associated with the disease in the subject. Such effective amounts can be gauged in clinical trials as well as animal studies for a given agent. [00159] The vector compositions can be administered directly to a particular site (e.g., intramuscular injection). It is also contemplated herein that the agents can also be delivered intravenously (by bolus or continuous infusion), or systemically, if so desired and provided that FKRP can be expressed in one or more muscle sites (e.g., skeletal and/or cardiac). [00160] Therapeutic compositions containing at least one agent can be conventionally administered in a unit dose. The term "unit dose" when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier, or vehicle. [00161] Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are particular to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more intervals by a subsequent injection or other administration. [00162] Administration of the doses recited above or as employed by a skilled clinician can be repeated for a limited and defined period of time. In some embodiments, the doses are given once a day, or multiple times a day, for example, but not limited to three times a day. Typically, the dosage regimen is informed by the half-life of the agent as well as the minimum therapeutic concentration of the agent in blood, serum or localized in a given biological tissue. In a preferred embodiment, the doses recited above are administered daily for several weeks or months. The duration of treatment depends upon the subject’s clinical progress and continued responsiveness to therapy. Continuous, relatively low maintenance doses are contemplated after an initial higher therapeutic dose. Efficacy [00163] In some embodiments, the methods and compositions described herein comprise a step of diagnosing a subject as having an FKRP-mediated disease. FKRP-mediated diseases can be initially diagnosed by a muscle MRI. FKRP-mediated diseases can show damage to the proximal muscles with relative preservation of the muscles of the anterior compartment of the thighs. A follow up with genetic analysis by specific sequencing of the FKRP gene or of a panel grouping together all the genes involved in the glycosylation of a α-dystroglycan, or a larger panel of genes can be used to confirm the diagnosis. [00164] The efficacy of a given treatment for reducing or preventing FKRP-mediated diseases can be determined by the skilled clinician. However, a treatment is considered “effective treatment," as the term is used herein, if any one or all of the signs or symptoms of muscle (e.g., muscle weakness, muscular atrophy), brain, and/or eye deterioration is/are altered in a beneficial manner, or other clinically accepted symptoms or markers of disease are improved, or ameliorated, e.g., by at least 10% following treatment with a therapeutic agent that increases expression of FKRP in muscle (e.g., skeletal and/or cardiac). Efficacy can also be measured by failure of an individual to worsen as assessed by stabilization of the disease, or the need for medical interventions (i.e., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing progression of muscle, brain, and/or eye deterioration or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of the disease, or preventing secondary diseases/disorders associated with the deterioration of the muscle, brain, and/or eye. [00165] The technology may be as described in any one of the following numbered paragraphs: [00166] Paragraph 1. A nucleic acid expression cassette comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a fukutin-related protein (FKRP) RNA transcript that comprises a modified 5’ and/or 3’ untranslated region (UTR). [00167] Paragraph 2. The nucleic acid expression cassette of paragraph 1, wherein the modified 5’ untranslated region (UTR) is truncated as compared to the 5’ UTR of wild-type FKRP. [00168] Paragraph 3. The nucleic acid expression cassette of paragraph 1, wherein the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 5’ UTR region. [00169] Paragraph 4. The nucleic acid expression cassette of paragraph 3, wherein the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR. [00170] Paragraph 5. The nucleic acid expression cassette of paragraph 4, wherein the modification comprises a modification to the Kozak consensus sequence. [00171] Paragraph 6. The nucleic acid expression cassette of paragraph 1, wherein the modified 3’ UTR is truncated compared to the 3’ UTR of wild-type FKRP. [00172] Paragraph 7. The nucleic acid expression cassette of paragraph 5, wherein the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region. [00173] Paragraph 8. The nucleic acid expression cassette of paragraph 1, wherein the nucleic acid encoding FKRP comprises a modification in each of the 5’ and 3’ UTRs. [00174] Paragraph 9. The nucleic acid expression cassette of any of paragraphs 1-8, wherein the modification in the 5’ and/or 3’ UTR of FKRP causes an increase or a decrease in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity expressed from a similar construct comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a FKRP RNA transcript that comprises a wild-type 5’ and/or 3’ untranslated region (UTR). [00175] Paragraph 10. The nucleic acid expression cassette of any one of paragraphs 1-9, wherein the transcriptional regulatory region comprises a muscle-specific expression cassette (MSEC). [00176] Paragraph 11. The nucleic acid expression cassette of paragraph 10, wherein the MSEC is selected from the group consisting of CK8e. [00177] Paragraph 12. The nucleic acid expression cassette of paragraph 11, wherein upon administration to a cell, expression level of an FKRP mRNA or protein is higher when operably linked to an MSEC than the expression level of the FKRP mRNA or protein when operably linked to a CK8e transcriptional regulatory region. [00178] Paragraph 13. The nucleic acid expression cassette of paragraph 11, wherein upon administration to a cell, expression level of an FKRP mRNA or protein is lower when operably linked to an MSEC than the expression level of the FKRP mRNA or protein when operably linked to a CK8e transcriptional regulatory region. [00179] Paragraph 14. An RNA transcript generated by transcription of the nucleic acid expression cassette of any one of paragraphs 1-13. [00180] Paragraph 15. An adeno-associated viral vector (AAV) comprising the nucleic acid expression cassette of any one of paragraphs 1- 13. [00181] Paragraph 16. The AAV vector of paragraph 15, wherein the adeno-associated viral vector is selected from the group consisting of: an AAVRh74 vector, an AAV8 vector, an AAV9 vector, an AAV6 vector, an AAV7 vector, an AAV2i8 vector, a NP vector, a NP 66 vector, a NP 22 vector, an AAVpo.1 vector, a MyoAAV vector, and an AAVMyo vector. [00182] Paragraph 17. The AAV vector of paragraph15, wherein the adeno-associated viral vector comprises an internal terminal repeat (ITR), a muscle-specific expression cassette, a nucleic acid encoding FKRP, a polyadenylation signal (pA+), and/or a second ITR. [00183] Paragraph 18. An engineered cell comprising or expressing a nucleic acid expression cassette of any one of paragraphs 1- 17. [00184] Paragraph 19. The engineered cell of paragraph 18, wherein the modified 5’ untranslated region (UTR) is truncated as compared to the wild-type 5’ UTR of FKRP. [00185] Paragraph 20. The engineered cell of paragraph 18 or 19, wherein the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 5’ UTR region. [00186] Paragraph 21. The engineered cell of paragraph 20, wherein the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR. [00187] Paragraph 22. The engineered cell of paragraph 20 or 21, wherein the modification comprises a modification to the Kozak consensus sequence. [00188] Paragraph 23.The engineered cell of paragraph 18, wherein the modified 3’ UTR is truncated compared to the 3’UTR of wild-type FKRP. [00189] Paragraph 24. The engineered cell of paragraph 23, wherein the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region. [00190] Paragraph 25. The engineered cell of paragraph 18, wherein the nucleic acid encoding FKRP comprises a modification in each of the 5’and 3’ UTRs. [00191] Paragraph 26. The engineered cell of any of paragraphs 18-25, wherein the modification in the 5’ and/or 3’ UTR of FKRP causes an increase or a decrease in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity expressed from a similar construct comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a FKRP RNA transcript that comprises a wild-type 5’ and/or 3’ untranslated region (UTR). [00192] Paragraph 27. A method of expressing an FKRP gene product in a subject comprising administering an adeno-associated viral vector of any one of paragraphs 15-17 to a subject in need thereof. [00193] Paragraph 28. The method of paragraph 27, wherein the FKRP gene product is a RNA transcript and/or a protein. [00194] Paragraph 29. The method of claim 27, wherein the subject in need thereof comprises limb girdle muscular dystrophy type 2I/R9 (LGMD2i), Walker-Warburg syndrome, or muscle-eye-brain disease (MED). [00195] Paragraph 30. The method of paragraph 27, wherein the modified 5’ untranslated region (UTR) is truncated as compared to the 5’ UTR of wild-type FKRP. [00196] Paragraph 31. The method of paragraph 27, wherein the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 5’ UTR region. [00197] Paragraph 32. The method of paragraph 31, wherein the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR. [00198] Paragraph 33. The method of paragraph 32, wherein the modification comprises a modification to the Kozak consensus sequence. [00199] Paragraph 34. The method of paragraph 27, wherein the modified 3’ UTR is truncated compared to the 3’ UTR of a wild-type FKRP. [00200] Paragraph 35. The method of paragraph 34, wherein the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region. [00201] Paragraph 36. The method of claim 27, wherein the nucleic acid encoding FKRP comprises a modification in each of the 5’ and 3’ UTRs. [00202] Paragraph 37. The method of any of paragraphs 27-36, wherein the modification in the 5’ and/or 3’ UTR of FKRP causes a reduction or inhibition in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity expressed from a similar construct comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a FKRP RNA transcript that comprises a wild-type 5’ and/or 3’ untranslated region (UTR). [00203] Paragraph 38. The method of paragraph 27, wherein the administration of the AAV vector comprises intravenous and/or intramuscular injection. [00204] Paragraph 39. The method of paragraph 27, wherein the subject is a human. [00205] Paragraph 40. A method for reducing at least one symptom of an FKRP-mediated disease or disorder, the method comprising administering an AAV vector of any one of paragraphs 15-17 to a subject in need thereof, thereby reducing at least one symptom of an FKRP-mediated disorder. [00206] Paragraph 41. The method of paragraph 40, wherein the FKRP-mediated disease or disorder comprises limb girdle muscular dystrophy type 2I/R9 (LGMD2i), Walker-Warburg syndrome, or muscle-eye-brain disease (MED). [00207] Paragraph 42. The method of paragraph 40, wherein the modified 5’ untranslated region (UTR) is truncated as compared to the wild-type 5’ UTR of FKRP. [00208] Paragraph 43. The method of paragraph 40, wherein the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 5’ UTR region. [00209] Paragraph 44. The method of paragraph 43, wherein the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR. [00210] Paragraph 45. The method of paragraph 44, wherein the modification comprises a modification to the Kozak consensus sequence. [00211] Paragraph 46.The method of paragraph 40, wherein the modified 3’ UTR is truncated compared to the 3’ UTR of a wild-type FKRP. [00212] Paragraph 47.The method of paragraph 46, wherein the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region. [00213] Paragraph 48. The method of paragraph 40, wherein the nucleic acid encoding FKRP comprises a modification in each of the 5’ and 3’ UTRs. [00214] Paragraph 49. The method of any of paragraphs 40-48, wherein the modification in the 5’ and/or 3’ UTR of FKRP causes an increase or a decrease in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity of a construct comprising wild type 5’ and 3’ FKRP UTRs under substantially similar conditions. [00215] Paragraph 50. The method of paragraph 40, wherein the administration of the AAV vector comprises intravenous and/or intramuscular injection. [00216] Paragraph 51. The method of paragraph 40, wherein the subject is a human. [00217] Paragraph 52. The method of paragraph 40, wherein at least one symptom of a FKRP-mediated disease or disorder comprises:  muscle pain, muscle weakness, muscle fatigue, muscle atrophy, inflammation, decrease in average myofiber diameter in skeletal muscle, loss of ambulation, abnormalities in the brain and/or eyes, eye problems, delay in development, intellectual disability, and seizures.   EXAMPLES [00218] It will be readily understood that the embodiments, as generally described herein, are exemplary. The following more detailed description of various embodiments is not intended to limit the scope of the present disclosure, but is merely representative of various embodiments, Moreover, the order of the steps or actions of the methods disclosed herein may be changed by those skilled in the art without departing from the scope of the present disclosure. In other words, unless a specific order of steps or actions in required for proper operation of the embodiment, the order or use of specific steps or actions may be modified. EXAMPLE 1: [00219] Mutations in the gene encoding fukutin-related protein (FKRP) lead to limb girdle muscular dystrophy type 2I/R9, and less frequently to congenital muscular dystrophy (MDC1C), Walker-Warburg syndrome (WWS) and rarely muscle-eye-brain disease (MEB). Provided herein are methods and compositions for delivering FKRP to a muscle cell. In some embodiments, FKRP is delivered to a cell by way of expression from a nucleic acid expression cassette. Such nucleic acid expression cassettes can be encoded in an AAV vector. [00220] The inventors have worked to optimize a vector system with the goal of achieving maximal functional benefit with the lowest possible dose. Exemplary vector backbones are shown in FIG.1A and FIG.2. Typically, an AAV plasmid used in the methods and compositions described herein comprises ITRs, a muscle-specific expression cassette (e.g., CK8e), the wild-type FKRP cDNA and a polyA signal. This vector can be adapted as desired and tested using initial dose escalation studies, for example, with the AAV6 capsid. [00221] The AAV vectors can be assessed and compared at varying intravascular doses between 5x10^12 vector genomes per kg (vg/kg) up to 2x10^14 vg/kg in different models. Without wishing to be bound by theory, the low dose of ~5x10^12 vg/kg may not be particularly useful for systemic delivery but can still be considered for direct administration to muscle. The high dose of ~2x10^14 vg/kg is the dose that can be used to obtain near saturating levels of gene transfer in several different models of muscular dystrophy, and is the dose being used in essentially all current human gene therapy trials for DMD, LGMD, SMA and MTM1. Thus, in one embodiment, a dose of 2x10^14 vg/kg of a given AAV vector is used with the methods and compositions described herein. [00222] It is specifically contemplated herein that additional studies can be designed to test and optimize the expression cassette needed for clinical gene transfer. Importantly, ongoing human gene therapy trials have highlighted the critical need for refined MSECs to appropriately target gene expression, while avoiding toxicity and immune activation. EXAMPLE 2: FKRP SEQUENCE OPTIMIZATION [00223] Analysis of the untranslated (UTR) regions of the FKRP gene indicates the presence of inhibitory sequences in the 5’ UTR of the FKRP gene. These include high GC content, an RNA G-quadruplex structure and other inverted repeats. Removal of these sequences in expression vectors as described herein has led to increased FKRP expression levels. [00224] The inventors have generated and tested expression cassettes with increasing truncations of the 5’ UTR in combination with altered Kozak consensus sequences to determine which regions maximally impact FKRP expression. In the context of FKRP gene delivery, these results can help identify ways to either enhance or dampen expression levels depending on whether the studies identify safety/toxicity/functionality issues with under- or over-expression of the native gene. [00225] Finally, the inventors have explored truncation of the large 3’ UTR of the FKRP gene with a goal of identifying potential regulatory sequences, which, if removed, permits shortening of the expression cassette to include auxiliary sequences for delivery or for tighter control of expression levels via MSECs. [00226] It is specifically contemplated herein that the sequences described herein can be codon-optimized for human use. Final designs can be tested in animal models, preferably the rat or mouse models. EXAMPLE 3: SELECTION OF AAV SEROTYPES [00227] Optimal gene therapy for LGMD2I and CMD can benefit from vectors that display enhanced targeting of striated muscles and muscle satellite (stem) cells, while minimizing transduction of the liver and other organs. Ongoing work in the field aims to develop synthetic capsids to either alter tropism or evade pre-existing immunity to natural serotypes. [00228] Three types of synthetic capsids have been developed: (a) insertion of short sequences from one serotype to another, (b) synthesis of novel capsids following ancestral AAV sequence reconstruction (c) shuffling sequences randomly and between different natural serotypes. [00229] Specific subsequences in these serotypes can also be shuffled between different capsids to further refine targeting, as can any promising ligands emerging from the nucleocapsid screens. EXAMPLE 4: IMMUNOLOGICAL CONSIDERATIONS [00230] One precaution with all exogenous therapeutics for genetic disorders is activation of immune responses that can limit therapies or lead to toxic adverse events such as liver dysfunction and thrombocytopenia. In some embodiments, the number or extent of immune events is monitored and approaches can be developed to ameliorate their occurrence. [00231] Efforts to limit immunity include the use of muscle-restricted (MSEC-mediated) gene expression, which has been shown to largely eliminate immune responses against foreign proteins. For example, by limiting expression to muscle, exposure of the modified genes and their products to antigen-presenting cells is limited. [00232] Monitoring immune responses can be performed using established assays for neutralizing antibody titers, cytokine release, complement activation, blood cell counts and chemistries and activation of cellular immune responses. EXAMPLE 6: TESTING GENE DELIVERY SYSTEMS [00233] A wide variety of testing systems can be used to evaluate the various vector and other gene delivery systems contemplated herein. These include conventional mouse models of FKRP disorders using dose escalation combined with functional and immunological testing. However, the inventors can also test expression cassettes and vectors in various in vitro systems, such as myogenic cultures (primary and iPSC-derived), 2D and 3D human myogenic systems as discussed elsewhere herein. [00234] The in vitro systems permit analysis of efficiency, expression levels and some functional readouts. The in vivo systems can also add tropism, efficiency, whole body function and safety studies including immune responses, serum chemistries, blood counts and complement activation. [00235] In some embodiments, the gene therapy vectors can be optimized to express FKRP at varying levels in muscle cells. The therapeutically effective amount of FKRP expression can be affected by the percentage of muscle fiber and cardiomyocyte myonuclei that are transduced, which varies by the use of different therapeutic vectors; and since new vector designs will improve transduction efficiencies, optimal therapeutic protein product levels may decrease as targeted transduction efficiencies improve. [00236] MSECs with attributes for treating LGMD2I have been designed and are currently being optimized, and can be tested for efficacy in FKRP mutant mouse and rat models. This work can be done in the following three exemplary phases. [00237] Phase 1 testing can entail a series of studies starting with the AAV gene therapy approaches outlined above. AAV-FKRP vectors can be tested using MSECs that are expected to produce low, medium and high levels of expression. A first goal can be to identify the strongest MSECs that generate therapeutic efficacy without toxicity. If toxicity is observed, the focus can be shifted to weaker MSECs. [00238] The Phase 2 testing can compare a series of MSEC cassettes in the general range of activity below those associated with adverse effects and focus on obtaining uniform expression levels in as many skeletal muscles and fiber types as possible, while also allowing good expression levels in cardiac muscle. These studies can involve sequential testing initially with reporter genes, followed by confirmatory and functional studies with the FKRP gene. [00239] Phase 3 testing can be performed to optimize MSECs for the potential expression of FKRP having modified 5’ and/or 3’ UTRs as described herein for up-regulating the expression of mutant FKRP in genetic situations in which LGMD2I patients produce low- activity FKRP. Each of these strategies can benefit from the use of existing MSECs, or pairing with additional MSECs, with transcriptional activities that are appropriate for the particular therapeutic strategy. EXAMPLE 7: CARDIAC AND SKELETAL MUSCLE PERFORMANCE [00240] For heart function, the inventors assess longitudinal systolic and diastolic performance by M-mode and Tissue Doppler echocardiographic imaging. Endpoint hemodynamics is assessed in situ by Millar catheter and in vitro by Langendorff perfusion. For skeletal muscle, in situ performance is assessed by direct nerve or zonal stimulation of rodent hindlimb muscle. For excised skeletal muscle, the inventors assess twitch and tetanic contraction, fatigue resistance and recovery. Intracellular calcium can be measured by microscopy after loading muscle with fluorescent calcium chelators. EXAMPLE 8: DEMEMBRANATED MUSCLE AND ISOLATED MYOFIBRIL CONTRACTILE PROPERTIES [00241] Dissolving the surface membrane of skeletal or cardiac muscle cells permits study of the contractile apparatus performance without the confounding influence of intracellular calcium dynamics that occurs during contractions. Tissue and single muscle cell preparations are robust, allowing the inventors to test several conditions in each preparation. Isolated myofibrils (single sub-cellular contractile organelles) allow study of the millisecond timescale kinetics of contractile activation and relaxation, with high fidelity and resolution of forces in the piconewton range. EXAMPLE 9: MUSCLE TISSUE, CELL AND MYOFIBRIL STRUCTURE [00242] Tissue-level structural analysis permits the examination of fibrosis, satellite cells and morphological features such as centralized nuclei and muscle damage. In muscle cells, the contractile units called sarcomeres align in the direction perpendicular to the long axis of the cell within myofibrils, and the boundaries of these structures (z-disks) align between myofibrils for high-order parallelization of the contractile apparatus. This order is often disrupted in diseased and damaged muscle, contributing to reduction of force production, altering contraction and relaxation kinetics, and furthering downward spiral muscle damage. Within individual myofibrils, electron microscopy can be used to examine the detailed structure of sarcomere thin and thick filaments and z-disks, and these approaches can be used to study structural effects of therapeutic interventions. EXAMPLE 10: ISOLATED CONTRACTILE PROTEIN MECHANICS [00243] An advantage in flow cell assays of isolated contractile protein mechanics is that effects on myosin, actin, troponin and tropomyosin can be assessed as the molecular target for therapeutics, and a large number of conditions can be rapidly assessed since proteins can be separately treated once they are all in a flow cell assay. EXAMPLE 11: METABOLIC PROFILING [00244] Targeted aqueous metabolite profiling analysis can be performed using, for example, the Agilent 1260/AB-Sciex 5500 Qtrap LC-MS/MS instrument and HILIC (hydrophilic interaction chromatography) protocols. The development of highly multiplexed, targeted methods using advanced LC-MS/MS instruments provides fast data acquisition and reasonably tight quality control, resulting in reproducible measurement of >200 metabolites located in >60 different metabolic pathways. EXAMPLE 12: MITOCHONDRIAL FUNCTION [00245] Isolated mitochondria and permeabilized cardiac tissue can undergo high-resolution respirometry (HRR) via Oxygraph for the measurement of respiration/respiratory complex analysis (oxygen consumption and flux), mitochondrial membrane potential, ROS and ATP production. Mitochondrial content can be assessed by Western blot to assay ETS (Mitosciences Oxphos profile) and VDAC (Santa Cruz Biotech) protein expression, citrate synthase activity and mtDNA/nuclear DNA. ATP synthesis rate in beating hearts can be assessed, if needed, using 31P NMR transfer compared to estimation by MVO2. EXAMPLE 13: Efficacy and Muscle Safety Assessment of Fukutin-Related Protein Gene Therapy [00246] INTRODUCTION: [00247] Dystroglycanopathies are a family of muscle disorders (> 20) that are caused by altered glycosylation of α-dystroglycan (α-DG), a peripheral membrane protein located on the extracellular side of the sarcolemma that normally binds to laminin. The laminin-α-DG association is a crucial portion of the dystrophin-glycoprotein complex (DGC), which provides a mechanical link between the intracellular actin cytoskeleton and the extracellular matrix. The DGC enables the lateral transmission of forces from within myofibers, allowing the muscle bundle to contract in unison and preventing cellular damage by internally maintained contractile energy. More than 11 glycosyltransferases are known to post- translationally modify α-DG, working sequentially to build long glycan chains onto the protein. One such glycosyltransferase, Fukutin-related protein (FKRP), catalyzes the transfer of ribitol 5-phosphate to a phosphorylated O-mannosyl trisaccharide on α-DG, but only after Fukutin has added a ribitol 5-phosphate to the growing chain. These sequential modifications lead up to the addition of a repeating glucuronic acid and xylose chain that serves as the laminin binding domain of α-DG. [00248] Altered α-DG glycosylation severely disrupts the DGC mechanics, leading to fragile sarcolemma membranes and muscular dystrophy. These resulting dystroglycanopathies are the primary cause for several forms of congenital muscular dystrophy as well as multiple limb-girdle muscular dystrophies. Limb-girdle muscular dystrophy type R9 (LGMDR9, previously LGMD2I) is one of the most common of these diseases. It is an autosomal recessive disorder caused by mutations in the FKRP gene which also can lead to congenital muscular dystrophy (MDC1C), Walker-Warburg syndrome (WWS) and muscle-eye-brain disease (MEB). The study is focused on developing treatment options for LGMDR9, although the results will also be relevant to the other, less prevalent FKRP disorders. LGMDR9 is slowly progressive, but patients still experience symptoms such as muscle weakness, muscle cramps, hypertrophy, joint contractures, and, in some cases, severe cardiomyopathy and respiratory issues. The age of LGMDR9 onset varies, with a spectrum of symptoms presenting in relation to specific mutations in FKRP. For example, affected LGMDR9 patients are often wheelchair-dependent by 25 years after age of onset. Diagnoses are typically made based on elevated serum creatine kinase (CK) levels and proximal muscle weakness followed by genotyping. There is no cure for LGMDR9, and treatments are limited to temporary symptom amelioration. [00249] LGMDR9 is often due to heterozygous and homozygous mutations in the 1.5 kb coding region of the FKRP gene, the most common of which is 826C>A (L276I). There is a strong genotype-phenotype correlation for this mutation with compound heterozygous patients displaying a more severe phenotype than homozygous patients. Another common mutation is 1343C>T (P448L), and both mutations interfere with the transfer of FKRP from the endoplasmic reticulum to the Golgi apparatus. As FKRP is a post-translational glycosyltransferase, mislocalization of this enzyme leads to decreased glycosylation and half- life, and results in increased targeting of α-DG by the proteosome. Additionally, other insertion, deletion, missense, and nonsense mutations have been reported in patients, though less commonly than the L276I and P448L point mutations. Interestingly, FKRP-null mutations are embryonic lethal, which explains why all patients genotyped to date have at least one mutant allele that leads to expression of a presumably partially functional protein. These and other data suggest that most, if not all, mutant FKRP enzymes found in patients still retain some enzymatic activity. [00250] Developing approaches for gene therapy of LGMDR9 have been promising. However, inconsistent vectors, mouse age, genotype, and transgene have resulted in contradictory toxicity evidence associated with FKRP-overexpression. For example, recent data have shown that adeno-associated viral vector (AAV) -mediated systemic delivery of FKRP can significantly ameliorate the dystrophic phenotype in a murine disease model, the FKRPP448L mouse, which will reasonably recapitulate LGMDR9. Mouse age varied widely across the studies and the regulatory cassettes used in the various studies similarly differed, as some groups used the strong and ubiquitously expressed CMV (human cytomegalovirus immediate early enhancer plus promoter) or CB (CMV enhancer/chicken ^-actin promoter) cassettes while others used a muscle-specific creatine kinase (CK7) cassette. The use of the mouse vs. the human FKRP cDNA also varied between studies, as did doses that ranged from 2x1012 vg/kg to 6x1013 vg/kg. The ability of gene therapy to treat LGMDR9 as well as its durability are also significantly dose-dependent, with lower doses displaying shorter-term, but still significant effects. [00251] Moreover, only one test of systemic muscle function has been performed on FKRPP448L mice receiving FKRP gene therapy, a treadmill exhaustion assay that, although useful, provides limited metrics for analysis. Other tests of systemic muscle function include running wheels for voluntary exercise and treadmills with respiratory chambers for assessing metabolic rate during forced exercise, all of which are recommended assessments for preclinical studies with dystrophic mice. Quantifying metabolic rate through indirect calorimetry assesses the combined functional integrity of both skeletal and cardiac muscle and differs from plethysmography. This latter measure of ventilation reflects diaphragm function only and has little effect on metabolic rate. Exercise-based assessments mimic those often used to assess dystrophic patients in the clinic (e.g., 6-minute-walk test) and can be used to exacerbate the dystrophic phenotype in preclinical studies. [00252] Therapeutic delivery method is yet another component that should be considered in the development of the safest possible therapeutic for LGMDR9 and related CMDs. Increased gene expression is often a critical part of gene therapy, as therapeutics are limited by the immune responses associated with high doses of AAVs in human patients. For example, the issue of systemically administered AAVs and the accompanying liver toxicity seen in clinical trials remain a barrier moving forward. Therefore, an ideal treatment would maximize gene expression while minimizing the AAV dose. The latest advancements in AAV vector designs have led to increased targeting and gene expression in specific tissues. The development of these capsid variants that permit muscle-specific gene delivery may alter dosing thresholds and therapeutic efficacy, and need to be examined in relation to current vectors used in the clinic. One such novel AAV capsid has been named AAVMYO1 for its myotropic properties, and has been shown to increase efficiency and specificity in heart, diaphragm, and skeletal muscle. These new developments not only address the issue of myotropism but also decrease liver-tropism, which will be critical to avoid the issues of innate immunity, and will affect the success of gene therapies using high dose AAV vectors. [00253] While it has been shown that FKRP gene therapy is generally an effective treatment in mouse models for LGMDR9, there has been little focus on transgene designs. Transgene optimization provides a tool to improve efficacy and lower necessary treatment doses. Evidence of potential secondary structures was discovered in the untranslated regions of FKRP mRNA. One particularly relevant structure is an RNA G-quadruplex (RGQ), a stable secondary mRNA configuration associated with the inhibition of translation. Repeats of a (CGG) motif in the 5’UTR of FKRP are suggestive of an RGQ, and further investigation into UTR regions can provide a tool for tunable gene expression (for FKRP and other genes). [00254] The current study tested the dose-dependent efficacy of a novel FKRP gene therapeutic. This particular therapeutic differed from the vectors previously tested as it used AAV6 instead of AAV9, as well as a miniaturized mouse muscle creatine kinase enhancer/promoter (CK8e) that is uniquely active and specific for striated muscle (AAV6- Ck8e-humanFKRP, A6.C8hF). To address the issue of potential FKRP toxicity, it was explored whether overexpression of FKRP in wild-type mice affected muscle physiology. These studies delivered the small FKRP cDNA using multiple doses of vectors pseudotyped with capsids from AAV6, AAV9, and the newer myotropic AAVMYO1. Cardiac and muscle physiology assays did not identify any adverse functional affects due to the exogenous FKRP in wild type mice when delivered with any of these serotypes. [00255] RESULTS: [00256] Removal of the untranslated regions from the FKRP cDNA increases protein expression. To explore FKRP expression, rabbit polyclonal antisera (named Ab607) was initially generated against a conserved fragment of the C-terminus of mouse and human FKRP (see methods). To verify the utility of the antisera after affinity-purification, C2C12 myotubes were transduced with 1x1012 vg, 1x1011 vg, or 1x1010 vg of AAV6-CK8e-mFKRP- FLAG (the murine Fkrp cDNA with a C-terminal FLAG-tag). Cell lysates revealed co- immunoreactivity with the FKRP and FLAG antibodies and provided confirmation of FKRP production by the AAV6-CK8e-FKRP vector (FIG.6A). To maximize protein expression, potential inhibitory sequences were located in the FKRP untranslated regions. Sequence analysis suggested there may be regulatory secondary mRNA structures, so both UTRs were removed from the Fkrp cDNA (A6.C8mF). Note, however, that CK8e, which is present in all these vectors, carries 49 bp from the mouse CKM 5’ UTR and that the polyA sequence used was also identical in all vectors (see methods). The effects of these modifications were then compared in vitro by transducing C2C12 myotubes with vectors either carrying or lacking both the Fkrp 5’ and 3’ UTRs. Western analysis with Ab607 showed a notable increase in FKRP expression upon the removal of the UTRs (FIG.6B). It is important to note that the minor bands at 55 kDa correspond to the predicted molecular weight of FKRP, while the larger bands at ~60 kDa are likely the N-glycosylated form of the protein. This vector was then tested in vivo via intramuscular injections into the tibialis anterior (TA) of wild-type mice (1x1011vg/muscle). Analysis of injected muscles revealed FKRP expression by western blot as well as mosaic distribution of the protein throughout the injected muscles (FIG.6C). [00257] To explore potential deleterious effects from high levels of exogenous FKRP expression, a similar vector was generated that expressed the human FKRP (also lacking the FKRP 5’ and 3’ UTRs). This CK8e-hFKRP construct was encapsulated into AAV6 (A6.C8hF), AAV9 (A9.C8hF), or AAVMYO1 (AM.C8hF) vectors and tested via systemic delivery (FIG. 6D and 6E). [00258] Wild-type animals treated with AAV-FKRP vectors displayed normal muscle physiology. To explore multiple approaches for delivering FKRP to skeletal muscles and simultaneously monitor for toxic effects, additional assays were performed in wild-type mice injected with vectors made with AAV6, AAV9 or AAVMYO1 capsids. These studies also provided a way to examine the effects of exogenous FKRP in muscle already expressing normal levels of the enzyme. In particular, one previous report suggested that overexpression of FKRP could impair the formation of a functional DGC. The studies used AAV6, as above, but also 2 additional vector types. Gene therapeutics featuring AAV9 capsids are commonly used in clinical trials for different neuromuscular disorders while the recently developed AAVMYO1 was reported to provide significantly enhanced muscle transduction. By using AAV vectors pseudotyped with multiple capsids, including a potent myotropic capsid (AAVMYO1), the results were not limited to the effects of a single type of AAV capsid. [00259] The first study consisted of wild-type mice injected with A6.C8hF at doses of 4x1013, 2x1014, or 4x1014 vg/kg to determine whether this vector caused an increase in susceptibility of muscles to contraction-induced injury. These high doses were a used in this preliminary safety assessment, as doses at or exceeding 1x1014 vg/kg have often led to serious adverse events in patients with various neuromuscular disorders. This is important because a high dose AAV-mediated FKRP therapeutic, plus endogenous FKRP, provides the ability to maximize potential expression. Some of these mice underwent gastrocnemius muscle physiology assays 5 weeks later and others were tested at 10 weeks. However, no deleterious impact on mechanical properties were observed at these doses (FIG.9A-9B). [00260] Finally, the AAV9 and AAVMYO1 vectors were injected into wild-type mice at doses of 6.4x1012, 2x1013, and 6.4x1013 vg/kg. The latter two cohort of mice were analyzed for a variety of properties at 4-, 6-, 8-, 10-, 12-, and 14- weeks post-injection to evaluate effects on muscle physiology (FIGs.7A-7I). At 6-, 10-, and 14- weeks post-injection, fatigue assays were performed in which mice ran on a treadmill at a speed of 10 meter/sec, with 1 meter/sec/minute increases (FIG. 7A-7C). No differences were detected between groups at each time point. Moreover, the absence of differences in relative change within and between groups similarly demonstrates an absence of detrimental effects on running fitness and fatiguability following vector delivery. Forelimb grip strength was also examined between 4-, 8-, and 12- weeks post- injection. Although mice treated with AAV9 displayed reduced force over time, there were no differences between groups at any time point. In fact, all the mice (treated and untreated) experienced a decrease in strength between 10- and 14-weeks post-injection groups, which typically occurs when mice habituate to the grip-strength assay. [00261] Another commonly used assay in mouse muscle analysis is the ankle plantarflexion assay, in which ankle torque over 20 eccentric contractions is quantified using a high yet physiologically relevant stimulation frequency that elicits a maximal response. The assay is designed to assess force generation in addition to fatigue caused by repeated eccentric contractions. At all time points, torque declined to 50% of starting baseline measurements, consistent with contraction-induced fatigue (FIG. 7G-7H). However, no differences were detected between any of the three groups at any timepoint. This includes potential differences in torque at a given contraction as well as the relative change from baseline. These results further suggest that A9.C8hF and AM.C8hF treatments at a dose of 6.4x1013 vg/kg did not compromise muscle force generation or enhance fatiguability. [00262] To identify potential adverse consequences of exogenous FKRP expression in cardiac and diaphragm muscles, ultrasound imaging was used to measure cardiac ejection fraction and fractional shortening as well as diaphragm displacement. Ejection fraction measures the percent of blood leaving the left ventricle, while fractional shortening is a measure of the heart’s contractility; both of which are common indicators of cardiac function. Diaphragm displacement is likewise an indicator of respiratory function. No differences were detected between untreated and treated groups, likely due to small sample size (n=3-4), however more data may highlight the increased injection fraction of AM.C8hF over A9.C8hF and untreated mice (FIG.8A-8C). At the experimental endpoint (18 weeks post-injection), skeletal muscle function was further assessed using a contraction-induced injury protocol on tibialis anterior and diaphragm muscles. Here, muscles were stretched by an additional 5% between contractions to measure specific force development and to examine the effects of contraction- induced injury on force. As with the other functional assays, no differences were detected between treated and untreated groups (FIGs.8D & 8E). These data complement those from previous tests, which together reveal no adverse consequences from exogenous FKRP overexpression in skeletal or cardiac muscles using the assays shown (FIGs.7 and 8). [00263] DISCUSSION: [00264] This study focused to develop a transgene with high expression levels in striated muscles. Use of such a transgene permits studies of safety while also facilitating therapeutic expression levels at lower vector doses. It should be noted that safety and toxicity concerns have been growing in the field, with adverse events from either the vector or transgene being observed in a variety of high dose, neuromuscular disease AAV trials. [00265] RNA G-quadruplexes (RGQ) are known to regulate gene expression and have been targeted as a strategy to ameliorate or worsen genetic disease pathologies. It was discovered that complete removal of both the 5’ and 3’ UTRs led to increased gene expression (FIG.6A- 6E). These data suggest the presence of inhibitory regulatory sequences within the UTRs of FKRP, which could provide a potential target for fine-tuning gene expression. The occurrence of toxicity associated with gene overexpression has sometimes been resolved by targeting UTRs, which continue to be evolved for even more influence over protein production. [00266] Demonstrating that the therapeutic can improve exercise capacity is especially meaningful, as the metrics measured are largely dependent upon cardiac rather than skeletal muscle function. Moreover, cardiac and respiratory impairment directly contribute to mortality in many individuals with different FKRP mutations and are predictors of long-term survival. It is not unreasonable, therefore, to presume that systemic FKRP gene therapy could ameliorate the primary cause of mortality in LGMDR9 patients. [00267] Safety assessment with exogenous FKRP expression [00268] While studies here and by others show improved phenotypic outcome from AAV- FKRP delivery, the effects in wild-type mice were explored to monitor potential adverse events from exogenous FKRP expression above normal muscle expression. These exogenous expression levels were augmented using a strong, muscle-specific regulatory cassette (CK8e), the removal of UTRs from the FKRP cDNA and the use of myotropic AAV capsids, especially AAVMYO1. The AAVMYO1 capsid in particular is one of a new class of potent muscle- targeting capsids and may allow lower doses to be used in clinical trials. Three doses of 4x1013, 2x1014, and 4x1014 vg/kg of A6.C8hF indicated that treated wild-type mice continued to gain strength, did not fatigue more rapidly, and showed no detrimental effects on mechanical properties (FIG. 9). A longer study was also performed with wild-type mice untreated or injected with A9.C8hF and AM.C8hF. These groups of mice showed no significant differences in distance run, forelimb grip strength, or TA, diaphragm muscle or hind limb eccentric contraction induced fatigue over time, nor were these measurements significantly different between groups (FIG.7). In these studies, treated and untreated mice also showed no change in ejection fraction, fractional shortening, nor diaphragm displacement over time (FIG. 8). Overall the data support the safety of A6.Ch8F, A9.C8hF, and AM.Ch8F as gene therapies for LGMDR9, as the assays show that none of these treatments led to adverse effects on wild-type mouse physiology. These results demonstrate that expression from the vectors used was not deleterious to muscle function and was able to ameliorate disease pathology in mutant mice. [00269] Conclusions and future directions [00270] The studies further support the feasibility of gene therapy for disorders resulting from FKRP mutations. Further, no detrimental effects from expression of FKRP were observed using a variety of AAV vectors at different doses in wild-type mice. A potential G-quadruplex in the 5’ UTR may serve to limit expression, although individual bases were dissected in the 5’ or 3’ UTRs of the FKRP mRNA that mediate this effect. The difficulty encountered by many labs in detecting endogenous FKRP expression suggests that extremely low levels of the enzyme is all that is needed for normal glycosylation of ^-DG. It is possible that inhibitory UTR sequences may serve a role in limiting overexpression during normal muscle activity, although further studies would be needed to clarify this issue. Importantly, the safety of this therapeutic observed in the studies supports continued advancement of methods for gene therapy to treat patients carrying mutations in the FKRP gene. EXAMPLE 14: MATERIALS AND METHODS [00271] Animals [00272] All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Washington Mouse studies were performed in C57BL6 wild- type male mice aged 6-35 weeks at age of injection. [00273] Plasmid construction and vector production [00274] The coding region of mouse Fkrp was PCR amplified (Forward primer: 5’ TTGTTAACATGCGGCTCACCC 3’ (SEQ ID NO: 30); Reverse primer: 5’ TACCGGTTCAACCGCCTGTC 3’ (SEQ ID NO: 31)) from mouse muscle cDNA. The resulting DNA fragment was digested with HpaI and AgeI and ligated into an AAV backbone vector containing a muscle specific CK8e promoter and synthetic poly A tail, as previously described. Briefly, a custom AAV transfer plasmid containing the previously described muscle-specific CK8e regulator cassette, a 1,482 bp cDNA expression construct for native mouse Fkrp mRNA (NCBI CCDS20853.1), a synthetic polyA signal and flanking AAV serotype 2 inverted terminal repeats was constructed using standard recombinant methodology. The resulting plasmid, pAAV-CK8e-FKRP, was then used to co-transfect HEK293 cells along with the pDGM6 helper plasmid to generate AAV6 vector as previously described. For AAV9 (pA9.C8hF) and AAVMYO1 (AM.C8hF) vectors the human FKRP cDNA was PCR amplified and cloned into pAAV-Ck8e-FKRP in place of the mouse cDNA. These latter vector preparations were produced and purified by Forge Biologics (Grove City, OH). [00275] Antibody production and purification [00276] Rabbit polyclonal antisera was generated against a peptide near the C-terminus of the FKRP sequence that was identical in the mouse and human proteins (as a fusion with KLH: KLH-C-APNNYRRFLELKFGPGVIENPQYPNP (SEQ ID NO: 32)) by Covance (Denver, PA). Affinity purification was performed using a maltose-binding protein fusion of the antigenic peptide on a MBPTrap HP columns (GE Healthcare) and coupled to Ultralink Biosupport polyacrylamide resin (Thermo Scientific) as per manufacturer’s instructions. The FKRP-C antibody (named Ab607) was affinity purified by HPLC through MBP-FKRP coupled beads and stored in BSA and NaN3. [00277] Cell culture [00278] Mouse C2C12 cells were plated at ~80% confluence on gelatin-coated 6-well plates with standard growth media (DMEM, 20% FBS, 1% penicillin-streptomycin (P/S)) overnight, then washed three times with 1x Saline G prior to infection with rAAV6-CK8- mFKRP. Vectors were diluted to the desired concentrations in differentiation media (DMEM, 2% HS, 1% Penicillin-Streptomycin). Cells in each well were incubated with 0.5 ml of diluted virus at 37oC for ~ 2 hours and brought to a final volume of 2 ml with differentiation media and incubated overnight prior to refeeding with fresh differentiation media. Western analysis was performed at day 3 post-differentiation to determine FKRP expression. [00279] Gene delivery [00280] Mice were randomly assigned to groups prior to treatment. They were then anesthetized by an intraperitoneal injection of 0.25 mg/g 2,2,2-tribromoethanol or isoflurane and subsequently injected retro-orbitally (RO) (150 µl at doses of 1.5x1013, 4x1013, 1.5x1014, 2x1014, or 4x1014 vg/kg), intramuscularly (30 µl at doses of 1x1010 vg and 1x1011 vg), or intravenously via the tail vein (150 µl at doses of 6.4x1013 vg/kg). [00281] Western analysis [00282] Frozen muscles were ground to fine powder and proteins extracted in 1x Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, protease inhibitor (Roche)). Lysates were centrifuged at 10,000x g for 10 min at 4oC, and supernatants transferred to fresh tubes. Total protein concentration was determined by BCA Assay (Pierce). Prior to loading, 50 mM DTT and 0.01% bromophenol blue were added to samples and heated at 94oC for 4 min. For each sample, 20 ^g of total protein was separated on 4-12% SDS-PAGE (Life Technologies), transferred to PVDF membranes (GE Healthcare), and blocked (5% skim milk, 1x PBS, 0.1% Tween-20) for 1hr at room temperature. Blots were probed with Ab607 (1:2000) overnight at 4oC, washed in 1x PBS-Tween, incubated with HRP-Rabbit secondary (1:20,000, Pierce) and developed with ECL chemiluminescent reagents (GE Healthcare). [00283] Statistical Analysis [00284] Data are presented as means + SEM and statistical comparisons of non-parametric data points were made using Prism (GraphPad Software, La Jolla, CA). Significant differences (p<0.05 unless otherwise noted) were determined using a Student’s t-test or with a 1- or 2-way analysis of variance coupled to Tukey’s post-hoc test for multiple mean comparisons. Significant differences are represented by different letters and a shared letter indicates no difference. For example, three data points labelled a, ab, and b represent a significant difference between a and b, as different letters indicate significance. There is no difference between a and ab or ab and b, as they share letters. EXAMPLE 16 [00285] Another way to analyze and verify the effects of gene therapy with FKRP constructs as described herein is to administer vectors as described herein to a mouse model of FKRP deficiency-mediated disease. To that end, FKRPP448L mice (which contain a mutation in the FKRP ORF at position 448 (P448L) between the ages of 6 months to a year (the preferred age is 10 months) can be injected with different doses of A6.C8hF (saline, at a range between 1x1012, and 1x1015 vg/kg) and monitored alongside age-matched wild-type controls. The purpose of using older mice is to address the effect of treatment in older mice with a more advanced phenotype. Forelimb grip strength of age from wild-type, untreated and treated FKRPP448L mice can be measured between 2-weeks and 16-weeks post-injection. It is expected that absolute forelimb grip strength will restore strength, e.g., between 30%-80% of wild-type levels or more, compared to untreated FKRPP448L mice. Additionally, it is expected that body weight and grip strength will increase modestly in all treated FKRPP448L mice. Such a change would indicate that changes in grip strength are unlikely to be due to compensatory responses to changes in body weight. [00286] Hindlimb grip-strength measurements are often highly variable until mice become accustomed to the assay. Based on this, it can be expected that grip-strength absolute force will increase in all groups before stabilizing between 7-10 weeks. However, it is also predicted that treated mice will also be stronger than untreated. These results will suggest that despite the innate variability of the assay, treatment with constructs as described herein, including but not limited to A6.C8hF, will increase hindlimb grip strength in aged mice. EXAMPLE 17 [00287] Exercise capacity of mice treated with FKRP viral vectors as described herein. Additional metrics that can be examined in treated mutant mice include measurements of VO2max, distance travelled, energy expended, and energy consumption rate. All of these can be measured using a metabolic treadmill and can include VO2max tests to assess changes in maximal O2 consumption, a measure of exercise capacity and cardiorespiratory function, and several intermittent training sessions meant to exacerbate the dystrophic phenotype. It is expected that training will have a beneficial effect in wild-type mice and will increase VO2max between 5%-15%. By contrast, it is expected that training will exacerbate the dystrophic phenotype in untreated FKRPP448L mice as VO2max will be reduced by over 5%- 20%. It is expected that this decline will be prevented by A6.C8hF treatment. Moreover, it is expected that the degree of relative change between tests will be significant among all three groups, indicating the effect of impact training and treatment. It is expected that changes in running distances and energy rates will reflect a similar relationship. These results will collectively suggest that although high dose treatment with A6.C8hF may not restore exercise capacity and cardiorespiratory function to wild-type levels, it prevents the deleterious effects of impact training and improves striated muscle functional efficiency. It should be noted that FKRP mutations in both animal models and human patients leads to a degenerative disease that progressively develops and worsens with age. As such, it would not be unexpected that even successful therapy in a model with advanced disease does not restore function to pre- or non-disease levels. Higher doses or ancillary treatments that enhance muscle mass and strength may be needed when degeneration is advanced. [00288] It is expected that both groups of mice will run shorter distances in the initial tests while expending similar calories. Although this will result in higher energy-consumption rates, these differences are not expected to be significantly different from wild-type mice. It is expected that the protective effect on exercise-induced impact will also be reflected in energy consumption rates. It is expected that all three groups will be significantly different from one another in the final test, with untreated FKRPP448L mice having the lowest VO2max and highest energy consumption rate. Treated FKRPP448L mice will display values closer to those of wild-type animals. It is expected that treatment with A6.C8hF will be able to at least partially restore exercise capacity. EXAMPLE 18 [00289] Effect of FKRP viral vectors as described herein on dystrophic respiratory pattern and endurance exercise capacity in FKRPP448L mice. Several pathological markers can be used for assessing exercise impact training for dystrophic mice. In addition to histological metrics, these include several exercise-induced markers in FKRPP448L mice such as variability in respiration (VO2cv), the respiratory exchange ratio (RER, VCO2/VO2), energy expended, and the accumulation of motivational shocks. It is expected that in some of training sessions, the VO2cv values for untreated FKRPP448L mice will be higher than those for wild-type mice, while the treated FKRPP448L animals will have values that will be either significantly lower than the untreated mice or not different from wild-type mice. A similar pattern is expected when comparing the overall differences between groups, which would be highly significant, and when assessing differences in maximal RER values. EXAMPLE 19 [00290] Evaluation of muscle degeneration, muscle fiber size distribution and creatine kinase levels. After assessing exercise impact training, all mice can be sacrificed, and different muscles will be collected. Compared to untreated FKRPP448L mice, it is expected that treated FKRPP448L mice will have larger skeletal muscle myofiber sizes and will have fewer centrally-nucleated myofibers and fewer total centrally-located nuclei, which are hallmarks of prior rounds of muscle necrosis and regeneration. It is expected that treatment will also impact serum creatine kinase levels in treated vs. untreated mice, with levels in treated mice being significantly lower than in untreated. Such a result would suggest that treatment with FKRP vectors as described herein could reduce exercise-induced muscle damage that activates cycles of necrosis and regeneration that lead to skeletal muscle atrophy. EXAMPLE 20: [00291] Combinations of G-quadruplex, hairpin, and 3’ + MSEC below based on the description from FIG.5. The term “FKRP ORF” refers to both wild-type FKRP and a mutated form of FKRP. The term “MSEC” refers to any MSEC as defined in the specification and referenced in PCT/US22/023915, which is incorporated by reference in its entirety. The following describes various combinations of 5’ UTR and 3’ UTR modifications in combination with MSECs contemplated for FKRP constructs as described herein. [00292] 5’ UTR truncations [00293] -01bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00294] -02bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00295] -03bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00296] -04bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00297] -05bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00298] -06bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00299] -07bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00300] -08bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00301] -09bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00302] -10bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00303] -11bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00304] -12bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00305] -13bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00306] -14bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00307] -15bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00308] -16bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00309] -17bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00310] -18bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00311] -19bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00312] -20bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00313] -21bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00314] -22bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00315] -23bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00316] -24bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00317] -25bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00318] -26bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00319] -27bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00320] -28bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00321] -29bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00322] -30bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00323] -31bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00324] -32bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00325] -33bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00326] -34bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00327] -35bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00328] -36bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00329] -37bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00330] -38bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00331] -39bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00332] -40bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00333] -41bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00334] -42bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00335] -43bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00336] -44bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00337] -45bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00338] -46bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00339] -47bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00340] -48bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00341] -49bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00342] -50bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00343] -51bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00344] -52bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00345] -53bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00346] -54bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00347] -55bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00348] -56bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00349] -57bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00350] -58bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00351] -59bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00352] -60bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00353] -61bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00354] -62bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00355] -63bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00356] -64bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00357] -65bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00358] -66bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00359] -67bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00360] -68bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00361] -69bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00362] -70bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00363] -71bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00364] -72bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00365] -73bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00366] -74bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00367] -75bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00368] -76bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00369] -77bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00370] -78bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00371] -79bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00372] -80bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00373] -81bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00374] -82bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00375] -83bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00376] -84bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00377] -85bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00378] -86bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00379] -87bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00380] -88bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00381] -89bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00382] -90bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00383] -91bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00384] -92bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00385] -93bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00386] -94bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00387] -95bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00388] -96bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00389] -97bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00390] -98bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00391] -99bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00392] -100bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00393] -101bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00394] -102bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00395] -103bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00396] -104bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00397] -105bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00398] -106bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00399] -107bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00400] -108bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00401] -109bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00402] -110bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00403] -111bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00404] -112bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00405] -113bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00406] -114bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00407] -115bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00408] -116bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00409] -117bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00410] -118bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00411] -119bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00412] -120bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00413] -121bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00414] -122bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00415] -123bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00416] -124bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00417] -125bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00418] -126bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00419] -127bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00420] -128bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00421] -129bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00422] -130bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00423] -131bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00424] -132bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00425] -133bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00426] -134bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00427] -135bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00428] -136bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00429] -137bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00430] -138bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00431] -139bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00432] -140bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00433] -141bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00434] -142bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00435] +1bp to -142bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00436] +1bp to -141bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00437] +1bp to -140bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00438] +1bp to -139bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00439] +1bp to -138bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00440] +1bp to -137bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00441] +1bp to -136bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00442] +1bp to -135bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00443] +1bp to -134bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00444] +1bp to -133bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00445] +1bp to -132bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00446] +1bp to -131bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00447] +1bp to -130bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00448] +1bp to -129bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00449] +1bp to -128bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00450] +1bp to -127bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00451] +1bp to -126bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00452] +1bp to -125bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00453] +1bp to -124bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00454] +1bp to -123bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00455] +1bp to -122bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00456] +1bp to -121bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00457] +1bp to -120bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00458] +1bp to -119bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00459] +1bp to -118bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00460] +1bp to -117bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00461] +1bp to -116bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00462] +1bp to -115bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00463] +1bp to -114bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00464] +1bp to -113bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00465] +1bp to -112bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00466] +1bp to -111bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00467] +1bp to -110bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00468] +1bp to -109bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00469] +1bp to -108bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00470] +1bp to -107bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00471] +1bp to -106bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00472] +1bp to -105bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00473] +1bp to -104bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00474] +1bp to -103bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00475] +1bp to -102bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00476] +1bp to -101bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00477] +1bp to -100bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00478] +1bp to -99bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00479] +1bp to -98bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00480] +1bp to -97bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00481] +1bp to -96bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00482] +1bp to -95bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00483] +1bp to -94bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00484] +1bp to -93bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00485] +1bp to -92bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00486] +1bp to -91bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00487] +1bp to -90bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00488] +1bp to -89bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00489] +1bp to -88bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00490] +1bp to -87bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00491] +1bp to -86bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00492] +1bp to -85bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00493] +1bp to -84bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00494] +1bp to -83bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00495] +1bp to -82bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00496] +1bp to -81bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00497] +1bp to -80bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00498] +1bp to -79bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00499] +1bp to -78bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00500] +1bp to -77bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00501] +1bp to -76bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00502] +1bp to -75bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00503] +1bp to -74bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00504] +1bp to -73bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00505] +1bp to -72bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00506] +1bp to -71bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00507] +1bp to -70bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00508] +1bp to -69bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00509] +1bp to -68bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00510] +1bp to -67bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00511] +1bp to -66bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00512] +1bp to -65bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00513] +1bp to -64bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00514] +1bp to -63bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00515] +1bp to -62bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00516] +1bp to -61bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00517] +1bp to -60bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00518] +1bp to -59bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00519] +1bp to -58bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00520] +1bp to -57bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00521] +1bp to -56bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00522] +1bp to -55bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00523] +1bp to -54bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00524] +1bp to -53bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00525] +1bp to -52bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00526] +1bp to -51bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00527] +1bp to -50bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00528] +1bp to -49bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00529] +1bp to -48bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00530] +1bp to -47bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00531] +1bp to -46bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00532] +1bp to -45bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00533] +1bp to -44bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00534] +1bp to -43bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00535] +1bp to -42bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00536] +1bp to -41bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00537] +1bp to -40bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00538] +1bp to -39bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00539] +1bp to -38bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00540] +1bp to -37bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00541] +1bp to -36bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00542] +1bp to -35bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00543] +1bp to -34bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00544] +1bp to -33bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00545] +1bp to -32bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00546] +1bp to -31bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00547] +1bp to -30bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00548] +1bp to -29bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00549] +1bp to -28bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00550] +1bp to -27bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00551] +1bp to -26bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00552] +1bp to -25bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00553] +1bp to -24bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00554] +1bp to -23bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00555] +1bp to -22bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00556] +1bp to -21bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00557] +1bp to -20bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00558] +1bp to -19bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00559] +1bp to -18bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00560] +1bp to -17bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00561] +1bp to -16bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00562] +1bp to -15bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00563] +1bp to -14bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00564] +1bp to -13bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00565] +1bp to -12bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00566] +1bp to -11bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00567] +1bp to -10bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00568] +1bp to -9bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00569] +1bp to -8bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00570] +1bp to -7bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00571] +1bp to -6bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00572] +1bp to -5bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00573] +1bp to -4bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00574] +1bp to -3bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00575] +1bp to -2bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00576] +1bp to -1bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00577] FKRP ORF + 3’ UTR + MSEC [00578] G-quadruplex [00579] +1bp to -39bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00580] -40bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00581] Secondary structures [00582] -39bp to -56bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00583] +1bp to -38bp and -57bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00584] Hairpin [00585] -56bp to -102bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00586] +1bp to -55bp and -103bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00587] Secondary structures [00588] -102bp to -123bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00589] +1bp to -101bp and -124bp to -143bp from 5’ UTR end + FKRP ORF + 3’ UTR + MSEC [00590] 3’ UTR truncations [00591] 5’ UTR + FKRP ORF + 1bp to +1600bp at 3’ UTR end + MSEC [00592] 5’ UTR + FKRP ORF + 1bp to +1500bp at 3’ UTR end + MSEC [00593] 5’ UTR + FKRP ORF + 1bp to +1400bp at 3’ UTR end + MSEC [00594] 5’ UTR + FKRP ORF + 1bp to +1300bp at 3’ UTR end + MSEC [00595] 5’ UTR + FKRP ORF + 1bp to +1200bp at 3’ UTR end + MSEC [00596] 5’ UTR + FKRP ORF + 1bp to +1100bp at 3’ UTR end + MSEC [00597] 5’ UTR + FKRP ORF + 1bp to +1000bp at 3’ UTR end + MSEC [00598] 5’ UTR + FKRP ORF + 1bp to +900bp at 3’ UTR end + MSEC [00599] 5’ UTR + FKRP ORF + 1bp to +800bp at 3’ UTR end + MSEC [00600] 5’ UTR + FKRP ORF + 1bp to +700bp at 3’ UTR end + MSEC [00601] 5’ UTR + FKRP ORF + 1bp to +600bp at 3’ UTR end + MSEC [00602] 5’ UTR + FKRP ORF + 1bp to +500bp at 3’ UTR end + MSEC [00603] 5’ UTR + FKRP ORF + 1bp to +400bp at 3’ UTR end + MSEC [00604] 5’ UTR + FKRP ORF + 1bp to +300bp at 3’ UTR end + MSEC [00605] 5’ UTR + FKRP ORF + 1bp to +200bp at 3’ UTR end + MSEC [00606] 5’ UTR + FKRP ORF + 1bp to +100bp at 3’ UTR end + MSEC [00607] 5’ UTR + FKRP ORF + 1bp to +01bp at 3’ UTR end + MSEC [00608] 5’ UTR + FKRP ORF + MSEC [00609] 5’ UTR + FKRP ORF + 01bp to +1600bp at 3’ UTR end + MSEC [00610] 5’ UTR + FKRP ORF + 100bp to +1600bp at 3’ UTR end + MSEC [00611] 5’ UTR + FKRP ORF + 200bp to +1600bp at 3’ UTR end + MSEC [00612] 5’ UTR + FKRP ORF + 300bp to +1600bp at 3’ UTR end + MSEC [00613] 5’ UTR + FKRP ORF + 400bp to+1600bp at 3’ UTR end + MSEC [00614] 5’ UTR + FKRP ORF + 500bp to+1600bp at 3’ UTR end + MSEC [00615] 5’ UTR + FKRP ORF + 600bp to+1600bp at 3’ UTR end + MSEC [00616] 5’ UTR + FKRP ORF + 700bp to+1600bp at 3’ UTR end + MSEC [00617] 5’ UTR + FKRP ORF + 800bp to+1600bp at 3’ UTR end + MSEC [00618] 5’ UTR + FKRP ORF + 900bp to+1600bp at 3’ UTR end + MSEC [00619] 5’ UTR + FKRP ORF + 1000bp to+1600bp at 3’ UTR end + MSEC [00620] 5’ UTR + FKRP ORF + 1100bp to+1600bp at 3’ UTR end + MSEC [00621] 5’ UTR + FKRP ORF + 1200bp to+1600bp at 3’ UTR end + MSEC [00622] 5’ UTR + FKRP ORF + 1300bp to+1600bp at 3’ UTR end + MSEC [00623] 5’ UTR + FKRP ORF + 1400bp to+1600bp at 3’ UTR end + MSEC [00624] 5’ UTR + FKRP ORF + 1500bp to+1600bp at 3’ UTR end + MSEC [00625] 5’ UTR + FKRP ORF + 1600bp to+1600bp at 3’ UTR end + MSEC [00626] FKRP ORF + MSEC [00627] REFERENCES: 1. 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Curr Top Dev Biol.2008;84:431-53. doi:10.1016/S0070-2153(08)00609-1 [00628] SEQ ID NO.1 is a human FKRP cDNA including 5’ and 3’ UTRs: attgctccaagatggcggcggcggcggcagcgggagcgcagctcagctgggctggaactgccctcctggaactcccccagcctac aacctaggaggtgcagggactgaggctcaggccaaatcgcaactcagacccagtgaacccaaggcctgaagagaatttggattcatt taccttgttttgtggggactggagagacaagtaaactctcagagtaactgtcccctctgactaccatttctaaggcaagccccctgtttcta ctcttgcgccccctgctggtttcctgccctgtctgatgccccggaggcccagctagccccagacttcggccccATGCGGCTCA CCCGCTGCCAGGCTGCCCTGGCGGCCGCCATCACCCTCAACCTTCTGGTCCTCTT CTATGTCTCGTGGCTGCAGCACCAGCCTAGGAATTCCCGGGCCCGGGGGCCCCGT CGTGCCTCTGCTGCCGGCCCCCGTGTCACCGTCCTGGTGCGGGAGTTCGAGGCAT TTGACAACGCGGTGCCCGAGCTGGTAGACTCCTTCCTGCAGCAAGACCCAGCCC AGCCCGTGGTGGTGGCAGCCGACACGCTCCCCTACCCGCCCCTGGCCCTGCCCCG CATCCCCAACGTGCGTCTGGCGCTGCTCCAGCCCGCCCTGGACCGGCCAGCCGCA GCCTCGCGCCCGGAGACCTACGTGGCCACCGAGTTTGTGGCCCTAGTACCTGATG GGGCGCGGGCTGAGGCACCTGGCCTGCTGGAGCGCATGGTGGAGGCGCTCCGCG CAGGAAGCGCACGTCTGGTGGCCGCCCCGGTTGCCACGGCCAACCCTGCCAGGT GCCTGGCCCTGAACGTCAGCCTGCGAGAGTGGACCGCCCGCTATGGCGCAGCCC CCGCCGCGCCCCGCTGCGACGCCCTGGACGGAGATGCTGTGGTGCTCCTGCGCGC CCGCGACCTCTTCAACCTCTCGGCGCCCCTGGCCCGGCCGGTGGGCACCAGCCTC TTTCTGCAGACCGCCCTTCGCGGCTGGGCGGTGCAGCTGCTGGACTTGACCTTCG CCGCGGCGCGCCAGCCCCCGCTGGCCACGGCCCACGCGCGCTGGAAGGCTGAGC GCGAGGGACGCGCTCGGCGGGCGGCGCTGCTCCGCGCGCTGGGCATCCGCCTAG TGAGCTGGGAAGGCGGGCGGCTGGAGTGGTTCGGCTGCAACAAGGAGACCACGC GCTGCTTCGGAACCGTGGTGGGCGACACGCCCGCCTACCTCTACGAGGAGCGCT GGACGCCCCCCTGCTGCCTGCGCGCGCTGCGCGAGACCGCCCGCTATGTGGTGG GCGTGCTGGAGGCTGCGGGCGTGCGCTACTGGCTCGAGGGCGGCTCACTGCTGG GGGCCGCCCGCCACGGGGACATCATCCCATGGGACTACGACGTGGACCTGGGCA TCTACTTGGAGGACGTGGGCAACTGCGAGCAGCTGCGGGGGGCAGAGGCCGGCT CGGTGGTGGATGAGCGCGGCTTCGTATGGGAGAAGGCGGTCGAGGGCGACTTTT TCCGCGTGCAGTACAGCGAAAGCAACCACTTGCACGTGGACCTGTGGCCCTTCTA CCCCCGCAATGGCGTCATGACCAAGGACACGTGGCTGGACCACCGGCAGGATGT GGAGTTTCCCGAGCACTTCCTGCAGCCGCTGGTGCCCCTGCCCTTTGCCGGCTTC GTGGCGCAGGCGCCTAACAACTACCGCCGCTTCCTGGAGCTCAAGTTCGGGCCC GGGGTCATCGAGAACCCCCAGTACCCCAACCCGGCACTGCTGAGTCTGACGGGA AGCGGCTGAagccctgataacctcgcctttgtttttcgggggtctgtctggatgtggagaagctctgtgtgagcggtgaggggt ggagggatgtcgcggagaggggaagggggaaactgaccaagaaagaaattctaaggagagcatgagagaaggctggcattggca ggaggagagcaccaggacgaggatgggaagcgacctccagatttatcaaatggtcatgcccactgggagccgtggatatgcgtgg ggacatcctgggtcatctcagtcatggagggagacggggatgtcacgccgtcccgcagggcccagcacagccccagacccgaaaa aagtgttctgcccaagattccgagagccctgcgctctagggcaggggcagagttttggaaacagtgcaggctctggagccagactgg cgagattcaaatcctggctctatcgcttcggagccaggtgggcctgggggggcgtcgcagtctctctgtgcctcagttgcttccaggat gcgggacccttggctgcaggggttgcttccgccactagagggcgcgccggtcccgctcctggtggcccactgtggctgcccgggcg acagtacgcccagggcctgtgttccatagccatctactctcttgagcctttggacttctctccaagcccctgtgggaggcggacagcagt gaccacctccccttcttttggactgcgacctccttccctcctgggagagccctgtgacctgcatgctactcttaactgttctattcaagactg aatagaagtatttcagtcttgcagaggaggaaatgctcagagctccgaggtgcggctgtggtcgagaaccgggtgctgggccgggc gcgggggctcacgcctgtaatcccagcactttgggaggccgaggtgggaggatcgcttgagcccaggagtctgagaccagcctcg gcaacatgccaagaccccgtctctatttttaaaaaagaaaaagaaccgacttctgaatcgcagctccactcatgactaatacctcattattt cagctgtctgcacctaattccccacttgcacggcagtgtagacaataaccatagctcacactcactgagcacctactgggtaccaggca ccattctcagtgtttcacctggatcaactaatgcgtccctcacctcagccctctgaagtgacagctgctattattttcattacacagatgaaa aagctgaggccagaatcgtgaagtcacttgctcaaggtcaggcagcttaggaaggggcagatcgggggcttgaacccaggtggtca ggctctggagcccacaattgtcttacccactatgcccctctctagtcatggtccccaagaggggcttggagacccacttagcaggtgaa agcaatggcagccttccttatttgattatgcacctaagaataaatggtatttgggcatgtattcccaatatgtgtatatttatttataaatatata cagatactattatctgtatgttagtaataaagcttaaattattccattttaaaattatgaatatgaatagggttttttttatgtttcttgcctcatccc aatgacttttgcacacccaggtgtgagcacccagcattcaagaccacg [00629] SEQ ID NO.2 is a murine FKRP cDNA including 5’ and 3’ UTRs: ATTGCTCCAAGATGGCGGCGGCGGCGGCGGCGGCAGGGCAGATGACCCAAGGTG AAACCACTCTTCTTGATCACCCTCAGCCTGAAACCTAGGGAGGATGCCCTGGACG CCCCAGCTAGGGTCTGACATCAGGCCCCATGCGGCTCACCCGCTGCTGGGCTGCC CTGGCAGCCGCCATCATCCTCAACCTCCTAGTCTTCTTCTATGTGTCATGGCTACA ACACCAGCCCAGAAACTCCCGGGCCCGGGGTCCCCGCCGGACTTCTGCCATTGG CCCCCGAGTCACCGTCCTGATTCGGGAATTTGAGGCTTTTGACAACGCGGTGCCA GAGCTAGTGGATTCCTTTTTGCAGCAGGACCCAGCCCAGCCCGTGGTAGTGGCGG CCGACACACTCCCTTACCCACCCCTGGCCTTGCCTCGCATCCCCAACGTTCGCCT GGCTCTGCTCCAGCCAGCCCTGGACCGGCCAGCGGCGGCCTCGCGCCCGGAGAC CTACGTAGCCACCGAGTTTGTGGCTCTAGTGCCTGATGGAGCGCGGGCCGAGTCA CCAGGCCACCTGGAGCGAATGGTGGAGGCGCTCCGAGGGAGCAGCGCGCGCCTA GTGGCCGCCCCGGTCGCCACCGCCAACCCAGCGCGGTGCCTAGCTCTGAACGTC AGCCTGCGGGAGTGGACTGCGCGCTACGACCCAGCTCCCAGCGCGCCCCGCTGC GACGCTTTGGATGGCGACGCTGTGCTGCTGATGCGCTCCCGCGACCTCTTCAACC TCTCGGTGCCCCTGGCGCGGCCGCTGGCCACCAGCCTCTTCCTACAGACCGCCCT GCGCGGCTGGGCAGTGCAGCTGCTGGACTTGACCTTCGCCGCGGCGCGCCAGCC ACCGCTGGCCACCGCCCACGCGCGCTGGAAGGCGGAACGTGAGGGGCGCTCACG GAGAGCGGCGCTGCTGCGCTCGTTGGGAATCCGTCTGGTGAGCTGGGAAGGCGG GCGGCTAGAGTGGTTTGGCTGCAGCAAGGAGAGCGCGCGCTGCTTCGGTACGGT GGCGGGCGACACACCCGCCTACCTGTATGAGGGCCGCTGGACCCCACCTTGTTGC CTGCGCGCGCTGCGCGAGACTGCGCGCTACGTGGTGGGCGTGCTGGAGGCGGCG GGCGTGCGCTACTGGTTGGAGGGCGGCTCGCTGCTGGGTGCAGCTCGCCACGGC GACATCATCCCTTGGGACTACGACGTAGATCTGGGCATCTACCTGGAGGACGTG GGCAACTGCGAGCAGTTGCGGGGTGCCGAAGCTGGCTCGGTAGTGGATGAACGC GGCTTTGTGTGGGAGAAGGCGGTGGAGGGCGACTTCTTCCGAGTACAGTACAGT GAGAACAACCACCTGCACGTGGACCTGTGGCCCTTTTACCCCCGCAATGGGGTTA TGACCAAGGACACGTGGCTGGACCACCGGCAGGATGTTGAGTTCCCAGAGCACT TCCTGCAGCCACTTGTCCCCCTGCCCTTTGCGGGTTTCATGGCACAGGCCCCTAA CAACTACCGCCGCTTCCTGGAGCTGAAGTTTGGGCCTGGGGTCATCGAGAACCCG GAGTACCCCAACCCCGCACTCTTAAGCTTGACAGGCGGTTGAAGCCCTGGTACCC ACATCTGGGGCTAGGTGAACAGCAGGGTACAAGAGTCTGTTCTTGCCTCGGATTG TGTATGGATCTGAACCAGTTTGTGATTGGGTAGTGGGGCTGTGCACTGGAGGGAA GAAAAAAAAAGGGTGAACTGGCCAAGGAAGATTGGAGGGAACACACAGGACAG TTTCAGCTTTGGCGGGAAGCAGAACCCAAAGACTGGAGCAACTCAGCCTCATGA GGGAGGAAAGTGGTGGCCTTTGATAGAAAAGCCCAGGGAGTCTTTAGGGCTCTG CATAGTCTCTGCCCTATATTCCAAGAGCTGTAGGCCCTTGGAGAGTGAGATCCGG AGAACAGACGAGCCAGCCTCAGTCTACCACATACAACTTAGGTATCCCTGGACC AGTTAGTTACCCAGTCTGTTTCATTTGAGATACCGAATGCCTCCCTTTGTGTTTAG GATCCTCAGGACGCTTGGCTCCAGTGCTTGCTTCCTCCACTAGAGGGCACTTCTG TCCCGCTTCAGTTCGTGTTTGATGACCGCTCGCGGCTCTGCTTCATCACCTTCATC TCTGGTTTTCTTGGAGTGGCTCTGGGAGGTGGGACAGCACTGGCCACCTCCTCTC CTCCCCGAATGTGGCACTTTCCTTTGAGAGTGCTGCTTGATGTATCTGCTGCTTCT AACCCCATCTTGCTCTTGGAGGACTGACTGGGCTCTCTCTGGTTTGGGATCTGGC CAATGCATTACCTCTTTATTTCAGCCCTCTGCACCTGTTCCCCGCCTTCCCCCTCC CTTCTAGGAAGGGATAAACCTTCGCCATAGCTCACATTTACAAAGAGCTTTCGGG CTCAGGCATACACTCGGAAGTGACAGCTGCTATTACGTCCATTTTCCGGGGAGGA AAACTGAGGCCAAAATGACAGTCACTTGCCAGTAATCAGATATTTTAGGAAGTG GCTAGTAAGGGACTTGAATTCAGATCACCGTTTCAAGGCCCACACTTCATTTCCT CTCTAGGTTCTGCTGTAGAAATAGTACCCAAGTGGAGCCTTGCCATGGCTGTGCC ACTCAAACAGGTTTAAAAAAAAATTCTCATTTAATTATGCATATAAATTATGTTC AAACCAGTCTGTATATTGTTAAATAAGACTTAAATTATGCTACTTTAAAGTTTAA AAATTCAAGACAAATAGAAAAAAAAAAAA [00630] SEQ ID NO.3 is a murine FKRP 5’ untranslated sequence: ATTGCTCCAAGATGGCGGCGGCGGCGGCGGCGGGGCAGATGACCCAAGGTGAA ACCACTCTTCTTGATCACCCTCAGCCTGAAACCTAGGGAGGATGCCCTGGACGCC CCAGCTAGGGTCTGACATCAGGCCCC [00631] SEQ ID NO.4 is a murine FKRP 3’ untranslated sequence: AGCCCTGGTACCCACATCTGGGGCTAGGTGAACAGCAGGGTACAAGAGTCTGTT CTTGCCTCGGATTGTGTATGGATCTGAACCAGTTTGTGATTGGGTAGTGGGGCTG TGCACTGGAGGGAAGAAAAAAAAAGGGTGAACTGGCCAAGGAAGATTGGAGGG AACACACAGGACAGTTTCAGCTTTGGCGGGAAGCAGAACCCAAAGACTGGAGCA ACTCAGCCTCATGAGGGAGGAAAGTGGTGGCCTTTGATAGAAAAGCCCAGGGAG TCTTTAGGGCTCTGCATAGTCTCTGCCCTATATTCCAAGAGCTGTAGGCCCTTGG AGAGTGAGATCCGGAGAACAGACGAGCCAGCCTCAGTCTACCACATACAACTTA GGTATCCCTGGACCAGTTAGTTACCCAGTCTGTTTCATTTGAGATACCGAATGCC TCCCTTTGTGTTTAGGATCCTCAGGACGCTTGGCTCCAGTGCTTGCTTCCTCCACT AGAGGGCACTTCTGTCCCGCTTCAGTTCGTGTTTGATGACCGCTCGCGGCTCTGC TTCATCACCTTCATCTCTGGTTTTCTTGGAGTGGCTCTGGGAGGTGGGACAGCAC TGGCCACCTCCTCTCCTCCCCGAATGTGGCACTTTCCTTTGAGAGTGCTGCTTGAT GTATCTGCTGCTTCTAACCCCATCTTGCTCTTGGAGGACTGACTGGGCTCTCTCTG GTTTGGGATCTGGCCAATGCATTACCTCTTTATTTCAGCCCTCTGCACCTGTTCCC CGCCTTCCCCCTCCCTTCTAGGAAGGGATAAACCTTCGCCATAGCTCACATTTAC AAAGAGCTTTCGGGCTCAGGCATACACTCGGAAGTGACAGCTGCTATTACGTCC ATTTTCCGGGGAGGAAAACTGAGGCCAAAATGACAGTCACTTGCCAGTAATCAG ATATTTTAGGAAGTGGCTAGTAAGGGACTTGAATTCAGATCACCGTTTCAAGGCC CACACTTCATTTCCTCTCTAGGTTCTGCTGTAGAAATAGTACCCAAGTGGAGCCT TGCCATGGCTGTGCCACTCAAACAGGTTTAAAAAAAAATTCTCATTTAATTATGC ATATAAATTATGTTCAAACCAGTCTGTATATTGTTAAATAAGACTTAAATTATGC TACTTTAAAGTTTAAAAATTCAAGACAAATAGAAAAAAAAAAAA [00632] SEQ ID NO.6 is a murine FKRP cDNA sequence containing a mutated Kozak sequence: ATTGCTCCAAGATGGCGGCGGCGGCGGCAGCGGGAGCGCAGCTCAGCTGGGCTG GAACTGCCCTCCTGGAACTCCCCCAGCCTACAACCTAGGAGGTGCAGGGACTGA GGCTCAGGCCAAATCGCAACTCAGACCCAGTGAACCCAAGGCCTGAAGAGAATT TGGATTCATTTACCTTGTTTTGTGGGGACTGGAGAGACAAGTAAACTCTCAGAGT AACTGTCCCCTCTGACTACCATTTCTAAGGATGCCCCGGAGGCCCAGCTAGCCCC AGACTTCTCCACCATGCGGCTCACCCGCTGCCAGGCTGCCCTGGCGGCCGCCATC ACCCTCAACCTTCTGGTCCTCTTCTATGTCTCGTGGCT... [00633] SEQ ID NO.7 is a murine FKRP cDNA sequence lacking the 5’ UTR: ...AGACTCAGCACTTAGTTTAGGAACCAGTGAGCAAGTCAGCCCTTGGGGCAGCC CATACAAGGCCATGGGGCTGGGCAAGCTGCACGCCTGGGTCCGGGGTGGGCACG GTGCCCGGGCAACGAGCTGAAAGCTCATCTGCTCTCAGGGGCCCCTCCCTGGGG ACAGCCCCTCCTGGCTAGTCACACCCTGTAGGCTCCTCTATATAACCCAGGGGCA CAGGGGCTGCCCTCATTCTACCACCACCTCCACAGCACAGACAGACACTCAGGA GCCAGCCAGCGTCGAGATGCGGCTCACCCGCTGCCAGGCTGCCCTGGCGGCCGC CATCACCCTCAACCTTCTGGTCCTCTTCTATGTCTCGTGGCT… [00634] SEQ ID NO.8 is the CK8e promoter: TGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTA ACCCAGACATGTGGCTGCCCCCCCCCCCCCAACACCTGCTGCCTCTAAAAATAAC CCTGCATGCCATGTTCCCGGCGAAGGGCCAGCTGTCCCCCGCCAGCTAGACTCAG CACTTAGTTTAGGAACCAGTGAGCAAGTCAGCCCTTGGGGCAGCCCATACAAGG CCATGGGGCTGGGCAAGCTGCACGCCTGGGTCCGGGGTGGGCACGGTGCCCGGG CAACGAGCTGAAAGCTCATCTGCTCTCAGGGGCCCCTCCCTGGGGACAGCCCCTC CTGGCTAGTCACACCCTGTAGGCTCCTCTATATAACCCAGGGGCACAGGGGCTGC CCTCATTCTACCACCACCTCCACAGCACAGACAGACACTCAGGAGCCAGCCAGC [00635] SEQ ID NO.9 is RNA G-quadruplex in exon 1 of human FKRP: attgctccaagatggcggcggcggcggcagcg [00636] SEQ ID NO 10 is RNA G-quadruplex in exon 1 of murine FKRP: tgtacaattgctccaagatggcggcggcggcggcggcggcag [00637] SEQ ID NO 11 is human FKRP mRNA transcript variant 2 mRNA, NM_001039885.3 CATTGCTCCAAGATGGCGGCGGCGGCGGCAGCGGGAGCGCAGCTCAGCTGGGCT GGAACTGCCCTCCTGGAACTCCCCCAGCCTACAACCTAGGAGGTGCAGGGACTG AGGCTCAGGCCAAATCGCAACTCAGACCCAGTGAACCCAAGGCCTGAAGAGAAT TTGGATTCATTTACCTTGTTTTGTGGGGACTGGAGAGACAAGTAAACTCTCAGAG TAACTGTCCCCTCTGACTACCATTTCTAAGGCAAGCCCCCTGTTTCTACTCTTGCG CCCCCTGCTGGTTTCCTGCCCTGTCTGATGCCCCGGAGGCCCAGCTAGCCCCAGA CTTCGGCCCCATGCGGCTCACCCGCTGCCAGGCTGCCCTGGCGGCCGCCATCACC CTCAACCTTCTGGTCCTCTTCTATGTCTCGTGGCTGCAGCACCAGCCTAGGAATTC CCGGGCCCGGGGGCCCCGTCGTGCCTCTGCTGCCGGCCCCCGTGTCACCGTCCTG GTGCGGGAGTTCGAGGCATTTGACAACGCGGTGCCCGAGCTGGTAGACTCCTTCC TGCAGCAAGACCCAGCCCAGCCCGTGGTGGTGGCAGCCGACACGCTCCCCTACC CGCCCCTGGCCCTGCCCCGCATCCCCAACGTGCGTCTGGCGCTGCTCCAGCCCGC CCTGGACCGGCCAGCCGCAGCCTCGCGCCCGGAGACCTACGTGGCCACCGAGTT TGTGGCCCTAGTACCTGATGGGGCGCGGGCTGAGGCACCTGGCCTGCTGGAGCG CATGGTGGAGGCGCTCCGCGCAGGAAGCGCACGTCTGGTGGCCGCCCCGGTTGC CACGGCCAACCCTGCCAGGTGCCTGGCCCTGAACGTCAGCCTGCGAGAGTGGAC CGCCCGCTATGGCGCAGCCCCCGCCGCGCCCCGCTGCGACGCCCTGGACGGAGA TGCTGTGGTGCTCCTGCGCGCCCGCGACCTCTTCAACCTCTCGGCGCCCCTGGCC CGGCCGGTGGGCACCAGCCTCTTTCTGCAGACCGCCCTTCGCGGCTGGGCGGTGC AGCTGCTGGACTTGACCTTCGCCGCGGCGCGCCAGCCCCCGCTGGCCACGGCCCA CGCGCGCTGGAAGGCTGAGCGCGAGGGACGCGCTCGGCGGGCGGCGCTGCTCCG CGCGCTGGGCATCCGCCTAGTGAGCTGGGAAGGCGGGCGGCTGGAGTGGTTCGG CTGCAACAAGGAGACCACGCGCTGCTTCGGAACCGTGGTGGGCGACACGCCCGC CTACCTCTACGAGGAGCGCTGGACGCCCCCCTGCTGCCTGCGCGCGCTGCGCGAG ACCGCCCGCTATGTGGTGGGCGTGCTGGAGGCTGCGGGCGTGCGCTACTGGCTC GAGGGCGGCTCACTGCTGGGGGCCGCCCGCCACGGGGACATCATCCCATGGGAC TACGACGTGGACCTGGGCATCTACTTGGAGGACGTGGGCAACTGCGAGCAGCTG CGGGGGGCAGAGGCCGGCTCGGTGGTGGATGAGCGCGGCTTCGTATGGGAGAAG GCGGTCGAGGGCGACTTTTTCCGCGTGCAGTACAGCGAAAGCAACCACTTGCAC GTGGACCTGTGGCCCTTCTACCCCCGCAATGGCGTCATGACCAAGGACACGTGGC TGGACCACCGGCAGGATGTGGAGTTTCCCGAGCACTTCCTGCAGCCGCTGGTGCC CCTGCCCTTTGCCGGCTTCGTGGCGCAGGCGCCTAACAACTACCGCCGCTTCCTG GAGCTCAAGTTCGGGCCCGGGGTCATCGAGAACCCCCAGTACCCCAACCCGGCA CTGCTGAGTCTGACGGGAAGCGGCTGAAGCCCTGATAACCTCGCCTTTGTTTTTC GGGGGTCTGTCTGGATGTGGAGAAGCTCTGTGTGAGCGGTGAGGGGTGGAGGGA TGTCGCGGAGAGGGGAAGGGGGAAACTGACCAAGAAAGAAATTCTAAGGAGAG CATGAGAGAAGGCTGGCATTGGCAGGAGGAGAGCACCAGGACGAGGATGGGAA GCGACCTCCAGATTTATCAAATGGTCATGCCCACTGGGAGCCGTGGATATGCGTG GGGACATCCTGGGTCATCTCAGTCATGGAGGGAGACGGGGATGTCACGCCGTCC CGCAGGGCCCAGCACAGCCCCAGACCCGAAAAAAGTGTTCTGCCCAAGATTCCG AGAGCCCTGCGCTCTAGGGCAGGGGCAGAGTTTTGGAAACAGTGCAGGCTCTGG AGCCAGACTGGCGAGATTCAAATCCTGGCTCTATCGCTTCGGAGCCAGGTGGGC CTGGGGGGGCGTCGCAGTCTCTCTGTGCCTCAGTTGCTTCCAGGATGCGGGACCC TTGGCTGCAGGGGTTGCTTCCGCCACTAGAGGGCGCGCCGGTCCCGCTCCTGGTG GCCCACTGTGGCTGCCCGGGCGACAGTACGCCCAGGGCCTGTGTTCCATAGCCAT CTACTCTCTTGAGCCTTTGGACTTCTCTCCAAGCCCCTGTGGGAGGCGGACAGCA GTGACCACCTCCCCTTCTTTTGGACTGCGACCTCCTTCCCTCCTGGGAGAGCCCTG TGACCTGCATGCTACTCTTAACTGTTCTATTCAAGACTGAATAGAAGTATTTCAGT CTTGCAGAGGAGGAAATGCTCAGAGCTCCGAGGTGCGGCTGTGGTCGAGAACCG GGTGCTGGGCCGGGCGCGGGGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGC CGAGGTGGGAGGATCGCTTGAGCCCAGGAGTCTGAGACCAGCCTCGGCAACATG CCAAGACCCCGTCTCTATTTTTAAAAAAGAAAAAGAACCGACTTCTGAATCGCA GCTCCACTCATGACTAATACCTCATTATTTCAGCTGTCTGCACCTAATTCCCCACT TGCACGGCAGTGTAGACAATAACCATAGCTCACACTCACTGAGCACCTACTGGG TACCAGGCACCATTCTCAGTGTTTCACCTGGATCAACTAATGCGTCCCTCACCTC AGCCCTCTGAAGTGACAGCTGCTATTATTTTCATTACACAGATGAAAAAGCTGAG GCCAGAATCGTGAAGTCACTTGCTCAAGGTCAGGCAGCTTAGGAAGGGGCAGAT CGGGGGCTTGAACCCAGGTGGTCAGGCTCTGGAGCCCACAATTGTCTTACCCACT ATGCCCCTCTCTAGTCATGGTCCCCAAGAGGGGCTTGGAGACCCACTTAGCAGGT GAAAGCAATGGCAGCCTTCCTTATTTGATTATGCACCTAAGAATAAATGGTATTT GGGCATGTATTCCCAATATGTGTATATTTATTTATAAATATATACAGATACTATTA TCTGTATGTTAGTAATAAAGCTTAAATTATTCCATTTTAAAATTATGAATATGAAT AGGGTTTTTTTTATGTTTCTTGCCTCATCCCAATGACTTTTGCACACCCAGGTGTG AGCACCCAGCATTCAAGACCACG [00638] SEQ ID NO.12 is FKRP mRNA transcript variant 1 mRNA, NM_024301.5 CATTGCTCCAAGATGGCGGCGGCGGCGGCAGCGGGAGCGCAGCTCAGCTGGGCT GGAACTGCCCTCCTGGAACTCCCCCAGCCTACAACCTAGGAGGTGCAGGGACTG AGGCTCAGGCCAAATCGCAACTCAGACCCAGTGAACCCAAGGCCTGAAGAGAAT TTGGATTCATTTACCTTGTTTTGTGGGGACTGGAGAGACAAGTAAACTCTCAGAG TAACTGTCCCCTCTGACTACCATTTCTAAGGATGCCCCGGAGGCCCAGCTAGCCC CAGACTTCGGCCCCATGCGGCTCACCCGCTGCCAGGCTGCCCTGGCGGCCGCCAT CACCCTCAACCTTCTGGTCCTCTTCTATGTCTCGTGGCTGCAGCACCAGCCTAGG AATTCCCGGGCCCGGGGGCCCCGTCGTGCCTCTGCTGCCGGCCCCCGTGTCACCG TCCTGGTGCGGGAGTTCGAGGCATTTGACAACGCGGTGCCCGAGCTGGTAGACT CCTTCCTGCAGCAAGACCCAGCCCAGCCCGTGGTGGTGGCAGCCGACACGCTCC CCTACCCGCCCCTGGCCCTGCCCCGCATCCCCAACGTGCGTCTGGCGCTGCTCCA GCCCGCCCTGGACCGGCCAGCCGCAGCCTCGCGCCCGGAGACCTACGTGGCCAC CGAGTTTGTGGCCCTAGTACCTGATGGGGCGCGGGCTGAGGCACCTGGCCTGCTG GAGCGCATGGTGGAGGCGCTCCGCGCAGGAAGCGCACGTCTGGTGGCCGCCCCG GTTGCCACGGCCAACCCTGCCAGGTGCCTGGCCCTGAACGTCAGCCTGCGAGAG TGGACCGCCCGCTATGGCGCAGCCCCCGCCGCGCCCCGCTGCGACGCCCTGGAC GGAGATGCTGTGGTGCTCCTGCGCGCCCGCGACCTCTTCAACCTCTCGGCGCCCC TGGCCCGGCCGGTGGGCACCAGCCTCTTTCTGCAGACCGCCCTTCGCGGCTGGGC GGTGCAGCTGCTGGACTTGACCTTCGCCGCGGCGCGCCAGCCCCCGCTGGCCACG GCCCACGCGCGCTGGAAGGCTGAGCGCGAGGGACGCGCTCGGCGGGCGGCGCTG CTCCGCGCGCTGGGCATCCGCCTAGTGAGCTGGGAAGGCGGGCGGCTGGAGTGG TTCGGCTGCAACAAGGAGACCACGCGCTGCTTCGGAACCGTGGTGGGCGACACG CCCGCCTACCTCTACGAGGAGCGCTGGACGCCCCCCTGCTGCCTGCGCGCGCTGC GCGAGACCGCCCGCTATGTGGTGGGCGTGCTGGAGGCTGCGGGCGTGCGCTACT GGCTCGAGGGCGGCTCACTGCTGGGGGCCGCCCGCCACGGGGACATCATCCCAT GGGACTACGACGTGGACCTGGGCATCTACTTGGAGGACGTGGGCAACTGCGAGC AGCTGCGGGGGGCAGAGGCCGGCTCGGTGGTGGATGAGCGCGGCTTCGTATGGG AGAAGGCGGTCGAGGGCGACTTTTTCCGCGTGCAGTACAGCGAAAGCAACCACT TGCACGTGGACCTGTGGCCCTTCTACCCCCGCAATGGCGTCATGACCAAGGACAC GTGGCTGGACCACCGGCAGGATGTGGAGTTTCCCGAGCACTTCCTGCAGCCGCTG GTGCCCCTGCCCTTTGCCGGCTTCGTGGCGCAGGCGCCTAACAACTACCGCCGCT TCCTGGAGCTCAAGTTCGGGCCCGGGGTCATCGAGAACCCCCAGTACCCCAACC CGGCACTGCTGAGTCTGACGGGAAGCGGCTGAAGCCCTGATAACCTCGCCTTTGT TTTTCGGGGGTCTGTCTGGATGTGGAGAAGCTCTGTGTGAGCGGTGAGGGGTGGA GGGATGTCGCGGAGAGGGGAAGGGGGAAACTGACCAAGAAAGAAATTCTAAGG AGAGCATGAGAGAAGGCTGGCATTGGCAGGAGGAGAGCACCAGGACGAGGATG GGAAGCGACCTCCAGATTTATCAAATGGTCATGCCCACTGGGAGCCGTGGATAT GCGTGGGGACATCCTGGGTCATCTCAGTCATGGAGGGAGACGGGGATGTCACGC CGTCCCGCAGGGCCCAGCACAGCCCCAGACCCGAAAAAAGTGTTCTGCCCAAGA TTCCGAGAGCCCTGCGCTCTAGGGCAGGGGCAGAGTTTTGGAAACAGTGCAGGC TCTGGAGCCAGACTGGCGAGATTCAAATCCTGGCTCTATCGCTTCGGAGCCAGGT GGGCCTGGGGGGGCGTCGCAGTCTCTCTGTGCCTCAGTTGCTTCCAGGATGCGGG ACCCTTGGCTGCAGGGGTTGCTTCCGCCACTAGAGGGCGCGCCGGTCCCGCTCCT GGTGGCCCACTGTGGCTGCCCGGGCGACAGTACGCCCAGGGCCTGTGTTCCATA GCCATCTACTCTCTTGAGCCTTTGGACTTCTCTCCAAGCCCCTGTGGGAGGCGGA CAGCAGTGACCACCTCCCCTTCTTTTGGACTGCGACCTCCTTCCCTCCTGGGAGA GCCCTGTGACCTGCATGCTACTCTTAACTGTTCTATTCAAGACTGAATAGAAGTA TTTCAGTCTTGCAGAGGAGGAAATGCTCAGAGCTCCGAGGTGCGGCTGTGGTCG AGAACCGGGTGCTGGGCCGGGCGCGGGGGCTCACGCCTGTAATCCCAGCACTTT GGGAGGCCGAGGTGGGAGGATCGCTTGAGCCCAGGAGTCTGAGACCAGCCTCGG CAACATGCCAAGACCCCGTCTCTATTTTTAAAAAAGAAAAAGAACCGACTTCTG AATCGCAGCTCCACTCATGACTAATACCTCATTATTTCAGCTGTCTGCACCTAATT CCCCACTTGCACGGCAGTGTAGACAATAACCATAGCTCACACTCACTGAGCACCT ACTGGGTACCAGGCACCATTCTCAGTGTTTCACCTGGATCAACTAATGCGTCCCT CACCTCAGCCCTCTGAAGTGACAGCTGCTATTATTTTCATTACACAGATGAAAAA GCTGAGGCCAGAATCGTGAAGTCACTTGCTCAAGGTCAGGCAGCTTAGGAAGGG GCAGATCGGGGGCTTGAACCCAGGTGGTCAGGCTCTGGAGCCCACAATTGTCTT ACCCACTATGCCCCTCTCTAGTCATGGTCCCCAAGAGGGGCTTGGAGACCCACTT AGCAGGTGAAAGCAATGGCAGCCTTCCTTATTTGATTATGCACCTAAGAATAAAT GGTATTTGGGCATGTATTCCCAATATGTGTATATTTATTTATAAATATATACAGAT ACTATTATCTGTATGTTAGTAATAAAGCTTAAATTATTCCATTTTAAAATTATGAA TATGAATAGGGTTTTTTTTATGTTTCTTGCCTCATCCCAATGACTTTTGCACACCC AGGTGTGAGCACCCAGCATTCAAGACCACG [00639] SEQ ID NO.13 is FKRP, transcript variant X10, mRNA, XM_047439426.1 GGGAAAGGTGGGCTGCTACAAGCAGGAGCGCAGCTCAGCTGGGCTGGAACTGCC CTCCTGGAACTCCCCCAGCCTACAACCTAGGAGGTGCAGGGACTGAGGCTCAGG CCAAATCGCAACTCAGACCCAGTGAACCCAAGGCCTGAAGAGAATTTGGATTCA TTTACCTTGTTTTGTGGGGACTGGAGAGACAAGTAAACTCTCAGAGTAACTGTCC CCTCTGACTACCATTTCTAAGGCAAGCCCCCTGTTTCTACTCTTGCGCCCCCTGCT GGTTTCCTGCCCTGTCTGTAAGTTGCATGGCTTTTGTCCGTCTTTTTTTTGTTTGTT TGTTTGTTTTGAGACAGGGTCTCACCCAGGCTAGAGTGAAGTGGAGCAATCTCGG CTCACTGCAACCTCCGCCTCCTGAGTTCAAGTGATTCTCACACCTCAGCCTCCCC AATAGCTGGGATTACAGGATGCCCCGGAGGCCCAGCTAGCCCCAGACTTCGGCC CCATGCGGCTCACCCGCTGCCAGGCTGCCCTGGCGGCCGCCATCACCCTCAACCT TCTGGTCCTCTTCTATGTCTCGTGGCTGCAGCACCAGCCTAGGAATTCCCGGGCC CGGGGGCCCCGTCGTGCCTCTGCTGCCGGCCCCCGTGTCACCGTCCTGGTGCGGG AGTTCGAGGCATTTGACAACGCGGTGCCCGAGCTGGTAGACTCCTTCCTGCAGCA AGACCCAGCCCAGCCCGTGGTGGTGGCAGCCGACACGCTCCCCTACCCGCCCCT GGCCCTGCCCCGCATCCCCAACGTGCGTCTGGCGCTGCTCCAGCCCGCCCTGGAC CGGCCAGCCGCAGCCTCGCGCCCGGAGACCTACGTGGCCACCGAGTTTGTGGCC CTAGTACCTGATGGGGCGCGGGCTGAGGCACCTGGCCTGCTGGAGCGCATGGTG GAGGCGCTCCGCGCAGGAAGCGCACGTCTGGTGGCCGCCCCGGTTGCCACGGCC AACCCTGCCAGGTGCCTGGCCCTGAACGTCAGCCTGCGAGAGTGGACCGCCCGC TATGGCGCAGCCCCCGCCGCGCCCCGCTGCGACGCCCTGGACGGAGATGCTGTG GTGCTCCTGCGCGCCCGCGACCTCTTCAACCTCTCGGCGCCCCTGGCCCGGCCGG TGGGCACCAGCCTCTTTCTGCAGACCGCCCTTCGCGGCTGGGCGGTGCAGCTGCT GGACTTGACCTTCGCCGCGGCGCGCCAGCCCCCGCTGGCCACGGCCCACGCGCG CTGGAAGGCTGAGCGCGAGGGACGCGCTCGGCGGGCGGCGCTGCTCCGCGCGCT GGGCATCCGCCTAGTGAGCTGGGAAGGCGGGCGGCTGGAGTGGTTCGGCTGCAA CAAGGAGACCACGCGCTGCTTCGGAACCGTGGTGGGCGACACGCCCGCCTACCT CTACGAGGAGCGCTGGACGCCCCCCTGCTGCCTGCGCGCGCTGCGCGAGACCGC CCGCTATGTGGTGGGCGTGCTGGAGGCTGCGGGCGTGCGCTACTGGCTCGAGGG CGGCTCACTGCTGGGGGCCGCCCGCCACGGGGACATCATCCCATGGGACTACGA CGTGGACCTGGGCATCTACTTGGAGGACGTGGGCAACTGCGAGCAGCTGCGGGG GGCAGAGGCCGGCTCGGTGGTGGATGAGCGCGGCTTCGTATGGGAGAAGGCGGT CGAGGGCGACTTTTTCCGCGTGCAGTACAGCGAAAGCAACCACTTGCACGTGGA CCTGTGGCCCTTCTACCCCCGCAATGGCGTCATGACCAAGGACACGTGGCTGGAC CACCGGCAGGATGTGGAGTTTCCCGAGCACTTCCTGCAGCCGCTGGTGCCCCTGC CCTTTGCCGGCTTCGTGGCGCAGGCGCCTAACAACTACCGCCGCTTCCTGGAGCT CAAGTTCGGGCCCGGGGTCATCGAGAACCCCCAGTACCCCAACCCGGCACTGCT GAGTCTGACGGGAAGCGGCTGAAGCCCTGATAACCTCGCCTTTGTTTTTCGGGGG TCTGTCTGGATGTGGAGAAGCTCTGTGTGAGCGGTGAGGGGTGGAGGGATGTCG CGGAGAGGGGAAGGGGGAAACTGACCAAGAAAGAAATTCTAAGGAGAGCATGA GAGAAGGCTGGCATTGGCAGGAGGAGAGCACCAGGACGAGGATGGGAAGCGAC CTCCAGATTTATCAAATGGTCATGCCCACTGGGAGCCGTGGATATGCGTGGGGAC ATCCTGGGTCATCTCAGTCATGGAGGGAGACGGGGATGTCACGCCGTCCCGCAG GGCCCAGCACAGCCCCAGACCCGAAAAAAGTGTTCTGCCCAAGATTCCGAGAGC CCTGCGCTCTAGGGCAGGGGCAGAGTTTTGGAAACAGTGCAGGCTCTGGAGCCA GACTGGCGAGATTCAAATCCTGGCTCTATCGCTTCGGAGCCAGGTGGGCCTGGG GGGGCGTCGCAGTCTCTCTGTGCCTCAGTTGCTTCCAGGATGCGGGACCCTTGGC TGCAGGGGTTGCTTCCGCCACTAGAGGGCGCGCCGGTCCCGCTCCTGGTGGCCCA CTGTGGCTGCCCGGGCGACAGTACGCCCAGGGCCTGTGTTCCATAGCCATCTACT CTCTTGAGCCTTTGGACTTCTCTCCAAGCCCCTGTGGGAGGCGGACAGCAGTGAC CACCTCCCCTTCTTTTGGACTGCGACCTCCTTCCCTCCTGGGAGAGCCCTGTGACC TGCATGCTACTCTTAACTGTTCTATTCAAGACTGAATAGAAGTATTTCAGTCTTGC AGAGGAGGAAATGCTCAGAGCTCCGAGGTGCGGCTGTGGTCGAGAACCGGGTGC TGGGCCGGGCGCGGGGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGG TGGGAGGATCGCTTGAGCCCAGGAGTCTGAGACCAGCCTCGGCAACATGCCAAG ACCCCGTCTCTATTTTTAAAAAAGAAAAAGAACCGACTTCTGAATCGCAGCTCCA CTCATGACTAATACCTCATTATTTCAGCTGTCTGCACCTAATTCCCCACTTGCACG GCAGTGTAGACAATAACCATAGCTCACACTCACTGAGCACCTACTGGGTACCAG GCACCATTCTCAGTGTTTCACCTGGATCAACTAATGCGTCCCTCACCTCAGCCCTC TGAAGTGACAGCTGCTATTATTTTCATTACACAGATGAAAAAGCTGAGGCCAGA ATCGTGAAGTCACTTGCTCAAGGTCAGGCAGCTTAGGAAGGGGCAGATCGGGGG CTTGAACCCAGGTGGTCAGGCTCTGGAGCCCACAATTGTCTTACCCACTATGCCC CTCTCTAGTCATGGTCCCCAAGAGGGGCTTGGAGACCCACTTAGCAGGTGAAAG CAATGGCAGCCTTCCTTATTTGATTATGCACCTAAGAATAAATGGTATTTGGGCA TGTATTCCCAATATGTGTATATTTATTTATAAATATATACAGATACTATTATCTGT ATGTTAGTAATAAAGCTTAAATTATTCCATTTTAAAATTATGAATATGAATAGGG TTTTTTTTATGTTTCTTGCCTCATCCCAATGACTTTTGCACACCCAGGTGTGAGCA CCCAGCATTCAAGACCACG [00640] SEQ ID NO.14 is FKRP, transcript variant X3, mRNA, XM_047439422.1 CATTGCTCCAAGATGGCGGCGGCGGCGGCAGCGGAGGAGATGCTCTGCTGAGGG GCAGTTGCTATGGTTACAGGGCCTGGACCTTCCTCCGGAAGGTGAGAAACAGGA GCGCAGCTCAGCTGGGCTGGAACTGCCCTCCTGGAACTCCCCCAGCCTACAACCT AGGAGGTGCAGGGACTGAGGCTCAGGCCAAATCGCAACTCAGACCCAGTGAACC CAAGGCCTGAAGAGAATTTGGATTCATTTACCTTGTTTTGTGGGGACTGGAGAGA CAAGTAAACTCTCAGAGTAACTGTCCCCTCTGACTACCATTTCTAAGGCAAGCCC CCTGTTTCTACTCTTGCGCCCCCTGCTGGTTTCCTGCCCTGTCTGTAAGTTGCATG GCTTTTGTCCGTCTTTTTTTTGTTTGTTTGTTTGTTTTGAGACAGGGTCTCACCCAG GCTAGAGTGAAGTGGAGCAATCTCGGCTCACTGCAACCTCCGCCTCCTGAGTTCA AGTGATTCTCACACCTCAGCCTCCCCAATAGCTGGGATTACAGGATGCCCCGGAG GCCCAGCTAGCCCCAGACTTCGGCCCCATGCGGCTCACCCGCTGCCAGGCTGCCC TGGCGGCCGCCATCACCCTCAACCTTCTGGTCCTCTTCTATGTCTCGTGGCTGCAG CACCAGCCTAGGAATTCCCGGGCCCGGGGGCCCCGTCGTGCCTCTGCTGCCGGCC CCCGTGTCACCGTCCTGGTGCGGGAGTTCGAGGCATTTGACAACGCGGTGCCCGA GCTGGTAGACTCCTTCCTGCAGCAAGACCCAGCCCAGCCCGTGGTGGTGGCAGC CGACACGCTCCCCTACCCGCCCCTGGCCCTGCCCCGCATCCCCAACGTGCGTCTG GCGCTGCTCCAGCCCGCCCTGGACCGGCCAGCCGCAGCCTCGCGCCCGGAGACC TACGTGGCCACCGAGTTTGTGGCCCTAGTACCTGATGGGGCGCGGGCTGAGGCA CCTGGCCTGCTGGAGCGCATGGTGGAGGCGCTCCGCGCAGGAAGCGCACGTCTG GTGGCCGCCCCGGTTGCCACGGCCAACCCTGCCAGGTGCCTGGCCCTGAACGTCA GCCTGCGAGAGTGGACCGCCCGCTATGGCGCAGCCCCCGCCGCGCCCCGCTGCG ACGCCCTGGACGGAGATGCTGTGGTGCTCCTGCGCGCCCGCGACCTCTTCAACCT CTCGGCGCCCCTGGCCCGGCCGGTGGGCACCAGCCTCTTTCTGCAGACCGCCCTT CGCGGCTGGGCGGTGCAGCTGCTGGACTTGACCTTCGCCGCGGCGCGCCAGCCC CCGCTGGCCACGGCCCACGCGCGCTGGAAGGCTGAGCGCGAGGGACGCGCTCGG CGGGCGGCGCTGCTCCGCGCGCTGGGCATCCGCCTAGTGAGCTGGGAAGGCGGG CGGCTGGAGTGGTTCGGCTGCAACAAGGAGACCACGCGCTGCTTCGGAACCGTG GTGGGCGACACGCCCGCCTACCTCTACGAGGAGCGCTGGACGCCCCCCTGCTGC CTGCGCGCGCTGCGCGAGACCGCCCGCTATGTGGTGGGCGTGCTGGAGGCTGCG GGCGTGCGCTACTGGCTCGAGGGCGGCTCACTGCTGGGGGCCGCCCGCCACGGG GACATCATCCCATGGGACTACGACGTGGACCTGGGCATCTACTTGGAGGACGTG GGCAACTGCGAGCAGCTGCGGGGGGCAGAGGCCGGCTCGGTGGTGGATGAGCG CGGCTTCGTATGGGAGAAGGCGGTCGAGGGCGACTTTTTCCGCGTGCAGTACAG CGAAAGCAACCACTTGCACGTGGACCTGTGGCCCTTCTACCCCCGCAATGGCGTC ATGACCAAGGACACGTGGCTGGACCACCGGCAGGATGTGGAGTTTCCCGAGCAC TTCCTGCAGCCGCTGGTGCCCCTGCCCTTTGCCGGCTTCGTGGCGCAGGCGCCTA ACAACTACCGCCGCTTCCTGGAGCTCAAGTTCGGGCCCGGGGTCATCGAGAACC CCCAGTACCCCAACCCGGCACTGCTGAGTCTGACGGGAAGCGGCTGAAGCCCTG ATAACCTCGCCTTTGTTTTTCGGGGGTCTGTCTGGATGTGGAGAAGCTCTGTGTG AGCGGTGAGGGGTGGAGGGATGTCGCGGAGAGGGGAAGGGGGAAACTGACCAA GAAAGAAATTCTAAGGAGAGCATGAGAGAAGGCTGGCATTGGCAGGAGGAGAG CACCAGGACGAGGATGGGAAGCGACCTCCAGATTTATCAAATGGTCATGCCCAC TGGGAGCCGTGGATATGCGTGGG GACATCCTGGGTCATCTCAGTCATGGAGGGAGACGGGGATGTCACGCCGTCCCG CAGGGCCCAGCACAGCCCCAGACCCGAAAAAAGTGTTCTGCCCAAGATTCCGAG AGCCCTGCGCTCTAGGGCAGGGGCAGAGTTTTGGAAACAGTGCAGGCTCTGGAG CCAGACTGGCGAGATTCAAATCCTGGCTCTATCGCTTCGGAGCCAGGTGGGCCTG GGGGGGCGTCGCAGTCTCTCTGTGCCTCAGTTGCTTCCAGGATGCGGGACCCTTG GCTGCAGG GGTTGCTTCCGCCACTAGAGGGCGCGCCGGTCCCGCTCCTGGTGGCCCACTGTGG CTGCCCGGGCGACAGTACGCCCAGGGCCTGTGTTCCATAGCCATCTACTCTCTTG AGCCTTTGGACTTCTCTCCAAGCCCCTGTGGGAGGCGGACAGCAGTGACCACCTC CCCTTCTTTTGGACTGCGACCTCCTTCCCTCCTGGGAGAGCCCTGTGACCTGCATG CTACTCTTAACTGTTCTATTCAAGACTGAATAGAAGTATTTCAGTCTTGCAGAGG AGGAAATGCTCAGAGCTCCGAGGTGCGGCTGTGGTCGAGAACCGGGTGCTGGGC CGGGCGCGGGGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGA GGATCGCTTGAGCCCAGGAGTCTGAGACCAGCCTCGGCAACATGCCAAGACCCC GTCTCTATTTTTAAAAAAGAAAAAGAACCGACTTCTGAATCGCAGCTCCACTCAT GACTAATACCTCATTATTTCAGCTGTCTGCACCTAATTCCCCACTTGCACGGCAGT GTAGACAATAACCATAGCTCACACTCACTGAGCACCTACTGGGTACCAGGCACC ATTCTCAGTGTTTCACCTGGATCAACTAATGCGTCCCTCACCTCAGCCCTCTGAA GTGACAGCTGCTATTATTTTCATTACACAGATGAAAAAGCTGAGGCCAGAATCGT GAAGTCACTTGCTCAAGGTCAGGCAGCTTAGGAAGGGGCAGATCGGGGGCTTGA ACCCAGGTGGTCAGGCTCTGGAGCCCACAATTGTCTTACCCACTATGCCCCTCTC TAGTCATGGTCCCCAAGAGGGGCTTGGAGACCCACTTAGCAGGTGAAAGCAATG GCAGCCTTCCTTATTTGATTATGCACCTAAGAAT AAATGGTATTTGGGCATGTATTCCCAATATGTGTATATTTATTTATAAATATATAC AGATACTATTATCTGTATGTTAGTAATAAAGCTTAAATTATTCCATTTTAAAATTA TGAATATGAATAGGGTTTTTTTTATGTTTCTTGCCTCATCCCAATGACTTTTGCAC ACCCAGGTGTGAGCACCCAGCATTCAAGACCACG [00641] SEQ ID NO.15 is FKRP, transcript variant X5, mRNA, XM_005259247.3 CATTGCTCCAAGATGGCGGCGGCGGCGGCAGCGGAGGAGATGCTCTGCTGAGGG GCAGTTGCTATGGTTACAGGGCCTGGACCTTCCTCCGGAAGGTGAGAAACAGGA GCGCAGCTCAGCTGGGCTGGAACTGCCCTCCTGGAACTCCCCCAGCCTACAACCT AGGAGGTGCAGGGACTGAGGCTCAGGCCAAATCGCAACTCAGACCCAGTGAACC CAAGGCCTGAAGAGAATTTGGATTCATTTACCTTGTTTTGTGGGGACTGGAGAGA CAAGTAAACTCTCAGAGTAACTGTCCCCTCTGACTACCATTTCTAAGGCAAGCCC CCTGTTTCTACTCTTGCGCCCCCTGCTGGTTTCCTGCCCTGTCTGATGCCCCGGAG GCCCAGCTAGCCCCAGACTTCGGCCCCATGCGGCTCACCCGCTGCCAGGCTGCCC TGGCGGCCGCCATCACCCTCAACCTTCTGGTCCTCTTCTATGTCTCGTGGCTGCAG CACCAGCCTAGGAATTCCCGGGCCCGGGGGCCCCGTCGTGCCTCTGCTGCCGGCC CCCGTGTCACCGTCCTGGTGCGGGAGTTCGAGGCATTTGACAACGCGGTGCCCGA GCTGGTAGACTCCTTCCTGCAGCAAGACCCAGCCCAGCCCGTGGTGGTGGCAGC CGACACGCTCCCCTACCCGCCCCTGGCCCTGCCCCGCATCCCCAACGTGCGTCTG GCGCTGCTCCAGCCCGCCCTGGACCGGCCAGCCGCAGCCTCGCGCCCGGAGACC TACGTGGCCACCGAGTTTGTGGCCCTAGTACCTGATGGGGCGCGGGCTGAGGCA CCTGGCCTGCTGGAGCGCATGGTGGAGGCGCTCCGCGCAGGAAGCGCACGTCTG GTGGCCGCCCCGGTTGCCACGGCCAACCCTGCCAGGTGCCTGGCCCTGAACGTCA GCCTGCGAGAGTGGACCGCCCGCTATGGCGCAGCCCCCGCCGCGCCCCGCTGCG ACGCCCTGGACGGAGATGCTGTGGTGCTCCTGCGCGCCCGCGACCTCTTCAACCT CTCGGCGCCCCTGGCCCGGCCGGTGGGCACCAGCCTCTTTCTGCAGACCGCCCTT CGCGGCTGGGCGGTGCAGCTGCTGGACTTGACCTTCGCCGCGGCGCGCCAGCCC CCGCTGGCCACGGCCCACGCGCGCTGGAAGGCTGAGCGCGAGGGACGCGCTCGG CGGGCGGCGCTGCTCCGCGCGCTGGGCATCCGCCTAGTGAGCTGGGAAGGCGGG CGGCTGGAGTGGTTCGGCTGCAACAAGGAGACCACGCGCTGCTTCGGAACCGTG GTGGGCGACACGCCCGCCTACCTCTACGAGGAGCGCTGGACGCCCCCCTGCTGC CTGCGCGCGCTGCGCGAGACCGCCCGCTATGTGGTGGGCGTGCTGGAGGCTGCG GGCGTGCGCTACTGGCTCGAGGGCGGCTCACTGCTGGGGGCCGCCCGCCACGGG GACATCATCCCATGGGACTACGACGTGGACCTGGGCATCTACTTGGAGGACGTG GGCAACTGCGAGCAGCTGCGGGGGGCAGAGGCCGGCTCGGTGGTGGATGAGCG CGGCTTCGTATGGGAGAAGGCGGTCGAGGGCGACTTTTTCCGCGTGCAGTACAG CGAAAGCAACCACTTGCACGTGGACCTGTGGCCCTTCTACCCCCGCAATGGCGTC ATGACCAAGGACACGTGGCTGGACCACCGGCAGGATGTGGAGTTTCCCGAGCAC TTCCTGCAGCCGCTGGTGCCCCTGCCCTTTGCCGGCTTCGTGGCGCAGGCGCCTA ACAACTACCGCCGCTTCCTGGAGCTCAAGTTCGGGCCCGGGGTCATCGAGAACC CCCAGTACCCCAACCCGGCACTGCTGAGTCTGACGGGAAGCGGCTGAAGCCCTG ATAACCTCGCCTTTGTTTTTCGGGGGTCTGTCTGGATGTGGAGAAGCTCTGTGTG AGCGGTGAGGGGTGGAGGGATGTCGCGGAGAGGGGAAGGGGGAAACTGACCAA GAAAGAAATTCTAAGGAGAGCATGAGAGAAGGCTGGCATTGGCAGGAGGAGAG CACCAGGACGAGGATGGGAAGCGACCTCCAGATTTATCAAATGGTCATGCCCAC TGGGAGCCGTGGATATGCGTGGGGACATCCTGGGTCATCTCAGTCATGGAGGGA GACGGGGATGTCACGCCGTCCCGCAGGGCCCAGCACAGCCCCAGACCCGAAAAA AGTGTTCTGCCCAAGATTCCGAGAGCCCTGCGCTCTAGGGCAGGGGCAGAGTTTT GGAAACAGTGCAGGCTCTGGAGCCAGACTGGCGAGATTCAAATCCTGGCTCTAT CGCTTCGGAGCCAGGTGGGCCTGGGGGGGCGTCGCAGTCTCTCTGTGCCTCAGTT GCTTCCAGGATGCGGGACCCTTGGCTGCAGGGGTTGCTTCCGCCACTAGAGGGC GCGCCGGTCCCGCTCCTGGTGGCCCACTGTGGCTGCCCGGGCGACAGTACGCCCA GGGCCTGTGTTCCATAGCCATCTACTCTCTTGAGCCTTTGGACTTCTCTCCAAGCC CCTGTGGGAGGCGGACAGCAGTGACCACCTCCCCTTCTTTTGGACTGCGACCTCC TTCCCTCCTGGGAGAGCCCTGTGACCTGCATGCTACTCTTAACTGTTCTATTCAAG ACTGAATAGAAGTATTTCAGTCTTGCAGAGGAGGAAATGCTCAGAGCTCCGAGG TGCGGCTGTGGTCGAGAACCGGGTGCTGGGCCGGGCGCGGGGGCTCACGCCTGT AATCCCAGCACTTTGGGAGGCCGAGGTGGGAGGATCGCTTGAGCCCAGGAGTCT GAGACCAGCCTCGGCAACATGCCAAGACCCCGTCTCTATTTTTAAAAAAGAAAA AGAACCGACTTCTGAATCGCAGCTCCACTCATGACTAATACCTCATTATTTCAGC TGTCTGCACCTAATTCCCCACTTGCACGGCAGTGTAGACAATAACCATAGCTCAC ACTCACTGAGCACCTACTGGGTACCAGGCACCATTCTCAGTGTTTCACCTGGATC AACTAATGCGTCCCTCACCTCAGCCCTCTGAAGTGACAGCTGCTATTATTTTCATT ACACAGATGAAAAAGCTGAGGCCAGAATCGTGAAGTCACTTGCTCAAGGTCAGG CAGCTTAGGAAGGGGCAGATCGGGGGCTTGAACCCAGGTGGTCAGGCTCTGGAG CCCACAATTGTCTTACCCACTATGCCCCTCTCTAGTCATGGTCCCCAAGAGGGGC TTGGAGACCCACTTAGCAGGTGAAAGCAATGGCAGCCTTCCTTATTTGATTATGC ACCTAAGAATAAATGGTATTTGGGCATGTATTCCCAATATGTGTATATTTATTTAT AAATATATACAGATACTATTATCTGTATGTTAGTAATAAAGCTTAAATTATTCCA TTTTAAAATTATGAATATGAATAGGGTTTTTTTTATGTTTCTTGCCTCATCCCAAT GACTTTTGCACACCCAGGTGTGAGCACCCAGCATTCAAGACCACG [00642] SEQ ID NO.16 is FKRP, transcript variant X4, mRNA, XM_005259248.3 CATTGCTCCAAGATGGCGGCGGCGGCGGCAGCGGAGGAGATGCTCTGCTGAGGG GCAGTTGCTATGGTTACAGGGCCTGGACCTTCCTCCGGAAGGTGAGAAACAGGA GCGCAGCTCAGCTGGGCTGGAACTGCCCTCCTGGAACTCCCCCAGCCTACAACCT AGGAGGTGCAGGGACTGAGGCTCAGGCCAAATCGCAACTCAGACCCAGTGAACC CAAGGCCTGAAGAGAATTTGGATTCATTTACCTTGTTTTGTGGGGACTGGAGAGA CAAGTAAACTCTCAGAGTAACTGTCCCCTCTGACTACCATTTCTAAGGATGCCCC GGAGGCCCAGCTAGCCCCAGACTTCGGCCCCATGCGGCTCACCCGCTGCCAGGC TGCCCTGGCGGCCGCCATCACCCTCAACCTTCTGGTCCTCTTCTATGTCTCGTGGC TGCAGCACCAGCCTAGGAATTCCCGGGCCCGGGGGCCCCGTCGTGCCTCTGCTGC CGGCCCCCGTGTCACCGTCCTGGTGCGGGAGTTCGAGGCATTTGACAACGCGGTG CCCGAGCTGGTAGACTCCTTCCTGCAGCAAGACCCAGCCCAGCCCGTGGTGGTG GCAGCCGACACGCTCCCCTACCCGCCCCTGGCCCTGCCCCGCATCCCCAACGTGC GTCTGGCGCTGCTCCAGCCCGCCCTGGACCGGCCAGCCGCAGCCTCGCGCCCGG AGACCTACGTGGCCACCGAGTTTGTGGCCCTAGTACCTGATGGGGCGCGGGCTG AGGCACCTGGCCTGCTGGAGCGCATGGTGGAGGCGCTCCGCGCAGGAAGCGCAC GTCTGGTGGCCGCCCCGGTTGCCACGGCCAACCCTGCCAGGTGCCTGGCCCTGAA CGTCAGCCTGCGAGAGTGGACCGCCCGCTATGGCGCAGCCCCCGCCGCGCCCCG CTGCGACGCCCTGGACGGAGATGCTGTGGTGCTCCTGCGCGCCCGCGACCTCTTC AACCTCTCGGCGCCCCTGGCCCGGCCGGTGGGCACCAGCCTCTTTCTGCAGACCG CCCTTCGCGGCTGGGCGGTGCAGCTGCTGGACTTGACCTTCGCCGCGGCGCGCCA GCCCCCGCTGGCCACGGCCCACGCGCGCTGGAAGGCTGAGCGCGAGGGACGCGC TCGGCGGGCGGCGCTGCTCCGCGCGCTGGGCATCCGCCTAGTGAGCTGGGAAGG CGGGCGGCTGGAGTGGTTCGGCTGCAACAAGGAGACCACGCGCTGCTTCGGAAC CGTGGTGGGCGACACGCCCGCCTACCTCTACGAGGAGCGCTGGACGCCCCCCTG CTGCCTGCGCGCGCTGCGCGAGACCGCCCGCTATGTGGTGGGCGTGCTGGAGGC TGCGGGCGTGCGCTACTGGCTCGAGGGCGGCTCACTGCTGGGGGCCGCCCGCCA CGGGGACATCATCCCATGGGACTACGACGTGGACCTGGGCATCTACTTGGAGGA CGTGGGCAACTGCGAGCAGCTGCGGGGGGCAGAGGCCGGCTCGGTGGTGGATGA GCGCGGCTTCGTATGGGAGAAGGCGGTCGAGGGCGACTTTTTCCGCGTGCAGTA CAGCGAAAGCAACCACTTGCACGTGGACCTGTGGCCCTTCTACCCCCGCAATGGC GTCATGACCAAGGACACGTGGCTGGACCACCGGCAGGATGTGGAGTTTCCCGAG CACTTCCTGCAGCCGCTGGTGCCCCTGCCCTTTGCCGGCTTCGTGGCGCAGGCGC CTAACAACTACCGCCGCTTCCTGGAGCTCAAGTTCGGGCCCGGGGTCATCGAGA ACCCCCAGTACCCCAACCCGGCACTGCTGAGTCTGACGGGAAGCGGCTGAAGCC CTGATAACCTCGCCTTTGTTTTTCGGGGGTCTGTCTGGATGTGGAGAAGCTCTGTG TGAGCGGTGAGGGGTGGAGGGATGTCGCGGAGAGGGGAAGGGGGAAACTGACC AAGAAAGAAATTCTAAGGAGAGCATGAGAGAAGGCTGGCATTGGCAGGAGGAG AGCACCAGGACGAGGATGGGAAGCGACCTCCAGATTTATCAAATGGTCATGCCC ACTGGGAGCCGTGGATATGCGTGGGGACATCCTGGGTCATCTCAGTCATGGAGG GAGACGGGGATGTCACGCCGTCCCGCAGGGCCCAGCACAGCCCCAGACCCGAAA AAAGTGTTCTGCCCAAGATTCCGAGAGCCCTGCGCTCTAGGGCAGGGGCAGAGT TTTGGAAACAGTGCAGGCTCTGGAGCCAGACTGGCGAGATTCAAATCCTGGCTCT ATCGCTTCGGAGCCAGGTGGGCCTGGGGGGGCGTCGCAGTCTCTCTGTGCCTCAG TTGCTTCCAGGATGCGGGACCCTTGGCTGCAGGGGTTGCTTCCGCCACTAGAGGG CGCGCCGGTCCCGCTCCTGGTGGCCCACTGTGGCTGCCCGGGCGACAGTACGCCC AGGGCCTGTGTTCCATAGCCATCTACTCTCTTGAGCCTTTGGACTTCTCTCCAAGC CCCTGTGGGAGGCGGACAGCAGTGACCACCTCCCCTTCTTTTGGACTGCGACCTC CTTCCCTCCTGGGAGAGCCCTGTGACCTGCATGCTACTCTTAACTGTTCTATTCAA GACTGAATAGAAGTATTTCAGTCTTGCAGAGGAGGAAATGCTCAGAGCTCCGAG GTGCGGCTGTGGTCGAGAACCGGGTGCTGGGCCGGGCGCGGGGGCTCACGCCTG TAATCCCAGCACTTTGGGAGGCCGAGGTGGGAGGATCGCTTGAGCCCAGGAGTC TGAGACCAGCCTCGGCAACATGCCAAGACCCCGTCTCTATTTTTAAAAAAGAAA AAGAACCGACTTCTGAATCGCAGCTCCACTCATGACTAATACCTCATTATTTCAG CTGTCTGCACCTAATTCCCCACTTGCACGGCAGTGTAGACAATAACCATAGCTCA CACTCACTGAGCACCTACTGGGTACCAGGCACCATTCTCAGTGTTTCACCTGGAT CAACTAATGCGTCCCTCACCTCAGCCCTCTGAAGTGACAGCTGCTATTATTTTCAT TACACAGATGAAAAAGCTGAGGCCAGAATCGTGAAGTCACTTGCTCAAGGTCAG GCAGCTTAGGAAGGGGCAGATCGGGGGCTTGAACCCAGGTGGTCAGGCTCTGGA GCCCACAATTGTCTTACCCACTATGCCCCTCTCTAGTCATGGTCCCCAAGAGGGG CTTGGAGACCCACTTAGCAGGTGAAAGCAATGGCAGCCTTCCTTATTTGATTATG CACCTAAGAATAAATGGTATTTGGGCATGTATTCCCAATATGTGTATATTTATTTA TAAATATATACAGATACTATTATCTGTATGTTAGTAATAAAGCTTAAATTATTCC ATTTTAAAATTATGAATATGAATAGGGTTTTTTTTATGTTTCTTGCCTCATCCCAA TGACTTTTGCACACCCAGGTGTGAGCACCCAGCATTCAAGACCACG [00643] SEQ ID NO.17 is FKRP, transcript variant X13, mRNA XM_024451707.2 CATTGCTCCAAGATGGCGGCGGCGGCGGCAGCGGAGGAGATGCTCTGCTGAGGG GCAGTTGCTATGGTTACAGGGCCTGGACCTTCCTCCGGAAGGTGAGAAACAGGA GCGCAGCTCAGCTGGGCTGGAACTGCCCTCCTGGAACTCCCCCAGCCTACAACCT AGGAGGATGCCCCGGAGGCCCAGCTAGCCCCAGACTTCGGCCCCATGCGGCTCA CCCGCTGCCAGGCTGCCCTGGCGGCCGCCATCACCCTCAACCTTCTGGTCCTCTT CTATGTCTCGTGGCTGCAGCACCAGCCTAGGAATTCCCGGGCCCGGGGGCCCCGT CGTGCCTCTGCTGCCGGCCCCCGTGTCACCGTCCTGGTGCGGGAGTTCGAGGCAT TTGACAACGCGGTGCCCGAGCTGGTAGACTCCTTCCTGCAGCAAGACCCAGCCC AGCCCGTGGTGGTGGCAGCCGACACGCTCCCCTACCCGCCCCTGGCCCTGCCCCG CATCCCCAACGTGCGTCTGGCGCTGCTCCAGCCCGCCCTGGACCGGCCAGCCGCA GCCTCGCGCCCGGAGACCTACGTGGCCACCGAGTTTGTGGCCCTAGTACCTGATG GGGCGCGGGCTGAGGCACCTGGCCTGCTGGAGCGCATGGTGGAGGCGCTCCGCG CAGGAAGCGCACGTCTGGTGGCCGCCCCGGTTGCCACGGCCAACCCTGCCAGGT GCCTGGCCCTGAACGTCAGCCTGCGAGAGTGGACCGCCCGCTATGGCGCAGCCC CCGCCGCGCCCCGCTGCGACGCCCTGGACGGAGATGCTGTGGTGCTCCTGCGCGC CCGCGACCTCTTCAACCTCTCGGCGCCCCTGGCCCGGCCGGTGGGCACCAGCCTC TTTCTGCAGACCGCCCTTCGCGGCTGGGCGGTGCAGCTGCTGGACTTGACCTTCG CCGCGGCGCGCCAGCCCCCGCTGGCCACGGCCCACGCGCGCTGGAAGGCTGAGC GCGAGGGACGCGCTCGGCGGGCGGCGCTGCTCCGCGCGCTGGGCATCCGCCTAG TGAGCTGGGAAGGCGGGCGGCTGGAGTGGTTCGGCTGCAACAAGGAGACCACGC GCTGCTTCGGAACCGTGGTGGGCGACACGCCCGCCTACCTCTACGAGGAGCGCT GGACGCCCCCCTGCTGCCTGCGCGCGCTGCGCGAGACCGCCCGCTATGTGGTGG GCGTGCTGGAGGCTGCGGGCGTGCGCTACTGGCTCGAGGGCGGCTCACTGCTGG GGGCCGCCCGCCACGGGGACATCATCCCATGGGACTACGACGTGGACCTGGGCA TCTACTTGGAGGACGTGGGCAACTGCGAGCAGCTGCGGGGGGCAGAGGCCGGCT CGGTGGTGGATGAGCGCGGCTTCGTATGGGAGAAGGCGGTCGAGGGCGACTTTT TCCGCGTGCAGTACAGCGAAAGCAACCACTTGCACGTGGACCTGTGGCCCTTCTA CCCCCGCAATGGCGTCATGACCAAGGACACGTGGCTGGACCACCGGCAGGATGT GGAGTTTCCCGAGCACTTCCTGCAGCCGCTGGTGCCCCTGCCCTTTGCCGGCTTC GTGGCGCAGGCGCCTAACAACTACCGCCGCTTCCTGGAGCTCAAGTTCGGGCCC GGGGTCATCGAGAACCCCCAGTACCCCAACCCGGCACTGCTGAGTCTGACGGGA AGCGGCTGAAGCCCTGATAACCTCGCCTTTGTTTTTCGGGGGTCTGTCTGGATGT GGAGAAGCTCTGTGTGAGCGGTGAGGGGTGGAGGGATGTCGCGGAGAGGGGAA GGGGGAAACTGACCAAGAAAGAAATTCTAAGGAGAGCATGAGAGAAGGCTGGC ATTGGCAGGAGGAGAGCACCAGGACGAGGATGGGAAGCGACCTCCAGATTTATC AAATGGTCATGCCCACTGGGAGCCGTGGATATGCGTGGGGACATCCTGGGTCAT CTCAGTCATGGAGGGAGACGGGGATGTCACGCCGTCCCGCAGGGCCCAGCACAG CCCCAGACCCGAAAAAAGTGTTCTGCCCAAGATTCCGAGAGCCCTGCGCTCTAG GGCAGGGGCAGAGTTTTGGAAACAGTGCAGGCTCTGGAGCCAGACTGGCGAGAT TCAAATCCTGGCTCTATCGCTTCGGAGCCAGGTGGGCCTGGGGGGGCGTCGCAGT CTCTCTGTGCCTCAGTTGCTTCCAGGATGCGGGACCCTTGGCTGCAGGGGTTGCT TCCGCCACTAGAGGGCGCGCCGGTCCCGCTCCTGGTGGCCCACTGTGGCTGCCCG GGCGACAGTACGCCCAGGGCCTGTGTTCCATAGCCATCTACTCTCTTGAGCCTTT GGACTTCTCTCCAAGCCCCTGTGGGAGGCGGACAGCAGTGACCACCTCCCCTTCT TTTGGACTGCGACCTCCTTCCCTCCTGGGAGAGCCCTGTGACCTGCATGCTACTCT TAACTGTTCTATTCAAGACTGAATAGAAGTATTTCAGTCTTGCAGAGGAGGAAAT GCTCAGAGCTCCGAGGTGCGGCTGTGGTCGAGAACCGGGTGCTGGGCCGGGCGC GGGGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGAGGATCGC TTGAGCCCAGGAGTCTGAGACCAGCCTCGGCAACATGCCAAGACCCCGTCTCTAT TTTTAAAAAAGAAAAAGAACCGACTTCTGAATCGCAGCTCCACTCATGACTAAT ACCTCATTATTTCAGCTGTCTGCACCTAATTCCCCACTTGCACGGCAGTGTAGAC AATAACCATAGCTCACACTCACTGAGCACCTACTGGGTACCAGGCACCATTCTCA GTGTTTCACCTGGATCAACTAATGCGTCCCTCACCTCAGCCCTCTGAAGTGACAG CTGCTATTATTTTCATTACACAGATGAAAAAGCTGAGGCCAGAATCGTGAAGTCA CTTGCTCAAGGTCAGGCAGCTTAGGAAGGGGCAGATCGGGGGCTTGAACCCAGG TGGTCAGGCTCTGGAGCCCACAATTGTCTTACCCACTATGCCCCTCTCTAGTCAT GGTCCCCAAGAGGGGCTTGGAGACCCACTTAGCAGGTGAAAGCAATGGCAGCCT TCCTTATTTGATTATGCACCTAAGAATAAATGGTATTTGGGCATGTATTCCCAATA TGTGTATATTTATTTATAAATATATACAGATACTATTATCTGTATGTTAGTAATAA AGCTTAAATTATTCCATTTTAAAATTATGAATATGAATAGGGTTTTTTTTATGTTT CTTGCCTCATCCCAATGACTTTTGCACACCCAGGTGTGAGCACCCAGCATTCAAG ACCACG [00644] SEQ ID NO.18 is FKRP, transcript variant X7, mRNA, XM_047439423.1 CATTGCTCCAAGATGGCGGCGGCGGCGGCAGCGGAGGAGATGCTCTGCTGAGGG GCAGTTGCTATGGTTACAGGGCCTGGACCTTCCTCCGGAAGGAGCGCAGCTCAG CTGGGCTGGAACTGCCCTCCTGGAACTCCCCCAGCCTACAACCTAGGAGGTGCA GGGACTGAGGCTCAGGCCAAATCGCAACTCAGACCCAGTGAACCCAAGGCCTGA AGAGAATTTGGATTCATTTACCTTGTTTTGTGGGGACTGGAGAGACAAGTAAACT CTCAGAGTAACTGTCCCCTCTGACTACCATTTCTAAGGCAAGCCCCCTGTTTCTAC TCTTGCGCCCCCTGCTGGTTTCCTGCCCTGTCTGATGCCCCGGAGGCCCAGCTAG CCCCAGACTTCGGCCCCATGCGGCTCACCCGCTGCCAGGCTGCCCTGGCGGCCGC CATCACCCTCAACCTTCTGGTCCTCTTCTATGTCTCGTGGCTGCAGCACCAGCCTA GGAATTCCCGGGCCCGGGGGCCCCGTCGTGCCTCTGCTGCCGGCCCCCGTGTCAC CGTCCTGGTGCGGGAGTTCGAGGCATTTGACAACGCGGTGCCCGAGCTGGTAGA CTCCTTCCTGCAGCAAGACCCAGCCCAGCCCGTGGTGGTGGCAGCCGACACGCTC CCCTACCCGCCCCTGGCCCTGCCCCGCATCCCCAACGTGCGTCTGGCGCTGCTCC AGCCCGCCCTGGACCGGCCAGCCGCAGCCTCGCGCCCGGAGACCTACGTGGCCA CCGAGTTTGTGGCCCTAGTACCTGATGGGGCGCGGGCTGAGGCACCTGGCCTGCT GGAGCGCATGGTGGAGGCGCTCCGCGCAGGAAGCGCACGTCTGGTGGCCGCCCC GGTTGCCACGGCCAACCCTGCCAGGTGCCTGGCCCTGAACGTCAGCCTGCGAGA GTGGACCGCCCGCTATGGCGCAGCCCCCGCCGCGCCCCGCTGCGACGCCCTGGA CGGAGATGCTGTGGTGCTCCTGCGCGCCCGCGACCTCTTCAACCTCTCGGCGCCC CTGGCCCGGCCGGTGGGCACCAGCCTCTTTCTGCAGACCGCCCTTCGCGGCTGGG CGGTGCAGCTGCTGGACTTGACCTTCGCCGCGGCGCGCCAGCCCCCGCTGGCCAC GGCCCACGCGCGCTGGAAGGCTGAGCGCGAGGGACGCGCTCGGCGGGCGGCGCT GCTCCGCGCGCTGGGCATCCGCCTAGTGAGCTGGGAAGGCGGGCGGCTGGAGTG GTTCGGCTGCAACAAGGAGACCACGCGCTGCTTCGGAACCGTGGTGGGCGACAC GCCCGCCTACCTCTACGAGGAGCGCTGGACGCCCCCCTGCTGCCTGCGCGCGCTG CGCGAGACCGCCCGCTATGTGGTGGGCGTGCTGGAGGCTGCGGGCGTGCGCTAC TGGCTCGAGGGCGGCTCACTGCTGGGGGCCGCCCGCCACGGGGACATCATCCCA TGGGACTACGACGTGGACCTGGGCATCTACTTGGAGGACGTGGGCAACTGCGAG CAGCTGCGGGGGGCAGAGGCCGGCTCGGTGGTGGATGAGCGCGGCTTCGTATGG GAGAAGGCGGTCGAGGGCGACTTTTTCCGCGTGCAGTACAGCGAAAGCAACCAC TTGCACGTGGACCTGTGGCCCTTCTACCCCCGCAATGGCGTCATGACCAAGGACA CGTGGCTGGACCACCGGCAGGATGTGGAGTTTCCCGAGCACTTCCTGCAGCCGCT GGTGCCCCTGCCCTTTGCCGGCTTCGTGGCGCAGGCGCCTAACAACTACCGCCGC TTCCTGGAGCTCAAGTTCGGGCCCGGGGTCATCGAGAACCCCCAGTACCCCAACC CGGCACTGCTGAGTCTGACGGGAAGCGGCTGAAGCCCTGATAACCTCGCCTTTGT TTTTCGGGGGTCTGTCTGGATGTGGAGAAGCTCTGTGTGAGCGGTGAGGGGTGGA GGGATGTCGCGGAGAGGGGAAGGGGGAAACTGACCAAGAAAGAAATTCTAAGG AGAGCATGAGAGAAGGCTGGCATTGGCAGGAGGAGAGCACCAGGACGAGGATG GGAAGCGACCTCCAGATTTATCAAATGGTCATGCCCACTGGGAGCCGTGGATAT GCGTGGGGACATCCTGGGTCATCTCAGTCATGGAGGGAGACGGGGATGTCACGC CGTCCCGCAGGGCCCAGCACAGCCCCAGACCCGAAAAAAGTGTTCTGCCCAAGA TTCCGAGAGCCCTGCGCTCTAGGGCAGGGGCAGAGTTTTGGAAACAGTGCAGGC TCTGGAGCCAGACTGGCGAGATTCAAATCCTGGCTCTATCGCTTCGGAGCCAGGT GGGCCTGGGGGGGCGTCGCAGTCTCTCTGTGCCTCAGTTGCTTCCAGGATGCGGG ACCCTTGGCTGCAGGGGTTGCTTCCGCCACTAGAGGGCGCGCCGGTCCCGCTCCT GGTGGCCCACTGTGGCTGCCCGGGCGACAGTACGCCCAGGGCCTGTGTTCCATA GCCATCTACTCTCTTGAGCCTTTGGACTTCTCTCCAAGCCCCTGTGGGAGGCGGA CAGCAGTGACCACCTCCCCTTCTTTTGGACTGCGACCTCCTTCCCTCCTGGGAGA GCCCTGTGACCTGCATGCTACTCTTAACTGTTCTATTCAAGACTGAATAGAAGTA TTTCAGTCTTGCAGAGGAGGAAATGCTCAGAGCTCCGAGGTGCGGCTGTGGTCG AGAACCGGGTGCTGGGCCGGGCGCGGGGGCTCACGCCTGTAATCCCAGCACTTT GGGAGGCCGAGGTGGGAGGATCGCTTGAGCCCAGGAGTCTGAGACCAGCCTCGG CAACATGCCAAGACCCCGTCTCTATTTTTAAAAAAGAAAAAGAACCGACTTCTG AATCGCAGCTCCACTCATGACTAATACCTCATTATTTCAGCTGTCTGCACCTAATT CCCCACTTGCACGGCAGTGTAGACAATAACCATAGCTCACACTCACTGAGCACCT ACTGGGTACCAGGCACCATTCTCAGTGTTTCACCTGGATCAACTAATGCGTCCCT CACCTCAGCCCTCTGAAGTGACAGCTGCTATTATTTTCATTACACAGATGAAAAA GCTGAGGCCAGAATCGTGAAGTCACTTGCTCAAGGTCAGGCAGCTTAGGAAGGG GCAGATCGGGGGCTTGAACCCAGGTGGTCAGGCTCTGGAGCCCACAATTGTCTT ACCCACTATGCCCCTCTCTAGTCATGGTCCCCAAGAGGGGCTTGGAGACCCACTT AGCAGGTGAAAGCAATGGCAGCCTTCCTTATTTGATTATGCACCTAAGAATAAAT GGTATTTGGGCATGTATTCCCAATATGTGTATATTTATTTATAAATATATACAGAT ACTATTATCTGTATGTTAGTAATAAAGCTTAAATTATTCCATTTTAAAATTATGAA TATGAATAGGGTTTTTTTTATGTTTCTTGCCTCATCCCAATGACTTTTGCACACCC AGGTGTGAGCACCCAGCATTCAAGACCACG [00645] SEQ ID NO.19 is FKRP, transcript variant X6, mRNA XM_005259249.5 CATTGCTCCAAGATGGCGGCGGCGGCGGCAGCGGAGGAGATGCTCTGCTGAGGG GCAGTTGCTATGGTTACAGGGCCTGGACCTTCCTCCGGAAGGAGCGCAGCTCAG CTGGGCTGGAACTGCCCTCCTGGAACTCCCCCAGCCTACAACCTAGGAGGTGCA GGGACTGAGGCTCAGGCCAAATCGCAACTCAGACCCAGTGAACCCAAGGCCTGA AGAGAATTTGGATTCATTTACCTTGTTTTGTGGGGACTGGAGAGACAAGTAAACT CTCAGAGTAACTGTCCCCTCTGACTACCATTTCTAAGGATGCCCCGGAGGCCCAG CTAGCCCCAGACTTCGGCCCCATGCGGCTCACCCGCTGCCAGGCTGCCCTGGCGG CCGCCATCACCCTCAACCTTCTGGTCCTCTTCTATGTCTCGTGGCTGCAGCACCAG CCTAGGAATTCCCGGGCCCGGGGGCCCCGTCGTGCCTCTGCTGCCGGCCCCCGTG TCACCGTCCTGGTGCGGGAGTTCGAGGCATTTGACAACGCGGTGCCCGAGCTGGT AGACTCCTTCCTGCAGCAAGACCCAGCCCAGCCCGTGGTGGTGGCAGCCGACAC GCTCCCCTACCCGCCCCTGGCCCTGCCCCGCATCCCCAACGTGCGTCTGGCGCTG CTCCAGCCCGCCCTGGACCGGCCAGCCGCAGCCTCGCGCCCGGAGACCTACGTG GCCACCGAGTTTGTGGCCCTAGTACCTGATGGGGCGCGGGCTGAGGCACCTGGC CTGCTGGAGCGCATGGTGGAGGCGCTCCGCGCAGGAAGCGCACGTCTGGTGGCC GCCCCGGTTGCCACGGCCAACCCTGCCAGGTGCCTGGCCCTGAACGTCAGCCTGC GAGAGTGGACCGCCCGCTATGGCGCAGCCCCCGCCGCGCCCCGCTGCGACGCCC TGGACGGAGATGCTGTGGTGCTCCTGCGCGCCCGCGACCTCTTCAACCTCTCGGC GCCCCTGGCCCGGCCGGTGGGCACCAGCCTCTTTCTGCAGACCGCCCTTCGCGGC TGGGCGGTGCAGCTGCTGGACTTGACCTTCGCCGCGGCGCGCCAGCCCCCGCTGG CCACGGCCCACGCGCGCTGGAAGGCTGAGCGCGAGGGACGCGCTCGGCGGGCG GCGCTGCTCCGCGCGCTGGGCATCCGCCTAGTGAGCTGGGAAGGCGGGCGGCTG GAGTGGTTCGGCTGCAACAAGGAGACCACGCGCTGCTTCGGAACCGTGGTGGGC GACACGCCCGCCTACCTCTACGAGGAGCGCTGGACGCCCCCCTGCTGCCTGCGCG CGCTGCGCGAGACCGCCCGCTATGTGGTGGGCGTGCTGGAGGCTGCGGGCGTGC GCTACTGGCTCGAGGGCGGCTCACTGCTGGGGGCCGCCCGCCACGGGGACATCA TCCCATGGGACTACGACGTGGACCTGGGCATCTACTTGGAGGACGTGGGCAACT GCGAGCAGCTGCGGGGGGCAGAGGCCGGCTCGGTGGTGGATGAGCGCGGCTTCG TATGGGAGAAGGCGGTCGAGGGCGACTTTTTCCGCGTGCAGTACAGCGAAAGCA ACCACTTGCACGTGGACCTGTGGCCCTTCTACCCCCGCAATGGCGTCATGACCAA GGACACGTGGCTGGACCACCGGCAGGATGTGGAGTTTCCCGAGCACTTCCTGCA GCCGCTGGTGCCCCTGCCCTTTGCCGGCTTCGTGGCGCAGGCGCCTAACAACTAC CGCCGCTTCCTGGAGCTCAAGTTCGGGCCCGGGGTCATCGAGAACCCCCAGTACC CCAACCCGGCACTGCTGAGTCTGACGGGAAGCGGCTGAAGCCCTGATAACCTCG CCTTTGTTTTTCGGGGGTCTGTCTGGATGTGGAGAAGCTCTGTGTGAGCGGTGAG GGGTGGAGGGATGTCGCGGAGAGGGGAAGGGGGAAACTGACCAAGAAAGAAAT TCTAAGGAGAGCATGAGAGAAGGCTGGCATTGGCAGGAGGAGAGCACCAGGAC GAGGATGGGAAGCGACCTCCAGATTTATCAAATGGTCATGCCCACTGGGAGCCG TGGATATGCGTGGGGACATCCTGGGTCATCTCAGTCATGGAGGGAGACGGGGAT GTCACGCCGTCCCGCAGGGCCCAGCACAGCCCCAGACCCGAAAAAAGTGTTCTG CCCAAGATTCCGAGAGCCCTGCGCTCTAGGGCAGGGGCAGAGTTTTGGAAACAG TGCAGGCTCTGGAGCCAGACTGGCGAGATTCAAATCCTGGCTCTATCGCTTCGGA GCCAGGTGGGCCTGGGGGGGCGTCGCAGTCTCTCTGTGCCTCAGTTGCTTCCAGG ATGCGGGACCCTTGGCTGCAGGGGTTGCTTCCGCCACTAGAGGGCGCGCCGGTC CCGCTCCTGGTGGCCCACTGTGGCTGCCCGGGCGACAGTACGCCCAGGGCCTGTG TTCCATAGCCATCTACTCTCTTGAGCCTTTGGACTTCTCTCCAAGCCCCTGTGGGA GGCGGACAGCAGTGACCACCTCCCCTTCTTTTGGACTGCGACCTCCTTCCCTCCT GGGAGAGCCCTGTGACCTGCATGCTACTCTTAACTGTTCTATTCAAGACTGAATA GAAGTATTTCAGTCTTGCAGAGGAGGAAATGCTCAGAGCTCCGAGGTGCGGCTG TGGTCGAGAACCGGGTGCTGGGCCGGGCGCGGGGGCTCACGCCTGTAATCCCAG CACTTTGGGAGGCCGAGGTGGGAGGATCGCTTGAGCCCAGGAGTCTGAGACCAG CCTCGGCAACATGCCAAGACCCCGTCTCTATTTTTAAAAAAGAAAAAGAACCGA CTTCTGAATCGCAGCTCCACTCATGACTAATACCTCATTATTTCAGCTGTCTGCAC CTAATTCCCCACTTGCACGGCAGTGTAGACAATAACCATAGCTCACACTCACTGA GCACCTACTGGGTACCAGGCACCATTCTCAGTGTTTCACCTGGATCAACTAATGC GTCCCTCACCTCAGCCCTCTGAAGTGACAGCTGCTATTATTTTCATTACACAGAT GAAAAAGCTGAGGCCAGAATCGTGAAGTCACTTGCTCAAGGTCAGGCAGCTTAG GAAGGGGCAGATCGGGGGCTTGAACCCAGGTGGTCAGGCTCTGGAGCCCACAAT TGTCTTACCCACTATGCCCCTCTCTAGTCATGGTCCCCAAGAGGGGCTTGGAGAC CCACTTAGCAGGTGAAAGCAATGGCAGCCTTCCTTATTTGATTATGCACCTAAGA ATAAATGGTATTTGGGCATGTATTCCCAATATGTGTATATTTATTTATAAATATAT ACAGATACTATTATCTGTATGTTAGTAATAAAGCTTAAATTATTCCATTTTAAAAT TATGAATATGAATAGGGTTTTTTTTATGTTTCTTGCCTCATCCCAATGACTTTTGC ACACCCAGGTGTGAGCACCCAGCATTCAAGACCACG [00646] SEQ ID NO.20 is FKRP, transcript variant X15, mRNA, XM_011527307.2 CATTGCTCCAAGATGGCGGCGGCGGCGGCAGCGGAGGAGATGCTCTGCTGAGGG GCAGTTGCTATGGTTACAGGGCCTGGACCTTCCTCCGGAAGGAGCGCAGCTCAG CTGGGCTGGAACTGCCCTCCTGGAACTCCCCCAGCCTACAACCTAGGAGGATGCC CCGGAGGCCCAGCTAGCCCCAGACTTCGGCCCCATGCGGCTCACCCGCTGCCAG GCTGCCCTGGCGGCCGCCATCACCCTCAACCTTCTGGTCCTCTTCTATGTCTCGTG GCTGCAGCACCAGCCTAGGAATTCCCGGGCCCGGGGGCCCCGTCGTGCCTCTGCT GCCGGCCCCCGTGTCACCGTCCTGGTGCGGGAGTTCGAGGCATTTGACAACGCG GTGCCCGAGCTGGTAGACTCCTTCCTGCAGCAAGACCCAGCCCAGCCCGTGGTG GTGGCAGCCGACACGCTCCCCTACCCGCCCCTGGCCCTGCCCCGCATCCCCAACG TGCGTCTGGCGCTGCTCCAGCCCGCCCTGGACCGGCCAGCCGCAGCCTCGCGCCC GGAGACCTACGTGGCCACCGAGTTTGTGGCCCTAGTACCTGATGGGGCGCGGGC TGAGGCACCTGGCCTGCTGGAGCGCATGGTGGAGGCGCTCCGCGCAGGAAGCGC ACGTCTGGTGGCCGCCCCGGTTGCCACGGCCAACCCTGCCAGGTGCCTGGCCCTG AACGTCAGCCTGCGAGAGTGGACCGCCCGCTATGGCGCAGCCCCCGCCGCGCCC CGCTGCGACGCCCTGGACGGAGATGCTGTGGTGCTCCTGCGCGCCCGCGACCTCT TCAACCTCTCGGCGCCCCTGGCCCGGCCGGTGGGCACCAGCCTCTTTCTGCAGAC CGCCCTTCGCGGCTGGGCGGTGCAGCTGCTGGACTTGACCTTCGCCGCGGCGCGC CAGCCCCCGCTGGCCACGGCCCACGCGCGCTGGAAGGCTGAGCGCGAGGGACGC GCTCGGCGGGCGGCGCTGCTCCGCGCGCTGGGCATCCGCCTAGTGAGCTGGGAA GGCGGGCGGCTGGAGTGGTTCGGCTGCAACAAGGAGACCACGCGCTGCTTCGGA ACCGTGGTGGGCGACACGCCCGCCTACCTCTACGAGGAGCGCTGGACGCCCCCC TGCTGCCTGCGCGCGCTGCGCGAGACCGCCCGCTATGTGGTGGGCGTGCTGGAG GCTGCGGGCGTGCGCTACTGGCTCGAGGGCGGCTCACTGCTGGGGGCCGCCCGC CACGGGGACATCATCCCATGGGACTACGACGTGGACCTGGGCATCTACTTGGAG GACGTGGGCAACTGCGAGCAGCTGCGGGGGGCAGAGGCCGGCTCGGTGGTGGAT GAGCGCGGCTTCGTATGGGAGAAGGCGGTCGAGGGCGACTTTTTCCGCGTGCAG TACAGCGAAAGCAACCACTTGCACGTGGACCTGTGGCCCTTCTACCCCCGCAATG GCGTCATGACCAAGGACACGTGGCTGGACCACCGGCAGGATGTGGAGTTTCCCG AGCACTTCCTGCAGCCGCTGGTGCCCCTGCCCTTTGCCGGCTTCGTGGCGCAGGC GCCTAACAACTACCGCCGCTTCCTGGAGCTCAAGTTCGGGCCCGGGGTCATCGAG AACCCCCAGTACCCCAACCCGGCACTGCTGAGTCTGACGGGAAGCGGCTGAAGC CCTGATAACCTCGCCTTTGTTTTTCGGGGGTCTGTCTGGATGTGGAGAAGCTCTGT GTGAGCGGTGAGGGGTGGAGGGATGTCGCGGAGAGGGGAAGGGGGAAACTGAC CAAGAAAGAAATTCTAAGGAGAGCATGAGAGAAGGCTGGCATTGGCAGGAGGA GAGCACCAGGACGAGGATGGGAAGCGACCTCCAGATTTATCAAATGGTCATGCC CACTGGGAGCCGTGGATATGCGTGGGGACATCCTGGGTCATCTCAGTCATGGAG GGAGACGGGGATGTCACGCCGTCCCGCAGGGCCCAGCACAGCCCCAGACCCGAA AAAAGTGTTCTGCCCAAGATTCCGAGAGCCCTGCGCTCTAGGGCAGGGGCAGAG TTTTGGAAACAGTGCAGGCTCTGGAGCCAGACTGGCGAGATTCAAATCCTGGCTC TATCGCTTCGGAGCCAGGTGGGCCTGGGGGGGCGTCGCAGTCTCTCTGTGCCTCA GTTGCTTCCAGGATGCGGGACCCTTGGCTGCAGGGGTTGCTTCCGCCACTAGAGG GCGCGCCGGTCCCGCTCCTGGTGGCCCACTGTGGCTGCCCGGGCGACAGTACGCC CAGGGCCTGTGTTCCATAGCCATCTACTCTCTTGAGCCTTTGGACTTCTCTCCAAG CCCCTGTGGGAGGCGGACAGCAGTGACCACCTCCCCTTCTTTTGGACTGCGACCT CCTTCCCTCCTGGGAGAGCCCTGTGACCTGCATGCTACTCTTAACTGTTCTATTCA AGACTGAATAGAAGTATTTCAGTCTTGCAGAGGAGGAAATGCTCAGAGCTCCGA GGTGCGGCTGTGGTCGAGAACCGGGTGCTGGGCCGGGCGCGGGGGCTCACGCCT GTAATCCCAGCACTTTGGGAGGCCGAGGTGGGAGGATCGCTTGAGCCCAGGAGT CTGAGACCAGCCTCGGCAACATGCCAAGACCCCGTCTCTATTTTTAAAAAAGAA AAAGAACCGACTTCTGAATCGCAGCTCCACTCATGACTAATACCTCATTATTTCA GCTGTCTGCACCTAATTCCCCACTTGCACGGCAGTGTAGACAATAACCATAGCTC ACACTCACTGAGCACCTACTGGGTACCAGGCACCATTCTCAGTGTTTCACCTGGA TCAACTAATGCGTCCCTCACCTCAGCCCTCTGAAGTGACAGCTGCTATTATTTTCA TTACACAGATGAAAAAGCTGAGGCCAGAATCGTGAAGTCACTTGCTCAAGGTCA GGCAGCTTAGGAAGGGGCAGATCGGGGGCTTGAACCCAGGTGGTCAGGCTCTGG AGCCCACAATTGTCTTACCCACTATGCCCCTCTCTAGTCATGGTCCCCAAGAGGG GCTTGGAGACCCACTTAGCAGGTGAAAGCAATGGCAGCCTTCCTTATTTGATTAT GCACCTAAGAATAAATGGTATTTGGGCATGTATTCCCAATATGTGTATATTTATTT ATAAATATATACAGATACTATTATCTGTATGTTAGTAATAAAGCTTAAATTATTC CATTTTAAAATTATGAATATGAATAGGGTTTTTTTTATGTTTCTTGCCTCATCCCA ATGACTTTTGCACACCCAGGTGTGAGCACCCAGCATTCAAGACCACG [00647] SEQ ID NO.21 is FKRP, transcript variant X1, mRNA XM_017027297.3 CATTGCTCCAAGATGGCGGCGGCGGCGGCAGCGGGAGCGCAGCTCAGCTGGGCT GGAACTGCCCTCCTGGAACTCCCCCAGCCTACAACCTAGGAGGTGCAGGGACTG AGGCTCAGGCCAAATCGCAACTCAGACCCAGTGAACCCAAGGCCTGAAGAGAAT TTGGATTCATTTACCTTGTTTTGTGGGGACTGGAGAGACAAGTAAACTCTCAGAG TAACTGTCCCCTCTGACTACCATTTCTAAGGCAAGCCCCCTGTTTCTACTCTTGCG CCCCCTGCTGGTTTCCTGCCCTGTCTGTAAGTTGCATGGCTTTTGTCCGTCTTTTTT TTGTTTGTTTGTTTGTTTTGAGACAGGGTCTCACCCAGGCTAGAGTGAAGTGGAG CAATCTCGGCTCACTGCAACCTCCGCCTCCTGAGTTCAAGTGATTCTCACACCTC AGCCTCCCCAATAGCTGGGATTACAGGATGCCCCGGAGGCCCAGCTAGCCCCAG ACTTCGGCCCCATGCGGCTCACCCGCTGCCAGGCTGCCCTGGCGGCCGCCATCAC CCTCAACCTTCTGGTCCTCTTCTATGTCTCGTGGCTGCAGCACCAGCCTAGGAATT CCCGGGCCCGGGGGCCCCGTCGTGCCTCTGCTGCCGGCCCCCGTGTCACCGTCCT GGTGCGGGAGTTCGAGGCATTTGACAACGCGGTGCCCGAGCTGGTAGACTCCTT CCTGCAGCAAGACCCAGCCCAGCCCGTGGTGGTGGCAGCCGACACGCTCCCCTA CCCGCCCCTGGCCCTGCCCCGCATCCCCAACGTGCGTCTGGCGCTGCTCCAGCCC GCCCTGGACCGGCCAGCCGCAGCCTCGCGCCCGGAGACCTACGTGGCCACCGAG TTTGTGGCCCTAGTACCTGATGGGGCGCGGGCTGAGGCACCTGGCCTGCTGGAGC GCATGGTGGAGGCGCTCCGCGCAGGAAGCGCACGTCTGGTGGCCGCCCCGGTTG CCACGGCCAACCCTGCCAGGTGCCTGGCCCTGAACGTCAGCCTGCGAGAGTGGA CCGCCCGCTATGGCGCAGCCCCCGCCGCGCCCCGCTGCGACGCCCTGGACGGAG ATGCTGTGGTGCTCCTGCGCGCCCGCGACCTCTTCAACCTCTCGGCGCCCCTGGC CCGGCCGGTGGGCACCAGCCTCTTTCTGCAGACCGCCCTTCGCGGCTGGGCGGTG CAGCTGCTGGACTTGACCTTCGCCGCGGCGCGCCAGCCCCCGCTGGCCACGGCCC ACGCGCGCTGGAAGGCTGAGCGCGAGGGACGCGCTCGGCGGGCGGCGCTGCTCC GCGCGCTGGGCATCCGCCTAGTGAGCTGGGAAGGCGGGCGGCTGGAGTGGTTCG GCTGCAACAAGGAGACCACGCGCTGCTTCGGAACCGTGGTGGGCGACACGCCCG CCTACCTCTACGAGGAGCGCTGGACGCCCCCCTGCTGCCTGCGCGCGCTGCGCGA GACCGCCCGCTATGTGGTGGGCGTGCTGGAGGCTGCGGGCGTCGCTACTGGCTC GAGGGCGGCTCACTGCTGGGGGCCGCCCGCCACGGGGACATCATCCCATGGGAC TACGACGTGGACCTGGGCATCTACTTGGAGGACGTGGGCAACTGCGAGCAGCTG CGGGGGGCAGAGGCCGGCTCGGTGGTGGATGAGCGCGGCTTCGTATGGGAGAAG GCGGTCGAGGGCGACTTTTTCCGCGTGCAGTACAGCGAAAGCAACCACTTGCAC GTGGACCTGTGGCCCTTCTACCCCCGCAATGGCGTCATGACCAAGGACACGTGGC TGGACCACCGGCAGGATGTGGAGTTTCCCGAGCACTTCCTGCAGCCGCTGGTGCC CCTGCCCTTTGCCGGCTTCGTGGCGCAGGCGCCTAACAACTACCGCCGCTTCCTG GAGCTCAAGTTCGGGCCCGGGGTCATCGAGAACCCCCAGTACCCCAACCCGGCA CTGCTGAGTCTGACGGGAAGCGGCTGAAGCCCTGATAACCTCGCCTTTGTTTTTC GGGGGTCTGTCTGGATGTGGAGAAGCTCTGTGTGAGCGGTGAGGGGTGGAGGGA TGTCGCGGAGAGGGGAAGGGGGAAACTGACCAAGAAAGAAATTCTAAGGAGAG CATGAGAGAAGGCTGGCATTGGCAGGAGGAGAGCACCAGGACGAGGATGGGAA GCGACCTCCAGATTTATCAAATGGTCATGCCCACTGGGAGCCGTGGATATGCGTG GGGACATCCTGGGTCATCTCAGTCATGGAGGGAGACGGGGATGTCACGCCGTCC CGCAGGGCCCAGCACAGCCCCAGACCCGAAAAAAGTGTTCTGCCCAAGATTCCG AGAGCCCTGCGCTCTAGGGCAGGGGCAGAGTTTTGGAAACAGTGCAGGCTCTGG AGCCAGACTGGCGAGATTCAAATCCTGGCTCTATCGCTTCGGAGCCAGGTGGGC CTGGGGGGGCGTCGCAGTCTCTCTGTGCCTCAGTTGCTTCCAGGATGCGGGACCC TTGGCTGCAGGGGTTGCTTCCGCCACTAGAGGGCGCGCCGGTCCCGCTCCTGGTG GCCCACTGTGGCTGCCCGGGCGACAGTACGCCCAGGGCCTGTGTTCCATAGCCAT CTACTCTCTTGAGCCTTTGGACTTCTCTCCAAGCCCCTGTGGGAGGCGGACAGCA GTGACCACCTCCCCTTCTTTTGGACTGCGACCTCCTTCCCTCCTGGGAGAGCCCTG TGACCTGCATGCTACTCTTAACTGTTCTATTCAAGACTGAATAGAAGTATTTCAGT CTTGCAGAGGAGGAAATGCTCAGAGCTCCGAGGTGCGGCTGTGGTCGAGAACCG GGTGCTGGGCCGGGCGCGGGGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGC CGAGGTGGGAGGATCGCTTGAGCCCAGGAGTCTGAGACCAGCCTCGGCAACATG CCAAGACCCCGTCTCTATTTTTAAAAAAGAAAAAGAACCGACTTCTGAATCGCA GCTCCACTCATGACTAATACCTCATTATTTCAGCTGTCTGCACCTAATTCCCCACT TGCACGGCAGTGTAGACAATAACCATAGCTCACACTCACTGAGCACCTACTGGG TACCAGGCACCATTCTCAGTGTTTCACCTGGATCAACTAATGCGTCCCTCACCTC AGCCCTCTGAAGTGACAGCTGCTATTATTTTCATTACACAGATGAAAAAGCTGAG GCCAGAATCGTGAAGTCACTTGCTCAAGGTCAGGCAGCTTAGGAAGGGGCAGAT CGGGGGCTTGAACCCAGGTGGTCAGGCTCTGGAGCCCACAATTGTCTTACCCACT ATGCCCCTCTCTAGTCATGGTCCCCAAGAGGGGCTTGGAGACCCACTTAGCAGGT GAAAGCAATGGCAGCCTTCCTTATTTGATTATGCACCTAAGAATAAATGGTATTT GGGCATGTATTCCCAATATGTGTATATTTATTTATAAATATATACAGATACTATTA TCTGTATGTTAGTAATAAAGCTTAAATTATTCCATTTTAAAATTATGAATATGAAT AGGGTTTTTTTTATGTTTCTTGCCTCATCCCAATGACTTTTGCACACCCAGGTGTG AGCACCCAGCATTCAAGACCACG [00648] SEQ ID NO.22 is FKRP, transcript variant X14, mRNA, XM_011527306.3 CATTGCTCCAAGATGGCGGCGGCGGCGGCAGCGGGAGCGCAGCTCAGCTGGGCT GGAACTGCCCTCCTGGAACTCCCCCAGCCTACAACCTAGGAGGATGCCCCGGAG GCCCAGCTAGCCCCAGACTTCGGCCCCATGCGGCTCACCCGCTGCCAGGCTGCCC TGGCGGCCGCCATCACCCTCAACCTTCTGGTCCTCTTCTATGTCTCGTGGCTGCAG CACCAGCCTAGGAATTCCCGGGCCCGGGGGCCCCGTCGTGCCTCTGCTGCCGGCC CCCGTGTCACCGTCCTGGTGCGGGAGTTCGAGGCATTTGACAACGCGGTGCCCGA GCTGGTAGACTCCTTCCTGCAGCAAGACCCAGCCCAGCCCGTGGTGGTGGCAGC CGACACGCTCCCCTACCCGCCCCTGGCCCTGCCCCGCATCCCCAACGTGCGTCTG GCGCTGCTCCAGCCCGCCCTGGACCGGCCAGCCGCAGCCTCGCGCCCGGAGACC TACGTGGCCACCGAGTTTGTGGCCCTAGTACCTGATGGGGCGCGGGCTGAGGCA CCTGGCCTGCTGGAGCGCATGGTGGAGGCGCTCCGCGCAGGAAGCGCACGTCTG GTGGCCGCCCCGGTTGCCACGGCCAACCCTGCCAGGTGCCTGGCCCTGAACGTCA GCCTGCGAGAGTGGACCGCCCGCTATGGCGCAGCCCCCGCCGCGCCCCGCTGCG ACGCCCTGGACGGAGATGCTGTGGTGCTCCTGCGCGCCCGCGACCTCTTCAACCT CTCGGCGCCCCTGGCCCGGCCGGTGGGCACCAGCCTCTTTCTGCAGACCGCCCTT CGCGGCTGGGCGGTGCAGCTGCTGGACTTGACCTTCGCCGCGGCGCGCCAGCCC CCGCTGGCCACGGCCCACGCGCGCTGGAAGGCTGAGCGCGAGGGACGCGCTCGG CGGGCGGCGCTGCTCCGCGCGCTGGGCATCCGCCTAGTGAGCTGGGAAGGCGGG CGGCTGGAGTGGTTCGGCTGCAACAAGGAGACCACGCGCTGCTTCGGAACCGTG GTGGGCGACACGCCCGCCTACCTCTACGAGGAGCGCTGGACGCCCCCCTGCTGC CTGCGCGCGCTGCGCGAGACCGCCCGCTATGTGGTGGGCGTGCTGGAGGCTGCG GGCGTGCGCTACTGGCTCGAGGGCGGCTCACTGCTGGGGGCCGCCCGCCACGGG GACATCATCCCATGGGACTACGACGTGGACCTGGGCATCTACTTGGAGGACGTG GGCAACTGCGAGCAGCTGCGGGGGGCAGAGGCCGGCTCGGTGGTGGATGAGCG CGGCTTCGTATGGGAGAAGGCGGTCGAGGGCGACTTTTTCCGCGTGCAGTACAG CGAAAGCAACCACTTGCACGTGGACCTGTGGCCCTTCTACCCCCGCAATGGCGTC ATGACCAAGGACACGTGGCTGGACCACCGGCAGGATGTGGAGTTTCCCGAGCAC TTCCTGCAGCCGCTGGTGCCCCTGCCCTTTGCCGGCTTCGTGGCGCAGGCGCCTA ACAACTACCGCCGCTTCCTGGAGCTCAAGTTCGGGCCCGGGGTCATCGAGAACC CCCAGTACCCCAACCCGGCACTGCTGAGTCTGACGGGAAGCGGCTGAAGCCCTG ATAACCTCGCCTTTGTTTTTCGGGGGTCTGTCTGGATGTGGAGAAGCTCTGTGTG AGCGGTGAGGGGTGGAGGGATGTCGCGGAGAGGGGAAGGGGGAAACTGACCAA GAAAGAAATTCTAAGGAGAGCATGAGAGAAGGCTGGCATTGGCAGGAGGAGAG CACCAGGACGAGGATGGGAAGCGACCTCCAGATTTATCAAATGGTCATGCCCAC TGGGAGCCGTGGATATGCGTGGGGACATCCTGGGTCATCTCAGTCATGGAGGGA GACGGGGATGTCACGCCGTCCCGCAGGGCCCAGCACAGCCCCAGACCCGAAAAA AGTGTTCTGCCCAAGATTCCGAGAGCCCTGCGCTCTAGGGCAGGGGCAGAGTTTT GGAAACAGTGCAGGCTCTGGAGCCAGACTGGCGAGATTCAAATCCTGGCTCTAT CGCTTCGGAGCCAGGTGGGCCTGGGGGGGCGTCGCAGTCTCTCTGTGCCTCAGTT GCTTCCAGGATGCGGGACCCTTGGCTGCAGGGGTTGCTTCCGCCACTAGAGGGC GCGCCGGTCCCGCTCCTGGTGGCCCACTGTGGCTGCCCGGGCGACAGTACGCCCA GGGCCTGTGTTCCATAGCCATCTACTCTCTTGAGCCTTTGGACTTCTCTCCAAGCC CCTGTGGGAGGCGGACAGCAGTGACCACCTCCCCTTCTTTTGGACTGCGACCTCC TTCCCTCCTGGGAGAGCCCTGTGACCTGCATGCTACTCTTAACTGTTCTATTCAAG ACTGAATAGAAGTATTTCAGTCTTGCAGAGGAGGAAATGCTCAGAGCTCCGAGG TGCGGCTGTGGTCGAGAACCGGGTGCTGGGCCGGGCGCGGGGGCTCACGCCTGT AATCCCAGCACTTTGGGAGGCCGAGGTGGGAGGATCGCTTGAGCCCAGGAGTCT GAGACCAGCCTCGGCAACATGCCAAGACCCCGTCTCTATTTTTAAAAAAGAAAA AGAACCGACTTCTGAATCGCAGCTCCACTCATGACTAATACCTCATTATTTCAGC TGTCTGCACCTAATTCCCCACTTGCACGGCAGTGTAGACAATAACCATAGCTCAC ACTCACTGAGCACCTACTGGGTACCAGGCACCATTCTCAGTGTTTCACCTGGATC AACTAATGCGTCCCTCACCTCAGCCCTCTGAAGTGACAGCTGCTATTATTTTCATT ACACAGATGAAAAAGCTGAGGCCAGAATCGTGAAGTCACTTGCTCAAGGTCAGG CAGCTTAGGAAGGGGCAGATCGGGGGCTTGAACCCAGGTGGTCAGGCTCTGGAG CCCACAATTGTCTTACCCACTATGCCCCTCTCTAGTCATGGTCCCCAAGAGGGGC TTGGAGACCCACTTAGCAGGTGAAAGCAATGGCAGCCTTCCTTATTTGATTATGC ACCTAAGAATAAATGGTATTTGGGCATGTATTCCCAATATGTGTATATTTATTTAT AAATATATACAGATACTATTATCTGTATGTTAGTAATAAAGCTTAAATTATTCCA TTTTAAAATTATGAATATGAATAGGGTTTTTTTTATGTTTCTTGCCTCATCCCAAT GACTTTTGCACACCCAGGTGTGAGCACCCAGCATTCAAGACCACG [00649] SEQ ID NO.23 is FKRP, transcript variant X12, mRNA XM_047439428.1 GGGGACAGTAGGAAGGGGGGCCGGCGGCGAGCGCGAGCCTCGCCCACCCGGGT CTACACCGAAGGGAGCGCAGCTCAGCTGGGCTGGAACTGCCCTCCTGGAACTCC CCCAGCCTACAACCTAGGAGGTGCAGGGACTGAGGCTCAGGCCAAATCGCAACT CAGACCCAGTGAACCCAAGGCCTGAAGAGAATTTGGATTCATTTACCTTGTTTTG TGGGGACTGGAGAGACAAGTAAACTCTCAGAGTAACTGTCCCCTCTGACTACCA TTTCTAAGGATGCCCCGGAGGCCCAGCTAGCCCCAGACTTCGGCCCCATGCGGCT CACCCGCTGCCAGGCTGCCCTGGCGGCCGCCATCACCCTCAACCTTCTGGTCCTC TTCTATGTCTCGTGGCTGCAGCACCAGCCTAGGAATTCCCGGGCCCGGGGGCCCC GTCGTGCCTCTGCTGCCGGCCCCCGTGTCACCGTCCTGGTGCGGGAGTTCGAGGC ATTTGACAACGCGGTGCCCGAGCTGGTAGACTCCTTCCTGCAGCAAGACCCAGCC CAGCCCGTGGTGGTGGCAGCCGACACGCTCCCCTACCCGCCCCTGGCCCTGCCCC GCATCCCCAACGTGCGTCTGGCGCTGCTCCAGCCCGCCCTGGACCGGCCAGCCGC AGCCTCGCGCCCGGAGACCTACGTGGCCACCGAGTTTGTGGCCCTAGTACCTGAT GGGGCGCGGGCTGAGGCACCTGGCCTGCTGGAGCGCATGGTGGAGGCGCTCCGC GCAGGAAGCGCACGTCTGGTGGCCGCCCCGGTTGCCACGGCCAACCCTGCCAGG TGCCTGGCCCTGAACGTCAGCCTGCGAGAGTGGACCGCCCGCTATGGCGCAGCC CCCGCCGCGCCCCGCTGCGACGCCCTGGACGGAGATGCTGTGGTGCTCCTGCGCG CCCGCGACCTCTTCAACCTCTCGGCGCCCCTGGCCCGGCCGGTGGGCACCAGCCT CTTTCTGCAGACCGCCCTTCGCGGCTGGGCGGTGCAGCTGCTGGACTTGACCTTC GCCGCGGCGCGCCAGCCCCCGCTGGCCACGGCCCACGCGCGCTGGAAGGCTGAG CGCGAGGGACGCGCTCGGCGGGCGGCGCTGCTCCGCGCGCTGGGCATCCGCCTA GTGAGCTGGGAAGGCGGGCGGCTGGAGTGGTTCGGCTGCAACAAGGAGACCAC GCGCTGCTTCGGAACCGTGGTGGGCGACACGCCCGCCTACCTCTACGAGGAGCG CTGGACGCCCCCCTGCTGCCTGCGCGCGCTGCGCGAGACCGCCCGCTATGTGGTG GGCGTGCTGGAGGCTGCGGGCGTGCGCTACTGGCTCGAGGGCGGCTCACTGCTG GGGGCCGCCCGCCACGGGGACATCATCCCATGGGACTACGACGTGGACCTGGGC ATCTACTTGGAGGACGTGGGCAACTGCGAGCAGCTGCGGGGGGCAGAGGCCGGC TCGGTGGTGGATGAGCGCGGCTTCGTATGGGAGAAGGCGGTCGAGGGCGACTTT TTCCGCGTGCAGTACAGCGAAAGCAACCACTTGCACGTGGACCTGTGGCCCTTCT ACCCCCGCAATGGCGTCATGACCAAGGACACGTGGCTGGACCACCGGCAGGATG TGGAGTTTCCCGAGCACTTCCTGCAGCCGCTGGTGCCCCTGCCCTTTGCCGGCTTC GTGGCGCAGGCGCCTAACAACTACCGCCGCTTCCTGGAGCTCAAGTTCGGGCCC GGGGTCATCGAGAACCCCCAGTACCCCAACCCGGCACTGCTGAGTCTGACGGGA AGCGGCTGAAGCCCTGATAACCTCGCCTTTGTTTTTCGGGGGTCTGTCTGGATGT GGAGAAGCTCTGTGTGAGCGGTGAGGGGTGGAGGGATGTCGCGGAGAGGGGAA GGGGGAAACTGACCAAGAAAGAAATTCTAAGGAGAGCATGAGAGAAGGCTGGC ATTGGCAGGAGGAGAGCACCAGGACGAGGATGGGAAGCGACCTCCAGATTTATC AAATGGTCATGCCCACTGGGAGCCGTGGATATGCGTGGGGACATCCTGGGTCAT CTCAGTCATGGAGGGAGACGGGGATGTCACGCCGTCCCGCAGGGCCCAGCACAG CCCCAGACCCGAAAAAAGTGTTCTGCCCAAGATTCCGAGAGCCCTGCGCTCTAG GGCAGGGGCAGAGTTTTGGAAACAGTGCAGGCTCTGGAGCCAGACTGGCGAGAT TCAAATCCTGGCTCTATCGCTTCGGAGCCAGGTGGGCCTGGGGGGGCGTCGCAGT CTCTCTGTGCCTCAGTTGCTTCCAGGATGCGGGACCCTTGGCTGCAGGGGTTGCT TCCGCCACTAGAGGGCGCGCCGGTCCCGCTCCTGGTGGCCCACTGTGGCTGCCCG GGCGACAGTACGCCCAGGGCCTGTGTTCCATAGCCATCTACTCTCTTGAGCCTTT GGACTTCTCTCCAAGCCCCTGTGGGAGGCGGACAGCAGTGACCACCTCCCCTTCT TTTGGACTGCGACCTCCTTCCCTCCTGGGAGAGCCCTGTGACCTGCATGCTACTCT TAACTGTTCTATTCAAGACTGAATAGAAGTATTTCAGTCTTGCAGAGGAGGAAAT GCTCAGAGCTCCGAGGTGCGGCTGTGGTCGAGAACCGGGTGCTGGGCCGGGCGC GGGGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGAGGATCGC TTGAGCCCAGGAGTCTGAGACCAGCCTCGGCAACATGCCAAGACCCCGTCTCTAT TTTTAAAAAAGAAAAAGAACCGACTTCTGAATCGCAGCTCCACTCATGACTAAT ACCTCATTATTTCAGCTGTCTGCACCTAATTCCCCACTTGCACGGCAGTGTAGAC AATAACCATAGCTCACACTCACTGAGCACCTACTGGGTACCAGGCACCATTCTCA GTGTTTCACCTGGATCAACTAATGCGTCCCTCACCTCAGCCCTCTGAAGTGACAG CTGCTATTATTTTCATTACACAGATGAAAAAGCTGAGGCCAGAATCGTGAAGTCA CTTGCTCAAGGTCAGGCAGCTTAGGAAGGGGCAGATCGGGGGCTTGAACCCAGG TGGTCAGGCTCTGGAGCCCACAATTGTCTTACCCACTATGCCCCTCTCTAGTCAT GGTCCCCAAGAGGGGCTTGGAGACCCACTTAGCAGGTGAAAGCAATGGCAGCCT TCCTTATTTGATTATGCACCTAAGAATAAATGGTATTTGGGCATGTATTCCCAATA TGTGTATATTTATTTATAAATATATACAGATACTATTATCTGTATGTTAGTAATAA AGCTTAAATTATTCCATTTTAAAATTATGAATATGAATAGGGTTTTTTTTATGTTT CTTGCCTCATCCCAATGACTTTTGCACACCCAGGTGTGAGCACCCAGCATTCAAG ACCACG [00650] SEQ ID NO.24 is FKRP, transcript variant X16, mRNA XM_047439429.1 GGGGCCGGCGGCGAGCGCGAGCCTCGCCCACCCGGGTCTACACCGAAGGGAGCG CAGCTCAGCTGGGCTGGAACTGCCCTCCTGGAACTCCCCCAGCCTACAACCTAGG AGGATGCCCCGGAGGCCCAGCTAGCCCCAGACTTCGGCCCCATGCGGCTCACCC GCTGCCAGGCTGCCCTGGCGGCCGCCATCACCCTCAACCTTCTGGTCCTCTTCTAT GTCTCGTGGCTGCAGCACCAGCCTAGGAATTCCCGGGCCCGGGGGCCCCGTCGT GCCTCTGCTGCCGGCCCCCGTGTCACCGTCCTGGTGCGGGAGTTCGAGGCATTTG ACAACGCGGTGCCCGAGCTGGTAGACTCCTTCCTGCAGCAAGACCCAGCCCAGC CCGTGGTGGTGGCAGCCGACACGCTCCCCTACCCGCCCCTGGCCCTGCCCCGCAT CCCCAACGTGCGTCTGGCGCTGCTCCAGCCCGCCCTGGACCGGCCAGCCGCAGCC TCGCGCCCGGAGACCTACGTGGCCACCGAGTTTGTGGCCCTAGTACCTGATGGGG CGCGGGCTGAGGCACCTGGCCTGCTGGAGCGCATGGTGGAGGCGCTCCGCGCAG GAAGCGCACGTCTGGTGGCCGCCCCGGTTGCCACGGCCAACCCTGCCAGGTGCC TGGCCCTGAACGTCAGCCTGCGAGAGTGGACCGCCCGCTATGGCGCAGCCCCCG CCGCGCCCCGCTGCGACGCCCTGGACGGAGATGCTGTGGTGCTCCTGCGCGCCCG CGACCTCTTCAACCTCTCGGCGCCCCTGGCCCGGCCGGTGGGCACCAGCCTCTTT CTGCAGACCGCCCTTCGCGGCTGGGCGGTGCAGCTGCTGGACTTGACCTTCGCCG CGGCGCGCCAGCCCCCGCTGGCCACGGCCCACGCGCGCTGGAAGGCTGAGCGCG AGGGACGCGCTCGGCGGGCGGCGCTGCTCCGCGCGCTGGGCATCCGCCTAGTGA GCTGGGAAGGCGGGCGGCTGGAGTGGTTCGGCTGCAACAAGGAGACCACGCGCT GCTTCGGAACCGTGGTGGGCGACACGCCCGCCTACCTCTACGAGGAGCGCTGGA CGCCCCCCTGCTGCCTGCGCGCGCTGCGCGAGACCGCCCGCTATGTGGTGGGCGT GCTGGAGGCTGCGGGCGTGCGCTACTGGCTCGAGGGCGGCTCACTGCTGGGGGC CGCCCGCCACGGGGACATCATCCCATGGGACTACGACGTGGACCTGGGCATCTA CTTGGAGGACGTGGGCAACTGCGAGCAGCTGCGGGGGGCAGAGGCCGGCTCGGT GGTGGATGAGCGCGGCTTCGTATGGGAGAAGGCGGTCGAGGGCGACTTTTTCCG CGTGCAGTACAGCGAAAGCAACCACTTGCACGTGGACCTGTGGCCCTTCTACCCC CGCAATGGCGTCATGACCAAGGACACGTGGCTGGACCACCGGCAGGATGTGGAG TTTCCCGAGCACTTCCTGCAGCCGCTGGTGCCCCTGCCCTTTGCCGGCTTCGTGGC GCAGGCGCCTAACAACTACCGCCGCTTCCTGGAGCTCAAGTTCGGGCCCGGGGT CATCGAGAACCCCCAGTACCCCAACCCGGCACTGCTGAGTCTGACGGGAAGCGG CTGAAGCCCTGATAACCTCGCCTTTGTTTTTCGGGGGTCTGTCTGGATGTGGAGA AGCTCTGTGTGAGCGGTGAGGGGTGGAGGGATGTCGCGGAGAGGGGAAGGGGG AAACTGACCAAGAAAGAAATTCTAAGGAGAGCATGAGAGAAGGCTGGCATTGG CAGGAGGAGAGCACCAGGACGAGGATGGGAAGCGACCTCCAGATTTATCAAAT GGTCATGCCCACTGGGAGCCGTGGATATGCGTGGGGACATCCTGGGTCATCTCA GTCATGGAGGGAGACGGGGATGTCACGCCGTCCCGCAGGGCCCAGCACAGCCCC AGACCCGAAAAAAGTGTTCTGCCCAAGATTCCGAGAGCCCTGCGCTCTAGGGCA GGGGCAGAGTTTTGGAAACAGTGCAGGCTCTGGAGCCAGACTGGCGAGATTCAA ATCCTGGCTCTATCGCTTCGGAGCCAGGTGGGCCTGGGGGGGCGTCGCAGTCTCT CTGTGCCTCAGTTGCTTCCAGGATGCGGGACCCTTGGCTGCAGGGGTTGCTTCCG CCACTAGAGGGCGCGCCGGTCCCGCTCCTGGTGGCCCACTGTGGCTGCCCGGGC GACAGTACGCCCAGGGCCTGTGTTCCATAGCCATCTACTCTCTTGAGCCTTTGGA CTTCTCTCCAAGCCCCTGTGGGAGGCGGACAGCAGTGACCACCTCCCCTTCTTTT GGACTGCGACCTCCTTCCCTCCTGGGAGAGCCCTGTGACCTGCATGCTACTCTTA ACTGTTCTATTCAAGACTGAATAGAAGTATTTCAGTCTTGCAGAGGAGGAAATGC TCAGAGCTCCGAGGTGCGGCTGTGGTCGAGAACCGGGTGCTGGGCCGGGCGCGG GGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGAGGATCGCTT GAGCCCAGGAGTCTGAGACCAGCCTCGGCAACATGCCAAGACCCCGTCTCTATTT TTAAAAAAGAAAAAGAACCGACTTCTGAATCGCAGCTCCACTCATGACTAATAC CTCATTATTTCAGCTGTCTGCACCTAATTCCCCACTTGCACGGCAGTGTAGACAA TAACCATAGCTCACACTCACTGAGCACCTACTGGGTACCAGGCACCATTCTCAGT GTTTCACCTGGATCAACTAATGCGTCCCTCACCTCAGCCCTCTGAAGTGACAGCT GCTATTATTTTCATTACACAGATGAAAAAGCTGAGGCCAGAATCGTGAAGTCACT TGCTCAAGGTCAGGCAGCTTAGGAAGGGGCAGATCGGGGGCTTGAACCCAGGTG GTCAGGCTCTGGAGCCCACAATTGTCTTACCCACTATGCCCCTCTCTAGTCATGG TCCCCAAGAGGGGCTTGGAGACCCACTTAGCAGGTGAAAGCAATGGCAGCCTTC CTTATTTGATTATGCACCTAAGAATAAATGGTATTTGGGCATGTATTCCCAATAT GTGTATATTTATTTATAAATATATACAGATACTATTATCTGTATGTTAGTAATAAA GCTTAAATTATTCCATTTTAAAATTATGAATATGAATAGGGTTTTTTTTATGTTTC TTGCCTCATCCCAATGACTTTTGCACACCCAGGTGTGAGCACCCAGCATTCAAGA CCACG [00651] SEQ ID NO.25 is FKRP, transcript variant X11, mRNA XM_047439427.1 CGCCCACCCGGGTCTACACCGAAGGGAGCGCAGCTCAGCTGGGCTGGAACTGCC CTCCTGGAACTCCCCCAGCCTACAACCTAGGAGGTGCAGGGACTGAGGCTCAGG CCAAATCGCAACTCAGACCCAGTGAACCCAAGGCCTGAAGAGAATTTGGATTCA TTTACCTTGTTTTGTGGGGACTGGAGAGACAAGTAAACTCTCAGAGTAACTGTCC CCTCTGACTACCATTTCTAAGGCAAGCCCCCTGTTTCTACTCTTGCGCCCCCTGCT GGTTTCCTGCCCTGTCTGTAAGTTGCATGGCTTTTGTCCGTCTTTTTTTTGTTTGTT TGTTTGTTTTGAGACAGGGTCTCACCCAGGCTAGAGTGAAGTGGAGCAATCTCGG CTCACTGCAACCTCCGCCTCCTGAGTTCAAGTGATTCTCACACCTCAGCCTCCCC AATAGCTGGGATTACAGGATGCCCCGGAGGCCCAGCTAGCCCCAGACTTCGGCC CCATGCGGCTCACCCGCTGCCAGGCTGCCCTGGCGGCCGCCATCACCCTCAACCT TCTGGTCCTCTTCTATGTCTCGTGGCTGCAGCACCAGCCTAGGAATTCCCGGGCC CGGGGGCCCCGTCGTGCCTCTGCTGCCGGCCCCCGTGTCACCGTCCTGGTGCGGG AGTTCGAGGCATTTGACAACGCGGTGCCCGAGCTGGTAGACTCCTTCCTGCAGCA AGACCCAGCCCAGCCCGTGGTGGTGGCAGCCGACACGCTCCCCTACCCGCCCCT GGCCCTGCCCCGCATCCCCAACGTGCGTCTGGCGCTGCTCCAGCCCGCCCTGGAC CGGCCAGCCGCAGCCTCGCGCCCGGAGACCTACGTGGCCACCGAGTTTGTGGCC CTAGTACCTGATGGGGCGCGGGCTGAGGCACCTGGCCTGCTGGAGCGCATGGTG GAGGCGCTCCGCGCAGGAAGCGCACGTCTGGTGGCCGCCCCGGTTGCCACGGCC AACCCTGCCAGGTGCCTGGCCCTGAACGTCAGCCTGCGAGAGTGGACCGCCCGC TATGGCGCAGCCCCCGCCGCGCCCCGCTGCGACGCCCTGGACGGAGATGCTGTG GTGCTCCTGCGCGCCCGCGACCTCTTCAACCTCTCGGCGCCCCTGGCCCGGCCGG TGGGCACCAGCCTCTTTCTGCAGACCGCCCTTCGCGGCTGGGCGGTGCAGCTGCT GGACTTGACCTTCGCCGCGGCGCGCCAGCCCCCGCTGGCCACGGCCCACGCGCG CTGGAAGGCTGAGCGCGAGGGACGCGCTCGGCGGGCGGCGCTGCTCCGCGCGCT GGGCATCCGCCTAGTGAGCTGGGAAGGCGGGCGGCTGGAGTGGTTCGGCTGCAA CAAGGAGACCACGCGCTGCTTCGGAACCGTGGTGGGCGACACGCCCGCCTACCT CTACGAGGAGCGCTGGACGCCCCCCTGCTGCCTGCGCGCGCTGCGCGAGACCGC CCGCTATGTGGTGGGCGTGCTGGAGGCTGCGGGCGTGCGCTACTGGCTCGAGGG CGGCTCACTGCTGGGGGCCGCCCGCCACGGGGACATCATCCCATGGGACTACGA CGTGGACCTGGGCATCTACTTGGAGGACGTGGGCAACTGCGAGCAGCTGCGGGG GGCAGAGGCCGGCTCGGTGGTGGATGAGCGCGGCTTCGTATGGGAGAAGGCGGT CGAGGGCGACTTTTTCCGCGTGCAGTACAGCGAAAGCAACCACTTGCACGTGGA CCTGTGGCCCTTCTACCCCCGCAATGGCGTCATGACCAAGGACACGTGGCTGGAC CACCGGCAGGATGTGGAGTTTCCCGAGCACTTCCTGCAGCCGCTGGTGCCCCTGC CCTTTGCCGGCTTCGTGGCGCAGGCGCCTAACAACTACCGCCGCTTCCTGGAGCT CAAGTTCGGGCCCGGGGTCATCGAGAACCCCCAGTACCCCAACCCGGCACTGCT GAGTCTGACGGGAAGCGGCTGAAGCCCTGATAACCTCGCCTTTGTTTTTCGGGGG TCTGTCTGGATGTGGAGAAGCTCTGTGTGAGCGGTGAGGGGTGGAGGGATGTCG CGGAGAGGGGAAGGGGGAAACTGACCAAGAAAGAAATTCTAAGGAGAGCATGA GAGAAGGCTGGCATTGGCAGGAGGAGAGCACCAGGACGAGGATGGGAAGCGAC CTCCAGATTTATCAAATGGTCATGCCCACTGGGAGCCGTGGATATGCGTGGGGAC ATCCTGGGTCATCTCAGTCATGGAGGGAGACGGGGATGTCACGCCGTCCCGCAG GGCCCAGCACAGCCCCAGACCCGAAAAAAGTGTTCTGCCCAAGATTCCGAGAGC CCTGCGCTCTAGGGCAGGGGCAGAGTTTTGGAAACAGTGCAGGCTCTGGAGCCA GACTGGCGAGATTCAAATCCTGGCTCTATCGCTTCGGAGCCAGGTGGGCCTGGG GGGGCGTCGCAGTCTCTCTGTGCCTCAGTTGCTTCCAGGATGCGGGACCCTTGGC TGCAGGGGTTGCTTCCGCCACTAGAGGGCGCGCCGGTCCCGCTCCTGGTGGCCCA CTGTGGCTGCCCGGGCGACAGTACGCCCAGGGCCTGTGTTCCATAGCCATCTACT CTCTTGAGCCTTTGGACTTCTCTCCAAGCCCCTGTGGGAGGCGGACAGCAGTGAC CACCTCCCCTTCTTTTGGACTGCGACCTCCTTCCCTCCTGGGAGAGCCCTGTGACC TGCATGCTACTCTTAACTGTTCTATTCAAGACTGAATAGAAGTATTTCAGTCTTGC AGAGGAGGAAATGCTCAGAGCTCCGAGGTGCGGCTGTGGTCGAGAACCGGGTGC TGGGCCGGGCGCGGGGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGG TGGGAGGATCGCTTGAGCCCAGGAGTCTGAGACCAGCCTCGGCAACATGCCAAG ACCCCGTCTCTATTTTTAAAAAAGAAAAAGAACCGACTTCTGAATCGCAGCTCCA CTCATGACTAATACCTCATTATTTCAGCTGTCTGCACCTAATTCCCCACTTGCACG GCAGTGTAGACAATAACCATAGCTCACACTCACTGAGCACCTACTGGGTACCAG GCACCATTCTCAGTGTTTCACCTGGATCAACTAATGCGTCCCTCACCTCAGCCCTC TGAAGTGACAGCTGCTATTATTTTCATTACACAGATGAAAAAGCTGAGGCCAGA ATC GTGAAGTCACTTGCTCAAGGTCAGGCAGCTTAGGAAGGGGCAGATCGGGGGCTT GAACCCAGGTGGTCAGGCTCTGGAGCCCACAATTGTCTTACCCACTATGCCCCTC TCTAGTCATGGTCCCCAAGAGGGGCTTGGAGACCCACTTAGCAGGTGAAAGCAA TGGCAGCCTTCCTTATTTGATTATGCACCTAAGAATAAATGGTATTTGGGCATGT ATTCCCAATATGTGTATATTTATTTATAAATATATACAGATACTATTATCTGTATG TTAGTAATAAAGCTTAAATTATTCCATTTTAAAATTATGAATATGAATAGGGTTTT TTTTATGTTTCTTGCCTCATCCCAATGACTTTTGCACACCCAGGTGTGAGCACCCA GCATTCAAGACCACG [00652] SEQ ID NO.26 is FKRP, transcript variant X8, mRNA XM_047439424.1 GGGGGCCGGCGGGTGGGAGATCGCGCCCCGAGGAGGGCGAGGGAGACCCAGGC CCCGCGCGACGGTCCCCCTCGCTGGAGTGAGACTGAGGAAGGGGCCGCTTTCTG CCCCCCTCCTCCCACTTCGCCCCAGAGAAAGAGCCTTACCCCTTCCACACCCAAA CACCACTCTCCTGGACAATTACTTTTACTCTGAGAGAAAGGGCGACCAGCAGTGG CCCCATCCTCTCTGATTGCCGGCCAGAGGAGATGCTCTGCTGAGGGGCAGTTGCT ATGGTTACAGGGCCTGGACCTTCCTCCGGAAGGAGCGCAGCTCAGCTGGGCTGG AACTGCCCTCCTGGAACTCCCCCAGCCTACAACCTAGGAGGTGCAGGGACTGAG GCTCAGGCCAAATCGCAACTCAGACCCAGTGAACCCAAGGCCTGAAGAGAATTT GGATTCATTTACCTTGTTTTGTGGGGACTGGAGAGACAAGTAAACTCTCAGAGTA ACTGTCCCCTCTGACTACCATTTCTAAGGCAAGCCCCCTGTTTCTACTCTTGCGCC CCCTGCTGGTTTCCTGCCCTGTCTGTAAGTTGCATGGCTTTTGTCCGTCTTTTTTTT GTTTGTTTGTTTGTTTTGAGACAGGGTCTCACCCAGGCTAGAGTGAAGTGGAGCA ATCTCGGCTCACTGCAACCTCCGCCTCCTGAGTTCAAGTGATTCTCACACCTCAG CCTCCCCAATAGCTGGGATTACAGGATGCCCCGGAGGCCCAGCTAGCCCCAGAC TTCGGCCCCATGCGGCTCACCCGCTGCCAGGCTGCCCTGGCGGCCGCCATCACCC TCAACCTTCTGGTCCTCTTCTATGTCTCGTGGCTGCAGCACCAGCCTAGGAATTCC CGGGCCCGGGGGCCCCGTCGTGCCTCTGCTGCCGGCCCCCGTGTCACCGTCCTGG TGCGGGAGTTCGAGGCATTTGACAACGCGGTGCCCGAGCTGGTAGACTCCTTCCT GCAGCAAGACCCAGCCCAGCCCGTGGTGGTGGCAGCCGACACGCTCCCCTACCC GCCCCTGGCCCTGCCCCGCATCCCCAACGTGCGTCTGGCGCTGCTCCAGCCCGCC CTGGACCGGCCAGCCGCAGCCTCGCGCCCGGAGACCTACGTGGCCACCGAGTTT GTGGCCCTAGTACCTGATGGGGCGCGGGCTGAGGCACCTGGCCTGCTGGAGCGC ATGGTGGAGGCGCTCCGCGCAGGAAGCGCACGTCTGGTGGCCGCCCCGGTTGCC ACGGCCAACCCTGCCAGGTGCCTGGCCCTGAACGTCAGCCTGCGAGAGTGGACC GCCCGCTATGGCGCAGCCCCCGCCGCGCCCCGCTGCGACGCCCTGGACGGAGAT GCTGTGGTGCTCCTGCGCGCCCGCGACCTCTTCAACCTCTCGGCGCCCCTGGCCC GGCCGGTGGGCACCAGCCTCTTTCTGCAGACCGCCCTTCGCGGCTGGGCGGTGCA GCTGCTGGACTTGACCTTCGCCGCGGCGCGCCAGCCCCCGCTGGCCACGGCCCAC GCGCGCTGGAAGGCTGAGCGCGAGGGACGCGCTCGGCGGGCGGCGCTGCTCCGC GCGCTGGGCATCCGCCTAGTGAGCTGGGAAGGCGGGCGGCTGGAGTGGTTCGGC TGCAACAAGGAGACCACGCGCTGCTTCGGAACCGTGGTGGGCGACACGCCCGCC TACCTCTACGAGGAGCGCTGGACGCCCCCCTGCTGCCTGCGCGCGCTGCGCGAG ACCGCCCGCTATGTGGTGGGCGTGCTGGAGGCTGCGGGCGTGCGCTACTGGCTC GAGGGCGGCTCACTGCTGGGGGCCGCCCGCCACGGGGACATCATCCCATGGGAC TACGACGTGGACCTGGGCATCTACTTGGAGGACGTGGGCAACTGCGAGCAGCTG CGGGGGGCAGAGGCCGGCTCGGTGGTGGATGAGCGCGGCTTCGTATGGGAGAAG GCGGTCGAGGGCGACTTTTTCCGCGTGCAGTACAGCGAAAGCAACCACTTGCAC GTGGACCTGTGGCCCTTCTACCCCCGCAATGGCGTCATGACCAAGGACACGTGGC TGGACCACCGGCAGGATGTGGAGTTTCCCGAGCACTTCCTGCAGCCGCTGGTGCC CCTGCCCTTTGCCGGCTTCGTGGCGCAGGCGCCTAACAACTACCGCCGCTTCCTG GAGCTCAAGTTCGGGCCCGGGGTCATCGAGAACCCCCAGTACCCCAACCCGGCA CTGCTGAGTCTGACGGGAAGCGGCTGAAGCCCTGATAACCTCGCCTTTGTTTTTC GGGGGTCTGTCTGGATGTGGAGAAGCTCTGTGTGAGCGGTGAGGGGTGGAGGGA TGTCGCGGAGAGGGGAAGGGGGAAACTGACCAAGAAAGAAATTCTAAGGAGAG CATGAGAGAAGGCTGGCATTGGCAGGAGGAGAGCACCAGGACGAGGATGGGAA GCGACCTCCAGATTTATCAAATGGTCATGCCCACTGGGAGCCGTGGATATGCGTG GGGACATCCTGGGTCATCTCAGTCATGGAGGGAGACGGGGATGTCACGCCGTCC CGCAGGGCCCAGCACAGCCCCAGACCCGAAAAAAGTGTTCTGCCCAAGATTCCG AGAGCCCTGCGCTCTAGGGCAGGGGCAGAGTTTTGGAAACAGTGCAGGCTCTGG AGCCAGACTGGCGAGATTCAAATCCTGGCTCTATCGCTTCGGAGCCAGGTGGGC CTGGGGGGGCGTCGCAGTCTCTCTGTGCCTCAGTTGCTTCCAGGATGCGGGACCC TTGGCTGCAGGGGTTGCTTCCGCCACTAGAGGGCGCGCCGGTCCCGCTCCTGGTG GCCCACTGTGGCTGCCCGGGCGACAGTACGCCCAGGGCCTGTGTTCCATAGCCAT CTACTCTCTTGAGCCTTTGGACTTCTCTCCAAGCCCCTGTGGGAGGCGGACAGCA GTGACCACCTCCCCTTCTTTTGGACTGCGACCTCCTTCCCTCCTGGGAGAGCCCTG TGACCTGCATGCTACTCTTAACTGTTCTATTCAAGACTGAATAGAAGTATTTCAGT CTTGCAGAGGAGGAAATGCTCAGAGCTCCGAGGTGCGGCTGTGGTCGAGAACCG GGTGCTGGGCCGGGCGCGGGGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGC CGAGGTGGGAGGATCGCTTGAGCCCAGGAGTCTGAGACCAGCCTCGGCAACATG CCAAGACCCCGTCTCTATTTTTAAAAAAGAAAAAGAACCGACTTCTGAATCGCA GCTCCACTCATGACTAATACCTCATTATTTCAGCTGTCTGCACCTAATTCCCCACT TGCACGGCAGTGTAGACAATAACCATAGCTCACACTCACTGAGCACCTACTGGG TACCAGGCACCATTCTCAGTGTTTCACCTGGATCAACTAATGCGTCCCTCACCTC AGCCCTCTGAAGTGACAGCTGCTATTATTTTCATTACACAGATGAAAAAGCTGAG GCCAGAATCGTGAAGTCACTTGCTCAAGGTCAGGCAGCTTAGGAAGGGGCAGAT CGGGGGCTTGAACCCAGGTGGTCAGGCTCTGGAGCCCACAATTGTCTTACCCACT ATGCCCCTCTCTAGTCATGGTCCCCAAGAGGGGCTTGGAGACCCACTTAGCAGGT GAAAGCAATGGCAGCCTTCCTTATTTGATTATGCACCTAAGAATAAATGGTATTT GGGCATGTATTCCCAATATGTGTATATTTATTTATAAATATATACAGATACTATTA TCTGTATGTTAGTAATAAAGCTTAAATTATTCCATTTTAAAATTATGAATATGAAT AGGGTTTTTTTTATGTTTCTTGCCTCATCCCAATGACTTTTGCACACCCAGGTGTG AGCACCCAGCATTCAAGACCACG [00653] SEQ ID NO.27 is FKRP, transcript variant X9, mRNA XM_047439425.1 GGGGGCCGGCGGGTGGGAGATCGCGCCCCGAGGAGGGCGAGGGAGACCCAGGC CCCGCGCGACGGTCCCCCTCGCTGGAGTGAGACTGAGGAAGGGGCCGCTTTCTG CCCCCCTCCTCCCACTTCGCCCCAGAGAAAGAGCCTTACCCCTTCCACACCCAAA CACCACTCTCCTGGACAATTACTTTTACTCTGAGAGAAAGGGCGACCAGCAGTGG CCCCATCCTCTCTGATTGCCGGCCAGGAGCGCAGCTCAGCTGGGCTGGAACTGCC CTCCTGGAACTCCCCCAGCCTACAACCTAGGAGGTGCAGGGACTGAGGCTCAGG CCAAATCGCAACTCAGACCCAGTGAACCCAAGGCCTGAAGAGAATTTGGATTCA TTTACCTTGTTTTGTGGGGACTGGAGAGACAAGTAAACTCTCAGAGTAACTGTCC CCTCTGACTACCATTTCTAAGGCAAGCCCCCTGTTTCTACTCTTGCGCCCCCTGCT GGTTTCCTGCCCTGTCTGTAAGTTGCATGGCTTTTGTCCGTCTTTTTTTTGTTTGTT TGTTTGTTTTGAGACAGGGTCTCACCCAGGCTAGAGTGAAGTGGAGCAATCTCGG CTCACTGCAACCTCCGCCTCCTGAGTTCAAGTGATTCTCACACCTCAGCCTCCCC AATAGCTGGGATTACAGGATGCCCCGGAGGCCCAGCTAGCCCCAGACTTCGGCC CCATGCGGCTCACCCGCTGCCAGGCTGCCCTGGCGGCCGCCATCACCCTCAACCT TCTGGTCCTCTTCTATGTCTCGTGGCTGCAGCACCAGCCTAGGAATTCCCGGGCC CGGGGGCCCCGTCGTGCCTCTGCTGCCGGCCCCCGTGTCACCGTCCTGGTGCGGG AGTTCGAGGCATTTGACAACGCGGTGCCCGAGCTGGTAGACTCCTTCCTGCAGCA AGACCCAGCCCAGCCCGTGGTGGTGGCAGCCGACACGCTCCCCTACCCGCCCCT GGCCCTGCCCCGCATCCCCAACGTGCGTCTGGCGCTGCTCCAGCCCGCCCTGGAC CGGCCAGCCGCAGCCTCGCGCCCGGAGACCTACGTGGCCACCGAGTTTGTGGCC CTAGTACCTGATGGGGCGCGGGCTGAGGCACCTGGCCTGCTGGAGCGCATGGTG GAGGCGCTCCGCGCAGGAAGCGCACGTCTGGTGGCCGCCCCGGTTGCCACGGCC AACCCTGCCAGGTGCCTGGCCCTGAACGTCAGCCTGCGAGAGTGGACCGCCCGC TATGGCGCAGCCCCCGCCGCGCCCCGCTGCGACGCCCTGGACGGAGATGCTGTG GTGCTCCTGCGCGCCCGCGACCTCTTCAACCTCTCGGCGCCCCTGGCCCGGCCGG TGGGCACCAGCCTCTTTCTGCAGACCGCCCTTCGCGGCTGGGCGGTGCAGCTGCT GGACTTGACCTTCGCCGCGGCGCGCCAGCCCCCGCTGGCCACGGCCCACGCGCG CTGGAAGGCTGAGCGCGAGGGACGCGCTCGGCGGGCGGCGCTGCTCCGCGCGCT GGGCATCCGCCTAGTGAGCTGGGAAGGCGGGCGGCTGGAGTGGTTCGGCTGCAA CAAGGAGACCACGCGCTGCTTCGGAACCGTGGTGGGCGACACGCCCGCCTACCT CTACGAGGAGCGCTGGACGCCCCCCTGCTGCCTGCGCGCGCTGCGCGAGACCGC CCGCTATGTGGTGGGCGTGCTGGAGGCTGCGGGCGTGCGCTACTGGCTCGAGGG CGGCTCACTGCTGGGGGCCGCCCGCCACGGGGACATCATCCCATGGGACTACGA CGTGGACCTGGGCATCTACTTGGAGGACGTGGGCAACTGCGAGCAGCTGCGGGG GGCAGAGGCCGGCTCGGTGGTGGATGAGCGCGGCTTCGTATGGGAGAAGGCGGT CGAGGGCGACTTTTTCCGCGTGCAGTACAGCGAAAGCAACCACTTGCACGTGGA CCTGTGGCCCTTCTACCCCCGCAATGGCGTCATGACCAAGGACACGTGGCTGGAC CACCGGCAGGATGTGGAGTTTCCCGAGCACTTCCTGCAGCCGCTGGTGCCCCTGC CCTTTGCCGGCTTCGTGGCGCAGGCGCCTAACAACTACCGCCGCTTCCTGGAGCT CAAGTTCGGGCCCGGGGTCATCGAGAACCCCCAGTACCCCAACCCGGCACTGCT GAGTCTGACGGGAAGCGGCTGAAGCCCTGATAACCTCGCCTTTGTTTTTCGGGGG TCTGTCTGGATGTGGAGAAGCTCTGTGTGAGCGGTGAGGGGTGGAGGGATGTCG CGGAGAGGGGAAGGGGGAAACTGACCAAGAAAGAAATTCTAAGGAGAGCATGA GAGAAGGCTGGCATTGGCAGGAGGAGAGCACCAGGACGAGGATGGGAAGCGAC CTCCAGATTTATCAAATGGTCATGCCCACTGGGAGCCGTGGATATGCGTGGGGAC ATCCTGGGTCATCTCAGTCATGGAGGGAGACGGGGATGTCACGCCGTCCCGCAG GGCCCAGCACAGCCCCAGACCCGAAAAAAGTGTTCTGCCCAAGATTCCGAGAGC CCTGCGCTCTAGGGCAGGGGCAGAGTTTTGGAAACAGTGCAGGCTCTGGAGCCA GACTGGCGAGATTCAAATCCTGGCTCTATCGCTTCGGAGCCAGGTGGGCCTGGG GGGGCGTCGCAGTCTCTCTGTGCCTCAGTTGCTTCCAGGATGCGGGACCCTTGGC TGCAGGGGTTGCTTCCGCCACTAGAGGGCGCGCCGGTCCCGCTCCTGGTGGCCCA CTGTGGCTGCCCGGGCGACAGTACGCCCAGGGCCTGTGTTCCATAGCCATCTACT CTCTTGAGCCTTTGGACTTCTCTCCAAGCCCCTGTGGGAGGCGGACAGCAGTGAC CACCTCCCCTTCTTTTGGACTGCGACCTCCTTCCCTCCTGGGAGAGCCCTGTGACC TGCATGCTACTCTTAACTGTTCTATTCAAGACTGAATAGAAGTATTTCAGTCTTGC AGAGGAGGAAATGCTCAGAGCTCCGAGGTGCGGCTGTGGTCGAGAACCGGGTGC TGGGCCGGGCGCGGGGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGG TGGGAGGATCGCTTGAGCCCAGGAGTCTGAGACCAGCCTCGGCAACATGCCAAG ACCCCGTCTCTATTTTTAAAAAAGAAAAAGAACCGACTTCTGAATCGCAGCTCCA CTCATGACTAATACCTCATTATTTCAGCTGTCTGCACCTAATTCCCCACTTGCACG GCAGTGTAGACAATAACCATAGCTCACACTCACTGAGCACCTACTGGGTACCAG GCACCATTCTCAGTGTTTCACCTGGATCAACTAATGCGTCCCTCACCTCAGCCCTC TGAAGTGACAGCTGCTATTATTTTCATTACACAGATGAAAAAGCTGAGGCCAGA ATCGTGAAGTCACTTGCTCAAGGTCAGGCAGCTTAGGAAGGGGCAGATCGGGGG CTTGAACCCAGGTGGTCAGGCTCTGGAGCCCACAATTGTCTTACCCACTATGCCC CTCTCTAGTCATGGTCCCCAAGAGGGGCTTGGAGACCCACTTAGCAGGTGAAAG CAATGGCAGCCTTCCTTATTTGATTATGCACCTAAGAATAAATGGTATTTGGGCA TGTATTCCCAATATGTGTATATTTATTTATAAATATATACAGATACTATTATCTGT ATGTTAGTAATAAAGCTTAAATTATTCCATTTTAAAATTATGAATATGAATAGGG TTTTTTTTATGTTTCTTGCCTCATCCCAATGACTTTTGCACACCCAGGTGTGAGCA CCCAGCATTCAAGACCACG [00654] SEQ ID NO.28 is FKRP, transcript variant X2, mRNA XM_047439421.1 TGTCATGGTAACCCTTCCCCAGCGGTGGGGGCGAGGTGGTGGGGAGCAGCCTTC CCCTCGCTTCCACCTTTGTGGTTATGGAGCCCCAAACCTCTTAGGACTCTGCACC AGGAGAAGGTTGCTAAGGTAACCTAAGTGTCCGTGGTGGTCCTTCTGCTCCTATC CTCATTGATTACTATGGCAACCTCCCCCCCACCTAGCATTCAGGTAGAAAGGACC CCAGCCCCAAGGACACTGATCTCTAAGAGAAGCACCTCTTCCCCAGCCCCTCCCC CATAGCCACCTAGTTGGCATTACCATGGTAACCGTCTTCCCCTTCCCATCGTGGG AGGCCCATGCACCAAACTAACCAGGTGTTTTTATTTAGAGGAGATGCTCTGCTGA GGGGCAGTTGCTATGGTTACAGGGCCTGGACCTTCCTCCGGAAGGTGAGAAACA GGAGCGCAGCTCAGCTGGGCTGGAACTGCCCTCCTGGAACTCCCCCAGCCTACA ACCTAGGAGGTGCAGGGACTGAGGCTCAGGCCAAATCGCAACTCAGACCCAGTG AACCCAAGGCCTGAAGAGAATTTGGATTCATTTACCTTGTTTTGTGGGGACTGGA GAGACAAGTAAACTCTCAGAGTAACTGTCCCCTCTGACTACCATTTCTAAGGATG CCCCGGAGGCCCAGCTAGCCCCAGACTTCGGCCCCATGCGGCTCACCCGCTGCCA GGCTGCCCTGGCGGCCGCCATCACCCTCAACCTTCTGGTCCTCTTCTATGTCTCGT GGCTGCAGCACCAGCCTAGGAATTCCCGGGCCCGGGGGCCCCGTCGTGCCTCTG CTGCCGGCCCCCGTGTCACCGTCCTGGTGCGGGAGTTCGAGGCATTTGACAACGC GGTGCCCGAGCTGGTAGACTCCTTCCTGCAGCAAGACCCAGCCCAGCCCGTGGT GGTGGCAGCCGACACGCTCCCCTACCCGCCCCTGGCCCTGCCCCGCATCCCCAAC GTGCGTCTGGCGCTGCTCCAGCCCGCCCTGGACCGGCCAGCCGCAGCCTCGCGCC CGGAGACCTACGTGGCCACCGAGTTTGTGGCCCTAGTACCTGATGGGGCGCGGG CTGAGGCACCTGGCCTGCTGGAGCGCATGGTGGAGGCGCTCCGCGCAGGAAGCG CACGTCTGGTGGCCGCCCCGGTTGCCACGGCCAACCCTGCCAGGTGCCTGGCCCT GAACGTCAGCCTGCGAGAGTGGACCGCCCGCTATGGCGCAGCCCCCGCCGCGCC CCGCTGCGACGCCCTGGACGGAGATGCTGTGGTGCTCCTGCGCGCCCGCGACCTC TTCAACCTCTCGGCGCCCCTGGCCCGGCCGGTGGGCACCAGCCTCTTTCTGCAGA CCGCCCTTCGCGGCTGGGCGGTGCAGCTGCTGGACTTGACCTTCGCCGCGGCGCG CCAGCCCCCGCTGGCCACGGCCCACGCGCGCTGGAAGGCTGAGCGCGAGGGACG CGCTCGGCGGGCGGCGCTGCTCCGCGCGCTGGGCATCCGCCTAGTGAGCTGGGA AGGCGGGCGGCTGGAGTGGTTCGGCTGCAACAAGGAGACCACGCGCTGCTTCGG AACCGTGGTGGGCGACACGCCCGCCTACCTCTACGAGGAGCGCTGGACGCCCCC CTGCTGCCTGCGCGCGCTGCGCGAGACCGCCCGCTATGTGGTGGGCGTGCTGGA GGCTGCGGGCGTGCGCTACTGGCTCGAGGGCGGCTCACTGCTGGGGGCCGCCCG CCACGGGGACATCATCCCATGGGACTACGACGTGGACCTGGGCATCTACTTGGA GGACGTGGGCAACTGCGAGCAGCTGCGGGGGGCAGAGGCCGGCTCGGTGGTGG ATGAGCGCGGCTTCGTATGGGAGAAGGCGGTCGAGGGCGACTTTTTCCGCGTGC AGTACAGCGAAAGCAACCACTTGCACGTGGACCTGTGGCCCTTCTACCCCCGCA ATGGCGTCATGACCAAGGACACGTGGCTGGACCACCGGCAGGATGTGGAGTTTC CCGAGCACTTCCTGCAGCCGCTGGTGCCCCTGCCCTTTGCCGGCTTCGTGGCGCA GGCGCCTAACAACTACCGCCGCTTCCTGGAGCTCAAGTTCGGGCCCGGGGTCATC GAGAACCCCCAGTACCCCAACCCGGCACTGCTGAGTCTGACGGGAAGCGGCTGA AGCCCTGATAACCTCGCCTTTGTTTTTCGGGGGTCTGTCTGGATGTGGAGAAGCT CTGTGTGAGCGGTGAGGGGTGGAGGGATGTCGCGGAGAGGGGAAGGGGGAAAC TGACCAAGAAAGAAATTCTAAGGAGAGCATGAGAGAAGGCTGGCATTGGCAGG AGGAGAGCACCAGGACGAGGATGGGAAGCGACCTCCAGATTTATCAAATGGTCA TGCCCACTGGGAGCCGTGGATATGCGTGGGGACATCCTGGGTCATCTCAGTCATG GAGGGAGACGGGGATGTCACGCCGTCCCGCAGGGCCCAGCACAGCCCCAGACCC GAAAAAAGTGTTCTGCCCAAGATTCCGAGAGCCCTGCGCTCTAGGGCAGGGGCA GAGTTTTGGAAACAGTGCAGGCTCTGGAGCCAGACTGGCGAGATTCAAATCCTG GCTCTATCGCTTCGGAGCCAGGTGGGCCTGGGGGGGCGTCGCAGTCTCTCTGTGC CTCAGTTGCTTCCAGGATGCGGGACCCTTGGCTGCAGGGGTTGCTTCCGCCACTA GAGGGCGCGCCGGTCCCGCTCCTGGTGGCCCACTGTGGCTGCCCGGGCGACAGT ACGCCCAGGGCCTGTGTTCCATAGCCATCTACTCTCTTGAGCCTTTGGACTTCTCT CCAAGCCCCTGTGGGAGGCGGACAGCAGTGACCACCTCCCCTTCTTTTGGACTGC GACCTCCTTCCCTCCTGGGAGAGCCCTGTGACCTGCATGCTACTCTTAACTGTTCT ATTCAAGACTGAATAGAAGTATTTCAGTCTTGCAGAGGAGGAAATGCTCAGAGC TCCGAGGTGCGGCTGTGGTCGAGAACCGGGTGCTGGGCCGGGCGCGGGGGCTCA CGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGAGGATCGCTTGAGCCCA GGAGTCTGAGACCAGCCTCGGCAACATGCCAAGACCCCGTCTCTATTTTTAAAAA AGAAAAAGAACCGACTTCTGAATCGCAGCTCCACTCATGACTAATACCTCATTAT TTCAGCTGTCTGCACCTAATTCCCCACTTGCACGGCAGTGTAGACAATAACCATA GCTCACACTCACTGAGCACCTACTGGGTACCAGGCACCATTCTCAGTGTTTCACC TGGATCAACTAATGCGTCCCTCACCTCAGCCCTCTGAAGTGACAGCTGCTATTAT TTTCATTACACAGATGAAAAAGCTGAGGCCAGAATCGTGAAGTCACTTGCTCAA GGTCAGGCAGCTTAGGAAGGGGCAGATCGGGGGCTTGAACCCAGGTGGTCAGGC TCTGGAGCCCACAATTGTCTTACCCACTATGCCCCTCTCTAGTCATGGTCCCCAA GAGGGGCTTGGAGACCCACTTAGCAGGTGAAAGCAATGGCAGCCTTCCTTATTT GATTATGCACCTAAGAATAAATGGTATTTGGGCATGTATTCCCAATATGTGTATA TTTATTTATAAATATATACAGATACTATTATCTGTATGTTAGTAATAAAGCTTAAA TTATTCCATTTTAAAATTATGAATATGAATAGGGTTTTTTTTATGTTTCTTGCCTCA TCCCAATGACTTTTGCACACCCAGGTGTGAGCACCCAGCATTCAAGACCACG [00655] SEQ ID NO.29 is the pseudoknot sequence in the 5’ UTR of human FKRP: AGCTCAGCTGGGCTGGAACTGCCCTCCTGGAACTCCCCCAGCCTACAACCTAGGA GGTGCAGGGACTGAGGCTCAGGCCAAATCGCAACTCAGACCCAGTGAACCCAAG GCCTGAAGAGAATTTGGATTCATT [00656] SEQ ID NO.30 is the PCR forward primer: TTGTTAACATGCGGCTCACCC [00657] SEQ ID NO.31 is the PCR reverse primer: TACCGGTTCAACCGCCTGTC [00658] SEQ ID NO.32 is a fusion with the C terminus of KLH: APNNYRRFLELKFGPGVIENPQYPNP [00659] SEQ ID NO.33 is the first 401 bases from a human FKRP cDNA including 5’ UTR which includes a predicted G-quadriplex, a predicted pseudoknot or an internal ribosome entry site, a predicted hairpin, and an encompassed Kozak consensus sequence: attgctccaagatggcggcggcggcggcagcgggagcgcagctcagctgggctggaactgccctcctggaactcccccagcctac aacctaggaggtgcagggactgaggctcaggccaaatcgcaactcagacccagtgaacccaaggcctgaagagaatttggattcatt taccttgttttgtggggactggagagacaagtaaactctcagagtaactgtcccctctgactaccatttctaaggcaagccccctgtttcta ctcttgcgccccctgctggtttcctgccctgtctgatgccccggaggcccagctagccccagacttcggccccATGCGGCTCA CCCGCTGCCAGGCTGCCCTGGCGGCCGCCATCACCCTCAACCTTCTGGTCCTCT

Claims

CLAIMS 1. A nucleic acid expression cassette comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a fukutin-related protein (FKRP) RNA transcript that comprises a modified 5’ and/or 3’ untranslated region (UTR).
2. The nucleic acid expression cassette of claim 1, wherein the modified 5’ untranslated region (UTR) is truncated as compared to the 5’ UTR of wild-type FKRP.
3. The nucleic acid expression cassette of claim 1, wherein the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 5’ UTR region.
4. The nucleic acid expression cassette of claim 3, wherein the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR.
5. The nucleic acid expression cassette of claim 4, wherein the modification comprises a modification to the Kozak consensus sequence.
6. The nucleic acid expression cassette of claim 1, wherein the modified 3’ UTR is truncated compared to the 3’ UTR of wild-type FKRP.
7. The nucleic acid expression cassette of claim 5, wherein the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region.
8. The nucleic acid expression cassette of claim 1, wherein the nucleic acid encoding FKRP comprises a modification in each of the 5’ and 3’ UTRs.
9. The nucleic acid expression cassette of any of claims 1-8, wherein the modification in the 5’ and/or 3’ UTR of FKRP causes an increase or a decrease in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity expressed from a similar construct comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a FKRP RNA transcript that comprises a wild-type 5’ and/or 3’ untranslated region (UTR).
10. The nucleic acid expression cassette of any one of claims 1-9, wherein the transcriptional regulatory region comprises a muscle-specific expression cassette (MSEC).
11. The nucleic acid expression cassette of claim 10, wherein the MSEC is selected from the group consisting of CK8e.
12. The nucleic acid expression cassette of claim 11, wherein upon administration to a cell, expression level of an FKRP mRNA or protein is higher when operably linked to an MSEC than the expression level of the FKRP mRNA or protein when operably linked to a CK8e transcriptional regulatory region.
13. The nucleic acid expression cassette of claim 11, wherein upon administration to a cell, expression level of an FKRP mRNA or protein is lower when operably linked to an MSEC than the expression level of the FKRP mRNA or protein when operably linked to a CK8e transcriptional regulatory region.
14. An RNA transcript generated by transcription of the nucleic acid expression cassette of any one of claims 1-13.
15. An adeno-associated viral vector (AAV) comprising the nucleic acid expression cassette of any one of claims 1- 13.
16. The AAV vector of claim 15, wherein the adeno-associated viral vector is selected from the group consisting of: an AAVRh74 vector, an AAV8 vector, an AAV9 vector, an AAV6 vector, an AAV7 vector, an AAV2i8 vector, a NP vector, a NP 66 vector, a NP 22 vector, an AAVpo.1 vector, a MyoAAV vector, and an AAVMyo vector.
17. The AAV vector of claim 15, wherein the adeno-associated viral vector comprises an internal terminal repeat (ITR), a muscle-specific expression cassette, a nucleic acid encoding FKRP, a polyadenylation signal (pA+), and/or a second ITR.
18. An engineered cell comprising or expressing a nucleic acid expression cassette of any one of claims 1- 17.
19. The engineered cell of claim 18, wherein the modified 5’ untranslated region (UTR) is truncated as compared to the wild-type 5’ UTR of FKRP.
20. The engineered cell of claim 18 or 19, wherein the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 5’ UTR region.
21. The engineered cell of claim 20, wherein the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR.
22. The engineered cell of claim 20 or 21, wherein the modification comprises a modification to the Kozak consensus sequence.
23. The engineered cell of claim 18, wherein the modified 3’ UTR is truncated compared to the 3’UTR of wild-type FKRP.
24. The engineered cell of claim 23, wherein the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region.
25. The engineered cell of claim 18, wherein the nucleic acid encoding FKRP comprises a modification in each of the 5’and 3’ UTRs.
26. The engineered cell of any of claims 18-25, wherein the modification in the 5’ and/or 3’ UTR of FKRP causes an increase or a decrease in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity expressed from a similar construct comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a FKRP RNA transcript that comprises a wild-type 5’ and/or 3’ untranslated region (UTR).
27. A method of expressing an FKRP gene product in a subject comprising administering an adeno-associated viral vector of any one of claims 15-17 to a subject in need thereof.
28. The method of claim 27, wherein the FKRP gene product is a RNA transcript and/or a protein.
29. The method of claim 27, wherein the subject in need thereof comprises limb girdle muscular dystrophy type 2I/R9 (LGMD2i), Walker-Warburg syndrome, or muscle-eye-brain disease (MED).
30. The method of claim 27, wherein the modified 5’ untranslated region (UTR) is truncated as compared to the 5’ UTR of wild-type FKRP.
31. The method of claim 27, wherein the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 5’ UTR region.
32. The method of claim 31, wherein the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR.
33. The method of claim 32, wherein the modification comprises a modification to the Kozak consensus sequence.
34. The method of claim 27, wherein the modified 3’ UTR is truncated compared to the 3’ UTR of a wild-type FKRP.
35. The method of claim 34, wherein the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region.
36. The method of claim 27, wherein the nucleic acid encoding FKRP comprises a modification in each of the 5’ and 3’ UTRs.
37. The method of any of claims 27-36, wherein the modification in the 5’ and/or 3’ UTR of FKRP causes a reduction or inhibition in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity expressed from a similar construct comprising a transcriptional regulatory region operably linked to a nucleic acid sequence encoding a FKRP RNA transcript that comprises a wild- type 5’ and/or 3’ untranslated region (UTR).
38. The method of claim 27, wherein the administration of the AAV vector comprises intravenous and/or intramuscular injection.
39. The method of claim 27, wherein the subject is a human.
40. A method for reducing at least one symptom of an FKRP-mediated disease or disorder, the method comprising administering an AAV vector of any one of claims 15-17 to a subject in need thereof, thereby reducing at least one symptom of an FKRP-mediated disorder.
41. The method of claim 40, wherein the FKRP-mediated disease or disorder comprises limb girdle muscular dystrophy type 2I/R9 (LGMD2i), Walker-Warburg syndrome, or muscle-eye-brain disease (MED).
42. The method of claim 40, wherein the modified 5’ untranslated region (UTR) is truncated as compared to the wild-type 5’ UTR of FKRP.
43. The method of claim 40, wherein the modified 5’ UTR comprises a deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 5’ UTR region.
44. The method of claim 43, wherein the modification of the 5’UTR comprises deletion or disruption of a G-quadruplex, or a hairpin in the 5’ UTR.
45. The method of claim 44, wherein the modification comprises a modification to the Kozak consensus sequence.
46. The method of claim 40, wherein the modified 3’ UTR is truncated compared to the 3’ UTR of a wild-type FKRP.
47. The method of claim 46, wherein the modification to the 3’ UTR comprises deletion of at least one nucleotide, a plurality of nucleotides or all of the nucleotides in the 3’ UTR region.
48. The method of claim 40, wherein the nucleic acid encoding FKRP comprises a modification in each of the 5’ and 3’ UTRs.
49. The method of any of claims 40-48, wherein the modification in the 5’ and/or 3’ UTR of FKRP causes an increase or a decrease in protein expression and/or enzymatic activity upon expression in a cell as compared to the protein expression and/or enzymatic activity of a construct comprising wild type 5’ and 3’ FKRP UTRs under substantially similar conditions.
50. The method of claim 40, wherein the administration of the AAV vector comprises intravenous and/or intramuscular injection.
51. The method of claim 40, wherein the subject is a human.
52. The method of claim 40, wherein at least one symptom of a FKRP-mediated disease or disorder comprises:  muscle pain, muscle weakness, muscle fatigue, muscle atrophy, inflammation, decrease in average myofiber diameter in skeletal muscle, loss of ambulation, abnormalities in the brain and/or eyes, eye problems, delay in development, intellectual disability, and seizures.
PCT/US2023/066296 2022-04-29 2023-04-27 Method for expressing a muscle-specific gene and cassettes for same Ceased WO2023212643A1 (en)

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WO2022076556A2 (en) * 2020-10-07 2022-04-14 Asklepios Biopharmaceutical, Inc. Therapeutic adeno-associated virus delivery of fukutin related protein (fkrp) for treating dystroglycanopathy disorders including limb girdle 2i (lgmd2i)

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WO2004092386A2 (en) * 2003-04-11 2004-10-28 The Government Of The United States Of America As Represented By The Secretary Of Health And Human Services Inducing a t cell response with recombinant pestivirus replicons or recombinant pestivirus replicon-transfected dendritic cells
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