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WO2025188993A2 - Thérapie génique pour le traitement de troubles liés à gne - Google Patents

Thérapie génique pour le traitement de troubles liés à gne

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Publication number
WO2025188993A2
WO2025188993A2 PCT/US2025/018739 US2025018739W WO2025188993A2 WO 2025188993 A2 WO2025188993 A2 WO 2025188993A2 US 2025018739 W US2025018739 W US 2025018739W WO 2025188993 A2 WO2025188993 A2 WO 2025188993A2
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Prior art keywords
gne
aav
raav
seq
sequence
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WO2025188993A8 (fr
WO2025188993A3 (fr
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Paul Martin
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Nationwide Childrens Hospital Inc
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Nationwide Childrens Hospital Inc
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Publication of WO2025188993A2 publication Critical patent/WO2025188993A2/fr
Publication of WO2025188993A8 publication Critical patent/WO2025188993A8/fr
Publication of WO2025188993A3 publication Critical patent/WO2025188993A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • 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
    • 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
    • A61K48/0075—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/20—Vector systems having a special element relevant for transcription transcription of more than one cistron

Definitions

  • the present invention relates to methods and materials for treating GNE-related disorders such as GNE myopathy, GNE-dependent ALS, sarcopenia and aging using a dual gene recombinant adeno-associated virus comprising the GNE gene and the follistatin gene under the control of two different transcriptional control sequences.
  • GNE Myopathy is an adult onset autosomal recessive genetic disease characterized by progressive muscle weakness that that can lead to loss of ambulation and loss of independent living. As its name implies, GNE myopathy is caused by loss of function pathogenic variants or mutations in the GNE gene.
  • Sialic acid is an acidic monosaccharide that modifies non-reducing terminal carbohydrate chains on glycoproteins and glycolipids and plays an important role in different processes such as cell-adhesion and cellular interactions. Sialic acid has been implicated in health and disease and is found in terminal sugar chains of proteins modulating their cellular functions.
  • GNE UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase
  • Sarcopenia is a condition marked by the wasting or loss muscle tissue and the replacement of muscle tissue with fibrosis tissue as the subject ages.
  • the etiology of sarcopenia is complex and can be attributed to a variety of factors, including oxidative stress, inflammation, apoptosis, and mitochondrial dysregulation (Roubenoff, 2003; Fulle et al., 2004), as well as genetic factors, inadequate diet, sedentary lifestyle, and the interplay between these factors (Rolland et al., 2008; Walrand et al., 2011).
  • Sarcopenia and age- related muscle wasting are associated with reduced GNE gene expression. Reduced GNE expression during aging is also associated with increased degeneration of brain neurons.
  • GNE myopathy leads to weakness and wasting of muscles in legs and arms. First symptoms usually occur in young adults (usually in the third decade of life), but a later onset has also been observed in some patients. A diagnosis of GNE myopathy should be considered primarily in patients presenting with distal weakness (foot drop) in early adulthood (other onset symptoms are possible too).
  • a GNE M712T variant knock-in mouse model showed premature death in the first few weeks of life due to kidney disease, a clinical phenotype that is not present in GNE Myopathy patients. Other lines of the same model were bred out to show no phenotype at all despite having the same genetic mutation.
  • Second is a lack of measurable natural history data from the rare and geographically diverse patient population.
  • GNE myopathy is a slow and variably progressing human disease and the lack of robust short-term clinical milestones make is difficult to treat effectively. For example, a current phase 3 clinical trial was 48 weeks in duration. In that time, there was not a significant drop in any of the strength measures for the patient population from pre-treatment baseline, though some measures did trend lower.
  • sialic acid or ManNAc a precursor of sialic acid
  • AAV8 viral vectors carrying wild type human GNE cDNA have been shown to transduce murine muscle cells and human GNE myopathy- derived muscle cells in culture and to express the transgene in these cells (Mitrani- Rosenbaum et al., Neuromuscul. Disord. 22(11): 1015-24, 2012).
  • the gene therapies in the prior art only focus on delivering wild-type GNE gene and do not utilize the dual gene function technology disclosed herein.
  • the disclosure provides for gene therapies which increase muscle strength at the same time as providing a transgene for gene replacement to prevent further muscle injury or to promote muscle growth are desired.
  • gene therapy vectors that provide GNE gene replacement are likely to be one of the only ways to prove clinical effectiveness for GNE myopathy in a period shorter than 5 years, as the natural history of disease progression is slow and quite variable. It will also be the one of the only ways to show clinical efficacy in all GNE myopathy patients, many of which have lost ambulation not long after diagnosis but that can still show significant arm function, for example self-feeding, which could still be preserved or improved by such a therapy. Because this disease is a myopathy and not a dystrophy, muscle, once repaired, should remain in place permanently. In patients with GNE-dependent ALS, building new muscle mass and strength is also indicated and should benefit patients even though the disease is caused by loss of motor neurons.
  • GNE myopathy GNE-dependent ALS
  • thrombocytopenia thrombocytopenia
  • sarcopenia sarcopenia
  • aging a dual gene therapy approach that will add a muscle building component to gene replacement. Such therapies have the potential to rebuild loss muscle strength while simultaneously arresting subsequent disease progression.
  • the goal of the GNE therapeutic methods provided herein is to create a tandem or dual gene therapy that expresses both the normal GNE gene and a muscle building protein, such as a follistatin.
  • Such an AAV vector will both correct the genetic defect of GNE myopathy and increase muscle strength, thus reversing rather than just arresting the decline of muscle strength clinical measures.
  • a dual gene therapy that builds new muscle and muscle strength while also preventing further disease by adding back the normal GNE gene will be of greater benefit to patients with GNE myopathy and will provide an easier means of demonstrating clinical improvement.
  • the dual gene therapy approach disclosed herein will also be effective for treating muscle wasting due to thrombocytopenia, sarcopenia and/or aging.
  • Treatment of muscle wasting may result in a reduction of muscle damage, prevention or inhibition of muscle damage and/or repair of muscle damage.
  • treatment of muscle wasting may build muscle mass, such as increasing the diameter of muscle fibers.
  • the disclosed dual gene rAAV vectors prevented muscles damage and/or built new muscle.
  • the rAAV vectors disclosed herein produce two mRNAs driven by two different transcriptional control sequences, e.g. promoters and/or enhancers, each with their own poly A tail (in a single AAV), and this gene therapy concept is vastly superior for producing large amounts of the second gene and the second protein, leading to improved therapeutic function.
  • the dual gene rAAV vectors disclosed herein allow for superior gene expression and superior function in inhibiting or preventing disease and building new muscle mass and strength (which will reverse disease).
  • the disclosure provides for a polynucleotide sequence comprising in 5’ to 3’ order: an AAV ITR, a first transcriptional control sequence operably linked to a first transgene sequence encoding GNE protein, such as a protien comprising the amino acid sequence of SEQ ID NO: 4, a polyadenylation signal sequence, a second transcriptional control sequence operably linked to a second (but different) transgene sequence encoding muscle building protein, a polyadenylation signal sequence and an AAV ITR.
  • the first transgene encodes the GNE protein.
  • the GNE gene sequence comprises the nucleotide sequence of SEQ ID NO: 3 (or nucleotides 2897-5065 of SEQ ID NO: 1 or nucleotides 2872-5040 of SEQ ID NO: 2) or comprises a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotides sequence of SEQ ID NO: 3 and encodes a functional GNE protein.
  • the GNE gene encodes a protein comprising amino acid sequence of SEQ ID NO: 4.
  • the disclosure provides for polynucleotides comprising a second transgene that is a gene that encodes a muscle building protein.
  • a muscle building protein is a protein that builds new muscle mass and inducing muscle growth, including a protein that stimulates muscle growth signals or inhibit repressive muscle growth signals.
  • exemplary transgenes are the follistatin gene (FST), e.g. follistatin 344 (FS344), follistatin 317 (FS317) or follistatin 314, Insulin-like growth factor 1 gene (IGF1), heparin binding Epidermal Growth Factor like Growth Factor gene (HB-EGF) or Mothers against decapentaplegic homolog 7 gene (SMAD7).
  • FST follistatin gene
  • IGF1 Insulin-like growth factor 1 gene
  • HB-EGF heparin binding Epidermal Growth Factor like Growth Factor gene
  • SMAD7 Mothers against decapentaplegic homolog 7 gene
  • the second transgene is the follistatin (FST) gene that encodes protein form FS344.
  • the FST (FS344) gene sequence comprises the nucleotide sequence of SEQ ID NO: 5 (or nucleotides 5406-6437 of SEQ ID NO: 1 or nucleotides 5381-6412 of SEQ ID NO: 2), or comprises a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of SEQ ID NO: 5 and encodes a functional FS344 protein.
  • a transcriptional control sequence is a promoter sequence that may also include an enhancer and/or intron.
  • Exemplary transcriptional control sequences include but are not limited to, promoters, enhancers and/or polyadenylation signal sequences.
  • transcriptional control sequences include the chicken ⁇ actin promoter (CBA), the hybrid form of the CBA promoter (Cbh), the cytomegaloviruses (CMV promoter), CMV enhancer, miniCMV promoter, MHCK7, the CK8 promoter, the CK8e promoter, the SPc5-12 promoter, a SP-301 promoter the P546 promoter simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor-1a promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • CBA chicken ⁇
  • the first or second transcriptional control sequence comprises one or more of the nucleotide sequences of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and the first and second transcriptional control sequence are different.
  • the first transcriptional control sequence comprises the one or more of the nucleotide sequences of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11. SEQ ID NO: 12, and the second transcriptional control sequence comprises the nucleotide sequence of SEQ ID NO: 13.
  • the first or second transcriptional control sequence is a constitutive control element, such CMV or Cbh.
  • exemplary muscle-specific promoter include one or more of a human skeletal actin gene element, a cardiac actin gene element, a desmin promoter, a skeletal alpha-actin (ASKA) promoter, a troponin I (TNNI2) promoter, a myocyte-specific enhancer binding factor MEF binding element, a muscle creatine kinase (MCK) promoter, a truncated MCK (tMCK) promoter, a myosin heavy chain (MHC) promoter, a hybrid a-myosin heavy chain enhancer- /MHC enhancer-promoter (MHCK7) promoter, a CK8 promoter, a CK8e promoter, a SPc5- 12 promoter, a SP-301 promoter, a murine
  • the first transcriptional control element sequence comprises an enhancer comprising the nucleotide sequence of SEQ ID NO: 10 and a promoter sequence comprising the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 9.
  • the transcriptional control element sequence further comprises an intron.
  • the intron comprises the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 15.
  • the disclosure provides for a polynucleotide sequence comprising in 5’ to 3’ order: an AAV ITR, a first transcriptional control sequence operably linked to a nucleotide sequence encoding the GNE protein (nucleotides 2897-5065 of SEQ ID NO: 1), wherein the first transcriptional control element comprises the CMV enhancer and the hybrid chicken beta-actin promoter (Cbh which is a hybrid CBA promoter and an intronic sequence ) (nucleotides 2060-2871 of SEQ ID NO: 1), and a second transcriptional control sequence operably that is the miniCMV promoter (nucleotides 5145-5371 of SEQ ID NO: 1) linked to a nucleotide sequence encoding the FS344 protein (nucleotides 5406-6437 of SEQ ID NO: 1), and an AAV ITR.
  • an AAV ITR a first transcriptional control sequence operably linked to a nucleotide sequence encoding the GNE
  • the disclosure provides for a polynucleotide sequence comprising in 5’ to 3’ order: an AAV ITR, a first transcriptional control sequence operably linked to a nucleotide sequence encoding the GNE protein (nucleotides 2872-5040 of SEQ ID NO: 2), wherein the first transcriptional control element comprises the CMV enhancer, the CMV promoter and a SV40 enhancer with an intron (nucleotides 2061-2852 of SEQ ID NO: 2), and a second transcriptional control sequence operably that is the miniCMV promoter (nucleotides 5120-5346 of SEQ ID NO: 2) linked to a nucleotide sequence encoding the FS344 protein (nucleotides 5381-6412 of SEQ ID NO: 2), and an AAV ITR.
  • an AAV ITR a first transcriptional control sequence operably linked to a nucleotide sequence encoding the GNE protein
  • the first transcriptional control element comprises the C
  • any of the polynucleotides disclosed herein further comprise a polyadenylation signal sequence, which optionally is a synthetic polyadenylation signal sequence.
  • exemplary polyadenylation sequences include SEQ ID NO: 16 or SEQ ID NO: 17.
  • the polynucleotide sequences disclosed herein comprise an inverted terminal repeat (ITR), such as a wild type ITR or a mutant ITR.
  • ITR inverted terminal repeat
  • Exemplary sequences of the ITRs includes the nucleotide sequence of SEQ ID NO: 18 and SEQ ID NO: 19.
  • the disclosure also provides for a polynucleotide sequence that is an AAV genome.
  • the disclosure provides an AAV genome or polynucleotide sequence comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to nucleotides 1847 to 6685 of SEQ ID NO: 1 or to nucleotides 1847 to 6660 of SEQ ID NO: 2.
  • the disclosure provides an AAV genome or polynucleotide sequence comprising nucleotides 1847 to 6685 of SEQ ID NO: 1 or to nucleotides 1847 to 6660 of SEQ ID NO: 2.
  • sequence identity in the context of nucleic acid or amino acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence.
  • the length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, is desired.
  • identity among smaller fragments e.g. of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired.
  • the rAAV genomes provided herein hybridizes under stringent conditions to the polynucleotide sequence of nucleotides 1847 to 6685 of SEQ ID NO: 1 or to nucleotides 1847 to 6660 of SEQ ID NO: 2 or the complement thereof.
  • the disclosure also provides for recombinant adeno-associated virus (rAAV) comprising any of the polynucleotide sequences described herein.
  • the rAAV comprises AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh.74, AAV rh.10, AAVMyo3, MYOAAV capsid protein, or a variant thereof.
  • the AAV comprises is AAVrh.74 capsid protein.
  • the disclosure also provides for a recombinant AAV particle comprising any of the polynucleotide sequences disclosed herein or any of the rAAV disclosed herein.
  • the disclosure provides for methods of producing a rAAV vector particle comprising culturing a cell that has been transfected with any rAAV vector of the disclosure and recovering rAAV particles from the supernatant of the transfected cells.
  • the disclosure also provides for viral particles comprising any of the recombinant AAV vectors of the disclosure.
  • the disclosure also provides for compositions comprising any of the rAAV disclosed herein or any of the rAAV particles described herein.
  • the compositions further comprise a pharmaceutically acceptable carrier.
  • the compositions may also comprise other ingredients such as diluents and adjuvants.
  • Acceptable carriers, diluents and adjuvants are nontoxic to recipients and are preferably inert at the dosages and concentrations employed and include buffers and surfactants such as pluronics.
  • the disclosure also provides for methods of treating a GNE-related disorder comprising administering any of the dual gene rAAV disclosed herein or any dual gene rAAV particles disclosed herein or any of the compositions disclosed herein to a subject in need thereof.
  • a GNE-related disorder is a disorder that is associated with a reduction the expression of the GNE gene or a dysfunction in GNE function or the subject has a mutation in the GNE gene.
  • the GNE dependent disorder is GNE-myopathy, GNE- dependent ALS, thrombocytopenia, sarcopenia or aging.
  • the dual gene rAAV, dual gene rAAV particle or the composition is administered using systemic administration, intramuscular injection or intravenous injection.
  • the disclosure also provides for compositions for treating a GNE-related disorder in a subject in need thereof, wherein the composition comprises any of the dual gene rAAV disclosed herein or any dual gene rAAV particles disclosed herein or any of the compositions disclosed herein.
  • the subject is suffering from GNE-myopathy, GNE-dependent ALS, sarcopenia or aging.
  • the dual gene rAAV, dual gene rAAV particle or the composition is administered using systemic administration, intramuscular injection or intravenous injection.
  • the disclosure also provides for use of any of the dual gene rAAV disclosed herein or any dual gene rAAV particles disclosed herein or any of the compositions disclosed herein for the preparation of a medicament for treating GNE-related disorder in a subject in need thereof.
  • the subject is suffering from GNE-myopathy, GNE-dependent ALS, sarcopenia or aging.
  • the dual gene rAAV, dual gene rAAV particle or the composition is administered using systemic administration, intramuscular injection or intravenous injection.
  • the disclosure also provides for methods of preventing or repairing muscle damage in a subject in need thereof comprising administering any of the dual gene rAAV disclosed herein or any dual gene rAAV particles disclosed herein or any of the compositions disclosed herein to a subject in need thereof.
  • the subject is suffering from a GNE-related disorder is a disorder that is associated with a reduction the expression of the GNE gene or a dysfunction in GNE function or the subject has a mutation in the GNE gene.
  • the GNE dependent disorder is GNE-myopathy, GNE-dependent ALS, thrombocytopenia, sarcopenia or aging.
  • the dual gene rAAV, dual gene rAAV particle or the composition is administered using systemic administration, intramuscular injection or intravenous injection.
  • the disclosure also provides for compositions for preventing or repairing muscle damage in a subject in need thereof, wherein the composition comprises any of the dual gene rAAV disclosed herein or any dual gene rAAV particles disclosed herein or any of the compositions disclosed herein.
  • the subject is suffering from GNE-myopathy, GNE-dependent ALS, sarcopenia or aging.
  • the dual gene rAAV, dual gene rAAV particle or the composition is administered using systemic administration, intramuscular injection or intravenous injection.
  • the disclosure also provides for use of any of the dual gene rAAV disclosed herein or any dual gene rAAV particles disclosed herein or any of the compositions disclosed herein for the preparation of a medicament for preventing or repairing muscle damage in a subject in need thereof.
  • the subject is suffering from GNE-myopathy, GNE-dependent ALS, sarcopenia or aging.
  • the dual gene rAAV, dual gene rAAV particle or the composition is administered using systemic administration, intramuscular injection or intravenous injection.
  • a "subject,” as used herein, can be any animal, and may also be referred to as the patient.
  • the subject is a vertebrate animal, and more preferably the subject is a mammal, such as a domesticated farm animal (e.g., cow, horse, pig) or pet (e.g., dog, cat). in some embodiments, the subject is a human.
  • Figure 1 is an annotated sequence of the plasmid sequence of pAAVCbh.GNE.spA.miniCMV.FST344.spA (SEQ ID NO: 1).
  • Figure 2 is an annotated sequence of the plasmid sequence of pAAVCMV.GNE.spA.miniCMV.FST344.Spa (SEQ ID NO: 2).
  • Figure 3 provides the muscle weights after intramuscular injection of AAV vectors expressing GNE transgene.
  • IVS refers to rAAV.CMV.GNE.FL-IRES.FST
  • CMV refers to rAAV.CMVf.GNE
  • Cbh refers to rAAV.Cbh.GNE
  • Bi CMV refers to the dual gene vector (also known as bicistronic vector) rAAV.CMV.GNE.pA.mCMV.FST
  • Bi Cbh refers to the dual gene vector (also known as bicistronic vector) rAAV.Cbh.GNE.pA.mCMV.FST.
  • Figure 5 demonstrates induction of myofiber growth and prevention of muscle pathology only occur simultaneously with dual gene AAV GNE/FST gene therapy in Gne- deficient muscle (aged Gne lox/lox mice). Hematoxylin and Eosin staining of myofibers sections is shown. Bar is 50 ⁇ m for all panels.
  • AAV.CMV.Cre (referred to as “CRE”) was injected with or without rAAV.CBh.GNE (referred to as “Cre+GNE”), rAAV.CMV.FST (referred to as “Cre+FST”) or dual gene rAAV.CBh.GNE.spA.mCMV.FST (referred to as “Cre+GNE/FST”).
  • FIG. 6 demonstrates that dual GNE/FST gene therapy prevents Cre-induced muscle damage and increases myofiber size in a dose-dependent manner. Hematoxylin and Eosin staining of myofibers sections is shown. Bar is 50 ⁇ m for all panels.
  • adult RosaCreER T2 Gne lox/lox mice were injected intravenously with PBS, 1x10 13 vg/kg, 5x10 13 vg/kg, or 2x10 14 vg/kg rAAV.CBh.GNE.mCMV.FST (GNE/FST).
  • AAV.CMV.Cre (referred to as “CRE”) was injected intramuscularly into the tibialis anterior muscle.
  • FIG. 7 demonstrates that muscle mass is increased by dual GNE/FST gene therapy. Muscle mass, as a percentage of body weight, was significantly increased at the high dose of dual gene therapy.
  • adult RosaCreER T2 Gne lox/lox mice were injected intravenously with PBS, 1x10 13 vg/kg, 5x10 13 vg/kg, or 2x10 14 vg/kg rAAV.CBh.GNE.mCMV.FST (GNE/FST).
  • AAV.CMV.Cre (referred to as “CRE”) was injected intramuscularly into the tibialis anterior muscle. Significance determined by ANOVA with Tukey post-hoc test. ***p ⁇ 0.001.
  • Figure 8 demonstrates the average myofiber diameter is increased by dual GNE/FST gene therapy. Average Mini-Feret diameter, as a percentage was significantly increased by the two higher doses of dual gene therapy.
  • adult RosaCreER T2 Gne lox/lox mice were injected intravenously with PBS, 1x10 13 vg/kg, 5x10 13 vg/kg, or 2x10 14 vg/kg rAAV.CBh.GNE.mCMV.FST (GNE/FST).
  • AAV.CMV.Cre (referred to as “CRE”) was injected intramuscularly into the tibialis anterior muscle. Significance determined by ANOVA with Tukey post-hoc test.
  • the rAAV vectors comprised an internal ribosomal entry site (IRES) to produce a second protein from a single AAV vector where a single mRNA was made by a single promote.
  • IRS internal ribosomal entry site
  • the dual gene rAAV vectors disclosed herein produce two mRNAs driven by two different promoters, each with their own poly A tail (in a single AAV), and this dual gene therapy concept is vastly superior for producing large amounts of the second gene and the second protein, leading to improved therapeutic function.
  • the dual gene rAAV vectors disclosed herein allow for superior gene expression and superior function in preventing or inhibiting disease and building new muscle mass and strength (which will reverse disease by repairing the muscle damage).
  • the disclosed dual gene therapy approach utilizes follistatin (FST) as a second gene component in the dual gene AAV vectors.
  • FST follistatin
  • FST encodes a secreted myostatin inhibitor protein that binds to and inhibits myostatin protein binding to its reception on muscle cells (Amthor et al, Dev Biol, 270(1): p.19-30, 2004).
  • Myostatin is a secreted muscle trophic factor that negatively regulates muscle growth and strength. Elimination of myostatin in mice, cows, or humans can double the size of skeletal muscles, with minimal to no effects on cardiac muscle or non-muscle tissues. Elimination of myostatin in mdx mice significantly increased muscle size and strength but did not improve weight-normalized grip strength or specific (weight-normalized) tetanic muscle force.
  • myostatin inhibition does not stabilize the muscle membrane or prevent muscle damage, but instead increases muscle strength by increasing muscle mass. In this sense then, myostatin inhibition therapy alone may be ineffective over the long term, as muscles expressing the inhibitor will eventually be destroyed if effective gene replacement is not also provided.
  • FST protein also activates Akt and mTOR signaling via a separate pathway. Each of these properties allows for the building of new muscle mass and strength, both by making pre-existing muscles bigger and by creating new muscle fibers. As GNE myopathy is a disease where muscle mass is lost, this dual gene therapy allows patients with this disease to recover lost muscle strength, providing them, for the first time, a therapy that may recover normal, pre-disease, muscle function.
  • the FST gene form used in the exemplary approach described herein, FS344, has been tested in two clinical trials: IM delivery of rAAV1.CMV.FST bilaterally in the quadriceps muscles of patients with Becker Muscular Dystrophy (BMD) yielded improvements in the 6- minute walk test (6MWT, as much as 125 meters) in 4 of 6 patients at 1 year post-treatment.
  • BMD Becker Muscular Dystrophy
  • 6MWT 6- minute walk test
  • Treated BMD patient muscle biopsies suggested muscle hypertrophy, decreased endomysial fibrosis, and more uniform myofiber size (Mendell et al., Mol Ther, 23(1): p.192- 201, 2015).
  • the dual gene therapy disclosed herein is useful for treating a GNE-related disorder, which is a disorder that is caused by GNE mutations or caused by reduced GNE expression or dysfunction in the GNE protein.
  • a GNE-related disorder which is a disorder that is caused by GNE mutations or caused by reduced GNE expression or dysfunction in the GNE protein.
  • the dual gene therapy disclosed herein is used to treat GNE myopathy, a muscle disease caused by missense mutations in the GNE gene.
  • Addition GNE-related disorders include GNE-dependent ALS, sarcopenia, and age-related neurodegeneration.
  • the disclosed dual gene therapy allows for expression of GNE in all cells, and as the hybrid form of the CBA promoter (referred to as Cbh) is particularly good at inducing expression in motor neurons (compared to CMV).
  • Cbh the hybrid form of the CBA promoter
  • Some GNE mutations are known to give rise to juvenile amyotrophic lateral sclerosis (ALS) in addition to GNE myopathy. While GNE myopathy is a muscle disease, ALS is a motor neuron disease where muscle strength is lost due to the death of motor neurons.
  • Cbh or CMV Cbh or CMV, promoters that allow for expression in all cells and tissues, provide a potential therapy for GNE dependent ALS as well as GNE myopathy.
  • GNE is expressed normally in all tissues and cells of the body. All cells make sialic acid, and GNE is the committed step in sialic acid biosynthesis. Thus, placement of a normal copy of GNE in all cells is the most likely approach to cure both of these diseases.
  • FST is specific for blocking myostatin, which is a muscle-specific protein
  • miniCMV miniCMV
  • Mini CMV must be used here instead of full length CMV because of the packaging limitations of the AAV capsid.
  • Cbh a chick beta actin with hybrid intron
  • Cbh a chick beta actin with hybrid intron
  • Cbh is published to be superior to CMV in allowing gene expression in motor neurons.
  • Muscle building proteins can include growth factors that induce muscle growth or increase muscle strength such as IGF, HB-EGF, Pax7, HGF (hepatocyte growth factor), HGH (human growth hormone), FGF19 (fibroblast growth factor 19), FGF21 (fibroblast growth factor 21), VEGF (vascular endothelial growth factor), IL6 (Interleukin 6), IL15 (Interleukin 15) and SMAD7 (mothers against decapentaplegic homolog 7 (MADH7)).
  • growth factors that induce muscle growth or increase muscle strength also include the follistatins (FST).
  • Follistatin is a secreted protein that inhibits the activity of TGF- ⁇ family members such as GDF-11/BMP-11.
  • Follistatin-344 is a follistatin precursor that undergoes peptide cleavage to form the circulating Follistatin-315 isoform which includes a C-terminal acidic region. It circulates with myostatin propeptide in a complex that includes two other proteins, follistatin related gene (FLRG) and GDF associated serum protein (GASP-1).
  • Follistatin-317 is another follistatin precursor that undergoes peptide cleavage to form the membrane-bound Follistatin-288 isoform.
  • FS344 contains a C-terminal protein domain lacking in FS288. The presence of this C-terminal domain reduces binding to activin and to heparan sulfate glycosaminoglycans, which in turn reduces non-muscle effects.
  • the Follistatin-288 isoform which lacks a C-terminal acidic region, exhibits strong affinity for heparin-sulfate-proteoglycans, is a potent suppressor of pituitary follicle stimulating hormone, is found in the follicular fluid of the ovary, and demonstrates high affinity for the granulose cells of the ovary.
  • the testis also produce Follistatin-288. Lack of follistatin results in reduced muscle mass at birth.
  • Examples of follistatins are provided in Shimasaki et al., U.S. Patent No. 5,041,538, other follistatin-like proteins are provided in U.S.
  • SMAD7 is known to inhibit TGF- ⁇ -activated signaling responses by associating with the active TGF- ⁇ complex, which results in reduced TGF- ⁇ signaling. Myostatin and TGF- ⁇ signaling induces SMAD7 expression establishing a negative feedback loop to inhibit TGF- ⁇ signaling.
  • MyoD is known to convert a number of cell types into muscle, including dermal fibroblasts, chondrocytes, smooth muscle, retinal pigmented epithelial cells, adipocytes, and melanoma, neuroblastoma, osteosarcoma, and hepatoma cells (Abraham & Tapscott, Curr. Opin. Genet. Dev.23(5): 568-573, 2013).
  • GNE myopathy is characterized by progressive muscle atrophy and weakness.
  • GNE myopathy is caused by mutations in the GNE gene, which encodes a bifunctional UDP-GlcNAc epimerase/ManNAc-6 kinase. GNE function is required for synthesis of all sialic acid (SA).
  • GNE myopathy incidence has recently been estimated to between 1 and 6 per million, a rare disease. There are, however, founder effect mutations that cause GNE myopathy to occur at much higher incidence in certain human populations, for example in patients of Japanese (D176V, D207V in the new nomenclature) and Middle Eastern (M712T, M743T in the new nomenclature) descent. Disease mutation carrier frequency in one study of 1000 Egyptian Jews was found to be 1 in 11. The partial reduction in GNE activity in patients leads to reduced, but not absent, SA expression.
  • IGF1R signaling has been shown to be a basis for muscle stem cell death in a model of GNE myopathy, making IGF1 a possible ideal growth factor element to the gene therapy design.
  • These tandem gene vectors are expected not only to inhibit disease progression (the function of GNE gene replacement) but also induce new muscle growth (thereby increasing muscle strength) and possibly prevent stem cell death.
  • These vectors are highly unique, as patients with GNE myopathy lose muscle and strength over decades, and the provided AAV are expected not only to slow this progression but to actually reverse it.
  • the provided dual function AAV will be able to show clinical efficacy, as this disease shows high clinical variability (between patient disease mutations and even amongst patients with the same disease mutation) and because it is slowly progressing (with major clinical changes occurring over decades).
  • GNE Myopathy Mutations [0070] In any of the provided methods of the subject is suffering from GNE myopathy.
  • the subject has a mutation in the GNE gene that results in reduced expression of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase.
  • a diagnosis of GNE myopathy is confirmed in a subject by the presence of pathogenic (mostly missense) mutations in both alleles of the GNE gene.
  • Table 1 provides of known mutations in the GNE gene that are associated with GNE myopathy is provided below.
  • the subjects of the claimed methods may comprise a mutation set out in this table.
  • Bold print indicates cDNA or protein truncating variants.
  • 1Amino acid substitutions are provided in the previously used hGNE1 (NP_005467.1) and in the preferred new hGNE2 (NP_001121699.1) nomenclature [Huizing et al.2014b].
  • updated nomenclature is provided extracted from the reference.
  • Amyotrophic lateral sclerosis [0072] Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder whose clinical features are determined by the progressive and inexorable degeneration of motor neurons.
  • Hallmarks of ALS are the sporadic nature, the great heterogeneity in age of onset, duration of disease and age of death. The average disease duration from diagnosis to death is 2-5 years, however, extremely aggressive or, on the opposite, mild variants where death follows as rapidly as 6 months or as late as 20 years after diagnosis, exist.
  • ALS cases are grouped into two different categories, sporadic (sALS) and familial (fALS). Around 10% of ALS cases are classified as fALS with a predominantly autosomal dominant pattern of inheritance, while the remaining 90% occur sporadically.
  • Sarcopenia and Aging are a condition marked by the wasting or loss muscle tissue and the replacement of muscle tissue with fibrosis tissue as the subject ages.
  • sarcopenia The etiology of sarcopenia is complex and can be attributed to a variety of factors, including oxidative stress, inflammation, apoptosis, and mitochondrial dysregulation (Roubenoff, 2003; Fulle et al., 2004), as well as genetic factors, inadequate diet, sedentary lifestyle, and the interplay between these factors (Rolland et al., 2008; Walrand et al., 2011). Reduction in expression of GNE naturally occurs with aging, and Gne +/- mice are shown to have aging dependent loss of muscle and brain function, along with reduced sialic acid as they age. [0075] Sarcopenia refers to the progressive deterioration in skeletal muscle mass, strength and physical function with advancing age.
  • Gne is an essential gene in mice; deletion causes embryonic lethality between embryonic (E) day 8.5 and 9.5.
  • Another mouse model is generated using Cas9-CRISPR, which will ultimately allow for the generation of a floxed allele into exon 3 of the mouse Gne gene, and introduction of this allele is sufficient to allow for Cre-mediated deletion.
  • Gne is essential in mice, leading to lethality between E8.5 and E9, creation of a floxed allele to delete the gene in the adult mouse should allow for creation of a robust body-wide or muscle-specific phenotypes using Cre-mediated deletion.
  • Gne lox/lox As well as Rosa26CreER T2 Gne lox/lox mice, using Cas9-CRISPR methods.
  • Gne lox/lox mice bear lox P sites flanking exon 3 of both alleles of the mouse Gne gene.
  • Cre recombinase When the Cre recombinase is introduced, it can induce Gne gene deletion in affected cells by deleting gene sequences within the loxP sites of the Gne gene.
  • Rosa26CreER T2 Gne lox/lox mice express a Cre- Estrogen Receptor (ER)T2 fusion protein in all tissues.
  • AAV AAV Gene Therapy
  • Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus.
  • AAV vector refers to a vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs).
  • AAV virion or "AAV viral particle” or “AAV vector particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "AAV vector particle” or simply an "AAV vector”.
  • a heterologous polynucleotide i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell
  • Adeno-associated virus is a replication-deficient parvovirus, the single- stranded DNA genome of which is about 4.7 kb in length including an inverted terminal repeat (ITRs). Exemplary ITR sequences may be 130 base pairs in length or 141 base pairs in length, such as the ITR sequence.
  • ITRs inverted terminal repeat
  • serotypes of AAV There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the AAV serotypes are known.
  • the nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45: 555-564 (1983) as corrected by Ruffing et al., J Gen Virol, 75: 3385-3392 (1994).
  • AAV-1 is provided in GenBank Accession No. NC_002077
  • the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829
  • the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829
  • the AAV-5 genome is provided in GenBank Accession No. AF085716
  • the complete genome of AAV-6 is provided in GenBank Accession No.
  • AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent Nos.7,282,199 and 7,790,449 relating to AAV-8); the AAV-9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). Cloning of the AAVrh.74 serotype is described in Rodino-Klapac., et al. Journal of translational medicine 5, 45 (2007).
  • Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the ITRs.
  • Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes.
  • the two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene.
  • Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome.
  • the cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins.
  • a single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992). [0085] AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic.
  • AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non- dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element).
  • the AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible.
  • the signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA such as a gene cassette containing a promoter, a DNA of interest and a polyadenylation signal.
  • the rep and cap proteins may be provided in trans.
  • Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56 o C to 65 o C for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized.
  • Recombinant AAV genomes of the disclosure comprise nucleic acid molecule of the disclosure and one or more AAV ITRs flanking a nucleic acid molecule.
  • AAV DNA in the rAAV genomes may be from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, or Anc80, AAV7m8 and their derivatives).
  • rAAV genome refers to a polynucleotide sequence that is derived from a native AAV genome that has been modified.
  • the rAAV genome has been modified to remove the native cap and rep genes.
  • the rAAV genome comprises the endogenous 5’ and 3’ inverted terminal repeats (ITRs).
  • the rAAV genome comprises ITRs from an AAV serotype that is different from the AAV serotype from which the AAV genome was derived.
  • the rAAV genome comprises a transgene of interest flanked on the 5’ and 3’ ends by inverted terminal repeat (ITR).
  • the rAAV genome comprises a “gene cassette.”
  • the genomes of both rAAV lack AAV rep and cap DNA, that is, there is no AAV rep or cap DNA between the ITRs of the genomes.
  • DNA plasmids of the disclosure comprise rAAV genomes of the disclosure. The DNA plasmids are transferred to cells permissible for infection with a helper virus of AAV (e.g., adenovirus, E1-deleted adenovirus or herpesvirus) for assembly of the rAAV genome into infectious viral particles.
  • a helper virus of AAV e.g., adenovirus, E1-deleted adenovirus or herpesvirus
  • rAAV particles in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions.
  • the AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-9, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-10, AAV-11, AAV-12 and AAV-13.
  • Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692 which is incorporated by reference herein in its entirety.
  • a method of generating a packaging cell is to create a cell line that stably expresses all the necessary components for AAV particle production.
  • a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell.
  • AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6.
  • the packaging cell line is then infected with a helper virus such as adenovirus.
  • a helper virus such as adenovirus.
  • packaging cells that produce infectious rAAV.
  • packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293 line).
  • packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with E1 of adenovirus), MRC-5 cells (human fetal fibroblasts), WI- 38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).
  • the rAAV may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper virus are known in the art and include methods disclosed in, for example, Clark et al., Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol.
  • compositions comprising rAAV of the present disclosure.
  • Compositions of the disclosure comprise rAAV and a pharmaceutically acceptable carrier.
  • the compositions may also comprise other ingredients such as diluents and adjuvants.
  • Acceptable carriers, diluents and adjuvants are nontoxic to recipients and are preferably inert at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate [e.g., phosphate-buffered saline (PBS)], citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, copolymers such as poloxamer 188, plu
  • Sterile injectable solutions are prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization.
  • dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.
  • Titers and dosages of rAAV to be administered in methods of the invention will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, the timing of administration, and the cell type(s) being targeted, and may be determined by methods standard in the art.
  • Titers of rAAV may range from about 1x10 6 , about 1x10 7 , about 1x10 8 , about 1x10 9 , about 1x10 10 , about 1x10 11 , about 1x10 12 , about 1x10 13 to about 2x10 14 or more DNase resistant particles (DRP) per kg of body weight. Dosages may also be expressed in units of viral genomes (vg).
  • These dosages of rAAV may range from about 1x10 9 vg or more, about 1x10 10 vg or more, about 1x10 11 vg or more, about 1x10 12 vg or more, about 6x10 12 or more, about 1x10 13 vg or more, about 1.3x10 13 vg or more, about 1.4x10 13 vg or more, about 2x10 13 vg or more, about 3x10 13 vg or more, about 6x10 13 vg or more, about 1x10 14 vg or more, about 3x10 14 or more, about 6x10 14 or more, about 1x10 15 vg or more, about 3x10 15 or more, about 6x10 15 or more, about 1x10 16 or more, about 3x10 16 or more, or about 6x10 16 or more.
  • the dosages of rAAV may range from about 1x10 9 vg or more, about 1x10 10 vg or more, about 1x10 11 vg or more, about 1x10 12 vg or more, about 6x10 12 or more, about 1x10 13 vg or more, about 1.3 x10 13 vg or more, about 1.4x10 13 vg or more, about 2x10 13 vg or more, about 3x10 13 vg or more, about 6x10 13 vg or more, about 1x10 14 vg or more, about 3x10 14 or more, about 6x10 14 or more, about 1x10 15 vg or more, about 3x10 15 or more, about 6x10 15 or more, about 1x10 16 or more, about 3x10 16 or more, or about 6x10 16 or more.
  • the in vivo methods comprise the step of administering an effective dose, or effective multiple doses, of a composition comprising a rAAV of the disclosure to an animal (including a human being) in need thereof. If the dose is administered prior to development of a disorder/disease, the administration is prophylactic. If the dose is administered after the development of a disorder/disease, the administration is therapeutic.
  • an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder/disease state being treated, that slows or prevents progression to a disorder/disease state, that slows or prevents progression of a disorder/disease state, that diminishes the extent of disease, that results in remission (partial or total) of disease, and/or that prolongs survival.
  • a disease contemplated for prevention or treatment with methods of the disclosure is limb girdle muscular dystrophy, e.g. LGMD2I, or congenital muscular dystrophy 1C.
  • transduction is used to refer to the administration/delivery of the coding region of the transgenes, e.g. FKRP gene and FST gene, to a recipient cell either in vivo or in vitro, via a replication-deficient rAAV of the disclosure resulting in expression of FKRP protein and FST protein in the recipient cell.
  • Transduction of cells with rAAV of the disclosure results in sustained expression of the proteins encoded by the first and second transgenes.
  • the present disclosure thus provides methods of administering/delivering rAAV to an animal, preferably a human being.
  • transducing tissues including, but not limited to, tissues such as muscle, organs such as liver and brain, and glands such as salivary glands
  • Transduction may be carried out with gene cassettes comprising tissue specific control elements.
  • one embodiment of the disclosure provides methods of transducing muscle cells and muscle tissues directed by muscle specific promoter elements, including, but not limited to, those derived from the actin and myosin gene families, such as from the myoD gene family (See Weintraub et al., Science, 251: 761-766 (1991)), the myocyte-specific enhancer binding factor MEF-2 (Cserjesi and Olson, Mol Cell Biol 11: 4854-4862 (1991)), control elements derived from the human skeletal actin gene (Muscat et al., Mol Cell Biol, 7: 4089-4099 (1987)), the cardiac actin gene, muscle creatine kinase sequence elements (See Johnson et al., Mol Cell Biol, 9:3393- 3399 (1989)) and the murine creatine kinase enhancer (MCK) element, MHCK7, control elements derived from the skeletal fast-twitch troponin C gene, the slow-twitch cardiac troponin C gene and
  • Muscle tissue is an attractive target for in vivo DNA delivery, because it is not a vital organ and is easy to access.
  • muscle cell or “muscle tissue” is meant a cell or group of cells derived from muscle of any kind (for example, skeletal muscle and smooth muscle, e.g. from the digestive tract, urinary bladder, blood vessels or cardiac tissue). Such muscle cells may be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes and cardiomyoblasts.
  • Combination therapies are also contemplated by the disclosure. Combination as used herein includes both simultaneous treatment and sequential treatments.
  • the combination therapy comprises administering an immunosuppressing agent in combination with the gene therapy disclosed herein.
  • Administration of an effective dose of the compositions may be by routes standard in the art including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal.
  • Route(s) of administration and serotype(s) of AAV components of the rAAV (in particular, the AAV ITRs and capsid protein) of the disclosure may be chosen and/or matched by those skilled in the art taking into account the disease state being treated and the target cells/tissue(s) that are to express the protein encoded by the first and/or second transgene..
  • the disclosure provides for local administration and systemic administration of an effective dose of rAAV and compositions of the disclosure.
  • systemic administration is administration into the circulatory system so that the entire body is affected.
  • Systemic administration includes enteral administration such as absorption through the gastrointestinal tract and parenteral administration through injection, infusion or implantation.
  • the immunosuppressing agent may be administered before or after the onset of an immune response to the rAAV in the subject after administration of the gene therapy.
  • the immunosuppressing agent may be administered simultaneously with the gene therapy or the protein replacement therapy.
  • the immune response in a subject includes an adverse immune response or an inflammatory response following or caused by the administration of rAAV to the subject.
  • the immune response may be the production of antibodies in the subject in response to the administered rAAV.
  • immunosuppressing agents include glucocorticosteroids, janus kinase inhibitors, calcineurin inhibitors, mTOR inhibitors, cyctostatic agents such as purine analogs, methotrexate and cyclophosphamide, inosine monophosphate dehydrogenase (IMDH) inhibitors, biologics such as monoclonal antibodies or fusion proteins.
  • the immunosuppressing agent may be an anti-inflammatory steroid, which is a steroid that decreases inflammation and suppresses or modulates the immune system of the subject.
  • Exemplary anti-inflammatory steroid are glucocorticoids such as prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, deflazacort, budesonide or prednisone.
  • Janus kinase inhibitors are inhibitors of the JAK/STAT signaling pathway by targeting one or more of the Janus kinase family of enzymes.
  • Exemplary janus kinase inhibitors include tofacitinib, baricitinib, upadacitinib, peficitinib, and oclacitinib.
  • Calcineurin inhibitors bind to cyclophilin and inhibits the activity of calcineurin
  • Exemplary calcineurine inhibitors includes cyclosporine, tacrolimus and picecrolimus.
  • mTOR inhibitors reduce or inhibit the serine/threonine-specific protein kinase mTOR.
  • Exemplary mTOR inhibitors include sirolimus, everolimus, and temsirolimus.
  • the immunosuppressing agents include immune suppressing macrolides.
  • immune suppressing macrolides refer to macrolide agents that suppresses or modulates the immune system of the subject.
  • a macrolide is a class of agents that comprise a large macrocyclic lactone ring to which one or more deoxy sugars, such as cladinose or desoamine, are attached.
  • the lactone rings are usually 14-, 15-, or 16-membered.
  • Macrolides belong to the polyketide class of agents and may be natural products. Examples of immunosuppressing macrolides include tacrolimus, pimecrolimus, and sirolimus.
  • Purine analogs block nucleotide synthesis and include IMDH inhibitors. Exemplary purine analogs include azathioprine, mycophenolate and lefunomide.
  • immunosuppressing biologics include abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinenumab, vedolizumab, basiliximab, belatacep, and daclizumab.
  • the immunosuppressing agent is an anti-CD20 antibody.
  • anti-CD20 specific antibody refers to an antibody that specifically binds to or inhibits or reduces the expression or activity of CD20.
  • anti-CD20 antibodies include rituximab, ocrelizumab or ofatumumab.
  • immuosuppressing antibodies include anti-CD25 antibodies (or anti-IL2 antibodies or anti-TAC antibodies) such as basiliximab and daclizumab, and anti-CD3 antibodies such as muromonab-CD3, otelixizumab, teplizumab and visilizumab, anti-CD52 antibodies such as alemtuzumab.
  • EXAMPLES are provided by way of illustration and not limitation. Described numerical ranges are inclusive of each integer value within each range and inclusive of the lowest and highest stated integer.
  • the dual gene expression cassette had a Kanamycin resistance gene, and an optimized Kozak sequence, which allows for more accurate and robust protein translation.
  • rAAV vectors were produced by a modified cross-packaging approach whereby the AAV type 2 vector genome can be packaged into multiple AAV capsid serotypes [Rabinowitz et al., J Virol.76 (2):791-801 (2002)]. Production was accomplished using a standard three plasmid DNA/CaPO4 precipitation method using HEK293 cells. HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin and streptomycin.
  • FBS fetal bovine serum
  • the production plasmids were: (i) plasmids encoding the therapeutic proteins, (ii) rep2-capX modified AAV helper plasmids encoding cap serotype AAVrh74 isolate, and (iii) an adenovirus type 5 helper plasmid (pAdhelper) expressing adenovirus E2A, E4 ORF6, and VA I/II RNA genes.
  • a quantitative PCR-based titration method was used to determine an encapsidated vector genome (vg) titer utilizing a Prism 7500 Taqman detector system (PE Applied Biosystems). [Clark et al., Hum Gene Ther.10 (6): 1031-1039 (1999)].
  • a final titer (vg ml ⁇ 1 ) was determined by quantitative reverse transcriptase PCR using the specific primers and probes utilizing a Prism 7500 Real-time detector system (PE Applied Biosystems, Grand Island, NY, USA). Aliquoted viruses were kept at ⁇ 80 °C until production. [00117] All plasmids used to make AAV genomes to be packaged also contain a Kanamycin resistance gene (KanR) outside of the ITR sequences used for packaging of the genome. This allows for the DNA encoding the AAV genome to be transformed into bacteria to produce large amounts of DNA in the presence of Kanamycin, which will kill all non- transformed bacteria.
  • KanR Kanamycin resistance gene
  • KanR is not packaged into the AAV capsid in the AAV genome used to treat patients, but its presence allows for DNA production in bacteria.
  • a description of pAAV.CBh.GNE.spA.miniCMV.FST344.spA plasmid (SEQ ID NO: 1) is provided in Table 2 below.
  • a description of pAAV.CMV.GNE.spA.miniCMV.FST344.spA plasmid (SEQ ID NO: 2) is provided in Table 3 below.
  • Gne haploinsufficiency (Gne +/- ) in mice also is associated with age-dependent losses in ambulation and neurodegeneration.
  • the disclosed rAAV are contemplated for use in treating sarcopenia, the normal age-dependent loss of muscle mass and strength, as well as age-dependent neurodegeneration.
  • Elderly humans lose 20-25% of their muscle mass as they age. This dramatically increases the risk of falls and bone fractures as well as impairment of ambulation as humans live past the age of 80.
  • the gene therapy disclosed herein can be used to offset and rebuild that lost muscle strength, while at the same time preventing neuronal cell death, which can contribute to cognitive loss (e.g. memory loss).
  • the disclosed gene therapies can also be used as an anti-aging therapy in humans without a genetic disease.
  • rAAV rAAVrh74.CMV.GNE.miniCMV.FST, rAAVrh74.Cbh.GNE.miniCMV.FST344, rAAVrh74.CMV.GNE.FL-IRES(full length internal ribosomal entry site from FGF1A), FST, rAAVrh74.CMV.GNE and rAAVrh74.Cbh.GNE.
  • Phosphate buffered saline was injected as a control.
  • Mice of 2 months of age were injected bilaterally into the tibialis anterior (TA) and the gastrocnemius (GS). At 4 months of age, 2 months after treatment, mice were euthanized and muscle dissected from tendon to tendon, weighed, and snap frozen for molecular and histological studies. Muscle weights for mice injected with rAAV.CMV.GNE.FL-IRES.FST showed no growth over this short interval relative to control mice injected with PBS.
  • the CMV or Cbh promoters were used to express the GNE gene alone (rAAV.CMV.GNE or rAAV.Cbh.GNE), and the same two promoters were used in duel gene constructs (either rAAV.CMV.GNE.pA.mCMV.FST or rAAV.Cbh.GNE.pA.mCMV.FST) to express both GNE and FST.
  • duel gene constructs either rAAV.CMV.GNE.pA.mCMV.FST or rAAV.Cbh.GNE.pA.mCMV.FST
  • each of the dual gene vectors allowed for a doubling of muscle size of both the tibialis anterior muscle and the gastrocnemius muscle compared to the PBS control, single gene constructs, or the IRES construct.
  • Muscles were injected with 1x10 11 vg AAV in the TA or 5x10 11 vg in the GS.
  • the effect of both human FST gene expression and human GNE expression, comparing the single gene and dual gene vectors was also assessed (see Fig.4).
  • Wild type (C57Bl/6J) mice were injected at 2 months of age, with analysis at 4 months of age, 2 months after injection.
  • RNA levels were measured by qRT-PCR, using an 18S rRNA internal control, and fold change in gene expression, relative to endogenous wild type mouse gene expression was calculated.
  • FST and GNE gene expression levels for the “IRES” construct were lower in the TA and GS than for either dual gene construct (with the exception of GNE in the gastrocnemius).
  • CMV rAAV.CMV.GNE
  • Cbh rAAV.Cbh.GNE
  • the two promoter versions of the dual gene vectors not only built more muscle mass (Fig.3) but amplified transgene expression (Fig.4) relative to the IRES vector or either single gene vector.
  • This study demonstrated that the use of a dual gene therapy vector using two different promoters to control gene expression, such as rAAV.Cbh.GNE.pA.mCMV.FST or rAAV.CMV.GNE.pA.mCMV.FST, are preferable to other single gene vectors or bicistronic vectors comprising an IRES for the treatment of GNE myopathy, GNE-dependent ALS, sarcopenia, and aging.
  • Example 3 Dual gene AAV GNE/FST Gene Therapy in Gne-deficient Mice
  • AAV.CMV.Cre (referred to as “CRE”) was injected with or without rAAV.CBh.GNE (referred to as “Cre+GNE”), rAAV.CMV.FST (referred to as “Cre+FST”) or dual gene rAAV.CBh.GNE.pA.mCMV.FST (referred to as “Cre+GNE/FST”).
  • FIG. 5 provides Hematoxylin and Eosin staining of myofibers sections obtained from the Gne-deficient treated mice.
  • Cre-induced Gne gene deletion caused muscle damage, as evidenced by the loss of muscle tissue with mononuclear cell infiltrates (indicative of inflammation), necrotic myofibers (indicative of muscle cell death), split myofibers (indicative of impaired muscle regeneration), and centrally located myofiber nuclei (indicative of a cycle of myofiber degeneration and regeneration).
  • GNE alone stopped all muscle damage caused by Cre, but did not significantly increase average myofiber size.
  • FST did not stop muscle damage, but modestly increased myofibers size.
  • Dual gene GNE/FST stopped all muscle damage and dramatically increased myofiber size, indicated coordinated correction of Gne gene deficiency (gene replacement) and induction of muscle growth.
  • Muscles were injected again intramuscularly with rAAV.CMV.Cre at 6 weeks (56 days), with analysis occurring 4 weeks later (at 10 weeks, or day 70). Hematoxylin and Eosin staining of myofibers sections obtained from the treated mice is shown in Figure 6. [00130] As shown in Figure 6, injection of Cre alone, which induces Gne gene deletion, resulted in muscle damage, as evidence by the presence of necrotic myofibers (indicative of muscle cell death), mononuclear infiltrates (indicative of inflammation), split myofibers (indicative of failed muscle regeneration), and myofibers with central nuclei (indicative of a cycle of myofiber degeneration and regeneration).

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Abstract

La présente invention concerne des méthodes et des substances visant à traiter des troubles liés à GNE tels que la myopathie GNE, la SLA dépendant de GNE, la thrombocytopénie, la sarcopénie et le vieillissement à l'aide d'un virus adéno-associé recombinant à double gène comprenant le gène GNE et le gène follistatine. Cette thérapie est unique en ce qu'elle peut reconstituer la force musculaire perdue en même temps qu'elle empêche la survenue d'une maladie musculaire ultérieure.
PCT/US2025/018739 2024-03-07 2025-03-06 Thérapie génique pour le traitement de troubles liés à gne Pending WO2025188993A2 (fr)

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