WO2024003687A1 - Acides nucléiques codant pour l'alpha-glucosidase acide (gaa) et vecteurs pour thérapie génique - Google Patents

Acides nucléiques codant pour l'alpha-glucosidase acide (gaa) et vecteurs pour thérapie génique Download PDF

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WO2024003687A1
WO2024003687A1 PCT/IB2023/056506 IB2023056506W WO2024003687A1 WO 2024003687 A1 WO2024003687 A1 WO 2024003687A1 IB 2023056506 W IB2023056506 W IB 2023056506W WO 2024003687 A1 WO2024003687 A1 WO 2024003687A1
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seq
nucleic acid
acid sequence
gaa
polypeptide
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Susan Adam BENARD
Waijiao CAI
Yuxing CHENG
Nicholas Andrew MARZE
Clark Qun Pan
Suryanarayan SOMANATHAN
Chihyi YU
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Pfizer Inc.
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Publication of WO2024003687A1 publication Critical patent/WO2024003687A1/fr

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    • 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
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    • A61P3/00—Drugs for disorders of the metabolism
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/0102—Alpha-glucosidase (3.2.1.20)
    • 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
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • Pompe disease also referred to as glycogen storage disease type II, type II glycogenosis or acid maltase deficiency
  • GAA glycogen storage disease type II, type II glycogenosis or acid maltase deficiency
  • Pompe disease There are three types of Pompe disease classified according to the onset and disease severity: classic infantile-onset, non-classic infantile-onset, and late onset. Infants affected with the infantile-onset form develop disease symptoms within a few months of birth experiencing hepatomegaly, myopathy, hypotonia and failure to thrive. Storage of glycogen within neurons and glial cells, brainstem motor and sensory neurons and sensory neurons, interneurons and motor neurons of the spinal cord is also observed. Without treatment, death due to heart failure generally occurs within the first year. The non-classic infantile-onset form usually presents by 1 year of age with delayed motor skills and progressive weakness. While patients with this form exhibit cardiomegaly, heart failure is not usually the cause of death.
  • Muscle weakness leads to breathing difficulties and a lifespan of only a few years. Late-onset Pompe disease may present later in childhood, adolescence or adulthood with progressive muscle weakness affecting the legs and trunk leading to respiratory failure. These patients also exhibit symptoms affecting the central, peripheral and autonomous nervous system, vascular malformation and cardiac involvement, musculoskeletal and bone changes, oro-gastrointestinal alterations and urinary tract alterations (Toscano et aL, Ann. TransL Med. (2019) 7(13) :284; Chan et aL, Mol. Genet. Metab. (2017) 120:163-172).
  • ERT enzyme replacement therapy
  • some patients treated with ERT exhibit weak correction of skeletal muscle function and a neurologic phenotype including bulbar muscular weakness. These persistent symptoms may be attributed to the inability of ERT to impact central nervous system glycogen stores due to failure to cross the blood brain barrier and failure to impact motor neuron glycogen stores.
  • a systemic therapy such as gene therapy, which is able to treat all tissues, including cardiac muscle, skeletal muscle and the CNS in patients with Pompe disease.
  • modified nucleic acids encoding a modified acid alpha-glucosidase (GAA) polypeptide
  • vectors e.g., rAAV vectors
  • GAA modified acid alpha-glucosidase
  • use of the vectors in the treatment of diseases, disorders and conditions associated with a decreased level of functional GAA including diseases, disorders and conditions associated with diminished cellular catabolism of glycogen, for example Pompe disease.
  • Modified GAA polypeptides are delivered to a subject as a gene therapy vector comprising a GAA transgene modified at least with a nucleic acid encoding a signal peptide that increases secretion of GAA polypeptide from a transduced cell (e.g., hepatocyte) and a nucleic acid encoding an amino-terminal peptide tag (“N-terminal peptide tag” or “NTPT”) that is associated with increased uptake of the expressed polypeptide by muscle tissue (e.g., cardiac, skeletal) and nervous system tissue (e.g., brain, spinal cord).
  • a transduced cell e.g., hepatocyte
  • N-terminal peptide tag amino-terminal peptide tag
  • muscle tissue e.g., cardiac, skeletal, smooth muscle
  • nervous system tissue e.g., brain, spinal cord
  • the amino-terminal peptide tag also facilitates transfer of the linked GAA polypeptide across the blood-brain-barrier such that GAA activity is increased in the brain and spinal cord and glycogen levels are decreased.
  • Such gene therapy vectors are demonstrated herein to correct symptoms of Pompe disease in GAA knockout mice, including, but not limited to an increase in GAA activity in serum, quadricep tissue, diaphragm tissue, heart, brain and spinal cord and a reduction in glycogen levels in quadricep tissue, diaphragm tissue, heart, brain and spinal cord.
  • amino-terminal peptide tags disclosed herein also exhibit reduced binding affinity to IGF1 receptors, insulin receptors or both, while maintaining binding affinity to IGF2 receptors as compared to wild type IGF2 polypeptides to advantageously reduce biological effects resulting from IGF1 receptor binding, insulin receptor binding or both, including cellular proliferation and blood glucose depletion.
  • the present disclosure provides a modified nucleic acid encoding an acid alpha-glucosidase (GAA) polypeptide comprising a nucleic acid sequence at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 , SEQ ID NO:52, SEQ ID N:53, SEQ ID NO:54 and SEQ ID NO:55.
  • GAA acid alpha-glucosidase
  • the present disclosure provides a modified nucleic acid encoding a polypeptide comprising an amino-terminal peptide tag (NTPT), a linker and an acid alphaglucosidase (GAA) polypeptide comprising a nucleic acid sequence at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NQ:60, SEQ ID NO:61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:118.
  • NTPT amino-terminal peptide tag
  • GAA acid alphaglucosidase
  • the present disclosure provides a modified nucleic acid encoding a polypeptide comprising a signal peptide, an amino-terminal peptide tag (NTPT), a linker and an acid alpha-glucosidase (GAA) polypeptide comprising a nucleic acid sequence at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:119 and SEQ ID NQ:120.
  • NTPT amino-terminal peptide tag
  • GAA acid alpha-glu
  • the present disclosure provides a vector genome comprising a modified nucleic acid encoding an acid alpha-glucosidase (GAA) polypeptide comprising a nucleic acid sequence at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:52, SEQ ID N:53, SEQ ID NO:54 and SEQ ID NO:55.
  • GAA acid alpha-glucosidase
  • the present disclosure provides a vector genome comprising a modified nucleic acid encoding a polypeptide comprising an amino-terminal peptide tag (NTPT), a linker and an acid alpha-glucosidase (GAA) polypeptide comprising a nucleic acid sequence at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NQ:60, SEQ ID NO:61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:118.
  • NTPT amino-terminal peptide tag
  • GAA acid alpha-glucosidase
  • the present disclosure provides a vector genome comprising a modified nucleic acid encoding a polypeptide comprising a signal peptide, an amino-terminal peptide tag (NTPT), a linker and an acid alpha-glucosidase (GAA) polypeptide comprising a nucleic acid sequence at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:119 and SEQ ID NQ:120.
  • NTPT amino-terminal peptide tag
  • the present disclosure provides a vector genome comprising a modified nucleic acid encoding a polypeptide comprising a signal peptide, an amino-terminal peptide tag (NTPT), a linker, an acid alpha-glucosidase (GAA) polypeptide and regulatory elements comprising a nucleic acid sequence at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:116 and SEQ ID NO:117.
  • NTPT amino-terminal peptide tag
  • GAA acid al
  • the vector genome further comprising at least one spacer sequence selected from the group consisting of SEQ ID NO:99, SEQ ID NQ:100, SEQ ID NQ:101 , SEQ ID NQ:102, SEQ ID NQ:103 and SEQ ID NQ:104.
  • the vector genome is a recombinant adeno-associated virus (rAAV) vector genome.
  • the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a vector genome comprising a modified nucleic acid, encoding a polypeptide comprising a signal peptide, an amino-terminal peptide tag (NTPT), a linker, an acid alpha-glucosidase (GAA) polypeptide, and regulatory elements comprising a nucleic acid sequence at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ
  • the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a vector genome comprising a modified nucleic acid, encoding a polypeptide comprising a signal peptide, an amino-terminal peptide tag (NTPT), a linker and an acid alpha-glucosidase (GAA) polypeptide, comprising a nucleic acid sequence at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87,
  • rAAV
  • the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a vector genome comprising a modified nucleic acid comprising i) a nucleic acid sequence encoding a signal peptide, wherein the nucleic acid sequence is at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22 and SEQ ID NO:23, ii) a nucleic acid sequence encoding a NTPT, wherein the nucleic acid sequence is at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ
  • the capsid is selected from the group consisting of a capsid of AAV1 , AAV2, AAV3 (including AAV3A and AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV12, AAVrh8, AAVrhIO, AAVrh39, AAVrh43, AAVrh74, AAVrh32.22, AAV1 .1 , AAV2.5, AAV6.1 , AAV6.2, AAV6.3.1 , AAV9.45, AAVShHIO, HSC15/17, RHM4-1 , RHM15-1 , RHM15-2, RHM15-3/RHM15- 5, RHM15-4, RHM15-6, AAVhu.26, AAV2i8, AAV29G, AAV2,8G9, AAV-LK03, AAV2-TT, AAV2- TT-S312N, AAV3B-S312N, avian AAV,
  • the capsid is an AAV9 capsid. In some embodiments, the capsid is an rh74 capsid. In some embodiments, the capsid is an AAV9 capsid comprising a viral protein 1 (VP1 ) and wherein the VP1 comprises an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:112. In some embodiments, the vector genome further comprises at least one element selected from the group consisting of at least one AAV inverted terminal repeat (ITR) sequence, an enhancer, a promoter, an intron, and a poly-adenylation (polyA) signal sequence.
  • ITR AAV inverted terminal repeat
  • polyA poly-adenylation
  • the vector genome further comprises at least one element selected from the group consisting of at least one AAV2 ITR, an apolipoprotein E (ApoE) enhancer, a human alpha-1 antitrypsin (hAAT) promoter, a chimeric intron, and a bovine growth hormone (BGH) polyA.
  • AAV2 ITR an apolipoprotein E (ApoE) enhancer
  • hAAT human alpha-1 antitrypsin
  • BGH bovine growth hormone
  • the vector genome further comprises a least one element selected from the group consisting of at least one ITR comprising the nucleic acid sequence of SEQ ID NO:109 or SEQ ID NO:110, an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105, a promoter comprising the nucleic acid sequence of SEQ ID NQ:106, an intron comprising the nucleic acid sequence of SEQ ID NO:107, and a polyA comprising the nucleic acid sequence of SEQ ID NO:108.
  • a least one element selected from the group consisting of at least one ITR comprising the nucleic acid sequence of SEQ ID NO:109 or SEQ ID NO:110, an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105, a promoter comprising the nucleic acid sequence of SEQ ID NQ:106, an intron comprising the nucleic acid sequence of SEQ ID NO:107, and a polyA comprising the nucleic acid sequence of SEQ ID NO:108.
  • the vector genome further comprises at least one spacer sequence, optionally wherein the spacer sequence is selected from the group consisting of SEQ ID NO:99, SEQ ID NQ:100, SEQ ID NQ:101 , SEQ ID NQ:102, SEQ ID NQ:103 and SEQ ID NQ:104.
  • the present disclosure provides a rAAV vector comprising a vector genome comprising from 5’ to 3’: a) an AAV inverted terminal repeat (ITR) comprising the nucleic acid sequence of SEQ ID NO:109 or SEQ ID NO:110; b) an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105; c) a promoter comprising the nucleic acid sequence of SEQ ID NQ:106; d) an intron comprising the nucleic acid sequence of SEQ ID NQ:107; e) a modified nucleic acid encoding a signal peptide, a NTPT, a linker and an alpha acid- glucosidase (GAA) polypeptide comprising the nucleic acid sequence of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:
  • ITR AAV in
  • the present disclosure provides a rAAV vector comprising a vector genome comprising from 5’ to 3’: a) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NO:109 or SEQ ID NO:110; b) an enhancer comprising the nucleic acid sequence of SEQ ID NO:105; c) a promoter comprising the nucleic acid sequence of SEQ ID NO:106; d) an intron comprising the nucleic acid sequence of SEQ ID NQ:107; e) a signal peptide comprising the nucleic acid sequence of any one of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24; f) a NTPT comprising the nucleic acid sequence of any one of SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33,
  • the present disclosure provides a rAAV vector comprising a vector genome comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:116 and SEQ ID NO:117.
  • the vector genome further comprises at least one spacer sequence, optionally wherein the spacer sequence is selected from the group consisting of SEQ ID NO:99, SEQ ID N0:100, SEQ ID NO:101 , SEQ ID NO:102, SEQ ID NQ:103 and SEQ ID NQ:104 and wherein the spacer sequence flanks an ITR sequence.
  • the vector comprises an AAV9 capsid.
  • the present disclosure provides a pharmaceutical composition comprising a rAAV vector disclosed herein.
  • the present disclosure provides methods of treating and/or preventing a disease, disorder or condition associated with deficiency or dysfunction of GAA, the method comprising administering a therapeutically effective amount of a rAAV vector or a pharmaceutical composition disclosed herein in a subject in need thereof.
  • the disease, disorder or condition associated with deficiency or dysfunction of GAA is Pompe disease.
  • the subject is a human subject with Pompe disease.
  • administering a therapeutically effective amount of an rAAV vector or a pharmaceutical composition disclosed herein to a subject decreases glycogen levels in quadricep muscle, diaphragm, heart, brain, spinal cord or a combination thereof as compared to glycogen levels in quadricep muscle, diaphragm, heart, brain, spinal cord or a combination thereof in the subject prior to administering the rAAV vector or the pharmaceutical composition.
  • administering a therapeutically effective amount of a rAAV vector or a pharmaceutical composition disclosed herein to a subject increases GAA activity levels in serum, quadricep muscle, diaphragm, heart, brain, spinal cord or a combination thereof as compared to GAA activity levels in serum quadricep muscle, diaphragm, heart, brain, spinal cord or a combination thereof in the subject prior to administering the rAAV vector or the pharmaceutical composition.
  • the rAAV vector or pharmaceutical composition is administered intravenously.
  • the disclosure provides a host cell comprising a modified nucleic acid, a vector genome or a rAAV vector disclosed herein.
  • a host cell is selected from the group consisting of VERO, WI38, MRC5, A549, HEK293, B-50 or any other HeLa cell, HepG2, Saos-2, HuH7, and HT 1080.
  • a host cell is a HEK293 cell adapted to growth in suspension culture.
  • a host cell is a HEK293 cell having American Type Culture Collection (ATCC) No. PTA 13274.
  • ATCC American Type Culture Collection
  • the disclosure provides a kit for the treatment of Pompe disease, comprising a therapeutically effective amount of a modified nucleic acid, a vector genome, a rAAV vector or a pharmaceutical composition disclosed herein.
  • the kit further comprises a label or insert including instructions for using one or more of the kit components.
  • the disclosure provides an amino-terminal peptide tag (NTPT) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42.
  • NTPT amino-terminal peptide tag
  • the NTPT is operably linked to a lysosomal enzyme polypeptide.
  • the NTPT is operably linked to a GAA polypeptide, optionally comprising an amino acid sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:114 and amino acids 70-952 of SEQ ID NO:114.
  • the NTPT increases uptake of the operably linked GAA polypeptide by a cell or tissue as compared to uptake of a GAA polypeptide without an operably linked NTPT by an otherwise identical cell or tissue.
  • the cell is a cell of skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord or a combination thereof.
  • the tissue is skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord or a combination thereof.
  • the NTPT facilitates transfer of the operably linked GAA polypeptide across the blood-brain-barrier.
  • transfer of the operably linked GAA polypeptide across the blood-brain-barrier is associated with increased GAA activity in the brain, spinal cord or both.
  • increased GAA activity in the brain, spinal cord or both is associated with decreased glycogen levels in the brain, spinal cord or both.
  • FIG. 1 depicts exemplary measurement of GAA activity in L6 myoblasts treated with conditioned median containing increasing amounts of wild type GAA (wtGAA) and NTPT-GAA polypeptides. Each data point represents the mean of three wells. Error bars represent the standard deviation.
  • FIG. 2 depicts exemplary quadricep GAA activity from mice transfected with a rAVV vector comprising either a wtGAA transgene or a NTPT-GAA transgene.
  • a rAVV vector comprising either a wtGAA transgene or a NTPT-GAA transgene.
  • Six to eight mice were dosed at each amount (2.5E12 vg/kg, 7.5E12 vg/kg and 2.25E13 vg/kg).
  • Serum GAA activity is shown on the x-axis
  • quadricep GAA activity is shown on the y-axis.
  • FIG. 3 depicts exemplary WesTM (automated capillary-based immunoassay) (ProteinSimple) analysis of serum GAA from GAA KO mice intravenously dosed with 7.5E12 vg/kg of a rAAV9 vector comprising a nucleic acid encoding wtGAA (animals #1 -3) or a rAAV9 vector comprising a nucleic acid encoding NTPT-GAA (animals #4-6) (top panel).
  • 1 .2 ng, 0.4 ng, 0.133 ng, 0.04 ng rhGAA R&D #8329-GH
  • the samples were probed with antihuman GAA (Invitrogen #PA5-96756).
  • the bottom panel depicts exemplary GAA concentration (left y-axis) and GAA activity (right y-axis) levels in the serum of the same animals.
  • FIG. 4 depicts exemplary WesTM (automated capillary-based immunoassay) (ProteinSimple) analysis of serum GAA from GAA KO mice intravenously dosed with 7.5E12 vg/kg of a rAAV9 vector comprising a nucleic acid encoding wtGAA or a rAAV9 vector comprising a nucleic acid encoding NTPT-GAA.
  • Two sets of serum samples were probed with anti-human GAA (Invitrogen #PA5-96756) and anti-human IGF2 (Abeam #ab9574) antibodies in parallel by WesTM analysis.
  • FIG. 5 depicts exemplary WesTM (automated capillary-based immunoassay) (ProteinSimple) analysis of cell lysates or conditioned media from HEK293 cells transfected with a wtGAA plasmid or a NTPT-GAA plasmid.
  • Cell lysates and conditioned media were probed with anti-human GAA antibody (Invitrogen #PA5-96756) and conditioned media was probed with anti-human IGF2 antibody (Abeam #ab9574).
  • FIG. 6A depicts exemplary serum GAA activity in rAAV9 wtGAA and rAAV9 NTPT- GAA treated GAA KO mice, untreated wild type (WT Untx) and untreated GAA KO mice (KO Untx).
  • FIG. 7A depicts exemplary quadricep tissue GAA activity levels in GAA KO mice administered rAAV9 wtGAA or rAAV9 NTPT-GAA relative to GAA activity in untreated wild type mice (WT Untx).
  • FIG. 7B depicts exemplary quadricep tissue glycogen levels in GAA KO mice administered rAAV9 wtGAA or rAAV9 NTPT-GAA relative to glycogen levels in untreated GAA KO (KO Untx) mice.
  • FIG. 8A depicts exemplary heart tissue glycogen levels in rAAV9 wtGAA and rAAV9 NTPT-GAA treated GAA KO mice, untreated GAA KO mice (KO Untx) and untreated wild type mice (WT Untx).
  • FIG. 8B depicts exemplary diaphragm glycogen levels in rAAV9 wtGAA and rAAV9 NTPT-GAA treated GAA KO mice, untreated GAA KO mice (KO Untx) and untreated wild type mice (WT Untx).
  • FIG. 9A depicts exemplary brain tissue glycogen levels in rAAV9 wt GAA and rAAV9 NTPT-GAA treated GAA KO mice, untreated GAA KO mice (KO Untx) and untreated wild type mice (WT Untx).
  • FIG. 9B depicts exemplary brain tissue GAA activity levels in rAAV9 wt GAA and rAAV9 NTPT-GAA treated GAA KO mice and untreated GAA KO mice (KO Untx) .
  • FIG. 10A depicts exemplary GAA activity of conditioned media from cells transfected with plasmids for the expression of GAA with various amino terminal signal peptides (SP). Each data point represents the mean of three wells. Error bars represent the standard deviation. Data was analyzed by one-way ANOVA followed by Dennett’s multiple comparisons vs SP17,
  • FIG. 10B depicts exemplary GAA activity in serum from wild type untreated mice (WT Untx), GAA KO untreated mice (KO Untx) and mice treated with 1 .5E13 vg/kg of rAAV NTPT-GAA vector, rAAV SP13-NTPT-GAA vector or rAAV IgVH-NTPT-GAA vector.
  • FIG. 11 B depicts exemplary GAA activity levels in serum from untreated GAA KO mice (KO Untx), from GAA KO mice administered the rAAV NTPT- GAACpGdl vector and from GAA KO mice administered the rAAV NTPT-GAA vector.
  • FIG. 12 depicts exemplary serum GAA activity levels in C57BL/6J male mice administrated 5E12 vg/kg of each of the following vectors: rAAV SP1 -NTPT-GAACpGd1 , rAAV SP7-NTPT-GAACpGd1 , rAAV SP13-NTPT-GAACpGd1 and rAAV SP13-NTPT-GAA.
  • One group of mice was administered a higher dose (1 .5E13 vg/kg) of the vector rAAV SP13-NTPT- GAA.
  • Each dose group comprised 5-6 mice.
  • blood was collected for serum GAA activity measurement.
  • FIG. 13A depicts exemplary serum GAA activity levels in GAA KO mice 3 weeks after administration of a rAAV vector encoding a polypeptide comprising a signal peptide, a N- terminal peptide tag and a GAA transgene.
  • FIG. 13B depicts exemplary serum GAA activity levels in GAA KO mice 12 weeks after administration of a rAAV vector encoding a polypeptide comprising a signal peptide, a N-terminal peptide tag and a GAA transgene.
  • n 8-10 per group, data represent mean and standard deviation.
  • FIG. 14A depicts exemplary latency to fall off a rotarod in seconds of untreated GAA KO (KO Untx) mice and GAA KO mice 11 weeks after administration of a rAAV vector encoding a polypeptide comprising a signal peptide, a N-terminal peptide tag and a GAA transgene. Data is expressed as a percentage of latency to fall of untreated wild type mice (WT UnTx).
  • FIG. 14B depicts exemplary muscle strength as maximum tetanic torque (grams) in untreated wild type mice (WT Untx), untreated GAA KO (KO Untx) mice and GAA KO mice 11 weeks after administration of a rAAV vector encoding a polypeptide comprising a signal peptide, a N- terminal peptide tag and a GAA transgene.
  • n 8-10 per group, data represent mean and standard deviation.
  • FIG. 15A depicts exemplary quadricep tissue GAA activity levels (nmol/hr/mg tissue) in untreated wild type mice (WT Untx), untreated GAA KO mice (KO Untx) and GAA KO mice 12 weeks after administration of a rAAV vector encoding a polypeptide comprising a signal peptide, a N-terminal peptide tag and a GAA transgene.
  • WT Untx wild type mice
  • KO Untx untreated GAA KO mice
  • 15B depicts exemplary quadricep tissue glycogen levels (pg/mg tissue) in untreated wild type mice (WT Untx), untreated GAA KO mice (KO Untx) and GAA KO mice 12 weeks after administration of a rAAV vector encoding a polypeptide comprising a signal peptide, a N-terminal peptide tag and a GAA transgene.
  • FIG. 16A depicts exemplary glycogen levels in diaphragm tissue of untreated wild type mice (WT Untx), untreated GAA KO mice (KO Untx) and GAA KO mice 12 weeks after administration of a rAAV vector encoding a polypeptide comprising a signal peptide, a N- terminal peptide tag and a GAA transgene.
  • WT Untx wild type mice
  • GAA KO mice untreated GAA KO mice
  • FIG. 16B depicts exemplary glycogen levels in heart tissue of untreated wild type mice (WT Untx), untreated GAA KO mice (KO Untx) and GAA KO mice 12 weeks after administration of a rAAV vector encoding a polypeptide comprising a signal peptide, a N-terminal peptide tag and a GAA transgene.
  • FIG. 17A depicts exemplary GAA activity levels in spinal cord tissue of untreated wild type mice (WT Untx), untreated GAA KO mice (KO Untx) and GAA KO mice 12 weeks after administration of a rAAV vector encoding a polypeptide comprising a signal peptide, a N- terminal peptide tag and a GAA transgene.
  • FIG. 17B depicts exemplary glycogen levels in spinal cord tissue of untreated wild type mice (WT Untx), untreated GAA KO mice (KO Untx) and GAA KO mice 12 weeks after administration of a rAAV vector encoding a polypeptide comprising a signal peptide, a N-terminal peptide tag and a GAA transgene.
  • FIG. 18A depicts exemplary GAA activity levels in brain tissue of untreated wild type mice (WT Untx), untreated GAA KO mice (KO Untx) and GAA KO mice 12 weeks after administration of a rAAV vector encoding a polypeptide comprising a signal peptide, a N- terminal peptide tag and a GAA transgene.
  • FIG. 18B depicts exemplary glycogen levels in brain tissue of untreated wild type mice (WT Untx), untreated GAA KO mice (KO Untx) and GAA KO mice 12 weeks after administration of a rAAV vector encoding a polypeptide comprising a signal peptide, a N-terminal peptide tag and a GAA transgene.
  • FIG. 19A depicts exemplary serum GAA activity in GAA KO mice treated with rAAV9 SP13-NTPT-GAACpGd1 (at doses1 E12, 5E12 and 2.5E13 vg/kg) and rAAV9 SP13- NTPTseq44-GAACpGd1 (at doses 1 E12 and 2.5E13 vg/kg), as well as in untreated GAA KO mice (KO Untx) and untreated wild type mice (WT Untx).
  • the 2.5E13 vg/kg groups had 14 animals per group, while other groups had 8-9 animals per group.
  • FIG. 19A depicts exemplary serum GAA activity in GAA KO mice treated with rAAV9 SP13-NTPT-GAACpGd1 (at doses1 E12, 5E12 and 2.5E13 vg/kg) and rAAV9 SP13- NTPTseq44-GAACpGd1 (at doses 1 E12 and 2.5E13 vg/
  • FIG. 19B depicts exemplary ratios of serum GAA activity to liver GAA activity in male GAA KO mice, which indicates liver secretion efficiency.
  • FIG. 19C depicts exemplary GAA activity levels in quadricep tissue as percentages of WT untreated.
  • Male GAA KO mice were treated with rAAV9 SP13-NTPT- GAACpGdl (at doses 1 E12, 5E12 and 2.5E13 vg/kg) and rAAV9 SP13-NTPTseq44- GAACpGdl (at doses 1 E12 and 2.5E13 vg/kg).
  • FIG. 19D depicts exemplary ratio of quadricep GAA activity to serum GAA activity, which indicates muscle enzyme uptake efficiency. The figure includes data from various groups in male GAA KO mice.
  • FIG. 19E depicts exemplary glycogen in tissues as percentages of KO untreated (Untx) in rAAV9 SP13-NTPT-GAACpGd1 (1 E12, 5E12 and 2.5E13 vg/kg) and rAAV9 SP13-NTPTseq44-GAACpGd1 (1 E12 and 2.5E13 vg/kg) treated GAA KO mice, untreated GAA KO mice (KO Untx) and untreated wild type mice (WT Untx).
  • the rAAV vectors disclosed herein include several advantageous properties including a modified nucleic acid encoding acid alpha-glucosidase (GAA).
  • the modified nucleic acids encoding GAA may be codon optimized.
  • a modified nucleic acid encoding GAA may be codon optimized to increase expression in a cell as compared to expression of a non-codon optimized nucleic acid encoding GAA in an otherwise identical cell.
  • Modified nucleic acids encoding GAA disclosed herein may be CpG modified.
  • CpG modification may improve transgene expression.
  • the nucleic acids are codon- optimized and CpG modified.
  • GAA polypeptides encoded by rAAV vectors of the disclosure may be modified by addition of an amino terminal signal peptide and an amino terminal peptide tag.
  • the amino terminal signal peptides disclosed herein advantageously increase secretion of the expressed GAA polypeptide from hepatocytes to increase the serum GAA level and thereby increase availability of the GAA enzyme to target tissue (e.g., skeletal muscle, heart, brain, spinal cord) uptake.
  • the amino terminal peptide tags (NTPT) disclosed herein surprisingly and advantageously increase uptake of the expressed GAA polypeptide by target cells and tissues (e.g., skeletal muscle, heart, brain, spinal cord) to increase the tissue GAA activity level.
  • target cells and tissues e.g., skeletal muscle, heart, brain, spinal cord
  • increased tissue GAA levels are associated with decreased glycogen stored in tissues useful to prevent or reduce pathogenic glycogen levels in subjects with Pompe disease.
  • the NTPTs disclosed herein function by targeting the GAA polypeptide to a lysosome within a muscle cell (e.g., skeletal, cardiac), within a cell of the central, peripheral and/or autonomic nervous systems (e.g., neuron, astrocyte, glial cell) or both.
  • the NTPTs disclosed herein surprisingly and advantageously facilitate transfer of the GAA polypeptide across the blood-brain-barrier.
  • the NTPTs facilitate transfer of the GAA polypeptide across the blood-brain barrier and target the GAA to a lysosome within a cell of the central, peripheral and/or autonomic nervous systems (e.g., neuron, astrocyte, glial cell).
  • a lysosome within a cell of the central, peripheral and/or autonomic nervous systems (e.g., neuron, astrocyte, glial cell).
  • rAAV vectors such as those disclosed herein, that encode modified GAA polypeptides with surprising and advantageous characteristics including increased secretion (and thus increase serum concentration), increased tissue enzyme activity and an ability to cross the blood-brain-barrier to be taken up by cells of the spinal cord and brain, would be beneficial for use in clinical gene therapy programs for the treatment of Pompe disease.
  • the term “about” refers to a measurable value such as an amount of the biological activity, length of polynucleotide or polypeptide sequence, content of G and C nucleotides, codon adaptation index, number of CpG dinucleotides, dose, time, temperature and the like and is meant to encompass +10% numerical range of a given value.
  • a dosage of about 50 milligrams per kilogram (mg/kg) refers to a range of 45 to 55 mg/kg.
  • the term “ameliorate” means a detectable or measurable improvement in a subject’s disease, disorder or condition, or symptom thereof, or an underlying cellular response.
  • a detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limit or control in the occurrence, frequency, severity, progression or duration of, complication caused by or associated with improvement in a symptom of, or a reversal of a disease, disorder or condition.
  • symptoms of Pompe disease such as increased glycogen levels in muscle and CNS tissue, are ameliorated by treatment with a rAAV vector encoding a modified GAA protein by decreasing glycogen levels in muscle and CNS tissue.
  • the term “associated with” refers to with one another, if the presence, level and/or form of one is correlated with that of the other.
  • a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc.
  • two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another.
  • two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example, by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and a combination thereof.
  • coding sequence or “encoding nucleic acid” refers to a nucleic acid sequence which encodes a protein or polypeptide and denotes a sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of (operably linked to) appropriate regulatory sequences. Boundaries of a coding sequence are generally determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.
  • flanked refers to a sequence that is flanked by other elements and indicates the presence of one or more flanking elements upstream and/or downstream, i.e., 5' and/or 3', relative to the sequence.
  • the term “flanked” is not intended to indicate that the sequences are necessarily contiguous. For example, there may be intervening sequences between a nucleic acid encoding a transgene and a flanking element.
  • a sequence e.g., a transgene
  • two other elements e.g., ITRs
  • fragment refers to a material or entity that has a structure that includes a discrete portion of the whole but lacks one or more moieties found in the whole. In some embodiments, a fragment consists of a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole.
  • a polymer fragment comprises, or consists of, at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., amino acid residues, nucleotides) found in the whole polymer.
  • monomeric units e.g., amino acid residues, nucleotides
  • formulation As used herein, the term “formulation,” “pharmaceutical formulation,” or “pharmaceutical composition” as they relate to a rAAV vector are meant to describe the rAAV vector in combination with a pharmaceutically acceptable excipient comprising, for example, a buffer, a salt, a cryoprotectant, a surfactant, and, wherein the pH is defined.
  • a pharmaceutically acceptable excipient comprising, for example, a buffer, a salt, a cryoprotectant, a surfactant, and, wherein the pH is defined.
  • a “pharmaceutical formulation” or “pharmaceutical composition” is a preparation which in in such form as to permit the biological activity of the active ingredients to be effective.
  • the term “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated. “Gene transfer” or “gene delivery” refers to methods or systems for reliably inserting foreign DNA into host cells. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g. episomes), and/or integration of transferred genetic material into the genomic DNA of host cells.
  • heterologous nucleic acid refers to a nucleic acid inserted into a vector (e.g., rAAV vector) for purposes of vector mediated transfer/delivery of the nucleic acid into a cell.
  • Heterologous nucleic acids are typically distinct from the vector (e.g., AAV) nucleic acid, that is, the heterologous nucleic acid is non-native with respect to the viral (e.g., AAV) nucleic acid found in the AAV in nature.
  • a heterologous nucleic acid contained within a vector, can be expressed (e.g., transcribed and translated if appropriate). Alternatively, a transferred (transduced) or delivered heterologous nucleic acid in a cell, contained within the vector, need not be expressed.
  • heterologous is not always used herein in reference to a nucleic acid, reference to a nucleic acid even in the absence of the modifier “heterologous” is intended to include a heterologous nucleic acid.
  • a heterologous nucleic acid would be a nucleic acid encoding a GAA polypeptide, for example a codon optimized nucleic acid encoding GAA used in the treatment of Pompe disease.
  • the terms “host cell,” refers to a cell into which an exogenous nucleic acid has been introduced and includes the progeny of such a cell.
  • a host cell includes a “transfectant,” “transformant,” “transformed cell,” and “transduced cell,” which includes the primary transfected, transformed or transduced cell, and progeny derived therefrom, without regard to the number of passages.
  • a host cell is a packaging cell for production of a rAAV vector.
  • a host cell is a “host cell line,” or “host cell culture” including it progeny derived therefrom.
  • identity refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. “Identity” measures the percent of identical matches between two or more sequences with gap alignments addressed by a particular mathematical model of computer programs (i.e. “algorithms”).
  • polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical.
  • Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of a reference sequence.
  • the nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST/.
  • Other alignment programs include MegAlign® program in the Lasergene® suite of bioinformatics software (DNASTAR®, Inc., Madison, Wl).
  • FASTA is available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA.
  • GCG Genetics Computing Group
  • the program has default parameters determined by the sequences inputted to be compared.
  • the sequence identity is determined using the default parameters determined by the program.
  • This program is available also from Genetics Computing Group (GCG) package, from Madison, Wl, USA.
  • GCG Genetics Computing Group
  • Another program of interest is the FastDB algorithm.
  • the terms “increase,” improve” or “reduce” indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein.
  • a “control individual” is an individual afflicted with the same form of disease or injury as an individual being treated.
  • inverted terminal repeat refers to palindromic terminal repeat sequences at or near the ends of the AAV virus genome, comprising mostly complementary, symmetrically arranged sequences. These ITRs can fold over to form T-shaped hairpin structures that function as primers during initiation of DNA replication. They are also needed for viral genome integration into host genome, for the rescue from the host genome; and for the encapsidation of viral nucleic acid into mature virions. The ITRs are required in cis for vector genome replication and its packaging into viral particles.
  • “5’ ITR” refers to the ITR at the 5’ end of the AAV genome and/or 5’ to a recombinant transgene.
  • “3’ ITR” refers to the ITR at the 3’ end of the AAV genome and/or 3’ to a recombinant transgene.
  • Wild type ITRs are approximately 145 bp in length.
  • a modified, or recombinant ITR may comprise a fragment or portion of a wild type AAV ITR sequence.
  • One of ordinary skill in the art will appreciate that during successive rounds of DNA replication ITR sequences may swap such that the 5’ ITR becomes the 3’ ITR, and vice versa.
  • At least one ITR is present at the 5’ and/or 3’ end of a recombinant vector genome such that the vector genome can be packaged into a capsid to produce a rAAV vector (also referred to herein as “rAAV vector particle” or “rAAV viral particle”) comprising the vector genome.
  • rAAV vector particle also referred to herein as “rAAV vector particle” or “rAAV viral particle”
  • nucleic acid construct refers to a non- naturally occurring nucleic acid molecule resulting from the use of recombinant DNA technology (e.g., a recombinant nucleic acid).
  • a nucleic acid construct is a nucleic acid molecule, either single or double stranded, which has been modified to contain segments of nucleic acid sequences, which are combined and arranged in a manner not found in nature.
  • a nucleic acid construct may be a “vector” (e.g., a plasmid, a rAAV vector genome, an expression vector, etc.), that is, a nucleic acid molecule designed to deliver exogenously created DNA into a host cell.
  • a vector e.g., a plasmid, a rAAV vector genome, an expression vector, etc.
  • operably linked refers to a linkage of nucleic acid (or polypeptide) sequence elements in a functional relationship.
  • a nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or other transcription regulatory sequence e.g., an enhancer
  • a polypeptide is operably linked with it is placed into a functional or structural relationship with another polypeptide sequence.
  • a signal peptide is operably linked to a polypeptide (e.g., an enzyme) when it is expressed with the polypeptide, facilitates secretion of the polypeptide from a cell or both.
  • operably linked means that nucleic acid (or polypeptide) sequences being linked are contiguous. In some embodiments, operably linked does not mean that nucleic acid (or polypeptide) sequences are contiguously linked, rather intervening sequences are between those nucleic acid (or polypeptide) sequences that are linked.
  • the term “pharmaceutically acceptable” and “physiologically acceptable” refers to a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact.
  • “Pharmaceutically acceptable excipients” are those, which can safely be administered to a subject to provide an effective dose of the active ingredient employed.
  • the term “excipient” or “carrier” as used herein refers to an inert substance, which is commonly used as a diluent, vehicle, preservative, binder or stabilizing agent for drugs.
  • diluent refers to a pharmaceutically acceptable (safe and non-toxic for administration to a human) solvent and is useful for the preparation of the liquid formulations herein.
  • exemplary diluents include, but are not limited to, sterile water and bacteriostatic water for injection (BWFI).
  • polynucleotide or “nucleic acid” refers to a polymeric form of nucleotides, either ribonucleotides or deoxynucleotides, or a modified form of either type of nucleotide, and may be single or double stranded forms.
  • a “polynucleotide” or a “nucleic acid” sequence encompasses its complement unless otherwise specified.
  • isolated polynucleotide means a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which by virtue of its origin or source of derivation, has one to three of the following: (1 ) is not associated with all or a portion of a polynucleotide with which the “isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.
  • polypeptide As used herein, the terms “polypeptide,” “protein,” “peptide” or “encoded by a nucleic acid sequence” (i.e., encode by a polynucleotide sequence, encoded by a nucleotide sequence) refer to full-length native sequences, as with naturally occurring proteins, as well as functional subsequences, modified forms or sequence variants so long as the subsequence, modified form or variant retains some degree of functionality of the native full-length protein.
  • polypeptides, proteins and peptides encoded by the nucleic acid sequences can be but are not required to be identical to the endogenous protein that is defective, or whose expression is insufficient, or deficient in a subject treated with gene therapy.
  • prevention refers to delay of onset , and/or reduction in frequency and/or severity of one or more sign or symptom of a particular disease, disorder or condition (e.g., a lysosomal storage disease, e.g., Pompe disease).
  • prevention is assessed on a population basis such that an agent is considered to “prevent” a particular disease, disorder or condition if a statistically significant decrease in the development, frequency and/or intensity of one or more sign or symptom of the disease, disorder or condition is observed in a population susceptible to the disease, disorder or condition.
  • Prevention may be considered complete when onset of disease, disorder or condition has been delayed for a predefined period of time.
  • the term “recombinant,” refers to a vector, polynucleotide (e.g., a recombinant nucleic acid), polypeptide or cell that is the product of various combinations of cloning, restriction or ligation steps (e.g. relating to a polynucleotide or polypeptide comprised therein), and/or other procedure that results in a construct that is distinct from a product found in nature.
  • polynucleotide e.g., a recombinant nucleic acid
  • polypeptide or cell that is the product of various combinations of cloning, restriction or ligation steps (e.g. relating to a polynucleotide or polypeptide comprised therein), and/or other procedure that results in a construct that is distinct from a product found in nature.
  • a recombinant virus or vector comprises a vector genome comprising a recombinant nucleic acid (e.g., a nucleic acid comprising a transgene and one or more regulatory elements, e.g., a codon optimized nucleic acid encoding an N-terminal peptide tag, a GAA and a hAAT promoter).
  • a recombinant nucleic acid e.g., a nucleic acid comprising a transgene and one or more regulatory elements, e.g., a codon optimized nucleic acid encoding an N-terminal peptide tag, a GAA and a hAAT promoter.
  • the terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.
  • the term “subject” refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog).
  • a subject is an acid alpha-glucosidase (GAA) knock out mouse.
  • GAA acid alpha-glucosidase
  • a human subject is an adult, adolescent, or pediatric subject.
  • a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder or condition that can be treated as provided herein.
  • a subject is suffering from a disease, disorder or condition associated with deficient or dysfunctional GAA activity, e.g., Pompe disease.
  • a subject is susceptible to a disease, disorder, or condition.
  • a susceptible subject is predisposed to and/or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing a disease, disorder or condition.
  • a subject displays one or more symptoms of a disease, disorder or condition.
  • a subject does not display a particular symptom (e.g., clinical manifestation of disease) or characteristic of a disease, disorder, or condition.
  • a subject does not display any symptom or characteristic of a disease, disorder, or condition.
  • a subject is a human patient.
  • a subject is an individual to whom diagnosis and/or therapy is and/or has been administered (e.g., gene therapy for Pompe disease).
  • a subject is a human patient with Pompe disease.
  • the term “substantial” or “substantially” refers to the qualitative condition of exhibition of total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the art will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve an absolute result.
  • the term “substantial” or “substantially” therefore captures the potential lack of completeness inherent in many biological and chemical phenomena.
  • symptoms are reduced” or “reduce symptoms” refers to when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g., intensity, severity etc.) and/or frequency. For purposes of clarity, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom.
  • therapeutic polypeptide is a peptide, polypeptide or protein (e.g., enzyme, structural protein, transmembrane protein, transport protein) that may alleviate or reduce symptoms that result from an absence or defect in a protein in a target cell (e.g., an isolated cell) or organism (e.g., a subject).
  • a therapeutic polypeptide or protein encoded by a transgene is one that confers a benefit to a subject, e.g., to correct a genetic defect, to correct a deficiency in a gene related to expression or function.
  • a “therapeutic transgene” is the transgene that encodes the therapeutic polypeptide.
  • a therapeutic polypeptide, expressed in a host cell is an enzyme expressed from a transgene (i.e., an exogenous nucleic acid that has been introduced into the host cell).
  • a therapeutic polypeptide is an GAA polypeptide (including a NTPT modified GAA polypeptide) expressed from a therapeutic transgene transduced into a liver cell (e.g., a hepatocyte) for the treatment of Pompe disease.
  • the term “therapeutically effective amount” refers to an amount that produces the desired therapeutic effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.
  • a gene therapy for the treatment disease includes transducing a vector genome comprising a modified nucleic acid encoding a therapeutic protein into a target cell.
  • a gene therapy for Pompe disease includes transducing a vector genome comprising a modified nucleic acid encoding GAA into a hepatocyte.
  • a cell into which a transgene has been introduced by a virus or a viral vector is referred to as a “transduced cell.”
  • a transduced cell is an isolated cell and transduction occurs ex vivo.
  • a transduced cell is a cell within an organism (e.g., a subject) and transduction occurs in vivo.
  • a transduced cell may be a target cell of an organism which has been transduced by a recombinant AAV vector such that the target cell of the organism expresses a polynucleotide (e.g., a transgene encoding a therapeutic protein, e.g., a modified nucleic acid encoding GAA).
  • a cell that may be transduced includes a cell of any tissue or organ type, or any origin (e.g., mesoderm, ectoderm or endoderm).
  • Non-limiting examples of cells include liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., beta islet cells, exocrine), lung, central or peripheral nervous system, such as brain (e.g., neural or ependymal cells, oligodendrocytes) or spine, kidney, eye (e.g., retinal), spleen, skin, thymus, testes, lung, diaphragm, heart (cardiac), muscle or psoas, or gut (e.g., endocrine), adipose tissue (white, brown or beige), muscle (e.g., fibroblasts, myocytes), synoviocytes, chondrocytes, osteoclasts, epithelial cells, end
  • stem cells such as pluripotent or multipotent progenitor cells that develop or differentiate into liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., beta islet cells, exocrine cells), lung, central or peripheral nervous system, such as brain (e.g., neural or ependymal cells, oligodendrocytes) or spine, kidney, eye (e.g., retinal), spleen, skin, thymus, testes, lung, diaphragm, heart (cardiac), muscle or psoas, or gut (e.g., endocrine), adipose tissue (white, brown or beige), muscle (e.g., fibroblast, myocytes), synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nervous cells or hematopoietic (e.g.
  • liver
  • cells present within particular areas of a tissue or organ may be transduced by vector (e.g., a rAAV comprising a therapeutic transgene, a reporter transgene) that is administered to the tissue or organ.
  • vector e.g., a rAAV comprising a therapeutic transgene, a reporter transgene
  • cells present within particular areas of a tissue or organ may be transduced by a rAAV vector (e.g., a rAAV comprising an GAA transgene) that is administered to the tissue or organ.
  • transfection refers to transfer of a recombinant nucleic acid (e.g., an expression plasmid) into a cell (e.g., a host cell) without use of a viral vector.
  • a cell into which a recombinant nucleic acid has been introduced is referred to as a “transfected cell.”
  • a transfected cell may be a host cell (e.g., a CHO cell, Pro10 cell, HEK293 cell) comprising an expression plasmid/vector for producing a recombinant AAV vector.
  • a transfected cell may comprise a plasmid comprising a transgene (e.g., a transgene encoding a therapeutic protein), a plasmid comprising an AAV rep gene and an AAV cap gene and a plasmid comprising a helper gene.
  • transfection techniques include, but are not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal.
  • transgene is used to mean any heterologous polynucleotide for delivery to and/or expression in a host cell, target cell or organism (e.g., a subject). Such “transgene” may be delivered to a host cell, target cell or organism using a vector (e.g., rAAV vector). A transgene may be operably linked to a control sequence, such as a promoter. It will be appreciated by those of skill in the art that expression control sequences can be selected based on an ability to promote expression of the transgene in a host cell, target cell or organism.
  • a transgene may be operably linked to an endogenous promoter associated with the transgene in nature, but more typically, the transgene is operably linked to a promoter with which the transgene is not associated in nature.
  • An example of a transgene is a nucleic acid encoding a therapeutic polypeptide, for example a GAA polypeptide, and an exemplary promoter is one not operably linked to a nucleotide encoding GAA in nature.
  • a non-endogenous promoter can include a human alpha 1 -antityrpsin (hAAT) promoter, among many others known in the art.
  • treat refers to administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder and/or condition.
  • the term “vector” refers to a plasmid, virus (e.g., a rAAV), cosmid, or other vehicle that can be manipulated by insertion or incorporation of a nucleic acid (e.g., a recombinant nucleic acid).
  • a vector can be used for various purposes including, e.g., genetic manipulation (e.g., cloning vector), to introduce/transfer a nucleic acid into a cell, to transcribe or translate an inserted nucleic acid in a cell.
  • a vector nucleic acid sequence contains at least an origin of replication for propagation in a cell.
  • a vector nucleic acid includes a heterologous nucleic acid sequence, an expression control element(s) (e.g., promoter, enhancer), a selectable marker (e.g., antibiotic resistance), a poly-adenosine (polyA) signal sequence and/or an ITR.
  • an expression control element(s) e.g., promoter, enhancer
  • a selectable marker e.g., antibiotic resistance
  • polyA poly-adenosine
  • ITR an ITR.
  • the nucleic acid sequence when delivered to a host cell, the nucleic acid sequence is propagated.
  • the cell when delivered to a host cell, either in vitro or in vivo, the cell expresses the polypeptide encoded by the heterologous nucleic acid sequence (e.g., a transgene).
  • the nucleic acid sequence, or a portion of the nucleic acid sequence is packaged into a capsid.
  • a host cell may be an isolated cell or a cell within a host organism.
  • additional sequences e.g., regulatory sequences
  • regulatory sequences may be present within the same vector (i.e., in cis to the gene) and flank the gene.
  • regulatory sequences may be present on a separate (e.g., a second) vector which acts in trans to regulate the expression of the gene.
  • Plasmid vectors may be referred to herein as “expression vectors.”
  • the term “vector genome” refers to a nucleic acid that that may, but need not, be packaged/ encapsidated in an AAV capsid to form a rAAV vector.
  • a vector genome includes a heterologous polynucleotide sequence (e.g., a transgene, regulatory elements, etc.) and at least one ITR.
  • a recombinant plasmid is used to construct or manufacture a recombinant vector (e.g., rAAV vector)
  • the vector genome does not include the entire plasmid but rather only the sequence intended for delivery by the viral vector.
  • the plasmid backbone This non-vector genome portion of the recombinant plasmid is referred to as the “plasmid backbone,” which is important for cloning, selection and amplification of the plasmid, a process that is needed for propagation of recombinant viral vector production, but which is not itself packaged or encapsidated into a rAAV vector.
  • the plasmid backbone may include spacer sequences.
  • the heterologous sequence to be packaged into the capsid is flanked by the ITRs such that when cleaved from the plasmid backbone, the heterologous sequence is packaged into the capsid.
  • viral vector generally refers to a viral particle that functions as a nucleic acid delivery vehicle and which comprises a vector genome (e.g., comprising a transgene which has replaced the wild type rep and cap) packaged within the viral particle (i.e., capsid) and includes, for example, lenti- and parvo- viruses, including AAV serotypes and variants (e.g., rAAV vectors).
  • a recombinant viral vector does not comprise a virus genome with a rep and/or a cap gene; rather, these sequences have been removed to provide capacity for the vector genome to carry a transgene of interest.
  • Adeno-associated virus is a 20-25 nm diameter non-enveloped single stranded DNA containing virus in the Dependovirus genus in the Parvoviridae family.
  • AAV adeno-associated virus
  • the term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise.
  • AAV is ubiquitously prevalent in the human population but has not been associated with disease. Helper virus infection or DNA damaging stress will trigger latent-AAV proviruses to become active leading to viral replication.
  • Parvoviruses are useful as gene therapy vectors as they can penetrate a cell and introduce a nucleic acid (e.g., transgene) into the nucleus.
  • a nucleic acid e.g., transgene
  • the introduced nucleic acid e.g., rAAV vector genome
  • a transgene is inserted in specific sites in the host cell genome, for example at a site on human chromosome 19. Site-specific integration, as opposed to random integration, is believed to likely result in a predictable long-term expression profile.
  • the insertion site of AAV into the human genome is referred to as AAVS1 .
  • nucleic acid Once introduced into a cell, polypeptides encoded by the nucleic acid can be expressed by the cell. Because AAV is not associated with any pathogenic disease in humans, a nucleic acid delivered by AAV can be used to express a therapeutic polypeptide for the treatment of a disease, disorder and/or condition in a human subject.
  • the canonical AAV wild type genome comprises 4681 bases (Berns et al. (1987) Advances in Virus Research 32:243-307) and includes terminal repeat sequences (e.g., inverted terminal repeats ( ITRs)) at each end which function in cis as origins of DNA replication and as packaging signals for the virus.
  • the genome includes two large open reading frames, known as AAV replication (“AAV rep” or “rep”) and capsid (“AAV cap” or “cap”) genes, respectively.
  • AAV rep and cap may also be referred to herein as AAV “packaging genes.” These genes code for the viral proteins involved in replication and packaging of the viral genome.
  • Wild type AAV comprises a small (20-25 nm) icosahedral virus capsid composed of three proteins, VP1 , VP2 and VP3, with 60 capsid proteins comprising the capsid.
  • Each viral particle has a 2-, 3- and 5-fold axis of symmetry.
  • the three capsid genes VP1 , VP2 and VP3 overlap each other within a single open reading frame and alternative splicing leads to production of VP1 , VP2 and VP3 (Grieger et al. (2005) J. Virol. 79(15):9933-9944.).
  • VP3 makes up to 80-90% of total subunits.
  • VP1 has essential functions including phospholipase activity and a nuclear localization signal.
  • a single P40 promoter allows all three capsid proteins to be expressed at a ratio of about 1 :1 :10 for VP1 , VP2, VP3, respectively, which complements AAV capsid production.
  • VP1 is the full-length protein, with VP2 and VP3 being increasingly shortened due to increasing truncation of the N-terminus.
  • a well-known example is the capsid of AAV9 as described in US Patent No. 7,906,111 , wherein VP1 comprises amino acid residues 1 to 736 of a sequence identified as number 123, VP2 comprises amino acid residues 138 to 736 of a sequence identified as number 123, and VP3 comprises amino acid residues 203 to 736 of a sequence identified as number 123.
  • the AAV2 capsid protein sequences are available in GenBank: VP1 (735 aa; GenBank Accession No. AAC03780), VP2 (598 aa; GenBank Accession No.
  • AAV Cap refers to AAV capsid proteins VP1 , VP2 and/or VP3, and variants and analogs thereof.
  • a second open reading frame of the capsid gene encodes an assembly factor, called assembly-activating protein (AAP), which is essential for the capsid assembly process (Sonntag et al. (2011) J. Virol. 85(23):12686-12697).
  • AAP assembly-activating protein
  • At least four viral proteins are synthesized from the AAV rep gene - Rep 78, Rep 68, Rep 52 and Rep 40 - named according to their apparent molecular weights.
  • AAV rep or “rep” means any one of AAV replication proteins Rep 78, Rep 68, Rep 52, Rep 40, as well as variants and analogs thereof.
  • rep and cap refer to both wild type and recombinant (e.g., modified chimeric, and the like) rep and cap genes as well as the polypeptides they encode.
  • a nucleic acid encoding a rep will comprise nucleotides from more than one AAV serotype.
  • a nucleic acid encoding a rep protein may comprise nucleotides from an AAV2 serotype and nucleotides from an AAV3 serotype (Rabinowitz etal. (2002) J. Virology 76(2):791-801).
  • AAV1-AAV15 Multiple serotypes of AAV exist in nature with at least fifteen wild type serotypes having been identified from humans thus far (i.e., AAV1-AAV15). Over 150 unique AAV serotypes have been identified. Naturally occurring and variant serotypes are distinguished by having a protein capsid that is serologically distinct from other AAV serotypes.
  • Naturally occurring and non-naturally occurring AAV serotypes include: AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3) including AAV type 3A (AAV3A) and AAV type 3B (AAV3B), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAV type 12 (AAV12), AAVrh8, AAVrhIO, AAVrh39, AAVrh43, AAVrh74 (see WO 2016/210170), AAVrh32.22, AAV1 .1 , AAV2.5, AAV6.1 , AAV6.2, AAV6.3.1 , AAV9.45, AAVShHIO, HSC15/17, RHM4-1 (SEQ ID NO:5 of WO 2015/013313), RHM15-1 ,
  • AAV variants isolated from human CD34+ cell include AAVHSC1 , AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11 , AAVHSC12, AAVHSC13, AAVHSC14 and AAVHSC15 (Smith et al.
  • Naturally occurring AAVs isolated from human tissues by long-read sequencing include AAVv66 with tropism for the CNS as well as AAVv33, AAVv37, AAVv40, AAVv67, AAVv70, AAVv72, AAVv84, AAVv86, AAVv87 and AAVv90 (Hsu etal. (2020) Nat. Comm. 11 :3279).
  • Serotype distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV.
  • Such cross-reactivity differences are usually due to differences in capsid protein sequences and antigenic determinants (e.g., due to VP1 , VP2, and/or VP3 sequence differences of AAV serotypes).
  • some naturally occurring AAV or man-made AAV mutants e.g., recombinant AAV may not exhibit serological difference with any of the currently known serotypes. These viruses may then be considered a subgroup of the corresponding type, or more simply a variant AAV.
  • serotype refers to both serologically distinct viruses, e.g., AAV, as well as viruses, e.g., AAV, that are not serologically distinct but that may be within a subgroup or a variant of a given serotype.
  • a comprehensive list and alignment of amino acid sequences of capsids of known AAV serotypes is provided by Marsic et al. (2014) Molecular Therapy 22(11):1900-1909, especially at supplementary Figure 1 ; the entire publication is hereby incorporated by reference.
  • Genomic sequences of various serotypes of AAV, as well as sequences of the native inverted terminal repeats (ITRs), rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank.
  • a capsid protein encoded by a nucleotide sequence derived from more than one AAV serotype is referred to as a “chimeric vector” or “chimeric capsid” (See U.S. Patent No. 6,491 ,907, the entire disclosure of which is incorporated herein by reference).
  • a chimeric capsid protein is encoded by a nucleic acid sequence derived from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more AAV serotypes.
  • a chimeric capsid sequence is derived from e.g., AAV1 , AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAVrh74, AAVrhIO, AAV2i8, or variant thereof, or any serotypes disclosed herein, resulting in a chimeric capsid protein comprising a combination of amino acids from any of the foregoing AAV serotypes (see, Viney et al. (2021) J. Virol. 95:1-15; Choi et al. (2005) Curr. Gene Ther. 5(3):299-310).
  • Chimeric capsids my comprise capsids with substitutions of the VP1 variable regions (e.g., VR I - VR IX) and p-sheet regions (e.g., A though I).
  • the amino acid sequence between -sheet G and p-sheet H (also referred to herein as the “GH loop”), encompasses variable region IV through variable region VIII and contains the highest level of diversity among AAV serotypes as well as among all Parvoviruses.
  • the GH loop is at the 3-fold axis of symmetry, constitutes about 30% of the capsid and interacts with primary glycan attachment receptor.
  • a chimeric AAV capsid polypeptide comprises an amino acid sequence of a parental AAV VP1 polypeptide, comprising a substitution of amino acids from a region between p-sheet G and p-sheet H with amino acids from a region between p-sheet G and p- sheet H of an alternative AAV VP1 polypeptide.
  • the substitution includes amino acids from the p-sheet G and/or p-sheet H of either the parental AAV VP1 polypeptide or the alternative AAV VP1 polypeptide.
  • Such chimeric capsids may be referred to as an “AAV capsid with a GH loop substitution” or a “GH loop substitution capsid.”
  • a chimeric AAV capsid polypeptide comprises an amino acid sequence of a parental AAV VP1 polypeptide, comprising a substitution of amino acids from a region between p-sheet G and p-sheet I with amino acids from a region between p-sheet G and P-sheet I of an alternative AAV VP1 polypeptide.
  • the substitution includes amino acids from the p-sheet G and/or p-sheet I.
  • chimeric capsids may be referred to an “AAV capsid with a Gl loop substitution” or a “Gl loop substitution capsid.”
  • chimeric vectors have been engineered to exhibit altered tropism or tropism for a particular tissue or cell type.
  • the term “tropism” refers to preferential entry of the virus into certain cell or tissue types and/or preferential interaction with the cell surface that facilitates entry into certain cell or tissue types.
  • AAV tropism is generally determined by the specific interaction between distinct viral capsid proteins and their cognate cellular receptors (Lykken et al. (2016) J. Neurodev. Disord. 10:16).
  • sequences e.g., heterologous sequences such as a transgene carried by the vector genome (e.g., a rAAV vector genome) are expressed.
  • a “tropism profile” refers to a pattern of transduction of one or more target cells, tissues and/or organs.
  • an AAV capsid may have a tropism profile characterized by efficient transduction of liver cells with only low transduction of, for example, brain cells.
  • a “recombinant adeno-associated virus,” or “rAAV” refers to an AAV capsid comprising a vector genome, unless specifically noted otherwise.
  • the vector genome comprises a polynucleotide sequence that is not, at least in part, derived from a naturally- occurring AAV (e.g., a heterologous polynucleotide not present in wild type AAV), and wherein the rep and/or cap genes of the wild type AAV genome have been removed from the vector genome. ITRs from an AAV have been added or remain in the vector genome.
  • rAAV vector encompasses a rAAV viral particle that comprises a capsid but does not comprise a complete wild type AAV genome; instead the recombinant viral particle can comprise a heterologous, (i.e., not originally present in the capsid), nucleic acid, the vector genome.
  • a “rAAV vector genome” refers to a heterologous polynucleotide sequence (including at least one ITR) that may, but need not, be contained within an AAV capsid.
  • a rAAV vector genome may be double-stranded (dsAAV), singlestranded (ssAAV) or self-complementary (scAAV).
  • a vector genome comprises a heterologous nucleic acid often encoding a therapeutic transgene, for example a lysosomal enzyme (e.g., GAA), a variant thereof, or a fragment thereof.
  • a vector genome comprises a heterologous nucleic acid comprising a nucleic acid of any one of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or nucleotides 550-3198 of SEQ ID NO:113 which encodes a therapeutic GAA polypeptide.
  • a vector genome comprises a heterologous nucleic acid encoding a GAA polypeptide comprising the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:58, SEQ ID NO:114 or amino acids 70- 952 of SEQ ID NO:114.
  • a rAAV vector and those terms provided above, are to be distinguished from an “AAV viral particle” or “AAV virus” that is not recombinant, contains a virus genome encoding rep and cap genes, and which AAV virus is capable of replicating when present in a cell also comprising a helper virus, such as an adenovirus and/or herpes simplex virus, and/or required helper genes therefrom.
  • AAV viral particle or “AAV virus” that is not recombinant, contains a virus genome encoding rep and cap genes, and which AAV virus is capable of replicating when present in a cell also comprising a helper virus, such as an adenovirus and/or herpes simplex virus, and/or required helper genes therefrom.
  • helper virus such as an adenovirus and/or herpes simplex virus, and/or required helper genes therefrom.
  • the present disclosure provides for a composition comprising a rAAV vector, and methods of use thereof.
  • the rAAV vector comprises an AAV9 capsid and a transgene encoding a polypeptide that is a target for therapeutic treatment.
  • a rAAV vector comprises an AAV9 capsid and a vector genome comprising a nucleic acid sequence comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 and nucleotides 550-3198 of SEQ ID NO:113 and encoding a GAA polypeptide for the treatment of Pompe disease.
  • a rAAV vector comprises an AAV9 capsid and a vector genome comprising a nucleic acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 and nucleotides 550-3198 of SEQ ID NO:113 and encoding a GAA polypeptide for the treatment of Pompe disease.
  • a rAAV vector comprises an AAV9 capsid and a vector genome comprising a nucleic acid encoding an GAA polypeptide comprising of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58 and amino acids 70-952 of SEQ ID NO:114 for the treatment of Pompe disease.
  • a rAAV vector comprises an AAV9 capsid and a vector genome comprising a nucleic acid encoding an GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58 or amino acids 70-952 and SEQ ID NO:114 for the treatment of Pompe disease.
  • a rAAV vector can be used to transfer/deliver a heterologous polynucleotide for expression for the treatment of diseases, disorders and/or conditions (e.g., Pompe disease).
  • a rAAV vector transfers a copy of a gene to a cell which expresses a lysosomal enzyme for the treatment of a lysosomal storage disease.
  • a rAAV vector transfers a copy of a GAA transgene (e.g., SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or nucleotides 550-3198 of SEQ ID NO:113), a variant thereof, or fragment thereof to hepatocytes which express a GAA polypeptide (e.g., SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:114), a variant thereof or a fragment thereof for the treatment of Pompe disease.
  • GAA transgene e.g., SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or nucleotides 550-3198 of SEQ ID NO:113
  • a rAAV vector genome generally retains 130 to 145 base ITRs in cis to the heterologous nucleic acid sequence that replaces the viral rep and cap genes. Such ITRs are necessary to produce a rAAV vector as they mediate AAV genome replication and packaging. However, modified AAV ITRs and non-AAV terminal repeats including partially or completely synthetic sequences can also serve this purpose. ITRs form hairpin structures and function to, for example, serve as primers for host-cell-mediated synthesis of the complementary DNA strand after infection. ITRs also play a role in viral packaging, integration, etc. ITRs are the only AAV viral elements which are required in cis for AAV genome replication and packaging into rAAV vectors.
  • a rAAV vector genome optionally comprises two ITRs which are generally at the 5’ and 3’ ends of the vector genome comprising a heterologous sequence (e.g., a transgene encoding a gene of interest, or a nucleic acid sequence of interest including, but not limited to, an antisense, an siRNA, a CRISPR molecule, among many others).
  • a 5’ and a 3’ ITR may both comprise the same sequence, or each may comprise a different sequence.
  • An AAV ITR may be from any AAV, including but not limited to, serotypes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 , or any other AAV serotype.
  • a rAAV vector genome comprises a 5’ and a 3’ ITR of the AAV2 serotype.
  • a rAAV vector genome may comprise an ITR from an AAV serotype (e.g., wild type AAV2, a fragment or variant thereof) that differs from the serotype of the capsid (e.g., AAV9).
  • AAV vector genome comprising at least one ITR from one serotype, but comprising a capsid from a different serotype, may be referred to as a hybrid viral vector (see U.S. Patent No. 7,172,893; Rabinowitz et al. (2002) J. Virology 76(2):791 -801 ).
  • a rAAV ITR may include the entire wild type ITR sequence, or be a variant, fragment, or modification thereof, but will retain functionality.
  • a heterologous polynucleotide comprises an ITR (e.g., an ITR from AAV2, but can comprise an ITR from any wild type AAV serotype, or a variant thereof) positioned at the left and right ends (i.e., 5’ and 3’ termini, respectively) of a vector genome.
  • a left (e.g., 5’) ITR comprises or consists of the nucleic acid sequence of SEQ ID NO:109 or SEQ ID NO:110.
  • a left (e.g., 5’) ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO:109 or SEQ ID NO:110.
  • a right (e.g., 3’) ITR comprises or consists of a nucleic acid sequence of SEQ ID NO:109 or SEQ ID NO:110.
  • a right (e.g., 3’) ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NQ:109 or SEQ ID NO:110.
  • Each ITR is in cis with each other, but may be separated from each other, or other elements in the vector genome, by a nucleic acid sequence of variable length, such as a recombinant nucleic acid comprising a modified nucleic acid encoding a lysosomal enzyme (e.g., NTPT modified GAA) and regulatory elements.
  • ITRs are AAV2 ITRs, or variants thereof, and flank a GAA transgene.
  • a rAAV comprises a GAA transgene (e.g., comprising the nucleic acid sequence any one of SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or nucleotides 550-3198 of SEQ ID NO:113) flanked by AAV2 ITRs (e.g., ITRs having the sequence as set forth in SEQ ID NO:109 or SEQ ID NO:110).
  • an expression vector (or cassette) selected from the group consisting of SEQ IQ NO:1 to SEQ ID NO: 16 comprises a 5’ ITR and a 3’ ITR of the AAV2 serotype. In some embodiments, an expression vector (or cassette) selected from the group consisting of SEQ IQ NO:1 to SEQ ID NO: 16 comprises a 5’ ITR and a 3’ ITR comprising or consisting of SEQ ID NQ:109 or SEQ ID NO:110.
  • a rAAV vector genome is linear, single-stranded and flanked by AAV ITRs.
  • a single stranded DNA genome of approximately 4700 nucleotides Prior to transcription and translation of the heterologous gene, a single stranded DNA genome of approximately 4700 nucleotides must be converted to a double-stranded form by DNA polymerases (e.g., DNA polymerases within the transduced cell) using the free 3’-OH of one of the self-priming ITRs to initiate second-strand synthesis.
  • DNA polymerases e.g., DNA polymerases within the transduced cell
  • full length-single stranded vector genomes i.e., sense and anti-sense
  • the efficiency of transgene expression from a rAAV vector can be hindered by the need to convert a single stranded rAAV genome (ssAAV) into double-stranded DNA prior to expression.
  • This step is circumvented by using a self-complementary AAV genome (scAAV) that can package an inverted repeat genome that can fold into double-stranded DNA without the need for DNA synthesis or base-pairing between multiple vector genomes (McCarty, (2008) Molec. Therapy 16(10):1648-1656; McCarty et aL, (2001 ) Gene Therapy 8:1248-1254; McCarty et aL, (2003) Gene Therapy 10:2112-2118).
  • scAAV self-complementary AAV genome
  • a limitation of a scAAV vector is that the size of the unique transgene, regulatory elements and IRTs to be package in the capsid is about half the size (i.e., -2,500 nucleotides of which 2,200 nucleotides may be a transgene and regulatory elements, plus two copies of the -145 nucleotide ITRs) of a ssAAV vector genome (i.e., - 4,900 nucleotides including two ITRs).
  • scAAV vector genomes may be made using a nucleic acid not comprising the terminal resolution site (TRS), or by altering the TRS, from one rAAV ITR of a vector, e.g., a plasmid, comprising the vector genome thereby preventing initiation of replication from that end (see U.S. Patent No. 8,784,799).
  • TRS terminal resolution site
  • AAV replication within a host cell is initiated at the wild type ITR of the scAAV vector genome and continues through the ITR lacking or comprising an altered terminal resolution site and then back across the genome to create a complementary strand.
  • the resulting complementary single nucleic acid molecule is thus a self-complementary nucleic acid molecule that results in a vector genome with a mutated (is not resolved) ITR in the middle, and wild type ITRs at each end.
  • a mutant ITR lacking a TRS or comprising an altered TRS is at the 5’ end of the vector genome.
  • a mutant ITR lacking a TRS or comprising an altered TRS that is not resolved (cleaved) is at the 3’ end of the vector genome.
  • a viral capsid of a rAAV vector may be a naturally occurring or non-naturally occurring AAV serotype including: AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3) including AAV type 3A (AAV3A) and AAV type 3B (AAV3B), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAV type 12 (AAV12), AAVrh8, AAVrhIO, AAVrh39, AAVrh43, AAVrh74 (see WO 2016/210170), AAVrh32.22, AAV1 .1 , AAV2.5, AAV6.1 , AAV6.2, AAV6.3.1 , AAV9.45, AAVShHIO, HSC15/17, RHM4-1 (SEQ
  • AAV variants isolated from human CD34+ cell include AAVHSC1 , AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11 , AAVHSC12, AAVHSC13, AAVHSC14 and AAVHSC15 (Smith et al.
  • Naturally occurring AAVs isolated from human tissues by long-read sequencing include AAVv66 with tropism for the CNS as well as AAVv33, AAVv37, AAVv40, AAVv67, AAVv70, AAVv72, AAVv84, AAVv86, AAVv87 and AAVv90 (Hsu etal. (2020) Nat. Comm. 11 :3279).
  • a viral capsid of a rAAV vector may be a chimeric AAV capsid as disclosed herein.
  • a rAAV vector disclosed herein comprises an AAV9 capsid.
  • Recombinant nucleic acids of the present disclosure include modified nucleic acids as well as plasmids and vector genomes that comprise a modified nucleic acid.
  • a recombinant nucleic acid, plasmid or vector genome may comprise regulatory sequences to modulate propagation (e.g., of a plasmid in vitro) and/or control expression of a modified nucleic acid (e.g., a transgene in vivo).
  • Recombinant nucleic acids may also be provided as a component of a viral vector (e.g., a rAAV vector).
  • a viral vector includes a vector genome comprising a recombinant nucleic acid packaged in an AAV capsid.
  • the present disclosure provides viral vectors with a vector genome comprising a transgene encoding a NTPT modified GAA with a signal peptide packaged in an AAV9 capsid.
  • a modified, or variant form, of a gene, nucleic acid or polynucleotide refers to a nucleic acid that deviates from a reference sequence.
  • a reference sequence may be a naturally occurring, wild type sequence (e.g., a gene) and may include naturally occurring variants (e.g., splice variants, alternative reading frames, mutants).
  • variants e.g., splice variants, alternative reading frames, mutants.
  • Modified/variant nucleic acids may have substantially the same, greater or lesser activity, function or expression as compared to a reference sequence.
  • a modified, or variant nucleic acid exhibits improved protein expression, e.g., a protein encoded thereby is expressed at a detectably greater level in a cell as compared to the level of expression of a protein provided by an endogenous gene (e.g., a wild type gene, a mutant gene) in an otherwise identical cell.
  • an endogenous gene e.g., a wild type gene, a mutant gene
  • a modified, or variant nucleic acid e.g., a modified nucleic acid encoding a lysosomal enzyme (e.g., GAA)
  • exhibits improved protein expression e.g., the lysosomal enzyme (e.g., GAA) encoded thereby is expressed at a detectably greater level in a cell as compared to the level of expression of a lysosomal enzyme (e.g., GAA) provided by an endogenous gene in an otherwise identical cell.
  • Modifications to nucleic acids may include one or more nucleotide substitutions (e.g., substitution of 1 -3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100 or more nucleotides), additions (e.g., insertion of 1 -3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40- 50, 50-100 or more nucleotides), deletions (e.g., deletion of 1 -3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100 or more nucleotides, deletion of a motif, domain, fragment, etc.) of a reference sequence.
  • nucleotide substitutions e.g., substitution of 1 -3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100 or more nucleotides
  • additions e.g., insertion of 1 -3, 3-5, 5
  • a modified nucleic acid may be at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% at least 97%, at least 98%, or at least 99% identical to a reference nucleic acid sequence.
  • a modified nucleic acid may be at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO: 53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:113 and nucleotides 550-3198 of SEQ ID NO:1 13.
  • a modified nucleic acid may be about 50% to about 99%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95% or about 90% to about 99% identical to a reference nucleic acid sequence.
  • a modified nucleic acid may be about 50% to about 99%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95% or about 90% to about 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO: 53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:113, and nucleotides 550-3198 of SEQ ID NO:1 13.
  • a modified nucleic acid comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO: 53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:1 13 and nucleotides 550-3198 of SEQ ID NO:113.
  • a modified nucleic acid may encode a polypeptide with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a reference polypeptide.
  • a modified nucleic acid may encode a polypeptide with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a polypeptide selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:114, or amino acids 70-952 of SEQ ID NO:1 14.
  • a modified nucleic acid may encode a polypeptide with about 50% to about 99%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 90% to about 99% identity to a reference polypeptide.
  • a modified nucleic acid may encode a polypeptide with about 50% to about 99%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 90% to about 99% identity to a polypeptide selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:114 or amino acids 70-952 of SEQ ID NO:114.
  • a modified nucleic acid encodes a GAA polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:114 or amino acids 70-952 of SEQ ID NO:114.
  • a modified nucleic acid encodes a lysosomal enzyme comprising or consisting of a reference polypeptide of GenBank Ref. No. NP 000143.2 (e.g., SEQ ID NO:114).
  • a modified nucleic acid encodes a lysosomal enzyme comprising or consisting of amino acids 25 to 952 of SEQ ID NO:114.
  • a modified nucleic acid encodes a lysosomal enzyme comprising or consisting of amino acids 27 to 952 of SEQ ID NO:114. In some embodiments, a modified nucleic acid encodes a lysosomal enzyme comprising or consisting of amino acids 70 to 952 of SEQ ID NO:114.
  • a modified nucleic acid encodes a wild type protein.
  • Such modified nucleic acid may be codon optimized.
  • Codon optimization refers to a coding sequence that has been optimized relative to a wild type coding sequence or reference sequence (e.g., a coding sequence for a lysosomal enzyme (e.g., GAA)).
  • codon optimization of a coding sequence increases expression of the polypeptide, e.g., by minimizing usage of rare codons, based on bias of an organism, in order to increase the translational efficiency of a gene of interest.
  • a level of expression of a polypeptide from a codon-optimized sequence is increased as compared to a level of expression of a polypeptide from a wild type gene in an otherwise identical cell.
  • a level of expression of a polypeptide from a codon- optimized sequence is not increased (e.g., expression is substantially similar) as compared to a level of expression of a polypeptide from a wild type gene in an otherwise identical cell.
  • a level of expression of a polypeptide from a codon- optimized sequence is increased as compared to a level of expression of a polypeptide from a mutant gene in an otherwise identical cell.
  • a codon- optimized sequence e.g., a modified nucleic acid encoding a lysosomal enzyme (e.g., GAA)
  • GAA lysosomal enzyme
  • a modified nucleic acid encoding a GAA polypeptide is codon optimized.
  • a modified nucleic acid encoding a GAA polypeptide is codon optimized and comprises or consists of the nucleic acid sequence of any one of SEQ ID NO:49, SEQ ID NQ:50, SEQ ID N0:51 , SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:56.
  • the nucleic acid that is codon-optimized comprises or consist of SEQ ID NO:113 (e.g., nucleotides 343-3198 of Gen Bank
  • the nucleic acid that is codon optimized comprises or consists of nucleotides 550-3198 of SEQ ID NO:113. In some embodiments, the nucleic acid that is codon optimized comprises or consists of nucleotides 415-3198 of SEQ ID NO:113. In some embodiments, the nucleic acid that is codon optimized comprises or consists of nucleotides 343-3198 of SEQ ID NO:113.
  • Modified nucleic acids can be optimized by decreasing the number of CpG dinucleotides, removing cryptic splice donor or acceptor sites, removing Kozak sequences, removing ribosomal entry sites, and the like. Examples of modifications include elimination of one or more c/s-acting motifs and introduction of one or more Kozak sequences. In some embodiments, one or more c/s-acting motifs are eliminated and one or more Kozak sequences are introduced.
  • Examples of c/s-acting motifs that may be eliminated include internal TATA-boxes; chi-sites; ribosomal entry sites; ARE, INS, and/or CRS sequence elements; repeat sequences and/or RNA secondary structures; (cryptic) splice donor and/or acceptor sites, branch points; and restriction sites.
  • Modified nucleic acids may have a modified GC content (e.g., the number of G and C nucleotides present in a nucleic acid sequence), a modified (e.g., increased or decreased) CpG dinucleotide content and/or a modified (e.g., increased or decreased) codon adaptation index (CAI) relative to a reference and/or wild type sequence (e.g., a wild type lysosomal enzyme (e.g., GAA) coding sequence).
  • modified refers to a decrease or an increase in a particular value, amount or effect.
  • a GC content of a modified nucleic acid sequence of the present disclosure is increased or decreased relative to a reference (e.g., a wild type) gene or coding sequence.
  • the GC content of a modified nucleic acid is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 15%, at least 17%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% greater than the GC content of a wild type coding sequence.
  • the GC content of a modified nucleic acid is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 15%, at least 17%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% less than the GC content of a wild type coding sequence
  • GC content is expressed as a percentage of G (guanine) and C (cytosine) nucleotides in the sequence. That is, the GC content of the wild type nucleic acid is about 25% to about 55% whereas the GC content of a modified transgene ranges from about 56% to about 70%. In one embodiment, the GC content of a modified nucleic acid of the invention is greater than the GC content of the wild type nucleic acid.
  • the amino acid sequence of the polypeptide expressed therefrom is, the amino acid sequence of the wild type polypeptide.
  • a CAI codon adaptation index
  • the index uses a reference set of highly expressed genes from a species to assess the relative values of each codon, and a score for a gene is calculated from the frequency of use of all codons in that gene. The index assesses the extent to which selection has been effective in selecting the pattern of codon usage. It can be utilized for predicting the level of expression of a gene and for making comparisons of codon usage in different organisms/species.
  • a codon adaptation index of a modified nucleic acid sequence of the present disclosure is at least 0.50, at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.76, at least 0.77, at least 0.78, at least 0.79, at least 0.80, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.90, at least 0.95, at least 0.96, at least 0.97 or at least 0.98.
  • methylation of CpG dinucleotides in eukaryotes essentially serves to silence gene expression through interfering with the transcriptional machinery.
  • nucleic acids and vectors having a reduced number of CpG dinucleotides may provide for high and longer-lasting transgene expression level.
  • Transgenes comprising CpG dinucleotides are also associated with increased immunogenicity of rAAV vectors that include such transgenes.
  • a modified nucleic acid sequence of the present disclosure has a reduced level of CpG dinucleotides, e.g., a reduction of at least 2%, at least 5%, at least
  • a modified nucleic acid of the present disclosure has a reduced level of CpG dinucleotide, e.g., a reduction of 2% to 100%, as compared to a wild type or reference nucleic acid sequence.
  • a reference nucleic acid sequence comprises or consists of the nucleic acid of SEQ ID NO:113, of nucleotides 343 to 3198 of SEQ ID NO:113, of nucleotides 415 to 3198 of SEQ ID NO:113 or of nucleotides 550-3198 of SEQ ID NO:113.
  • a modified nucleic acid sequence of the present disclosure has a reduced level of CpG dinucleotides and comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55.
  • a modified nucleic acid has 1-5 fewer, 5-10 fewer, 10-15 fewer, 15-20 fewer, 20-25 fewer, 25-30 fewer, 30-40 fewer, 40-45 fewer, 45-50 fewer, 1 -25, 1 - 50, 1-75, 1-100 fewer or even fewer CpG dinucleotides than a reference sequence (e.g., a wild type sequence).
  • the present disclosure provides modified GAA nucleic acid sequences that encode a GAA polypeptide and which comprise at least one modification as compared to a wild type nucleic acid sequence (e.g. SEQ ID NO:113; GenBank Accession Number NM 000152.5 and more specifically nucleotides 343 to 3198 of SEQ ID NO:113, nucleotides 415 to 3198 of SEQ ID NO:113 or nucleotides 550-3198 of SEQ ID NO:113).
  • a wild type nucleic acid sequence e.g. SEQ ID NO:113; GenBank Accession Number NM 000152.5 and more specifically nucleotides 343 to 3198 of SEQ ID NO:113, nucleotides 415 to 3198 of SEQ ID NO:113 or nucleotides 550-3198 of SEQ ID NO:113.
  • a modified nucleic acid as disclosed herein may be modified by more than one method or parameter.
  • a modified nucleic acid may be codon optimized, CpG modified or both.
  • a modified nucleic acid encoding GAA is a codon optimized sequence (e.g., SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55).ln some embodiments, a modified nucleic acid encoding GAA is codon optimized and CpG modified (e.g., SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55).
  • a modified nucleic acid encoding GAA is a codon-optimized nucleic acid encoding a GAA polypeptide (e.g., SEQ ID NO:57) and comprises or consists of a sequence of SEQ ID NO:49 or SEQ ID NQ:50.
  • a modified nucleic acid encoding a GAA polypeptide is a codon-optimized nucleic acid and comprises a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence of SEQ ID NO:49 or SEQ ID NQ:50.
  • a cell comprising a modified nucleic acid encoding a lysosomal enzyme exhibits increased protein expression, e.g., the protein encoded thereby is expressed at a detectably greater level in a cell as compared to the level of expression of the protein in an otherwise identical cell comprising a wild type lysosomal enzyme (e.g., GAA) nucleic acid, or an otherwise identical cell comprising a mutant nucleic acid encoding a lysosomal enzyme (e.g., GAA).
  • a level of lysosomal enzyme (e.g., GAA) expression in a cell comprising a modified nucleic acid (e.g., SEQ ID NO:49, SEQ ID NQ:50: SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56) encoding the lysosomal enzyme is increased by 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 120%, at least 140%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500% or more as compared to the level of lysosomal enzyme (e.g., GAA) expression in an otherwise identical cell comprising a nucleic acid encoding a wild type lysosomal enzyme (e.g.,
  • a level of lysosomal enzyme (e.g., GAA) expression in a cell comprising a modified nucleic acid encoding the lysosomal enzyme is increased by 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 120%, at least 140%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500% or more as compared to the level of lysosomal enzyme (e.g., GAA) expression in an otherwise identical cell comprising a nucleic acid encoding a mutant or variant lysosomal enzyme with decreased activity.
  • a modified nucleic acid encoding the lysosomal enzyme e.g., SEQ ID NO:49, SEQ ID NQ:50: SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ
  • this can be referred to as an “expression optimized” or “enhanced expression” nucleic acid, or simply, as a “modified nucleic acid.”
  • modified means that the nucleic acid sequence encoding a polypeptide that exits in nature has been altered such that in one embodiment, the modified nucleic acid sequence drives a higher level of expression of the protein in a cell as compared to the level of expression of the protein from the unmodified (i.e., occurring in nature (including mutant form of a gene)) nucleic acid sequence in an otherwise identical cell.
  • a modified nucleic acid encodes a modified or variant polypeptide (e.g., a modified lysosomal enzyme).
  • a modified polypeptide encoded by a modified nucleic acid may retain all or a part of the function or activity of a polypeptide encoded by a wild type coding or reference sequence.
  • a modified polypeptide has one or more non-conservative or conservative amino acid changes.
  • certain domains that have been demonstrated to play a limited or no role in a function of a polypeptide are not present in a modified polypeptide (e.g., certain binding domains) (e.g., WO 2016/097219).
  • Modified nucleic acids present in rAAV vectors may comprise fewer nucleotides than the wild type coding, or reference sequence, due to the packaging capacity of a rAAV capsid (e.g., shortened minidystrophin transgene, see WO 2001/83695; a B-domain deleted human Factor VIII transgene, see WO 2017/074526), and also include shortened transgenes that are both truncated and codon-optimized (e.g., a codon optimized mini-dystrophin transgene described in WO 2017/221145).
  • a polypeptide encoded by a modified nucleic acid has less than, the same, or greater, but at least a part of, a function or activity of a polypeptide encoded by a reference sequence.
  • the present disclosure includes fragments of any one of the nucleic acid sequences set forth in SEQ ID NO:49, SEQ ID NQ:50: SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:113 and which encode a functionally active fragment of the GAA polypeptide.
  • a “functionally active” or “functional GAA polypeptide” indicates that the fragment provides the same or similar biological function and/or activity as a full-length GAA polypeptide. That is, the fragment provides the same activity including, but not limited to, the ability to breakdown glycogen to glucose.
  • the biological activity of GAA, or a functional fragment thereof also encompasses reversing or preventing at least the skeletal muscle, cardiac or CNS phenotype associated with Pompe disease, as demonstrated elsewhere herein, and in GAA knock out (KO) mice.
  • a polypeptide encoded by a modified nucleic acid is a “functional GAA polypeptide” that provides the same or similar biological function and/or activity as a GAA polypeptide (e.g., SEQ ID NO:114, amino acids 70-952 of SEQ ID NO:114) encoded by a wild type nucleic acid.
  • a GAA polypeptide encoded by a modified nucleic acid provides the same activity including, but not limited to, the ability to breakdown glycogen to glucose.
  • the biological activity of GAA encompasses reversing or preventing at least the skeletal muscle, cardiac or CNS phenotype associated with Pompe disease as demonstrated elsewhere herein in GAA KO mice including, but not limited to, improved performance of rotarod latency to fall, improved muscle tension, decreased glycogen levels in quadricep muscle tissue, diaphragm, heart, brain and spinal cord, and increased GAA activity level in serum, quadricep muscle tissue, diaphragm, heart, brain and spinal cord.
  • a modified nucleic acid encodes a polypeptide which exhibits improved secretion from the cell in which it is expressed, e.g., a polypeptide encoded thereby is secreted at a detectably greater level from a cell as compared to the level of secretion of a protein provided by a reference nucleic acid (e.g., a wild type, variant or mutant transgene) in an otherwise identical cell.
  • a variant transgene is a nucleic acid with a different modification than that of the modified nucleic acid.
  • the modified nucleic acid is a transgene depleted of 10 CpG dinucleotides and the variant transgene is depleted of 12 CpG dinucleotides, or vice versa.
  • a modified nucleic acid encodes a polypeptide (e.g., a lysosomal enzyme (e.g., GAA)) which exhibits improved secretion from the cell in which is expressed (e.g., a hepatocyte) as compared to the level of secretion of a polypeptide (e.g., a lysosomal enzyme (e.g., GAA)) provided by a reference nucleic acid (e.g., a wild type, variant or mutant transgene) in an otherwise identical cell.
  • a polypeptide e.g., a lysosomal enzyme (e.g., GAA)
  • GAA lysosomal enzyme
  • a modified nucleic acid encodes a GAA polypeptide which exhibits improved secretion from the cell in which is expressed (e.g., a hepatocyte) as compared to the level of secretion of a GAA polypeptide provided by a reference nucleic acid (e.g., a wild type gene, a variant or mutant gene) in an otherwise identical cell.
  • a reference nucleic acid e.g., a wild type gene, a variant or mutant gene
  • a polypeptide that exhibits increased secretion from the cell in which it is expressed is encoded by a modified nucleic acid which encodes a signal peptide operably linked to the nucleic acid encoding the polypeptide.
  • a signal peptide that increases secretion of a polypeptide from a cell in which it is expressed may be an endogenous signal peptide, that is the naturally occurring signal peptide associated with the polypeptide.
  • an GAA signal peptide e.g., SEQ ID NO:30
  • a signal peptide that increases secretion of a polypeptide from a cell in which it is expressed may be a naturally occurring signal peptide, but not the naturally occurring signal peptide associated with the protein.
  • an IGF2 signal peptide e.g., SEQ ID NO:25
  • GAA lysosomal enzyme
  • a signal peptide is a naturally occurring signal peptide selected from the group consisting of serum albumin (e.g., GenBank NP 000468), IgG heavy chain (e.g., GenBank AAA59018), IgG light chain (e.g., GenBank AAA59018), interleukin 2 (IL2), azurocidin (e.g., GenBank NP 001691 ; ), complement 6, chromtrypsinogen, IL-2, trypsinogen 2, AB2, AB3, AB4, granulocyte-macrophage colony-stimulating factor (GMCSF), an antibody, cystatin (e.g., GenBank NP 001890), ApoCII, alpha glucosidase (GAA) and insulinlike growth factor II (IGFII or IGF2, amino acids 1 -24 of GenBank NP 000603.1 ; SEQ ID NO:25).
  • serum albumin e.g., GenBank NP 000468
  • Signal peptides such as these may be used to increase secretion of their endogenous polypeptide (that is, a GAA signal peptide increases secretion of GAA) or to increase secretion of a polypeptide (e.g., a heterologous polypeptide) that the signal peptide is not naturally associated with (that is, an IGF2 signal peptide increases secretion of a GAA polypeptide).
  • a signal peptide that increases secretion of a polypeptide from a cell in which it is expressed may be a modified signal peptide, that is a modification of a naturally occurring signal peptide.
  • a signal peptide that increases secretion of a polypeptide from a cell in which it is expressed may be encoded by a modified nucleic acid, such as a codon-optimized nucleic acid, a CpG modified nucleic acid or both.
  • a signal peptide is encoded by a nucleic acid of SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:22.
  • a signal peptide that increases secretion of a polypeptide from a cell in which it is expressed may be a modified endogenous signal peptide, that is a modification of the naturally occurring signal peptide associated with the protein.
  • a modified GAA signal peptide may be used to increase the secretion of GAA from a cell that expresses the protein (e.g., hepatocyte).
  • a signal peptide that increases secretion of a peptide from a cell in which it is expressed may be a modified signal peptide, that is a modification of a naturally occurring peptide.
  • a modified IGF2 signal peptide may be used to increase the secretion of a lysosomal enzyme (e.g., GAA) from the cell that expresses the protein (e.g., hepatocyte).
  • GAA lysosomal enzyme
  • a signal peptide is a modification of a naturally occurring signal peptide selected from the group consisting of serum albumin, IgG heavy chain, IgG light chain, interleukin 2 (IL2), azurocidin, complement 6, chromtrypsinogen, IL-2, trypsinogen 2, AB2, AB3, AB4, granulocyte-macrophage colony-stimulating factor (GMCSF), an antibody, cystatin, ApoCII, alpha glucosidase (GAA) and insulin-like growth factor II (IGFII or IGF2).
  • IL2 interleukin 2
  • GMCSF granulocyte-macrophage colony-stimulating factor
  • GACSF granulocyte-macrophage colony-stimulating factor
  • Naturally occurring signal peptides, and modifications thereof, that may be used to advantageously increase secretion of a protein from a cell in which it is expressed, are provided in Zhang et al. (J. Gene Med. (2005) 7:354-365) and incorporated herein by reference.
  • a signal peptide that increases secretion of a polypeptide e.g., a lysosomal enzyme (e.g., GAA)
  • a polypeptide e.g., a lysosomal enzyme (e.g., GAA)
  • GAA lysosomal enzyme
  • a nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:20 (encodes an albumin signal peptide referred to herein as SP1 ), SEQ ID NO:21 (encodes a chymotrypsinogen signal peptide referred to herein as SP7), SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:22 (each of which encodes an IGF2 signal peptide referred to herein as SP18), SEQ ID NO:18 (encodes a modified IL2 signal peptide referred to herein as SP13), SEQ ID NO:23 (encodes
  • a signal peptide that increases secretion of a GAA polypeptide including a NTPT modified GAA polypeptide from a cell (as compared to a GAA polypeptide without a signal peptide) is encoded by a nucleic acid comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.
  • a signal peptide that increases secretion of a GAA polypeptide including a NTPT modified GAA polypeptide from a cell (as compared to a GAA polypeptide without a signal peptide) is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.
  • a signal peptide that increases secretion of a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:114, or amino acids 70-952 of SEQ ID NO:114 from a cell is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.
  • a nucleic acid encodes a signal peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:29 and SEQ ID NQ:30, and said signal peptide increases secretion of an operably linked polypeptide (e.g., a lysosomal enzyme (e.g., GAA)) from a cell as compared to secretion of a polypeptide (e.g., a lysosomal enzyme (e.g., GAA)) without a signal peptide from an otherwise identical cell.
  • an operably linked polypeptide e.g., a lysosomal enzyme (e.g., GAA)
  • a nucleic acid encodes a signal peptide comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NQ:30 and said signal peptide increases secretion of an operably linked GAA polypeptide from a cell as compared to secretion of a GAA polypeptide without a signal peptide from an otherwise identical cell.
  • a nucleic acid encodes a signal peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NQ:30 and said signal peptide increases secretion of an operably linked polypeptide GAA polypeptide from a cell as compared to secretion of a GAA polypeptide without a signal peptide from an otherwise identical cell.
  • a nucleic acid encodes a signal peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NQ:30 and said signal peptide increases secretion of an operably linked polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:114 or amino acids 70-952 of SEQ ID NO:114 from a cell as compared to secretion of the polypeptide without a signal peptide from an otherwise identical cell.
  • a modified nucleic acid encodes a GAA polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:57, SEQ ID NO:69, SEQ ID NQ:70, SEQ ID NO:71 and SEQ ID NO:72 operably linked to a signal peptide comprising or consisting of the amino acid sequence of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29 which exhibits improved secretion from a cell in which it is expressed (e.g., a hepatocyte) as compared to the level of secretion of a GAA polypeptide provided by a reference nucleic acid (e.g., a wild type gene, a variant or mutant gene) in an otherwise identical cell.
  • a reference nucleic acid e.g., a wild type gene, a variant or mutant gene
  • a modified nucleic acid encodes a GAA polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:57, SEQ ID NO:69, SEQ ID NO:70, SEQ ID N0:71 or SEQ ID NO:72 operably linked to a signal peptide comprising or consisting of the amino acid sequence of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29 which exhibits improved secretion from the cell in which is expressed (e.g., a hepatocyte) as compared to the level of secretion of a GAA polypeptide provided by a reference nucleic acid (e.g., SEQ ID NO:56, SEQ ID NO:113, nucleotides 415- 3198 of SEQ ID NO:113 or nucleotides 550-3198 of SEQ ID NO:113) in an otherwise identical cell.
  • a reference nucleic acid e.g., SEQ ID NO:56,
  • a signal peptide that increases secretion of a polypeptide e.g., a lysosomal enzyme (e.g., GAA)
  • a polypeptide e.g., a lysosomal enzyme (e.g., GAA)
  • a nucleic acid comprising a nucleic acid sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.
  • a signal peptide that increases secretion of a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:114 or amino acids 70-952 of SEQ ID NO:114 from a cell is encoded by a nucleic acid comprising a nucleic acid sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.
  • a nucleic acid encodes a polypeptide comprising an amino acid sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NQ:30 and said polypeptide increases secretion of an operably linked polypeptide (e.g., a lysosomal enzyme (e.g., GAA)) from a cell.
  • an operably linked polypeptide e.g., a lysosomal enzyme (e.g., GAA)
  • a nucleic acid encodes a polypeptide comprising an amino acid sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NQ:30, and said polypeptide increases secretion of an operably linked polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:114 amino acids 70-952 of SEQ ID NO:114 from a cell.
  • a modified nucleic acid comprises or consists of i) a nucleic acid sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24 which encodes a signal peptide that increases secretion of an operably linked polypeptide from a cell, and ii) a nucleic acid at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
  • a modified nucleic acid encodes i) a signal peptide that increase secretion of an operably linked polypeptide from a cell and comprises an amino acid sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NQ:30, and ii) an operably linked GAA polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:114 and amino acids 70-952 of SEQ ID NO:114.
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:17 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:49 (encodes a GAA).
  • a modified nucleic acid comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:25 (a signal peptide) operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO:57 (a GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:18 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:49 (encodes a GAA polypeptide).
  • a modified nucleic acid comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:26 (a signal peptide) operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO:57 (a GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:19 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NQ:50 (encodes a GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:18 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:50 (encodes a GAA polypeptide).
  • a modified nucleic acid comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:26 (a signal peptide) operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO:57 (a GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NQ:20 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NQ:50 (encodes a GAA polypeptide).
  • a modified nucleic acid comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:27 (a signal peptide) operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO:57 (a GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:21 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NQ:50 (encodes a GAA polypeptide).
  • a modified nucleic acid comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:28 (a signal peptide) operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO:57 (a GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:22 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:51 (encodes a GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:19 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:52 (encodes a GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:22 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:53 (encodes a GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:19 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:54 (encodes a GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:22 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:55 (encodes a GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:23 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:49 (encodes a GAA polypeptide).
  • a modified nucleic acid comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:29 (a signal peptide) operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO:57 (a GAA polypeptide).
  • a modified nucleic acid comprising the sequence of SEQ ID NO:24 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:56 (encodes a GAA polypeptide).
  • a modified nucleic acid comprises a nucleic acid encoding the amino acid sequence of SEQ ID NQ:30 (a signal peptide) operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO:58 (a GAA polypeptide).
  • a modified nucleic acid encodes a polypeptide which exhibits improved or increased uptake by a target cell, e.g., a polypeptide encoded thereby is taken up by a target cell at a detectably greater level as compared to the level of uptake by a target cell of a polypeptide provided by a reference nucleic acid (e.g., a wild type, variant or mutant transgene) in an otherwise identical cell.
  • a variant transgene is a nucleic acid with a different modification than that of the modified nucleic acid.
  • the modified nucleic acid is a transgene depleted of 10 CpG dinucleotides and the variant transgene is depleted of 12 CpG dinucleotides, or vice versa.
  • a modified nucleic acid encodes a polypeptide (e.g., a lysosomal enzyme (e.g., GAA)) which exhibits improved or increased uptake by a target cell or tissue (e.g., skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord) as compared to the level of uptake of a polypeptide (e.g., a lysosomal enzyme (e.g., GAA)) provided by a reference nucleic acid (e.g., a wild type, variant or mutant transgene) in an otherwise identical cell.
  • a polypeptide e.g., a lysosomal enzyme (e.g., GAA)
  • GAA lysosomal enzyme
  • a modified nucleic acid encodes a GAA polypeptide which exhibits an improved or increased level of uptake by a target cell or tissue selected from the group consisting of skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord and a combination thereof, as compared to the level of uptake of a GAA polypeptide provided by a reference nucleic acid (e.g., a wild type, variant or mutant transgene) in an otherwise identical cell.
  • a reference nucleic acid e.g., a wild type, variant or mutant transgene
  • a polypeptide that exhibits an improved or increased level of uptake by a target cell is encoded by a modified nucleic acid that also encodes a peptide tag.
  • a polypeptide that exhibits an improved or increased level of uptake by a target cell is operably linked to a peptide tag.
  • a polypeptide may be linked directly to a peptide tag by a peptide bond.
  • a polypeptide may be linked to a peptide tag by a linker comprising 1 to 50 amino acids.
  • a polypeptide linked to a peptide tag may be referred to herein as a fusion protein.
  • a linker may comprise or consist of the amino acid sequence GAP.
  • a linker comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten or more consecutive units of the amino acid sequence GAP.
  • a linker comprises a sequence GAPGAP (SEQ ID NO:47).
  • a linker comprising or consisting of the amino acid sequence GAP is encoded by a nucleic acid sequence of GGCGCCCCA (“L1”), GGAGCCCCA (“L2”) or GGTGCCCCA (“L3”).
  • a linker comprises or consists of the amino acid sequence GAPGAP (SEQ ID NO:47) and is encoded by a nucleic acid sequence of GGCGCCCCAGGCGCCCCA (SEQ ID NO:43), GGTGCCCCAGGTGCCCCA (SEQ ID NO:44) or GGAGCCCCAGGAGCCCCA (SEQ ID NO:45).
  • a linker may comprise or consist of the amino acid sequence GGGGS (SEQ ID NO:48). In some embodiments, a linker may comprise or consist of one, two, three, four, five, six, seven, eight, nine, ten or more units of the amino acid sequence GGGGS. In some embodiments, a linker comprising or consisting of the amino acid sequence GGGGS is encoded by the nucleic acid sequence of SEQ ID NO:46.
  • a peptide tag may be located at the amino terminus of a polypeptide which exhibits an improved or increased level of uptake by a target cell or tissue and the tag may be referred to as an N-terminal peptide tag (“NTPT”).
  • NTPT N-terminal peptide tag
  • a polypeptide with a peptide tag at the N-terminus may be referred to as a NTPT modified polypeptide, for example a NTPT modified GAA polypeptide.
  • a peptide tag may be located at the carboxy terminus of a polypeptide which exhibits an improved or increased level of uptake by a target cell or tissue and the tag may be referred to as a C-terminal peptide tag (“CTPT”).
  • CTT C-terminal peptide tag
  • Peptide tags may be located at the amino terminus and at the carboxy terminus of a polypeptide which exhibits and improved or increased level of uptake by a target cell or tissue.
  • a NTPT, a CTPT or both can function to at least modulate binding of an operably linked polypeptide to a cellular receptor.
  • a NTPT, a CTPT or both that is operably linked to a polypeptide binds to a cellular receptor and modulates interaction of the polypeptide with the cell.
  • a NTPT is an IGF2 polypeptide, a truncated and/or a modified IGF2 polypeptide (e.g., amino acids 32-91 of GenBank NP 000603.1) that is operably linked to GAA polypeptide or a modified GAA polypeptide.
  • a NTPT is a truncated IGF2 polypeptide, a modified IGF2 polypeptide or both (e.g., SEQ ID NO:39, SEQ ID NQ:40, SEQ ID NO:41 , SEQ ID NO:42) that is operably linked to a GAA polypeptide or a modified GAA polypeptide.
  • a NTPT, a CTPT or both that is operably linked to a polypeptide binds to a cellular receptor and modulates interaction of the polypeptide with the cell.
  • a NTPT, a CTPT or both that is operably linked to a polypeptide binds a cellular receptor and modulates uptake of the polypeptide by the cell.
  • a NTPT, a CTPT or both that comprises or consists of an IGF2 polypeptide or a truncated IGF2 polypeptide, a modified IGF2 polypeptide or both, that is operably linked to a second polypeptide (e.g., a lysosomal enzyme, e.g., GAA) binds a cellular receptor and increases uptake of the second polypeptide by the cell (e.g., a cell of skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord) as compared to the uptake of the second polypeptide without an NTPT or CTPT by an otherwise identical cell.
  • a NTPT, a CTPT or both that is operably linked to a polypeptide increases transfer of the polypeptide through the blood-brain-barrier.
  • a second polypeptide e.g., a lysosomal enzyme, e.g., GAA
  • a NTPT, a CTPT or both comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NQ:40 (NTPTseqO2), SEQ ID NO:41 (NTPTseq43) and SEQ ID NO:42 (NTPTseq44).
  • a NTPT, a CTPT or both comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NQ:40, SEQ ID NO:41 and SEQ ID NO:42.
  • a NTPT, CTPT or both are encoded by a nucleic acid comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO:31 , SEQ ID NO:32 (NTPTseqO2), SEQ ID NO:33 (NTPTseq43), SEQ ID NO:34 (NTPTseq44), SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38.
  • a NTPT, CTPT or both are encoded by a nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38.
  • a NTPT, a CTPT or both that comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NQ:40, SEQ ID NO:41 and SEQ ID NO:42 and that are operably linked to a GAA polypeptide, bind a cellular receptor and increase uptake of the GAA polypeptide by the cell (e.g., a cell of skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord) as compared to the uptake of a GAA polypeptide without either an operably linked NTPT or CTPT by an otherwise identical cell.
  • the cell e.g., a cell of skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord
  • a NTPT, a CTPT or both that comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NQ:40, SEQ ID NO:41 and SEQ ID NO:42 and that are operably linked to a GAA polypeptide, increase transfer of the GAA polypeptide across the blood-brain-barrier as compared to the transfer of a GAA polypeptide without either an operably linked NTPT or CTPT.
  • a NTPT comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42 that is operably linked to a GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:114 and amino acids 70-952 of SEQ ID NO:114, binds a cellular receptor and increases uptake of the GAA polypeptide by a cell selected from the group consisting of a cell of skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord and a combination thereof, as compared to the uptake of a GAA polypeptide without an operably linked NTPT by an otherwise identical cell.
  • a NTPT encoded by a nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, operably linked to a GAA polypeptide encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 and nucleotides 550-3198 of SEQ ID NO:113, binds a cellular receptor and increases uptake of the GAA polypeptide by a cell selected from the group consisting of a cell of skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord and a combination thereof, as
  • a NTPT encoded by a nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, operably linked to a GAA polypeptide encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 and nucleotides 550-3198 of SEQ ID NO:113, increases transfer of the GAA polypeptide across the blood-brain-barrier, as compared to the transfer of a GAA polypeptide without an operably linked NTPT.
  • a NTPT, a CTPT or both that are operably linked to a polypeptide (e.g., a lysosomal enzyme, e.g., GAA) bind a cellular receptor and activate an intracellular signaling pathway.
  • a polypeptide e.g., a lysosomal enzyme, e.g., GAA
  • a NTPT, a CTPT or both that comprise an IGF2 polypeptide or a modified IGF2 polypeptide and that is operably linked to a second polypeptide (e.g., a lysosomal enzyme, e.g., GAA), bind an IGF2 receptor and activate an intracellular signaling pathway.
  • a NTPT, a CTPT or both that comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NQ:40, SEQ ID NO:41 and SEQ ID NO:42 and that are operably linked to a second polypeptide (e.g., a lysosomal enzyme, e.g., GAA) bind an IGF2 cellular receptor and activate an intracellular signaling pathway.
  • a second polypeptide e.g., a lysosomal enzyme, e.g., GAA
  • a NTPT, a CTPT or both that comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42 and that are operably linked to a GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58 or amino acids 70-952 of SEQ ID NO:114 bind an IGF2 cellular receptor and activate an intracellular signaling pathway.
  • a NTPT, a CTPT or both that are operably linked to a polypeptide (e.g., a lysosomal enzyme, e.g., GAA) bind an IGF1 receptor and activate an intracellular signaling pathway.
  • a polypeptide e.g., a lysosomal enzyme, e.g., GAA
  • the intracellular signally pathway stimulates cellular proliferation, blood glucose depletion or both.
  • a NTPT, a CTPT or both that comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NQ:40, SEQ ID NO:41 and SEQ ID NO:42 and that are operably linked to a polypeptide (e.g., a lysosomal enzyme, e.g., GAA) bind an IGF1 cellular receptor and activate an intracellular signaling pathway.
  • a polypeptide e.g., a lysosomal enzyme, e.g., GAA
  • a NTPT, a CTPT or both that are operably linked to a polypeptide (e.g., a lysosomal enzyme, e.g., GAA), bind an insulin receptor (INS) and activate an intracellular signaling pathway.
  • a polypeptide e.g., a lysosomal enzyme, e.g., GAA
  • INS insulin receptor
  • the intracellular signally pathway stimulates cellular proliferation, blood glucose depletion or both.
  • a NTPT, a CTPT or both that comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NQ:40, SEQ ID NO:41 and SEQ ID NO:42 and that are operably linked to a polypeptide (e.g., a lysosomal enzyme, e.g., GAA) bind an INS cellular receptor and activate an intracellular signaling pathway.
  • a polypeptide e.g., a lysosomal enzyme, e.g., GAA
  • a NTPT, a CTPT or both that comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NQ:40, SEQ ID NO:41 and SEQ ID NO:42 and that are operably linked to a GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, or amino acids 70-952 of SEQ ID NO:114, exhibit a decreased binding affinity to a cellular IGF1 receptor, an insulin receptor or both as compared to a binding affinity of an wild type IGF2 polypeptide that is operably linked to a GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:114 or amino acids 70-952 of SEQ ID NO:114 on an otherwise identical cell.
  • a NTPT comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42 that is operably linked to a GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, or amino acids 70-952 of SEQ ID NO:114, exhibits a decreased binding affinity to a cellular IGF1 receptor, an insulin receptor or both as compared to the binding affinity of a NTPT comprising or consisting of the amino acid sequence of SEQ ID NO:39 that is operably linked to a GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, or amino acids 70-952 of SEQ ID NO:114 on an otherwise identical cell.
  • a NTPT encoded by a nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34 that is operably linked to a GAA encoded by a nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 and nucleotides 550-3198 of SEQ ID NO:113, exhibits a decreased binding affinity to a cellular IGF1 receptor, an insulin receptor or both as compared to a binding affinity of a NTPT encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:31 , SEQ ID NO:35, SEQ ID NO:36; SEQ ID NO:37 and SEQ
  • a NTPT, a CTPT or both that comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NQ:40, SEQ ID NO:41 and SEQ ID NO:42 and that are operably linked to a GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, and amino acids 70-952 of SEQ ID NO:114, exhibit a decreased binding affinity to a cellular IGF1 receptor, an insulin receptor or both, but maintain a binding affinity to a cellular IGF2 receptor as compared to a binding affinity of a wild type IGF2 polypeptide that is operably linked to a GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:114 and amino acids 70-952 of SEQ ID NO:114 on an otherwise identical cell.
  • a NTPT comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NQ:40, SEQ ID NO:41 and SEQ ID NO:42 that is operably linked to a GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, and amino acids 70-952 of SEQ ID NO:114, exhibits a decreased binding affinity to a cellular IGF1 receptor, an insulin receptor or both, but maintains its binding affinity to a cellular IGF2 receptor, as compared to a binding affinity of a NTPT comprising or consisting of the amino acid sequence of SEQ ID NO:39 that is operably linked to a GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, or amino acids 70-952 of SEQ ID NO:114 on an otherwise identical cell.
  • a NTPT encoded by a nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34 that is operably linked to a GAA encoded by a nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 and nucleotides 550-3198 of SEQ ID NO:113, exhibits a decreased binding affinity to a cellular IGF1 receptor, an insulin receptor or both, but maintains its binding affinity to a cellular IGF2 receptor, as compared to a binding affinity of a NTPT encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:31 , SEQ ID NO:35
  • a NTPT encoded by a nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38 that is operably linked to a GAA encoded by a nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 and nucleotides 550-3198 of SEQ ID NO:113, exhibits a decreased binding affinity to a cellular IGF1 receptor, an insulin receptor or both, but maintains its binding affinity to a cellular IGF2 receptor, as compared to a binding affinity of
  • a modified nucleic acid comprises or consists of i) a nucleic acid sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38 which encodes a NTPT that increases uptake of an operably linked polypeptide by a cell, and ii) a nucleic acid at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%
  • a modified nucleic acid comprises or consists of i) a nucleic acid sequence comprising or consisting of sequence of SEQ ID NO:31 which encodes a NTPT that increases uptake of an operably linked polypeptide by a cell, and ii) a nucleic acid sequence comprising or consisting of a sequence of SEQ ID NO:49 which encodes an operably linked GAA polypeptide.
  • a modified nucleic acid comprises or consists of i) a nucleic acid sequence comprising or consisting of sequence of SEQ ID NO:34 which encodes a NTPT that increases uptake of an operably linked polypeptide by a cell, and ii) a nucleic acid sequence comprising or consisting of a sequence of SEQ ID NO:49 which encodes an operably linked GAA polypeptide.
  • a modified nucleic acid comprises or consists of i) a nucleic acid sequence comprising or consisting of sequence of SEQ ID NO:35 which encodes a NTPT that increases uptake of an operably linked polypeptide by a cell, and ii) a nucleic acid sequence comprising or consisting of a sequence of SEQ ID NQ:50 which encodes an operably linked GAA polypeptide.
  • a modified nucleic acid encodes i) a NTPT that increases uptake of an operably linked polypeptide by a cell and comprises an amino acid sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NQ:40, SEQ ID NO:4, and SEQ ID NO:42, and ii) an operably linked GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:114 and amino acids 70-952 of SEQ ID NO:114.
  • a modified nucleic acid encodes i) a NTPT that increases uptake of an operably linked polypeptide by a cell and comprises or consists of an amino acid sequence of SEQ ID NO:39, , and ii) an operably linked GAA polypeptide comprising or consisting of an amino acid sequence of SEQ ID NO:57.
  • a modified nucleic acid encodes i) a NTPT that increases uptake of an operably linked polypeptide by a cell and comprises or consists of an amino acid sequence of SEQ ID NO:42, and ii) an operably linked GAA polypeptide comprising or consisting of an amino acid sequence of SEQ ID NO:57.
  • a recombinant nucleic acid encodes a NTPT, a linker and a GAA polypeptide and comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:118.
  • a recombinant nucleic acid encodes a NTPT, a linker and a GAA polypeptide and comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NQ:60, SEQ ID NO:61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:118.
  • a recombinant nucleic acid encodes a NTPT, a linker and a GAA polypeptide and comprises or consists of a nucleic acid sequence of SEQ ID NO:59. In some embodiments, a recombinant nucleic acid encodes a NTPT, a linker and a GAA polypeptide and comprises or consists of a nucleic acid sequence of SEQ ID NO:62. In some embodiments, a recombinant nucleic acid encodes a NTPT, a linker and a GAA polypeptide and comprises or consists of a nucleic acid sequence of SEQ ID NO:63. In some embodiments, a recombinant nucleic acid encodes a NTPT, a linker and a GAA polypeptide and comprises or consists of a nucleic acid sequence of SEQ ID NO:118.
  • a polypeptide comprising a NTPT, a linker and GAA polypeptide comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO:69, SEQ ID NQ:70, SEQ ID NO:71 and SEQ ID NO:72.
  • polypeptide comprising a NTPT, a linker and GAA polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:69, SEQ ID NQ:70, SEQ ID NO:71 and SEQ ID NO:72.
  • polypeptide comprising a NTPT, a linker and GAA polypeptide comprises or consists of an amino acid sequence of SEQ ID NO:69.
  • polypeptide comprising a NTPT, a linker and GAA polypeptide comprises or consists of an amino acid sequence of SEQ ID NQ:70.
  • a polypeptide comprising a NTPT, a linker and GAA polypeptide comprises or consists of an amino acid sequence of SEQ ID NO:71 . In some embodiments, a polypeptide comprising a NTPT, a linker and GAA polypeptide comprises or consists of an amino acid sequence of SEQ ID NO:72.
  • a recombinant nucleic acid encodes i) a signal peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NQ:30, ii) a NTPT comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NQ:40, SEQ ID NO:41 and SEQ ID NO:42, iii) a linker comprising or consisting of the amino acid sequence GAP or GGGGS (SEQ ID NO:48), and iv) a GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58 and amino acids 70-952 of SEQ ID NO:114.
  • a recombinant nucleic acid encodes i) a signal peptide comprising the amino acid sequence of SEQ ID NO:26, ii) a NTPT comprising the amino acid sequence of SEQ ID NO:39 iii) a linker comprising the amino acid sequence GAP, and iv) a GAA polypeptide comprising the amino acid sequence of SEQ ID NO:57.
  • a recombinant nucleic acid encodes i) a signal peptide comprising the amino acid sequence of SEQ ID NO:26, ii) a NTPT comprising the amino acid sequence of SEQ ID NO:42 iii) a linker comprising the amino acid sequence of GAP, and iv) a GAA polypeptide comprising the amino acid sequence of SEQ ID NO:57.
  • a recombinant nucleic acid comprises i) a nucleic acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24 which encodes a signal peptide, ii) a nucleic acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34: SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 which encodes a NTPT, iii) a nucleic acid sequence comprising or consisting of a sequence selected from the group consisting of L1 , L2 and L3 which encodes a linker, and iv
  • a recombinant nucleic acid comprises i) a nucleic acid sequence comprising the sequence of SEQ ID NO:18, which encodes a signal peptide, ii) a nucleic acid sequence comprising the sequence of SEQ ID NO:31 which encodes a NTPT, iii) a nucleic acid sequence comprising the sequence of L1 , which encodes a linker, and iv) a nucleic acid comprising the sequence of SEQ ID NO:49 which encodes an operably linked GAA polypeptide.
  • a recombinant nucleic acid comprises i) a nucleic acid sequence comprising the sequence of SEQ ID NO:18, which encodes a signal peptide, ii) a nucleic acid sequence comprising the sequence of SEQ ID NO:34 which encodes a NTPT, iii) a nucleic acid sequence comprising the sequence of L1 , which encodes a linker, and iv) a nucleic acid comprising the sequence of SEQ ID NO:49 which encodes an operably linked GAA polypeptide.
  • a recombinant nucleic acid comprises i) a nucleic acid sequence comprising the sequence of SEQ ID NO:18, which encodes a signal peptide, ii) a nucleic acid sequence comprising the sequence of SEQ ID NO:35 which encodes a NTPT, iii) a nucleic acid sequence comprising the sequence of L3, which encodes a linker, and iv) a nucleic acid comprising the sequence of SEQ ID NO:50 which encodes an operably linked GAA polypeptide.
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:17 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:59 (encodes a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:25 (a signal peptide) operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO:69 (a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:18 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:59 (encodes a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:26 (a signal peptide) operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO:69 (a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:18 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NQ:60 (encodes a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:26 (a signal peptide) operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NQ:70 (a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:18 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:61 (encodes a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:26 (a signal peptide) operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO:71 (a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:18 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:62 (encodes a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:26 (a signal peptide) operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO:72 (a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:19 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:63 (encodes a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:18 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:63 (encodes a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:20 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:63 (encodes a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:27 (e.g., SP1) operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO:69 a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:21 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:63 (encodes a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:28 (a signal peptide) operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO:69 (a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:22 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:64: (encodes a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:19 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:65 (encodes a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:22 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:66 (encodes a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:19 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:67 (encodes a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:22 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:68 (encodes a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:23 (encodes a signal peptide) operably linked to the nucleic acid sequence of SEQ ID NO:59 (encodes a NTPT modified GAA polypeptide).
  • a modified nucleic acid comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:29 (a signal peptide) operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO:69 (a NTPT modified GAA polypeptide).
  • a recombinant nucleic acid encodes a signal peptide, NTPT, a linker and a GAA polypeptide and comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:119 and SEQ ID NO:120.
  • a recombinant nucleic acid encodes a signal peptide, NTPT, a linker and a GAA polypeptide and comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:119 and SEQ ID NQ:120.
  • a recombinant nucleic acid encodes a signal peptide, NTPT, a linker and a GAA polypeptide and comprises or consists of the nucleic acid sequence of SEQ ID NO:74. In some embodiments, a recombinant nucleic acid encodes a signal peptide, NTPT, a linker and a GAA polypeptide and comprises or consists of the nucleic acid sequence of SEQ ID NO:77. In some embodiments, a recombinant nucleic acid encodes a signal peptide, NTPT, a linker and a GAA polypeptide and comprises or consists of the nucleic acid sequence of SEQ ID NO:79. In some embodiments, a recombinant nucleic acid encodes a signal peptide, NTPT, a linker and a GAA polypeptide and comprises or consists of the nucleic acid sequence of SEQ ID NQ:120.
  • a recombinant nucleic acid encodes a signal peptide, NTPT, a linker and a GAA polypeptide comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO:89, SEQ ID NQ:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:121.
  • a recombinant nucleic acid encodes a signal peptide, NTPT, a linker and a GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:89, SEQ ID NQ:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:121.
  • a recombinant nucleic acid encodes a signal peptide, NTPT, a linker and a GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NQ:90. In some embodiments, a recombinant nucleic acid encodes a signal peptide, NTPT, a linker and a GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:93. ii. Regulatory elements
  • the present disclosure includes a recombinant nucleic acid including a modified nucleic acid encoding a lysosomal enzyme (e.g., GAA, NTPT modified GAA) and various regulatory or control elements.
  • regulatory elements are nucleic acid sequence(s) that influence expression of an operably linked polynucleotide.
  • the precise nature of regulatory elements useful for gene expression will vary from organism to organism and from cell type to cell type including, for example, a promoter, enhancer, intron etc., with the intent to facilitate proper heterologous polynucleotide transcription and translation. Regulatory control can be affected at the level of transcription, translation, splicing, message stability, etc.
  • a regulatory control element that modulates transcription is juxtaposed near the 5’ end of the transcribed polynucleotide (i.e., upstream). Regulatory control elements may also be located at the 3’ end of the transcribed sequence (i.e., downstream) or within the transcript (e.g., in an intron). Regulatory control elements can be located at a distance away from the transcribed sequence (e.g., 1 to 100, 100 to 500, 500 to 1000, 1000 to 5000, 5000 to 10,000 or more nucleotides). However, due to the length of an AAV vector genome, regulatory control elements are typically within 1 to 1000 nucleotides from the polynucleotide.
  • promoter refers to a nucleotide sequence that initiates transcription of a particular gene, or one or more coding sequences (e.g., a GAA coding sequence), in eukaryotic cells (e.g., a hepatocyte).
  • a promoter can work with other regulatory elements or regions to direct the level of transcription of the gene or coding sequence(s). These regulatory elements include, for example, transcription binding sites, repressor and activator protein binding sites, and other nucleotide sequences known to act directly or indirectly to regulate the amount of transcription from the promoter, including, for example, attenuators, enhances and silencers.
  • the promoter is most often located on the same strand and near the transcription start site, 5’ of the gene or coding sequence to which it is operably linked.
  • a promoter is generally 100 - 1000 nucleotides in length.
  • a promoter typically increases gene expression relative to expression of the same gene in the absence of a promoter.
  • a “core promoter” or “minimal promoter” refers to the minimal portion of a promoter sequence required to properly initiate transcription. It may include any of the following: a transcription start site, a binding site for RNA polymerase and a general transcription factor binding site.
  • a promoter may also comprise a proximal promoter sequence (5’ of a core promoter) that contains other primary regulatory elements (e.g., enhancer, silencer, boundary element, insulator) as well as a distal promoter sequence (3’ of a core promoter).
  • adenoviral promoters such as the adenoviral major late promoter; heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus promoter; the Rous Sarcoma Virus (RSV) promoter; the albumin promoter; inducible promoters, such as the Mouse Mammary Tumor Virus (MMTV) promoter; the metallothionein promoter; heat shock promoters; the human a-1 -antitrypsin (hAAT) promoter; the hepatitis B surface antigen promoter; the transferrin promoter; the apolipoprotein A-1 promoter; chicken p-actin (CBA) promoter, the elongation factor 1 a promoter (EF1a), the hybrid form of the CBA promoter (CBh promoter), and the CAG promoter (cytomegalovirus early enhancer element and the promoter, the first exon, and the
  • a recombinant nucleic acid comprises a eukaryotic promoter sequence (e.g., a hAAT promoter) operably linked to a modified nucleic acid encoding GAA or a GAA with a signal peptide, a NTPT or both.
  • a eukaryotic promoter sequence e.g., a hAAT promoter
  • a recombinant nucleic acid comprises a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NO:106 (e.g., a hAAT promoter) operably linked to a modified nucleic acid encoding a GAA polypeptide or a GAA polypeptide with a signal peptide, a NTPT or both.
  • a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NO:106 (e.g., a hAAT promoter) operably linked to a modified nucleic acid encoding a GAA polypeptide or a GAA polypeptide with a signal peptide, a NTPT or both.
  • a recombinant nucleic acid comprises a promoter comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence of SEQ ID NQ:106 operably linked to i) a nucleic acid encoding a NTPT -GAA polypeptide and comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NQ:60, SEQ ID NO:61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:118, a ii) a nucleic acid encoding
  • a recombinant nucleic acid comprises a promoter comprising or consisting of a nucleic acid sequence of SEQ ID NQ:106 operably linked to i) a nucleic acid encoding a NTPT -GAA polypeptide and comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NQ:60, SEQ ID NO:61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:118, a ii) a nucleic acid encoding a signal peptide and comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ
  • a recombinant nucleic acid comprises a promoter comprising a nucleic acid sequence at least 98% identical to SEQ ID NO:106 operably linked to i) a nucleic acid encoding a NTPT-GAA polypeptide and comprising a nucleic acid sequence at least 98% identical to SEQ ID NO:59, a ii) a nucleic acid encoding a signal peptide and comprising a nucleic acid sequence at least 98% identical to SEQ ID NO:18 or iii) both.
  • a recombinant nucleic acid comprises a promoter comprising a nucleic acid sequence at least 98% identical to SEQ ID NQ:106 operably linked to i) a nucleic acid encoding a NTPT-GAA polypeptide and comprising a nucleic acid sequence at least 98% identical to SEQ ID NO:62, a ii) a nucleic acid encoding a signal peptide and comprising a nucleic acid sequence at least 98% identical to SEQ ID NO:18 or iii) both.
  • a recombinant nucleic acid comprises a promoter comprising a nucleic acid sequence at least 98% identical to SEQ ID NQ:106 operably linked to i) a nucleic acid encoding a NTPT-GAA polypeptide and comprising a nucleic acid sequence at least 98% identical to SEQ ID NO:63, a ii) a nucleic acid encoding a signal peptide and comprising a nucleic acid sequence at least 98% identical to SEQ ID NO:18 or iii) both.
  • a recombinant nucleic acid comprises a promoter comprising or consisting of a nucleic acid sequence of SEQ ID NQ:106 operably linked to i) a nucleic acid encoding a NTPT-GAA polypeptide and comprising or consisting of a nucleic acid sequence of SEQ ID NO:59, a ii) a nucleic acid encoding a signal peptide and comprising or consisting of a nucleic acid sequence of SEQ ID NO:18 or iii) both.
  • a recombinant nucleic acid comprises a promoter comprising or consisting of a nucleic acid sequence of SEQ ID NQ:106 operably linked to i) a nucleic acid encoding a NTPT-GAA polypeptide and comprising or consisting of a nucleic acid sequence of SEQ ID NO:62, a ii) a nucleic acid encoding a signal peptide and comprising or consisting of a nucleic acid sequence of SEQ ID NO:18 or iii) both.
  • a recombinant nucleic acid comprises a promoter comprising or consisting of a nucleic acid sequence of SEQ ID NQ:106 operably linked to i) a nucleic acid encoding a NTPT-GAA polypeptide and comprising or consisting of a nucleic acid sequence at of SEQ ID NO:63, a ii) a nucleic acid encoding a signal peptide and comprising or consisting of a nucleic acid sequence of SEQ ID NO:18 or iii) both.
  • a recombinant nucleic acid comprises a promoter comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence of SEQ ID NQ:106 operably linked to a modified nucleic acid encoding a signal peptide-NTPT-GAA (SP-NTPT-GAA) polypeptide and comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID N0:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:
  • a recombinant nucleic acid comprises a promoter comprising or consisting of a nucleic acid sequence of SEQ ID NQ:106 operably linked to a modified nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:119 and SEQ ID NQ:120.
  • a recombinant nucleic acid comprises a promoter comprising a nucleic acid sequence at least 98% identical to SEQ ID NQ:106 operably linked to a modified nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising a nucleic acid sequence at least 98% identical to SEQ ID NO:74.
  • a recombinant nucleic acid comprises a promoter comprising a nucleic acid sequence at least 98% identical to SEQ ID NQ:106 operably linked to a modified nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising a nucleic acid sequence at least 98% identical to SEQ ID NO:77.
  • a recombinant nucleic acid comprises a promoter comprising a nucleic acid sequence at least 98% identical to SEQ ID NQ:106 operably linked to a modified nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising a nucleic acid sequence at least 98% identical to SEQ ID NO:79.
  • a recombinant nucleic acid comprises a promoter comprising a nucleic acid sequence at least 98% identical to SEQ ID NQ:106 operably linked to a modified nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising a nucleic acid sequence at least 98% identical to SEQ ID NQ:120.
  • a recombinant nucleic acid comprises a promoter comprising or consisting of a nucleic acid sequence of SEQ ID NQ:106 operably linked to a modified nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising or consisting of a nucleic acid sequence of SEQ ID NO:74.
  • a recombinant nucleic acid comprises a promoter comprising or consisting of a nucleic acid sequence of SEQ ID NQ:106 operably linked to a modified nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising or consisting of a nucleic acid sequence of SEQ ID NO:77.
  • a recombinant nucleic acid comprises a promoter comprising or consisting of a nucleic acid sequence of SEQ ID NQ:106 operably linked to a modified nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising or consisting of a nucleic acid sequence of SEQ ID NO:79.
  • a recombinant nucleic acid comprises a promoter comprising or consisting of a nucleic acid sequence of SEQ ID NO:106 operably linked to a modified nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising or consisting of a nucleic acid sequence of SEQ ID NQ:120.
  • a recombinant nucleic acid comprises a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:106 operably linked to one or both nucleic acids: i) a nucleic acid encoding a NTPT-GAA polypeptide and comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NQ:60, SEQ ID NO:61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:118, and ii) a nucleic acid encoding a signal peptide and comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID N
  • a recombinant nucleic acid comprises a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:106 operably linked to a nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:119 and SEQ ID NO:120 and induces expression of the SP-NTPT-GAA polypeptide encoded by the nucleic acid sequence in hepatocytes.
  • expression of a polypeptide encoded by a nucleic acid comprising or consisting of a nucleic acid sequence encoding a SP-NTPT-GAA polypeptide and comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:119 and SEQ ID NO:120, operably linked to a promoter comprising or consisting of a nucleic acid comprising SEQ ID NO:106, is at a detectably greater level in a cell as compared to the level of expression of a polypeptide encoded by a nucleic acid
  • a recombinant nucleic acid comprises a modified nucleic acid encoding a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:89, SEQ ID NQ:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:121 , operably linked to a regulatory element comprising a promoter sequence (e.g., SEQ ID NQ:106) for the expression of said polypeptide in a cell (e.g., a hepatocyte).
  • a promoter sequence e.g., SEQ ID NQ:106
  • a recombinant nucleic acid comprises a modified nucleic acid encoding a SP-NTPT-GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:89, SEQ ID NQ:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:121 , operably linked to a promoter comprising or consisting of a nucleic acid comprising SEQ ID NQ:106, is expressed at a detectably greater level in a cell as compared to the level of expression of a SP-NPTP-GAA polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:89, SEQ ID NQ:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:
  • a promoter may be constitutive, tissue-specific or regulated. Constitutive promoters are those which cause an operably linked gene to be expressed essentially at all times. In some embodiments, a constitutive promoter is active in most eukaryotic tissues under most physiological and developmental conditions.
  • Regulated promoters are those which can be activated or deactivated. Regulated promoters include inducible promoters, which are usually “off” but which may be induced to turn “on,” and “repressible” promoters, which are usually “on” but may be turned “off.” Many different regulators are known, including temperature, hormones, cytokines, heavy metals and regulatory proteins. The distinctions are not absolute; a constitutive promoter may often be regulated to some degree. In some cases, an endogenous pathway may be utilized to provide regulation of the transgene expression, e.g., using a promoter that is naturally downregulated when the pathological condition improves.
  • a tissue-specific promoter is a promoter that is active in only specific types of tissues, cells or organs.
  • a tissue-specific promoter is recognized by transcriptional activator elements that are specific to a particular tissue, cell and/or organ.
  • a tissue-specific promoter may be more active in one or several particular tissues (e.g., two, three or four) than in other tissues.
  • expression of a gene modulated by a tissue-specific promoter is much higher in the tissue for which the promoter is specific than in other tissues.
  • a promoter may be a tissuespecific promoter, such as the mouse albumin promoter, or the transthyretin promoter (TTR), which are active in liver cells.
  • tissue specific promoters include promoters from genes encoding skeletal a-actin, myosin light chain 2A, dystrophin, muscle creatine kinase which induce expression in skeletal muscle (Li et al. (1999) Nat. Biotech. 17:241-245).
  • Liver specific expression may be induced using promoters from the albumin gene (Miyatake et al. (1997) J. Virol. 71 :5124-5132), hepatitis B. virus core promoter (Sandig, et al. (1996) Gene Ther. 3:1002-1009) and alpha-fetoprotein (Arbuthnot et aL, (1996) Hum. Gene. Ther. 7:1503- 1514).
  • a modified nucleic acid encoding a therapeutic polypeptide further comprises an enhancer to increase expression of the therapeutic polypeptide (e.g., a GAA polypeptide, or a GAA polypeptide with a signal peptide, a NTPT or both).
  • an enhancer element is located upstream of a promoter element but may also be located downstream or within another sequence (e.g., a transgene).
  • An enhancer may be located 100 nucleotides, 200 nucleotides, 300 nucleotides or more upstream or downstream of a modified nucleic acid.
  • An enhancer typically increases expression of a modified nucleic acid (e.g., encoding a therapeutic polypeptide, e.g., encoding GAA) beyond the increased expression provided by a promoter element alone.
  • CMV MIE promoter comprises three regions: the modulator, the unique region and the enhancer (Isomura and Stinski (2003) J. Virol. 77(6):3602-3614).
  • the CMV enhancer region can be combined with another promoter, or a portion thereof, to form a hybrid promoter to further increase expression of a nucleic acid operably linked thereto.
  • a chicken p- actin (CBA) promoter can be combined with a CMV promoter/enhancer, or a portion thereof, to make a version of CBA termed the “CBh” promoter, which stands for chicken beta-actin hybrid promoter, as described in Gray et al. (2011 , Human Gene Therapy 22:1143-1153).
  • CBA chicken p- actin
  • CBh chicken beta-actin hybrid promoter
  • an enhancer sequence e.g., a apolipoprotein E (ApoE) enhancer
  • a apolipoprotein E (ApoE) enhancer is operably linked to a modified nucleic acid encoding GAA or a GAA with a signal peptide, a NTPT or both.
  • an enhancer sequence e.g., a ApoE enhancer
  • a promoter e.g., hAAT promoter
  • an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NO:105 is operably linked to a modified nucleic acid encoding GAA or a GAA with a signal peptide, a NTPT or both.
  • an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NO:105 e.g., an ApoE enhancer
  • a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NO:106 are operably linked to a modified nucleic acid encoding GAA or a GAA with a signal peptide, a NTPT or both.
  • a recombinant nucleic acid comprises an enhancer comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence of SEQ ID NQ:105 operably linked to i) a promoter comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence of SEQ ID NQ:106, ii) a nucleic acid encoding a signal peptide and comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:
  • a recombinant nucleic acid comprises an enhancer comprising or consisting of a nucleic acid sequence of SEQ ID NQ:105 operably linked to i) a promoter comprising or consisting of a nucleic acid sequence of SEQ ID NQ:106, ii) a nucleic acid encoding a signal peptide and comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24 iii) a nucleic acid encoding a NTPT-GAA polypeptide and comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NQ:60; SEQ ID NO:61 , SEQ ID NO:62: SEQ ID NO
  • recombinant nucleic acid comprises an enhancer comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:105 operably linked to i) a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:106, ii) a nucleic acid encoding a signal peptide and comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:18, iii) a nucleic acid encoding a NTPT-GAA polypeptide and comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:59 or iv) a combination thereof.
  • recombinant nucleic acid comprises an enhancer comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:105 operably linked to i) a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:106, ii) a nucleic acid encoding a signal peptide and comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:18, iii) a nucleic acid encoding a NTPT-GAA polypeptide and comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:62 or iv) a combination thereof.
  • recombinant nucleic acid comprises an enhancer comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:105 operably linked to i) a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:106, ii) a nucleic acid encoding a signal peptide and comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:18, iii) a nucleic acid encoding a NTPT-GAA polypeptide and comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:63 or iv) a combination thereof.
  • a recombinant nucleic acid comprises an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NQ:105 operably linked to i) a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NQ:106, ii) a nucleic acid encoding a signal peptide and comprising or consisting of the nucleic acid sequence of SEQ ID NO:18, iii) a nucleic acid encoding a NTPT-GAA polypeptide and comprising or consisting of the nucleic acid sequence of SEQ ID NO:59 or iv) a combination thereof.
  • a recombinant nucleic acid comprises an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NQ:105 operably linked to i) a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NQ:106, ii) a nucleic acid encoding a signal peptide and comprising or consisting of the nucleic acid sequence of SEQ ID NO:18, iii) a nucleic acid encoding a NTPT-GAA polypeptide and comprising or consisting of the nucleic acid sequence of SEQ ID NO:62 or iv) a combination thereof.
  • a recombinant nucleic acid comprises an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NQ:105 operably linked to i) a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NQ:106, ii) a nucleic acid encoding a signal peptide and comprising or consisting of the nucleic acid sequence of SEQ ID NO:18, iii) a nucleic acid encoding a NTPT-GAA polypeptide and comprising or consisting of the nucleic acid sequence of SEQ ID NO:63 or iv) a combination thereof.
  • a recombinant nucleic acid comprises an enhancer comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence of SEQ ID NO:105 operably linked to i) a promoter comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence of SEQ ID NO:106, ii) a nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ
  • a recombinant nucleic acid comprises an enhancer comprising or consisting of a nucleic acid sequence of SEQ ID NQ:105 operably linked to i) a promoter comprising or consisting of a nucleic acid sequence of SEQ ID NQ:106, ii) a nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:119 and SEQ ID NO:120 or iii) both.
  • a recombinant nucleic acid comprises an enhancer comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:105 is operably linked to i) a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:106, ii) a nucleic acid encoding a SP- NTPT-GAA polypeptide and comprising a sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:74 or iii) both.
  • a recombinant nucleic acid comprises an enhancer comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:105 operably linked to i) a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:106, ii) a nucleic acid encoding a SP- NTPT-GAA polypeptide and comprising a nucleic sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:77 or iii) both.
  • a recombinant nucleic acid comprises an enhancer comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:105 operably linked to i) a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:106, ii) a nucleic acid encoding a SP- NTPT-GAA polypeptide and comprising a nucleic sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:79 or iii) both.
  • a recombinant nucleic acid comprises an enhancer comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:105 operably linked to i) a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:106, ii) a nucleic acid encoding a SP- NTPT-GAA polypeptide and comprising a nucleic sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:120 or iii) both.
  • a recombinant nucleic acid comprises an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NQ:105 operably linked to i) a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NQ:106, ii) a nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising or consisting of the nucleic acid sequence of SEQ ID NO:74 or iii) both.
  • a recombinant nucleic acid comprises an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NQ:105 operably linked to i) a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NQ:106, ii) a nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising or consisting of the nucleic acid sequence of SEQ ID NO:77 or iii) both.
  • a recombinant nucleic acid comprises an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NQ:105 operably linked to i) a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NQ:106, ii) a nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising or consisting of the nucleic sequence of SEQ ID NO:79 or iii) both.
  • a recombinant nucleic acid comprises an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NQ:105 operably linked to i) a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NQ:106, ii) a nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising or consisting of the nucleic sequence of SEQ ID NQ:120 or iii) both.
  • recombinant nucleic acid comprising an enhancer comprising or consisting of a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NQ:105 and a promoter comprising or consisting of a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NQ:106 induce expression of a polypeptide encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:119 and SEQ ID NQ:120 in hepatocytes.
  • a recombinant nucleic acid comprising an enhancer comprising a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NQ:105, operably linked to a promoter comprising a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NQ:106, together induce expression of a polypeptide encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:119 and SEQ ID NQ:120 in hepatocytes.
  • a recombinant nucleic acid comprising an enhancer comprising a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NQ:105, operably linked to a promoter comprising a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NQ:106, together induce expression of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:89, SEQ ID NQ:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:121 in hepatocytes.
  • a recombinant nucleic acid comprising an enhancer comprising a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NQ:105, operably linked to a promoter comprising a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NQ:106, together induce expression of a polypeptide encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:79 and SEQ ID NQ:120 in hepatocytes.
  • a recombinant nucleic acid comprises a nucleic acid encoding a polypeptide selected from the group consisting of SEQ ID NO:89, SEQ ID NQ:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:121 , operably linked to a regulatory element comprising at least one of: an enhancer sequence (e.g., SEQ ID NO:105) and a promoter sequence (e.g., SEQ ID NO:106) for the expression of said polypeptide in a cell (e.g., a hepatocyte).
  • an enhancer sequence e.g., SEQ ID NO:105
  • a promoter sequence e.g., SEQ ID NO:106
  • a recombinant nucleic acid includes, for example, an intron, exon and/or a portion thereof.
  • An intron may function as a filler or staffer polynucleotide sequence to achieve an appropriate length for vector genome packaging into a rAAV vector.
  • An intron and/or an exon sequence can also enhance expression of a polypeptide (e.g., a transgene) as compared to expression in the absence of the intron and/or exon element (Karachi et al. (1995) J. Biol. Chem. 270 (10):576-5281 ; WO 2017/074526).
  • filler/stuffer polynucleotide sequences also referred to as “insulators” are well known in the art and include, but are not limited to, those described in WO 2014/144486 and WO 2017/074526.
  • An intron element may be derived from the same gene as a heterologous polynucleotide, or derived from a completely different gene or other DNA sequence (e.g., chicken p-actin gene, minute virus of mice (MVM)).
  • a recombinant nucleic acid includes at least one intron derived from a non-cognate gene (i.e., not derived from the modified nucleic acid, e.g., transgene).
  • an intron comprises or consists of the nucleic acid sequence of SEQ ID NO: 107.
  • an intron comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence of SEQ ID NO:107.
  • a recombinant nucleic acid is comprised of at least one of: an enhancer sequence (e.g., SEQ ID NO:105), a promoter sequence (e.g., SEQ ID NQ:106), and an intron (e.g., SEQ ID NQ:107) and modulates expression of a heterologous polypeptide encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:119 and SEQ ID NQ:120.
  • an enhancer sequence e.g., SEQ ID NO:105
  • a recombinant nucleic acid is comprised of at least one of: an enhancer sequence (e.g., SEQ ID NQ:105), a promoter sequence (e.g., SEQ ID NQ:106), and an intron (e.g., SEQ ID NQ:107) and modulates expression of a heterologous polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:89, SEQ ID NQ:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:121 .
  • an enhancer sequence e.g., SEQ ID NQ:105
  • a promoter sequence e.g., SEQ ID NQ:106
  • an intron e.g., SEQ ID NQ:107
  • a recombinant nucleic acid comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:119 and SEQ ID NO:120, operably linked to a regulatory element comprising at least one of: an enhancer sequence (e.g., SEQ ID NQ:105), a promoter sequence (e.g., SEQ ID NQ:106) and an intron (e.g., SEQ ID NQ:107).
  • an enhancer sequence e.g., SEQ ID NQ:105
  • a promoter sequence e.g
  • a recombinant nucleic acid comprises a nucleic acid encoding a polypeptide selected from the group consisting of SEQ ID NO:89, SEQ ID NQ:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:121 , operably linked to a regulatory element comprising at least one of: an enhancer sequence (e.g., SEQ ID NQ:105), a promoter sequence (e.g., SEQ ID NQ:106) and an intron (e.g., SEQ ID NQ:107) for the expression of said polypeptide in a cell (e.g., a hepatocyte).
  • an enhancer sequence e.g., SEQ ID NQ:105
  • a promoter sequence e.g., SEQ ID NQ:106
  • an intron e
  • a recombinant nucleic acid comprises an intron comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:107 operably linked to i) an enhancer comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:105 ii) a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:106, iii) a nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising a sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:74 or iv) or a combination thereof.
  • a recombinant nucleic acid comprises an intron comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:107 operably linked to i) an enhancer comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:105 ii) a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:106, iii) a nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising a sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:77 or iv) or a combination thereof.
  • a recombinant nucleic acid comprises an intron comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:107 operably linked to i) an enhancer comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:105 ii) a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:106, iii) a nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising a sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:79 or iv) or a combination thereof.
  • a recombinant nucleic acid comprises an intron comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:107 operably linked to i) an enhancer comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:105 ii) a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:106, iii) a nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising a sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:120 or iv) or a combination thereof.
  • a recombinant nucleic acid comprises an intron comprising or consisting of a nucleic acid sequence of SEQ ID NQ:107 operably linked to i) an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NO:105, ii) a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NO:106, iii) a nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising or consisting of the nucleic acid sequence of SEQ ID NO:74 or iv) a combination thereof.
  • a recombinant nucleic acid comprises an intron comprising or consisting of a nucleic acid sequence of SEQ ID NQ:107 operably linked to i) an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NQ:105, ii) a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NO:106, iii) a nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising or consisting of the nucleic acid sequence of SEQ ID NO:77 or iv) a combination thereof.
  • a recombinant nucleic acid comprises an intron comprising or consisting of a nucleic acid sequence of SEQ ID NQ:107 operably linked to i) an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NO:105, ii) a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NO:106, iii) a nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising or consisting of the nucleic acid sequence of SEQ ID NO:79 or iv) a combination thereof.
  • a recombinant nucleic acid comprises an intron comprising or consisting of a nucleic acid sequence of SEQ ID NQ:107 operably linked to i) an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NO:105, ii) a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NO:106, iii) a nucleic acid encoding a SP-NTPT-GAA polypeptide and comprising or consisting of the nucleic acid sequence of SEQ ID NO:120 or iv) a combination thereof.
  • Further regulatory elements may include a stop codon, a termination sequence, and a polyadenylation (polyA) signal sequence, such as, but not limited to a bovine growth hormone polyA signal sequence (BHG polyA).
  • a polyA signal sequence drives efficient addition of a polyadenosine “tail” at the 3’ end of a eukaryotic mRNA which guides termination of gene transcription (see, e.g., Goodwin and Rottman J. Biol. Chem. (1992) 267(23):16330-16334).
  • a polyA signal acts as a signal for the endonucleolytic cleavage of the newly formed precursor mRNA at its 3’ end and for addition to this 3’ end of an RNA stretch consisting only of adenine bases.
  • a polyA tail is important for the nuclear export, translation and stability of mRNA.
  • a polyA is a SV40 early polyadenylation signal, a SV40 late polyadenylation signal, an HSV thymidine kinase polyadenylation signal, a protamine gene polyadenylation signal, an adenovirus 5 E1 b polyadenylation signal, a growth hormone polyadenylation signal, a PBGD polyadenylation signal or an in silico designed polyadenylation signal.
  • a polyA signal sequence of a recombinant nucleic acid is a polyA signal that is capable of directing and effecting the endonucleolytic cleavage and polyadenylation of the precursor mRNA resulting from the transcription of a modified nucleic acid encoding a modified GAA polypeptide (e.g., SEQ ID NO: SEQ ID NO:89, SEQ ID NQ:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:121 ).
  • a modified GAA polypeptide e.g., SEQ ID NO: SEQ ID NO:89, SEQ ID NQ:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO
  • a polyA sequence comprises or consists of the nucleic acid sequence of SEQ ID NQ:108. In some embodiments, a polyA sequence comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence of SEQ ID NQ:108.
  • a recombinant nucleic acid comprises at least one of: an enhancer sequence (e.g., SEQ ID NQ:105), a promoter sequence (e.g., SEQ ID NQ:106), an intron (e.g., SEQ ID NQ:107) and a polyA (SEQ ID NQ:108) and modulates the expression of a heterologous polypeptide, encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:1 19 and SEQ ID NQ:120.
  • an enhancer sequence
  • a recombinant nucleic acid comprises i) an intron comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:107, ii) an enhancer comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:105 iii) a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:106, iv) a nucleic acid encoding a modified GAA polypeptide and comprising a sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:74, v) a polyA sequence comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:108 or vi) or a combination thereof.
  • a recombinant nucleic acid comprises i) an intron comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:107, ii) an enhancer comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:105 iii) a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:106, iv) a nucleic acid encoding a modified GAA polypeptide and comprising a sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:77, v) a polyA sequence comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:108 or vi) or a combination thereof.
  • a recombinant nucleic acid comprises i) an intron comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:107, ii) an enhancer comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:105 iii) a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:106, iv) a nucleic acid encoding a modified GAA polypeptide and comprising a sequence at least 98% identical to the nucleic acid sequence of SEQ ID NO:79, v) a polyA sequence comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:108 or vi) or a combination thereof.
  • a recombinant nucleic acid comprises i) an intron comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:107, ii) an enhancer comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:105 iii) a promoter comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:106, iv) a nucleic acid encoding a modified GAA polypeptide and comprising a sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:120, v) a polyA sequence comprising a nucleic acid sequence at least 98% identical to the nucleic acid sequence of SEQ ID NQ:108 or vi) or a combination thereof.
  • a recombinant nucleic acid comprises i) an intron comprising or consisting of the nucleic acid sequence of SEQ ID NQ:107, ii) an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NQ:105 iii) a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NQ:106, iv) a nucleic acid encoding a modified GAA polypeptide and comprising or consisting of the nucleic acid sequence of SEQ ID NO:74, v) a polyA sequence comprising or consisting of the nucleic acid sequence of SEQ ID NQ:108, or vi) a combination thereof.
  • a recombinant nucleic acid comprises i) an intron comprising or consisting of the nucleic acid sequence of SEQ ID NQ:107, ii) an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NO:105 iii) a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NO:106, iv) a nucleic acid encoding a modified GAA polypeptide and comprising or consisting of the nucleic acid sequence of SEQ ID NO:77, v) a polyA sequence comprising or consisting of the nucleic acid sequence of SEQ ID NQ:108, or vi) a combination thereof.
  • a recombinant nucleic acid comprises i) an intron comprising or consisting of a nucleic acid sequence of SEQ ID NQ:107, ii) an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NQ:105 iii) a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NQ:106, iv) a nucleic acid encoding a modified GAA polypeptide and comprising or consisting of the nucleic acid sequence of SEQ ID NO:79 v) a polyA sequence comprising or consisting of the nucleic acid sequence of SEQ ID NQ:108, or vi) a combination thereof.
  • a recombinant nucleic acid comprises i) an intron comprising or consisting of a nucleic acid sequence of SEQ ID NQ:107, ii) an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NQ:105 iii) a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NQ:106, iv) a nucleic acid encoding a modified GAA polypeptide and comprising or consisting of the nucleic acid sequence of SEQ ID NQ:120 v) a polyA sequence comprising or consisting of the nucleic acid sequence of SEQ ID NQ:108, or vi) a combination thereof.
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA), with tropism for hepatocytes, contains a vector genome comprising AAV ITRs (e.g., AAV2 ITRs) and a recombinant nucleic acid comprising a modified (e.g., codon-optimized, CpG modified) nucleic acid encoding a modified GAA polypeptide (e.g., SP-NTPT-GAA) and at least one of the following regulatory elements: an enhancer (e.g., an ApoE enhancer), a promoter (e.g., a hAAT promoter), an intron (e.g., a chimeric intron) and a polyA (e.g., a BHG polyA).
  • AAV ITRs e.g., AAV2 ITRs
  • a recombinant nucleic acid comprising a modified (e.g., codon-optimized
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA), with tropism for hepatocytes, contains a vector genome comprising AAV ITRs (e.g., SEQ ID NQ:109, SEQ ID NQ:110) and a recombinant nucleic acid comprising a modified (e.g., codon-optimized, CpG modified) nucleic acid (e.g., SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:119 and SEQ ID NO:120) encoding
  • AAV ITRs
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA), with tropism for hepatocytes, contains a vector genome comprising AAV ITRs comprising or consisting of the ITRs of SEQ ID NO:109 or SEQ ID NO:110, and a recombinant nucleic acid comprising a modified (e.g., codon-optimized, CpG modified) nucleic acid comprising or consisting of the nucleic acid sequence of SEQ ID NO:74 encoding a modified GAA polypeptide (e.g., SP-NTPT- GAA) and at least one of the following regulatory elements: an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NO:105, a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NO:106, an intron comprising or consisting of the nucleic acid sequence of SEQ ID NO:107 and a polyA comprising or consisting of
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA), with tropism for hepatocytes, contains a vector genome comprising AAV ITRs comprising or consisting of the ITRs of SEQ ID NO:109 and/or SEQ ID NO:110, and a recombinant nucleic acid comprising a modified (e.g., codon-optimized, CpG modified) nucleic acid comprising or consisting of the nucleic acid sequence of SEQ ID NO:77 encoding a modified GAA polypeptide (e.g., SP-NTPT- GAA) and at least one of the following regulatory elements: an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NO:105, a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NO:106, an intron comprising or consisting of the nucleic acid sequence of SEQ ID NO:107 and a polyA comprising or consist
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA), with tropism for hepatocytes, contains a vector genome comprising AAV ITRs comprising or consisting of the ITRs of SEQ ID NO:109 and/or SEQ ID NO:110, and a recombinant nucleic acid comprising a modified (e.g., codon-optimized, CpG modified) nucleic acid comprising or consisting of the nucleic acid sequence of SEQ ID NO:79 encoding a modified GAA polypeptide (e.g., SP-NTPT- GAA) and at least one of the following regulatory elements: an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NO:105, a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NO:106, an intron comprising or consisting of the nucleic acid sequence of SEQ ID NO:107 and a polyA comprising or consist
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA), with tropism for hepatocytes, contains a vector genome comprising AAV ITRs comprising or consisting of the ITRs of SEQ ID NO:109 and/or SEQ ID NO:110, and a recombinant nucleic acid comprising a modified (e.g., codon-optimized, CpG modified) nucleic acid comprising or consisting of the nucleic acid sequence of SEQ ID NQ:120 encoding a modified GAA polypeptide (e.g., SP- NTPT-GAA) and at least one of the following regulatory elements: an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NQ:105, a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NQ:106, an intron comprising or consisting of the nucleic acid sequence of SEQ ID NO:107 and a polyA
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA), with tropism for hepatocytes, contains a vector genome comprising AAV ITRs (e.g., SEQ ID NO:109, SEQ ID NO:110) and a recombinant nucleic acid encoding a modified GAA polypeptide (e.g., SP-NTPT- GAA) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO:89, SEQ ID NQ:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:121 and at least one of the following regulatory elements: an enhancer (e.g., SEQ ID NQ:105), a promoter (e.g., SEQ ID NQ:106), an intron (e.g
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA), with tropism for hepatocytes, contains a vector genome comprising AAV ITRs comprising or consisting of the ITRs of SEQ ID NO:109 and/or SEQ ID NO:110, and a recombinant nucleic acid encoding a modified GAA polypeptide (e.g., SP-NTPT-GAA) comprising or consisting of the amino acid sequence of SEQ ID NQ:90 and at least one of the following regulatory elements: an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NQ:105, a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NQ:106, an intron comprising or consisting of the nucleic acid sequence of SEQ ID NO:107 and a polyA comprising or consisting of a nucleic acid sequence of SEQ ID NQ:108.
  • AAV9-SP-NTPT-GAA e.
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA), with tropism for hepatocytes, contains a vector genome comprising AAV ITRs comprising or consisting of the ITRs of SEQ ID NO:109 and/or SEQ ID NO:110, and a recombinant nucleic acid encoding a modified GAA polypeptide (e.g., SP-NTPT-GAA) comprising or consisting of the amino acid sequence of SEQ ID NO:93 and at least one of the following regulatory elements: an enhancer comprising or consisting of the nucleic acid sequence of SEQ ID NO:105, a promoter comprising or consisting of the nucleic acid sequence of SEQ ID NO:106, an intron comprising or consisting of the nucleic acid sequence of SEQ ID NO:107 and a polyA comprising or consisting of a nucleic acid sequence of SEQ ID NQ:108.
  • AAV9-SP-NTPT-GAA a vector genome comprising
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA) contains a vector genome comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:116 and SEQ ID NO:117.
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA) contains a vector genome comprising a nucleic acid sequence at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID N0:11 , SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:16, SEQ ID N0:116 and SEQ ID N0:117.
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA) contains a vector genome comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:116 and SEQ ID NO:117.
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA) contains a vector genome comprising a nucleic acid sequence at least 98% identical to SEQ ID NO:2.
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA) contains a vector genome comprising a nucleic acid sequence at least 98% identical to SEQ ID NO:5.
  • a rAAV vector e.g., AAV9-SP-NTPT-GAA
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA) contains a vector genome comprising a nucleic acid sequence at least 98% identical to SEQ ID NO:116. In some embodiments, a rAAV vector (e.g., AAV9-SP-NTPT-GAA) contains a vector genome comprising a nucleic acid sequence at least 98% identical to SEQ ID NO:117.
  • a recombinant nucleic acid intended for use in a rAAV vector may include an additional nucleic acid element to adjust the length of the nucleic acid to near, or at the normal size (e.g., approximately 4.7 to 4.9 kilobases), of the viral genomic sequence acceptable for AAV packaging into a rAAV vector (Grieger and Samulski (2005) J. Virol. 79(15):9933-9944). Such a sequence may be referred to interchangeably as filler, spacer or staffer.
  • spacer DNA is an untranslated (non-protein coding) segment of nucleic acid.
  • a spacer polynucleotide sequence is a sequence between about 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90-90-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1000, 1000-1500, 1500-2000, 2000-3000 or more in length.
  • one or more spacer sequences flank an ITR sequence.
  • AAV vectors typically accept inserts of DNA having a size ranging from about 4 kb to about 5.2 kb or about 4.1 to 4.9 kb for optimal packaging of the nucleic acid into the AAV capsid.
  • a rAAV vector comprises a vector genome having a total length between about 3.0 kb to about 3.5 kb, about 3.5 kb to about 4.0 kb, about 4.0 kb to about 4.5kb, about 4.5 kb to about 5.0 kb or about 5.0 kb to about 5.2 kb.
  • a rAAV vector comprises a vector genome having a total length of about 4.3 kb.
  • a rAAV vector comprises a vector genome that is self-complementary. While the total length of a self-complementary (sc) vector genome in a rAAV vector is equivalent to a single-stranded (ss) vector genome (i.e., from about 4 kb to about 5.2 kb), the nucleic acid sequence (i.e., comprising the transgene, regulatory elements and ITRs) encoding the sc vector genome must be only half as long as a nucleic acid sequence encoding a ss vector genome in order for the sc vector genome to be packaged in the capsid.
  • sc self-complementary
  • a recombinant nucleic acid comprises one or more spacers comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:99, SEQ ID N0:100, SEQ ID NO:101 , SEQ ID NO:102, SEQ ID NO:103 and SEQ ID NQ:104.
  • a recombinant nucleic acid comprises one or more spacers comprising a nucleic acid sequence at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 , SEQ ID NQ:102, SEQ ID NQ:103 and SEQ ID NQ:104.
  • a recombinant nucleic acid comprises one or more spacers comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:99, SEQ ID NQ:100, SEQ ID NQ:101 , SEQ ID NQ:102, SEQ ID NQ:103 and SEQ ID NQ:104.
  • one or more spacer sequences comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:99, SEQ ID NQ:100, SEQ ID NQ:101 , SEQ ID NQ:102, SEQ ID NQ:103, SEQ ID NQ:104 flank at least one ITR sequence.
  • one or more spacer sequences comprising a nucleic acid sequence at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:99, SEQ ID NQ:100, SEQ ID NQ:101 , SEQ ID NQ:102, SEQ ID NQ:103, SEQ ID NQ:104 flank a recombinant nucleic acid comprising a nucleic acid sequence at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:116 and SEQ ID NO:117
  • one or more spacer sequences comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:99, SEQ ID NQ:100, SEQ ID NQ:101 , SEQ ID NQ:102, SEQ ID NQ:103, SEQ ID NQ:104 flank a recombinant nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:16, SEQ ID N0:116 and SEQ ID N0:117.
  • spacer sequences comprising or consisting of the nucleic acid sequence of SEQ ID NQ:100, SEQ ID NQ:103 or both flank a recombinant nucleic acid comprising or consisting of the nucleic acid sequence of SEQ ID NO:2.
  • spacer sequences comprising or consisting of the nucleic acid sequence of SEQ ID NO:99, SEQ ID NQ:102 or both flank a recombinant nucleic acid comprising or consisting of the nucleic acid sequence of SEQ ID NO:5.
  • spacer sequences comprising or consisting of the nucleic acid sequence of SEQ ID NO:99, SEQ ID NQ:102 or both flank a recombinant nucleic acid comprising or consisting of the nucleic acid sequence of SEQ ID NO:7. In some embodiments, spacer sequences comprising or consisting of the nucleic acid sequence of SEQ ID NO:99, SEQ ID NQ:102 or both flank a recombinant nucleic acid comprising or consisting of the nucleic acid sequence of SEQ ID NO:116.
  • spacer sequences comprising or consisting of the nucleic acid sequence of SEQ ID NO:99, SEQ ID NQ:102 or both flank a recombinant nucleic acid comprising or consisting of the nucleic acid sequence of SEQ ID NO:117.
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA) contains a vector genome comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:116 and SEQ ID NO:117 flanked by at least one spacer comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA) contains a vector genome comprising a nucleic acid sequence at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:116 and SEQ ID NO:117 flanked by at least one spacer comprising a nucleic acid sequence at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:99, SEQ ID NQ:100, SEQ ID NQ:101 , SEQ ID
  • a rAAV vector (e.g., AAV9-SP-NTPT-GAA) contains a vector genome comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID N0:11 , SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:16, SEQ ID N0:116 and SEQ ID N0:117 flanked by at least one spacer comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 , SEQ ID NQ:102, S
  • a viral vector e.g., rAAV vector
  • a transgene e.g., a modified GAA transgene
  • a viral vector include but are not limited to adenoviral, retroviral, lentiviral, herpesvirus and adeno-associated virus (AAV) vectors, and in particular rAAV vector (as discussed, supra).
  • a vector genome component of a rAAV vector produced according to the methods of the disclosure include at least one transgene, e.g., a modified nucleic acid encoding a GAA polypeptide or a NTPT modified GAA polypeptide, and associated expression control sequences for controlling expression of the modified nucleic acid encoding a GAA polypeptide or a NTPT modified GAA polypeptide.
  • transgene e.g., a modified nucleic acid encoding a GAA polypeptide or a NTPT modified GAA polypeptide
  • associated expression control sequences for controlling expression of the modified nucleic acid encoding a GAA polypeptide or a NTPT modified GAA polypeptide.
  • a vector genome includes a portion of a parvovirus genome, such as an AAV genome with rep and cap deleted and/or replaced by a modified nucleic acid (e.g., transgene, e.g., modified nucleic acid encoding a GAA polypeptide) and its associated expression control sequences.
  • a modified nucleic acid encoding a GAA polypeptide is typically inserted adjacent to one or two (i.e., is flanked by) AAV ITRs or ITR elements adequate for viral replication (Xiao et al. (1997) J. Virol. 71 (2): 941-948), in place of the nucleic acid encoding viral rep and cap proteins.
  • Other regulatory sequences suitable for use in facilitating tissue-specific expression of a modified nucleic acid encoding a GAA polypeptide in the target cell may also be included.
  • Cap and rep genes may be supplied to a cell (e.g., a host cell, e.g., a packaging cell) as part of a plasmid that is separate from a plasmid supplying the vector genome with the transgene.
  • Packaging cell or “producer cell” means a cell or cell line which may be transfected with a vector, plasmid or DNA construct, and provides in trans all the missing functions which are required for the complete replication and packaging of a viral vector.
  • the required genes for rAAV vector assembly include the vector genome (e.g., a modified nucleic acid encoding a GAA polypeptide, regulatory elements, and ITRs), AAV rep gene, AAV cap gene, and certain helper genes from other viruses such as, e.g., adenovirus.
  • the requisite genes for AAV production can be introduced into a packaging cell in various ways including, for example, transfection of one or more plasmids.
  • some genes may already be present in a packaging cell, either integrated into the genome or carried on an episome.
  • a packaging cell expresses, in a constitutive or inducible manner, one or more missing viral functions.
  • Any suitable packaging cell known in the art may be employed in the production of a packaged viral vector.
  • Mammalian cells or insect cells are preferred.
  • Examples of cells useful for the production of a packaging cell in the practice of the disclosure include, for example, human cell lines, such as PER.C6, WI38, MRC5, A549, HEK293 cells (which express functional adenoviral E1 under the control of a constitutive promoter), B-50 or any other HeLa cell, HepG2, Saos-2, HuH7, and HT1080 cell lines.
  • Suitable non-human mammalian cell lines include, for example, VERO, COS-1 , COS-7, MDCK, BHK21 -F, HKCC or CHO cells.
  • Insect cell lines include Spodoptera frugiperda, such as the Sf9 or Sf21 cell lines, Drosophila spp. cell lines, or mosquito cell lines, e.g., Aedes albopictus derived cell lines.
  • a preferred cell line is the Spodoptera frugiperda Sf9 cell line.
  • AAV is a Dependovirus in that it cannot replicate in a cell without co-infection of the cell by a helper virus.
  • Helper functions include helper virus elements needed for establishing active infection of a packaging cell, which is required to initiate packaging of the viral vector.
  • Helper viruses include, typically, adenovirus or herpes simplex virus.
  • Adenovirus helper functions typically include adenovirus components adenovirus early region 1A (E1a), E1b, E2a, E4, and viral associated (VA) RNA.
  • Helper functions can be provided to a packaging cell by transfecting the cell with one or more nucleic acids encoding various helper elements.
  • a host cell e.g., a packaging cell
  • a host cell can comprise a nucleic acid encoding the helper protein.
  • HEK293 cells were generated by transforming human cells with adenovirus 5 DNA and now express a number of adenoviral genes, including, but not limited to E1 and E3 (see, e.g., Graham et al. (1977) J. Gen. Virol. 36:59-72).
  • a packaging cell is transfected with at least one of the following: i) a plasmid comprising a vector genome comprising a modified GAA transgene (including a signal peptide and NTPT) (e.g., SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:119 and SEQ ID NO:120) and AAV ITRs (e.g., SEQ ID NO:109 and/or SEQ ID NO:109 and/or SEQ ID NO:109 and/or SEQ ID NO:109 and/or SEQ ID NO:109 and/or SEQ ID NO:109 and/or SEQ ID NO:109 and
  • rAAV capsids of the disclosure may be produced by methods known to skilled artisans (see, e.g., WO 2013/063379). An exemplary non-limiting method is described in Grieger, et al. (2015) Molecular Therapy 24(2):287-297, the contents of which are incorporated by reference herein for all purposes.
  • Chimeric AAV capsids can be made by mammalian cell AAV production systems (e.g., those based on 293T or HEK293 cells) and insect cell AAV production systems (e.g., those based on sf9 insect cells and/or those using baculoviral helper vectors).
  • Transfection of HEK293 cells allows for rapid and scalable AAV and rAAV production.
  • a triple transfection method e.g., WO 96/40240
  • a packaging cell such as HEK293, a plasmid encoding an AAV rep and chimeric capsid of the disclosure, a plasmid encoding helper functions (e.g., adenovirus or HSV proteins such as E1 a, E1b, E2a, E4, and VA RNA)
  • a transgene e.g., a therapeutic transgene, a reporter transgene (e.g., green fluorescent protein) and various elements to control expression of the transgene
  • rAAV vectors comprising a chimeric capsid of the disclosure can be produced.
  • a rAAV vector may be purified by methods standard in the art such as by any number of column chromatography methods (e.g., affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography) or cesium chloride gradients. Methods for purifying rAAV vectors are known in the art and include methods described in Clark et al. (1999) Human Gene Therapy 10(6):1031 -1039; Schenpp etal. (2002) Methods Mol. Med. 69:427-443; US Patent No. 6,566,118, WO 98/09657 and WO 2022/097008.
  • rAAV vectors After rAAV vectors have been produced and purified, they can be titered (e.g., the amount of rAAV vector in a sample can be quantified) to prepare compositions for administration to subjects, such as human subjects with a disease. rAAV vector titering can be accomplished using methods know in the art.
  • a rAAV vector of the present disclosure transduces a target cell (e.g., an hepatocyte) and mediates a biological activity.
  • a rAAV vector comprising a transgene encoding a modified GAA polypeptide e.g., including a signal peptide and a NTPT
  • a target cell e.g., a hepatocyte
  • a rAAV vector comprising a transgene encoding a modified GAA polypeptide (e.g., including a signal peptide and a NTPT) transduces a target cell (e.g., a hepatocyte) for expression and secretion of the modified GAA polypeptide which demonstrates or mediates at least one detectable activity selected from the group consisting of:
  • (xvii) demonstrates no significant difference in glycogen levels in diaphragm tissue of GAA knockout mice treated with a NTPT modified GAA as compared to glycogen levels in diaphragm tissue of untreated wild type mice; (xviii) no significant difference in glycogen levels in brain tissue of GAA knockout mice treated with a NTPT modified GAA as compared to glycogen levels in brain tissue of untreated wild type mice;
  • (xix) inhibits, decreases and/or reduces glycogen levels in brain tissue of GAA knockout mice treated with a NTPT modified GAA as compared to glycogen levels in brain tissue of GAA knock out mice treated with wild type GAA;
  • a rAAV vector which transduces a target cell comprises an AAV9 capsid and a GAA transgene comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:119 and SEQ ID NO:120.
  • a rAAV vector which transduces a target cell comprises an AAV9 capsid and a GAA transgene cassette comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:116 and SEQ ID NO:117.
  • a target cell e.g., a hepatocyte
  • GAA transgene cassette comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ
  • a cell e.g., a myocyte
  • a cell takes up a NTPT modified GAA polypeptide comprising an amino acid sequence of SEQ ID NO:69 more efficiently than an otherwise identical cell takes up a wild type GAA polypeptide (SEQ ID NO:58).
  • a cell e.g., a myocyte
  • a cell expressing a modified GAA polypeptide encoded by a nucleic acid sequence of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68 has increased GAA activity level as compared to a cell expressing a modified GAA polypeptide encoded by a nucleic acid sequence of SEQ ID NO:59.
  • serum GAA activity level is sustained for up to 100 days in a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence of SEQ ID NO:73 is administered.
  • a dose dependent increase in serum GAA activity level is demonstrated in a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence of SEQ ID NO:73 is administered.
  • a quadricep tissue GAA activity level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence of SEQ ID NO:73 is administered is significantly increased as compared to a quadricep tissue GAA activity level of an otherwise identical subject with a non-functional GAA gene to whom the rAAV vector is not administered.
  • a quadricep tissue glycogen level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence of SEQ ID NO:73 is administered is significantly decreased as compared to a quadricep tissue glycogen level of an otherwise identical subject with a non-functional GAA gene to whom the rAAV vector is not administered.
  • a quadricep tissue glycogen level of a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence of SEQ ID NO:73 is administered is not significantly different from a quadricep tissue glycogen level of a subject with a wild type GAA gene to whom the rAAV vector is not administered [0399]
  • a heart tissue glycogen level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence of SEQ ID NO:73 is administered is significantly decreased as compared to a heart tissue glycogen level of an otherwise identical subject with a non-functional GAA gene to whom the rAAV vector is not administered.
  • a diaphragm tissue glycogen level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence of SEQ ID NO:73 is administered is significantly decreased as compared to a diaphragm tissue glycogen level of an otherwise identical subject with a non-functional GAA gene to whom the rAAV vector is not administered.
  • a brain tissue glycogen level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide is significantly decreased as compared to a brain tissue glycogen level of an otherwise identical subject with a non-functional GAA gene to whom the rAAV9 vector is not administered.
  • a brain tissue glycogen level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide is significantly decreased as compared to a brain tissue glycogen level of an otherwise identical subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a wild type GAA polypeptide (e.g., SEQ ID NO:88) is administered.
  • a brain tissue glycogen level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide is not significantly different from a brain tissue glycogen level of a subject with a wild type GAA gene to whom the rAAV vector is not administered.
  • a brain tissue GAA activity level of a subject with a non- functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide is significantly increased as compared to the brain tissue GAA activity level of an otherwise identical subject with a non-functional GAA gene to whom the rAAV9 vector is not administered.
  • a brain tissue GAA activity level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (e.g., SEQ ID NO:73, SEQ ID NO:119, SEQ ID NQ:120) is administered is significantly increased as compared to a brain tissue GAA activity level of an otherwise identical subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a wild type GAA polypeptide (e.g., SEQ ID NO:88) is administered.
  • a serum GAA activity level of a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:74) is administered is significantly increased as compared to a serum GAA activity level of an otherwise identical subject with a non-functional GAA gene to whom the rAAV9 vector is not administered.
  • a serum GAA activity level of a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:74) is administered is significantly increased as compared to the serum GAA activity level of a subject with a functional GAA gene to whom the rAAV9 vector is not administered.
  • a serum GAA activity level of a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:74) is administered is significantly increased as compared to a serum GAA activity level of an otherwise identical subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence of SEQ ID NO:87 encoding a NTPT-GAA polypeptide is administered.
  • a serum GAA activity level of a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:73, SEQ ID NO:78, SEQ ID NO:119, SEQ ID NQ:120) is administered is significantly increased as compared to the serum GAA activity level of an otherwise identical subject with a non-functional GAA gene to whom the rAAV9 vector is not administered.
  • a serum GAA activity level of a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:78) is administered is significantly increased as compared to a serum GAA activity level of an otherwise identical subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence of SEQ ID NO:73 and encoding a NTPT-GAA polypeptide is administered.
  • a serum GAA activity level of a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (e.g., SEQ ID NO:74, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:120) is administered is significantly increased as compared to the serum GAA activity level of an otherwise identical subject with a functional GAA gene to whom the rAAV9 vector is not administered.
  • a serum GAA activity level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:79) is administered is significantly increased as compared to a serum GAA activity level of an otherwise identical subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence of SEQ ID NO:74, SEQ ID NQ:80 or SEQ ID NO:81 and encoding a NTPT-GAA polypeptide is administered.
  • a quadricep tissue GAA activity level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78) is administered is significantly increased as compared to a quadricep tissue GAA activity level of an otherwise identical subject with a non-functional GAA gene to whom the rAAV9 vector is not administered.
  • a quadricep tissue GAA activity level of a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77) is administered is not significantly different as compared to a quadricep tissue GAA level of a subject with a functional GAA gene to whom the rAAV9 vector is not administered.
  • a quadricep tissue GAA activity level of a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:78) is administered is significantly increased as compared to a quadricep tissue GAA activity level of an otherwise identical subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence of SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 or SEQ ID NO:77 and encoding a NTPT-GAA polypeptide is administered.
  • a quadricep tissue glycogen level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78) is administered is significantly decreased as compared to a quadricep tissue glycogen level of an otherwise identical subject with a non-functional GAA gene to whom the rAAV9 vector is not administered.
  • a quadricep tissue glycogen level of a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78) is administered is not significantly different as compared to a quadricep tissue glycogen level of a subject with a functional GAA gene to whom the rAAV9 vector is not administered.
  • a diaphragm tissue glycogen level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78) is administered is significantly decreased as compared to a diaphragm glycogen level of an otherwise identical subject with a non-functional GAA gene to whom the rAAV9 vector is not administered.
  • a diaphragm tissue glycogen level of a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide is not significantly different as compared to a diaphragm tissue glycogen level of a subject with a functional GAA gene to whom the rAAV9 vector is not administered.
  • a heart tissue glycogen level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78) is administered is significantly decreased as compared to a heart tissue glycogen level of an otherwise identical subject with a non-functional GAA gene to whom the rAAV9 vector is not administered.
  • a heart tissue glycogen level of a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78) is administered is not significantly different as compared to a heart tissue glycogen level of a subject with a functional GAA gene to whom the rAAV9 vector is not administered.
  • a spinal cord tissue GAA activity level of a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78) is administered is significantly increased as compared to a spinal cord tissue GAA activity level of an otherwise identical subject with a non-functional GAA gene to whom the rAAV9 vector is not administered.
  • a spinal cord tissue GAA activity level of a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78) is administered is not significantly different as compared to a spinal cord tissue GAA activity level of a subject with a functional GAA gene to whom the rAAV9 vector is not administered.
  • a spinal cord tissue glycogen level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78) is administered is significantly decreased as compared to a spinal cord tissue glycogen level of an otherwise identical subject with a non-functional GAA gene to whom the rAAV9 vector is not administered.
  • a spinal cord tissue glycogen level of a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:74, SEQ ID NO:78) is administered is not significantly different as compared to a spinal cord tissue glycogen level of a subject with a functional GAA gene to whom the rAAV9 vector is not administered.
  • a brain tissue GAA activity level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:78) is administered is not significantly different as compared to a brain tissue GAA activity level of a subject with a functional GAA gene to whom the rAAV9 vector is not administered.
  • a brain tissue glycogen level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78) is administered is significantly decreased as compared to a brain tissue glycogen level of an otherwise identical subject with a non-functional GAA gene to whom the rAAV9 vector is not administered.
  • a brain tissue glycogen level of a subject with a non-functional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:74, SEQ ID NO:78) is administered is not significantly different as compared to a brain tissue glycogen level of a subject with a functional GAA gene to whom the rAAV9 vector is not administered.
  • a maximum tetanic torque level of a subject with a nonfunctional GAA gene to whom a rAAV9 vector comprising a nucleic acid sequence encoding a NTPT-GAA polypeptide (SEQ ID NO:77, SEQ ID NO:78) is administered is significantly increased as compared to a maximum tetanic torque level of an otherwise identical subject with a non-functional GAA gene to whom the rAAV9 vector is not administered.
  • a modified nucleic acid such as a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA), as disclosed herein, may be used for gene therapy treatment and/or prevention of a disease, disorder or condition associated with deficiency or dysfunction of a GAA polypeptide (e.g., Pompe disease), and of any other condition and or illness in which an upregulation of a GAA gene may produce a therapeutic benefit or improvement, e.g., a disease, disorder or condition mediated by, or associated with, a decrease in the level or function of a GAA polypeptide compared with the level or function of a GAA polypeptide in an otherwise healthy individual.
  • a disease, disorder or condition associated with deficiency or dysfunction of a GAA polypeptide e.g., Pompe disease
  • a vector genome and/or a rAAV vector comprising a modified nucleic acid encoding a modified GAA may be used for gene therapy treatment and/or prevention of a disease, disorder or condition associated with or caused by deficiency or dysfunction of a GAA enzyme (e.g., Pompe disease), and of any other condition and/or illness in which an upregulation of a GAA enzyme may produce a therapeutic benefit or improvement.
  • methods of the disclosure include use of a rAAV vector, or a pharmaceutical composition thereof, in the treatment of Pompe disease in a subject.
  • methods of the disclosure include use of a rAAV vector (e.g., rAAV9 NTPT -GAA), or pharmaceutical composition thereof, to increase the level of GAA in a subject in need thereof.
  • a modified nucleic encoding a modified GAA (e.g., a NTPT modified GAA), a vector genome comprising a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA) and/or a rAAV vector (e.g., AAV9 NTPT-GAA) comprising a modified nucleic acid encoding a modified GAA of the disclosure, may be used in the preparation of a medicament for use in the treatment and/or prevention of a disease, disorder or condition associated with or caused by deficiency or dysfunction of GAA (e.g., a decreased level of functional GAA enzyme such as in Pompe disease) and of any other condition or illness in which an upregulation of GAA may produce a therapeutic benefit or improvement.
  • a disease, disorder or condition associated with or caused by deficiency or dysfunction of GAA e.g., a decreased level of functional GAA enzyme such as in Pompe disease
  • gene therapy treatment and/or prevention of a disease, disorder or condition associated with deficiency or dysfunction of a GAA enzyme comprises administration of a therapeutically effective amount of a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA), a vector genome comprising a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA) and/or a rAAV vector (e.g., AAV9 NTPT-GAA) comprising a modified nucleic acid encoding GAA of the disclosure to a subject (e.g., a patient) in need of treatment.
  • a modified nucleic acid encoding a modified GAA e.g., a NTPT modified GAA
  • a vector genome comprising a modified nucleic acid encoding a modified GAA
  • a rAAV vector e.g., AAV9 NTPT-GAA
  • Treatment of a subject with a therapeutically effective amount of a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA), a vector genome comprising a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA) and/or a rAAV vector (e.g., AAV9 NTPT-GAA) comprising a modified nucleic acid encoding a modified GAA of the disclosure may alleviate, ameliorate, treat, prevent or reduce the severity of one or more symptoms of Pompe disease as compared to a baseline measurement, such as a measurement in the same individual prior to initiation of treatment described herein, or a measurement in a control individual (or multiple control individuals thereby establishing a level for comparison) in the absence of the treatment described herein.
  • a “control individual” is an individual afflicted with the same form of disease or injury as an individual being treated,
  • treatment of a subject with a therapeutically effective amount of a modified nucleic acid encoding a modified GAA e.g., a NTPT modified GAA
  • a vector genome comprising a modified nucleic acid encoding a modified GAA e.g., a NTPT modified GAA
  • a rAAV vector e.g., AAV9 NTPT-GAA
  • glycogen accumulation in brain tissue, spinal cord tissue, heart tissue, diaphragm tissue, quadricep tissue or a combination thereof is reduced by 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% or by 100% in a subject who is treated as compared to a control individual, or as compared with the same individual prior to treatment.
  • treatment of a subject with a therapeutically effective amount of a modified nucleic acid encoding a modified GAA e.g., a NTPT modified GAA
  • a vector genome comprising a modified nucleic acid encoding a modified GAA e.g., a NTPT modified GAA
  • a rAAV vector e.g., AAV9 NTPT-GAA
  • AAV9 NTPT-GAA may increase a GAA activity level as compared to a GAA activity level in a control individual, or as compared to a GAA activity level in the same individual prior to treatment.
  • a GAA activity level in brain tissue, spinal cord tissue, heart tissue, diaphragm tissue, quadricep tissue, serum or a combination thereof is increased by 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% or by 100% in a subject who is treated as compared to a control individual, or as compared with the same individual prior to treatment.
  • treatment may also alleviate, ameliorate, treat, prevent or reduce the severity of hepatomegaly, myopathy, hypotonia, heart failure, failure to thrive, delay in delayed motor skills, progressive weakness, cardiomegaly, muscle weakness leading to breathing difficulties, muscle weakness affecting the legs and trunk, symptoms affecting the central, peripheral and autonomous nervous system, vascular malformation, musculoskeletal changes, bone changes, oro-gastrointestinal alterations and urinary tract alterations as compared to the same in a control individual, or in a subject prior to treatment.
  • a subject appropriate for treatment includes any subject having, or at risk of, producing an insufficient amount, or having a deficiency of, a functional gene product (protein), or that produces an aberrant, partially functional or non-function gene product (protein, e.g., an enzyme), which can lead to disease.
  • a patient is treated with a vector or pharmaceutical composition of the present disclosure prior to exhibiting any symptoms of a disease, disorder or condition (e.g., Pompe disease).
  • a patient who has been diagnosed as at-risk for a disease, disorder or condition (e.g., Pompe disease) by genetic analysis is treated with a rAAV vector or composition of the present disclosure prior to exhibiting symptoms.
  • a subject to be treated may be mammal, and in particular a subject is a human patient, for example, a patient with Pompe disease.
  • a subject may be in need of treatment because, as a result of one or more mutations in the coding sequence of the GAA gene, the GAA protein has an incorrect amino acid sequence, and thereby has decreased or no function, is expressed in the wrong tissues or at the wrong time, is under expressed or not expressed at all.
  • a modified nucleic acid encoding GAA of the present invention may be administered to enhance, improve or provide production of a functional GAA enzyme which can, in turn, catalyze the breakdown of glycogen to glucose, among other biological functions as discussed elsewhere herein.
  • a target cell of the rAAV vector of the instant invention is a cell, in particular a hepatocyte, that is normally, endogenously capable of expressing the GAA enzyme, such as those of in the liver of a mammal.
  • a pharmaceutical composition for preventing or treating a disease, disorder or condition mediated by or associated with decreased expression and/or activity of GAA, e.g., Pompe disease.
  • a pharmaceutical composition comprises a modified nucleic acid, a recombinant nucleic acid, a viral vector genome, an expression vector, a host cell or a rAAV vector, and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises a therapeutically effective amount of a vector (e.g., viral vector genome, expression vector, rAAV vector) or host cell comprising a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA) which can increase the level of expression and/or the level of activity of GAA in a cell.
  • a vector e.g., viral vector genome, expression vector, rAAV vector
  • host cell comprising a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA) which can increase the level of expression and/or the level of activity of GAA in a cell.
  • a modified GAA e.g., a NTPT modified GAA
  • a pharmaceutical composition comprises a therapeutically effective amount of a vector (e.g., viral vector genome, expression vector, rAAV vector) or host cell (e.g., for ex vivo gene therapy) comprising a modified, nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA) wherein the composition further comprises a pharmaceutically-acceptable carrier, adjuvant, diluent, excipient and/or other medicinal agents.
  • a vector e.g., viral vector genome, expression vector, rAAV vector
  • host cell e.g., for ex vivo gene therapy
  • the composition further comprises a pharmaceutically-acceptable carrier, adjuvant, diluent, excipient and/or other medicinal agents.
  • a pharmaceutically acceptable carrier, adjuvant, diluent, excipient or other medicinal agent is one that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing undesirable biological effects which outweigh the advantageous biological effects of the material.
  • Any suitable pharmaceutically acceptable carrier or excipient can be used in the preparation of a pharmaceutical composition according to the invention (See e.g., Remington The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April 1997).
  • a pharmaceutical composition is typically sterile, pyrogen-free and stable under the conditions of manufacture and storage.
  • a pharmaceutical composition may be formulated as a solution (e.g., water, saline, dextrose solution, buffered solution, or other pharmaceutically sterile fluid), microemulsion, liposome, or other ordered structure suitable to accommodate a high product (e.g., viral vector particles, microparticles or nanoparticles) concentration.
  • a pharmaceutical composition comprising a modified nucleic acid, vector genome comprising the modified nucleic acid, host cell or rAAV vector of the disclosure is formulated in water or a buffered saline solution.
  • a carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • Proper fluidity can be maintained, for example, by use of a coating such as lecithin, by maintenance of a required particle size, in the case of dispersion, and by the use of surfactants.
  • Prolonged adsorption of an injectable composition can be brought about by including, in the composition, an agent which delays absorption, e.g., a monostearate salt and gelatin.
  • a nucleic acid, vector and/or host cell of the disclosure may be administered in a controlled release formulation, for example, in a composition which includes a slow release polymer or other carrier that protects the product against rapid release, including an implant and microencapsulated delivery system.
  • a pharmaceutical composition of the disclosure is a parenteral pharmaceutical composition, including a composition suitable for intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intraarticular, intraparenchymal, intrathecal, intracerebroventricular and/or intracisternal magna administration.
  • a pharmaceutical composition comprising a rAAV vector comprising a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA) is formulated for administration by intravenous injection.
  • a modified nucleic acid encoding a transgene e.g., GAA
  • a vector e.g., vector genome, rAAV vector
  • a target cell of a vector of the present disclosure includes cells of the liver, preferably a hepatocyte.
  • a vector can be administered in addition to, and as an adjunct to, the standard of care treatment. That is, the vector can be co-administered with another agent, compound, drug, treatment or therapeutic regimen, either simultaneously, contemporaneously, or at a determined dosing interval as would be determined by one skilled in the art using routine methods. Uses disclosed herein include administration of a rAAV vector of the disclosure at the same time, in addition to and/or on a dosing schedule concurrent with, the standard of care for Pompe disease as known in the art.
  • a combination composition includes one or more immunosuppressive agents.
  • a combination composition includes a rAAV vector comprising a transgene (e.g., a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA)) and one or more immunosuppressive agents.
  • a method includes administering or delivering a rAAV vector comprising a transgene (e.g., a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA)) to a subject and administering an immunosuppressive agent to the subject either prophylactically prior to administration of the vector, or after administration of the vector (i.e., either before or after symptoms of a response against the vector and/or the protein provided thereby are evident).
  • a transgene e.g., a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA)
  • a rAAV of the invention can be co-administered with empty capsids (i.e., a virus capsid that does not contain a nucleic acid molecule or vector genome) comprising the same, or a different, capsid protein as a rAAV vector comprising a modified nucleic acid (e.g., encoding a modified GAA (e.g., a NTPT modified GAA)).
  • empty capsids i.e., a virus capsid that does not contain a nucleic acid molecule or vector genome
  • a modified nucleic acid e.g., encoding a modified GAA (e.g., a NTPT modified GAA)
  • an immune response e.g., a neutralizing response
  • an empty capsid may serve as an immune decoy allowing a rAAV vector comprising a modified nucleic acid (e.g., encoding a modified GAA (e.g., a NTPT modified GAA)) to avoid a neutralizing antibody (Nab) immune response as discussed in, e.g., WO 2015/013313.
  • a modified nucleic acid e.g., encoding a modified GAA (e.g., a NTPT modified GAA)
  • Nab neutralizing antibody
  • a vector of the disclosure e.g., a rAAV vector comprising a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA)
  • a vector of the disclosure is administered systemically.
  • exemplary methods of systemic administration include, but are not limited to, intravenous (e.g., portal vein), intraarterial (e.g., femoral artery, hepatic artery), intravascular, subcutaneous, intradermal, intraperitoneal, transmucosal, intrapulmonary, intralymphatic and intramuscular administration, and the like, as well as direct tissue or organ injection.
  • systemic administration can deliver a modified nucleic acid (e.g., a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA)) to all tissues.
  • direct tissue or organ administration includes administration to the liver.
  • direct tissue or organ administration includes administration to areas directly affected by GAA deficiency (e.g., brain and/or central nervous system).
  • vectors of the disclosure, and pharmaceutical compositions thereof are administered to the brain parenchyma (i.e., by intraparenchymal administration), to the spinal canal or the subarachnoid space so that it reaches the cerebrospinal fluid (CSF) (i.e., by intrathecal administration), to a ventricle of the brain (i.e., by intracerebroventricular administration) and/or to the cisterna magna of the brain (i.e., by intracisternal magna administration).
  • CSF cerebrospinal fluid
  • a vector of the disclosure is administered by at least two routes.
  • a vector is administered systemically and also directly into the brain.
  • a vector is administered systemically and also directly into the liver. If administered via at least two routes, the administration of a vector can be, but need not be, simultaneous or contemporaneous. Instead, administration via different routes can be performed separately with an interval of time between each administration.
  • a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA), a vector genome comprising a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA) and/or a rAAV vector comprising a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA) of the disclosure, may be used for transduction of a cell ex vivo or for administration directly to a subject (e.g., directly to the liver of a patient with Pompe disease).
  • a transduced cell e.g., a host cell
  • a disease, disorder or condition e.g., cell therapy for Pompe disease
  • a rAAV vector comprising a modified therapeutic nucleic acid e.g., encoding a modified GAA (e.g., a NTPT modified GAA)
  • a modified GAA e.g., a NTPT modified GAA
  • a biolog ically-effective amount of a vector is an amount that is sufficient to result in transduction and expression of a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA) (i.e., a transgene) in a target cell (e.g., a hepatocyte).
  • a modified GAA e.g., a NTPT modified GAA
  • a target cell e.g., a hepatocyte
  • the disclosure includes a method of increasing the level and/or activity of GAA in a cell by administering to a cell (/n vivo, in vitro or ex vivo) a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA), either alone or in a vector (including a plasmid, a virus vector, a nanoparticle, a liposome, or any known method for providing a nucleic acid to a cell).
  • a modified nucleic acid encoding a modified GAA e.g., a NTPT modified GAA
  • a vector including a plasmid, a virus vector, a nanoparticle, a liposome, or any known method for providing a nucleic acid to a cell.
  • the dosage amount of a rAAV vector depends upon, e.g., the mode of administration, disease or condition to be treated, the stage and/or aggressiveness of the disease, individual subject's condition (age, sex, weight, etc.), particular viral vector, stability of protein to be expressed, host immune response to the vector, and/or gene to be delivered.
  • a dose ranges from at least 1 x 10 8 vector genomes (vg) per kilogram (kg) total body weight of the subject, or more, e.g., 1 x 10 9 vg/kg, 1 x 10 1 ° vg/kg, 1 x 10 11 vg/kg, 1 x 10 12 vg/kg, 1 x 10 13 vg/kg, 1 x 10 14 vg/kg, 1 x 10 15 vg/kg or more to achieve a therapeutic effect.
  • vg vector genomes
  • a dose may range from 1 x 10 9 vg/kg to 1 x 10 15 vg/kg, from 1 x 1 O 10 vg/kg to 1 x 10 14 vg/kg, from 1 x 10 11 vg/kg to 1 x 10 13 vg/kg or from 1 x 10 11 vg/kg to 1 x 10 12 vg/kg total body weight of the subject.
  • a dose ranges from at least 1 x 10 8 vector genomes (vg) per kilogram (kg) lean body mass of the subject, or more, e.g., 1 x 10 9 vg/kg, 1 x 1 O 10 vg/kg, 1 x 10 11 vg/kg, 1 x 10 12 vg/kg, 1 x 10 13 vg/kg, 1 x 10 14 vg/kg, 1 x 10 15 vg/kg or more to achieve a therapeutic effect.
  • vg vector genomes
  • a dose may range from 1 x 10 9 vg/kg to 1 x 10 15 vg/kg, from 1 x 1 O 10 vg/kg to 1 x 10 14 vg/kg, from 1 x 10 11 vg/kg to 1 x 10 13 vg/kg or from 1 x 10 11 vg/kg to 1 x 10 12 vg/kg lean body mass of the subject.
  • a subject may be administered a single dose of a rAAV vector.
  • a subject may be administered more than one dose of a rAAV vector.
  • an rAAV vector is administered two times, three times, four times, five times or more.
  • the period of time between doses may vary.
  • a subject may receive a first dose of a rAAV vector and a second dose of the rAAV vector 1 year later.
  • the time period between doses may be 1 week, 1 month, 6 months, 9 months, 1 year, 1.5 years, 2 years, 3 year, 5 year, 10 years or more.
  • a modified nucleic acid encoding a modified GAA may be administered as a component of a DNA molecule (e.g., a recombinant nucleic acid) having a regulatory element (e.g., a promoter) appropriate for expression in a target cell (e.g., hepatocyte).
  • the modified nucleic acid encoding a modified GAA may be administered as a component of a plasmid or a viral vector, such as a rAAV vector.
  • a rAAV vector may be administered in vivo by delivery of the vector intravenously to a patient (e.g., a Pompe patient) in need of treatment.
  • a rAAV vector may be administered to a patient ex vivo by administration of the vector in vitro to a cell from a donor patient in need of treatment, followed by introduction of the transduced cell back into the donor (e.g., cell therapy).
  • the present disclosure includes a method of administration that results in a level of mRNA encoding a modified GAA (e.g., a NTPT modified GAA), a level of GAA protein expression, and/or a level of GAA activity that is detectably greater than the level of GAA expression (mRNA and/or protein) or GAA activity in an otherwise identical cell that is not administered a modified nucleic acid (e.g., a modified nucleic acid encoding GAA).
  • a modified GAA e.g., a NTPT modified GAA
  • a level of GAA protein expression e.g., a level of GAA protein expression
  • a level of GAA activity that is detectably greater than the level of GAA expression (mRNA and/or protein) or GAA activity in an otherwise identical cell that is not administered a modified nucleic acid (e.g., a modified nucleic acid encoding GAA).
  • the present disclosure includes a method of administration that results in a level of mRNA encoding a functional a modified GAA (e.g., a NTPT modified GAA), and/or a level of functional (e.g., biologically active) GAA protein expression, that is detectably greater than the level of functional GAA (mRNA and/or protein) present in an otherwise identical cell that is not administered the modified nucleic acid (e.g., a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA)). That is, the present invention includes method of increasing the level of functional GAA in a cell where the cell produces a normal level of GAA but the GAA protein lacks activity or demonstrates decreased activity compared with normal wild type GAA.
  • a level of functional GAA e.g., biologically active
  • kits typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo or ex vivo, of the components therein.
  • a kit can contain a collection of such components, e.g., a modified nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA), a recombinant nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA), a vector genome encoding a modified GAA (e.g., a NTPT modified GAA), a rAAV vector comprising a nucleic acid encoding a modified GAA (e.g., a NTPT modified GAA), and optionally a second active agent such as a compound, therapeutic agent, drug or composition.
  • a second active agent may be an immunosuppressive agent.
  • a kit refers to a physical structure that contains one or more components of the kit.
  • Packaging material can maintain the components in a sterile manner and can be made of material commonly used for such purposes (e.g., paper, glass, plastic, foil, ampules, vials, tubes, etc.).
  • a label or insert can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredients(s) including mechanism of action, pharmacokinetics and pharmacodynamics.
  • a label or insert can include information identifying manufacture, lot numbers, manufacture location and date, expiration dates.
  • a label or insert can include information on a disease (e.g., Pompe disease) for which a kit component may be used.
  • a label or insert can include instructions for a clinician or subject for using one or more of the kit components in a method, use or treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency of duration and instructions for practicing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimens described herein.
  • a label or insert can include information on potential adverse side effects, complications or reaction, such as a warning to a subject or clinician regarding situations where it would not be appropriate to use a particular composition.
  • An isolated nucleic acid encoding acid alpha-glucosidase comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55.
  • GAA acid alpha-glucosidase
  • An isolated nucleic acid encoding acid alpha-glucosidase comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence of SEQ ID NO:49 or SEQ ID NQ:50.
  • An isolated nucleic acid encoding acid alpha-glucosidase comprising a nucleic acid sequence comprising or consisting of a sequence of SEQ ID NO:49 or SEQ ID NQ:50.
  • GAA acid alpha-glucosidase
  • E6 An isolated nucleic acid wherein the nucleic acid encodes a GAA polypeptide comprising or consisting of an amino acid sequence of SEQ ID NO:57.
  • a modified nucleic acid encoding acid alpha-glucosidase comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55.
  • GAA acid alpha-glucosidase
  • a modified nucleic acid encoding acid alpha-glucosidase comprising a nucleic acid sequence comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55.
  • a modified nucleic acid encoding acid alpha-glucosidase comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence of SEQ ID NO:49 or SEQ ID NQ:50.
  • a modified nucleic acid encoding acid alpha-glucosidase comprising a nucleic acid sequence comprising or consisting of a sequence of SEQ ID NO:49 or SEQ ID NQ:50.
  • a modified nucleic acid encoding acid alpha-glucosidase comprising or consisting of a codon optimized nucleic acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55.
  • a modified nucleic acid encoding acid alpha-glucosidase comprising or consisting of a codon optimized and CpG modified nucleic acid sequence selected from the group consisting of SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55.
  • a modified nucleic acid wherein the nucleic acid encodes a GAA polypeptide at least 80%, at least 85%, at least 90%, at least 91%, at least, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:57.
  • E14 A modified nucleic acid wherein the nucleic acid encodes a GAA polypeptide comprising or consisting of an amino acid sequence of SEQ ID NO:57.
  • E15 The modified nucleic acid of any one of E7-E14, wherein a cell comprising the modified nucleic acid exhibits increased GAA polypeptide expression.
  • E16 The modified nucleic acid of E15, wherein the increased GAA polypeptide expression is at a detectably greater level in a cell as compared to the level of expression of a GAA polypeptide in an otherwise identical cell comprising a wild type GAA nucleic acid, or an otherwise identical cell comprising a mutant GAA nucleic acid.
  • E17 The modified nucleic acid of E15, wherein the increased GAA polypeptide expression is at a detectably greater level in a cell as compared to the level of expression of a GAA polypeptide in an otherwise identical cell comprising a wild type GAA nucleic acid, or an otherwise identical cell comprising a mutant GAA nucleic acid.
  • the modified nucleic acid of E15 or E16 wherein the level of GAA polypeptide expression in a cell comprising the modified nucleic acid is increased by 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 120%, at least 140%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500% or more as compared to a level of GAA polypeptide expression in an otherwise identical cell comprising a nucleic acid encoding a wild type GAA.
  • E18 The modified nucleic acid of any one of E15-E17, wherein the cell is a hepatocyte.
  • E19 The modified nucleic acid of any one of E15-E18, wherein the GAA polypeptide is secreted from the cell.
  • E20 A modified nucleic acid of any one of E7-E19, further comprising a nucleic acid sequence encoding a signal peptide.
  • E21 The modified nucleic acid of E20, wherein the modified nucleic acid encodes a GAA polypeptide operably linked to a signal peptide.
  • E22 The modified nucleic acid of E20 or E21 , wherein the signal peptide is encoded by a nucleic acid at least 80%, at least 85%, at least 90%, at least 91%, at least, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22 and SEQ ID NO:23.
  • E23 The modified nucleic acid of any one of E20-E22, wherein the signal peptide is encoded by a nucleic acid comprising of consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22 and SEQ ID NO:23.
  • E24 The modified nucleic acid of any one of E20-E23, wherein the signal peptide is encoded by a nucleic acid at least 80%, at least 85%, at least 90%, at least 91%, at least, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence of SEQ ID NO:18.
  • E25 The modified nucleic acid of any one of E20-E24, wherein the signal peptide is encoded by a nucleic acid comprising of consisting of the nucleic acid sequence of SEQ ID NO:18.
  • E26 The modified nucleic acid of any one of E20-E25, wherein the nucleic acid encodes a signal peptide comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29.
  • E27 The modified nucleic acid of any one of E20-E26, wherein the nucleic acid encodes a signal peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29.
  • E28 The modified nucleic acid of any one of E20-E27, wherein the nucleic acid encodes a signal peptide comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:26.
  • E29 The modified nucleic acid of any one of E20-E28, wherein the nucleic acid encodes a signal peptide comprising or consisting of the amino acid sequence of SEQ ID NO:26.
  • E30 The modified nucleic acid of any one of E20-E29, wherein the nucleic acid sequence encoding the signal peptide is codon-optimized, CpG modified or both
  • E31 The modified nucleic acid of any one of E20-E29, wherein the signal peptide is a heterologous signal peptide.
  • E32 The modified nucleic acid of any one of E20-E31 , wherein the signal peptide is a IGF2 signal peptide or a variant thereof.
  • E33 The modified nucleic acid of any one of E20-E32, wherein a cell comprising the modified nucleic acid expresses the GAA polypeptide encoded by the modified nucleic acid and exhibits increased secretion of the expressed GAA polypeptide from the cell.
  • E34 The modified nucleic acid of E33, wherein the cell is a hepatocyte.
  • E35 The modified nucleic acid of any one of E20-E34, wherein the increased secretion of the expressed GAA polypeptide is at a detectably greater level in the cell as compared to the level of secretion of an expressed GAA polypeptide from an otherwise identical cell comprising a wild type GAA nucleic acid, or an otherwise identical cell comprising a mutant GAA nucleic acid.
  • E36 The modified nucleic acid of any one of E20-E35, wherein the increased secretion of the expressed GAA polypeptide is increased by 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 120%, at least 140%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500% or more.
  • E37 The modified nucleic acid of any one of E20-E36, wherein the modified nucleic acid comprises or consists of i) a nucleic acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22 and SEQ ID NO:23 encoding a signal peptide and ii) a nucleic acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55 encoding an operably linked GAA polypeptide for expression and secretion from a cell and.
  • E39 The modified nucleic acid of any one of E20-E38, comprising the nucleic acid sequence of SEQ ID NO:18 operably linked to the nucleic acid sequence of SEQ ID NO:49.
  • E40 The modified nucleic acid of any one of E20-E38, comprising the nucleic acid sequence of SEQ ID NO:18 operably linked to the nucleic acid sequence of SEQ ID NQ:50.
  • E41 The modified nucleic acid of E39 or E40, comprising a nucleic acid encoding the amino acid sequence of SEQ ID NO:26 operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO:57.
  • a signal peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29.
  • a signal peptide encoded by a nucleic acid sequence comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22 and SEQ ID NO:23.
  • NTPT amino-terminal peptide tag
  • E45 The modified nucleic acid of E44, wherein the modified nucleic acid encodes a NTPT operably linked to a GAA polypeptide and a signal peptide.
  • E46 The modified nucleic acid of E44 or E45, wherein the NTPT is encoded by a nucleic acid at least 80%, at least 85%, at least 90%, at least 91%, at least, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38.
  • E47 The modified nucleic acid of any one of E44-E46, wherein the NTPT is encoded by a nucleic acid comprising of consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38.
  • E48 The modified nucleic acid of any one of E44-E47, wherein the NTPT is encoded by a nucleic acid at least 80%, at least 85%, at least 90%, at least 91%, at least, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:31 , SEQ ID NO:34 and SEQ ID NO:35.
  • E49 The modified nucleic acid of any one of E44-E48, wherein the NTPT is encoded by a nucleic acid comprising of consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:31 , SEQ ID NO:34 and SEQ ID NO:35.
  • E50 The modified nucleic acid of any one of E44-E49, wherein the nucleic acid encodes a NTPT comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NQ:40, SEQ ID NO:41 and SEQ ID NO:42.
  • E51 The modified nucleic acid of any one of E44-E50, wherein the nucleic acid encodes a NTPT comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NQ:40, SEQ ID NO:41 and SEQ ID NO:42.
  • E52 The modified nucleic acid of any one of E44-E51 , wherein the nucleic acid encodes a NTPT comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:39 or SEQ ID NO:42.
  • E53 The modified nucleic acid of any one of E44-E52, wherein the nucleic acid encodes a NTPT comprising or consisting of an amino acid sequence of SEQ ID NO:39 or SEQ ID NO:42.
  • E54 The modified nucleic acid of any one of E44-E53, wherein the nucleic acid sequence encoding the NTPT is codon-optimized, CpG modified or both.
  • E55 The modified nucleic acid of any one of E44-E54, wherein the modified nucleic acid encodes a polypeptide comprising a GAA polypeptide and an operably linked NTPT which exhibits increased uptake by a target cell or tissue as compared to the level of uptake of a GAA polypeptide encoded by a reference nucleic acid (e.g., a wild type gene, a mutant gene) in an otherwise identical cell.
  • a reference nucleic acid e.g., a wild type gene, a mutant gene
  • E56 The modified nucleic acid of E55, wherein the target cell is a cell from a tissue selected from the group consisting of skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord and a combination thereof.
  • E57 The modified nucleic acid of E55, wherein the target tissue is selected from the group consisting of skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord and a combination thereof.
  • E58 The modified nucleic acid of any one of E55-E57, wherein the reference nucleic acid comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NO:56, SEQ ID NO:113, amino acids 343-3198 of SEQ ID NO:113, amino acids 415-3198 of SEQ ID NO:113 and amino acids 550-3198 of SEQ ID NO:113.
  • E59 The modified nucleic acid of any one of E55-E58, wherein the increased uptake by a target cell or tissue is an increase of 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 120%, at least 140%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500% or more.
  • E60 The modified nucleic acid of any one of E45-E59, wherein the GAA polypeptide is operably linked to the NTPT by a peptide bond.
  • E61 The modified nucleic acid of any one of E45-E60, wherein the GAA polypeptide is operably linked to the NTPT by a linker.
  • E62 The modified nucleic acid of E61 , wherein the linker comprises or consists of the amino acid sequence GAP or GGGGS (SEQ ID NO:48).
  • E63 The modified nucleic acid of E62, wherein the linker comprises two or more consecutive units of the amino acid sequence GAP or GGGGS.
  • E64 The modified nucleic acid of E62, wherein the GAP linker is encoded by a nucleic acid sequence selected from the group consisting of L1 , L2 and L3.
  • E65 The modified nucleic acid of any one of E44-E64, wherein the NTPT modulates binding of an operably linked polypeptide to a cellular receptor.
  • E66 The modified nucleic acid of any one of E44-E65, wherein the NTPT is an IGF2 polypeptide or a modified IGF2 polypeptide (e.g., SEQ ID NO: 39, SEQ ID NO:40, SEQ ID NO:41 , SEQ ID NO:42) and the polypeptide is a GAA polypeptide (e.g., SEQ ID NO:57).
  • the NTPT is an IGF2 polypeptide or a modified IGF2 polypeptide (e.g., SEQ ID NO: 39, SEQ ID NO:40, SEQ ID NO:41 , SEQ ID NO:42) and the polypeptide is a GAA polypeptide (e.g., SEQ ID NO:57).
  • E67 The modified nucleic acid of any one of E44-E66, comprising i) a nucleic acid sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38 and encoding a NTPT, and ii) a nucleic acid at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting
  • E68 The modified nucleic acid of any one of E44-E67, encoding i) a NTPT comprising an amino acid sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NQ:40, SEQ ID NO:41 , and SEQ ID NO:42, and ii) an operably linked GAA polypeptide comprising an amino acid sequence of SEQ ID NO:57.
  • E69 The modified nucleic acid of any one of E44-E68, comprising i) a nucleic acid comprising or consisting of a nucleic acid sequence of SEQ ID NO:31 and encoding a NTPT, and ii) a nucleic acid comprising or consisting of a nucleic acid sequence of SEQ ID NO:49 and encoding an operably linked GAA polypeptide.
  • E70 The modified nucleic acid of E69, encoding i) a NTPT comprising or consisting of an amino acid sequence of SEQ ID NO:39 and ii) an operably linked GAA polypeptide comprising or consisting of an amino acid sequence of SEQ ID NO:57.
  • E71 The modified nucleic acid of any one of E44-E68, comprising i) a nucleic acid comprising or consisting of a nucleic acid sequence of SEQ ID NO:34 and encoding a NTPT, and ii) a nucleic acid comprising or consisting of a nucleic acid sequence of SEQ ID NO:49 and encoding an operably linked GAA polypeptide.
  • E72 The modified nucleic acid of E71 , encoding i) a NTPT comprising or consisting of an amino acid sequence of SEQ ID NO:42 and ii) an operably linked GAA polypeptide comprising or consisting of an amino acid sequence of SEQ ID NO:57.
  • E73 The modified nucleic acid of any one of E44-E68, comprising i) a nucleic acid comprising or consisting of a nucleic acid sequence of SEQ ID NO:35 and encoding a NTPT, and ii) a nucleic acid comprising or consisting of a nucleic acid sequence of SEQ ID NQ:50 and encoding an operably linked GAA polypeptide.
  • E74 The modified nucleic acid of any one of E44-E73, wherein the NTPT increases the uptake of the operably linked GAA polypeptide by a target cell or tissue as compared to the level of uptake of a GAA polypeptide encoded by a reference nucleic acid (e.g., a wild type gene, a mutant gene) in an otherwise identical cell.
  • a reference nucleic acid e.g., a wild type gene, a mutant gene
  • E75 The modified nucleic acid of E74, wherein the target cell is a cell from a tissue selected from the group consisting of skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord and a combination thereof.
  • E76 The modified nucleic acid of E74, wherein the target tissue is selected from the group consisting of skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord and a combination thereof.
  • E77 The modified nucleic acid of any one of E74-E76, wherein the reference nucleic acid comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NO:56, SEQ ID NO:113, amino acids 343-3198 of SEQ ID NO:113, amino acids 415-3198 of SEQ ID NO:113 and amino acids 550-3198 of SEQ ID NO:113.
  • E78 The modified nucleic acid of any one of E74-E77, wherein the increased uptake by a target cell or tissue is an increase of 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 120%, at least 140%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500% or more.
  • a modified nucleic acid comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NQ:60, SEQ ID NO:61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 and SEQ ID NO:68, wherein the nucleic acid encodes a polypeptide comprising a NTPT, a linker and a GAA polypeptide.
  • a modified nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NQ:60, SEQ ID NO:61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 and SEQ ID NO:68, wherein the nucleic acid encodes a polypeptide comprising a NTPT, a linker and a GAA polypeptide.
  • a modified nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NO:62 and SEQ ID NO:63, wherein the nucleic acid encodes a polypeptide comprising a NTPT, a linker and a GAA polypeptide.
  • E84 The modified nucleic acid of any one of E79-E83, wherein a level of uptake of the polypeptide by a target cell or tissue is increased as compared to a level of uptake of a GAA polypeptide encoded by a reference nucleic acid (e.g., a wild type gene, mutant gene) in an otherwise identical cell or tissue.
  • a reference nucleic acid e.g., a wild type gene, mutant gene
  • E86 The modified nucleic acid of E84, wherein the target tissue is selected from the group consisting of skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord and a combination thereof.
  • E87 The modified nucleic acid of any one of E84-E86, wherein the reference nucleic acid comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NO:56, SEQ ID NO:113, amino acids 343-3198 of SEQ ID NO:113, amino acids 415-3198 of SEQ ID NO:113 and amino acids 550-3198 of SEQ ID NO:113.
  • E88 The modified nucleic acid of any one of E84-E7, wherein the increased uptake by a target cell or tissue is an increase of 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 120%, at least 140%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500% or more.
  • a modified nucleic acid comprising a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:119 and SEQ ID NO:120, wherein the nucleic acid encodes a polypeptide comprising a signal peptide, a NTPT, a linker and a GAA polypeptide
  • a modified nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:119 and SEQ ID NQ:120, wherein the nucleic acid encodes a polypeptide comprising a signal peptide, a NTPT, a linker and a GAA polypeptide.
  • a modified nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:79 and SEQ ID NQ:120, wherein the nucleic acid encodes a polypeptide comprising a signal peptide, a NTPT, a linker and a GAA polypeptide.
  • E92 A modified nucleic acid encoding a polypeptide comprising a signal peptide, a NTPT, a linker and GAA polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:89, SEQ ID NQ:90, SEQ ID NO:91 , SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:121.
  • E95 The modified nucleic acid of any one of E89-E94, wherein the level of uptake of the polypeptide by a target cell or tissue is increased as compared to the level of uptake of a GAA polypeptide encoded by a reference nucleic acid (e.g., a wild type gene, a mutant gene) in an otherwise identical cell.
  • a reference nucleic acid e.g., a wild type gene, a mutant gene
  • E96 The modified nucleic acid of E95, wherein the target cell is a cell from a tissue selected from the group consisting of skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord and a combination thereof.
  • E97 The modified nucleic acid of E95, wherein the target tissue is selected from the group consisting of skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord and a combination thereof.
  • E98 The modified nucleic acid of any one of E95-E97, wherein the reference nucleic acid comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NO:56, SEQ ID NO:113, amino acids 343-3198 of SEQ ID NO:113, amino acids 415-3198 of SEQ ID NO:113 and amino acids 550-3198 of SEQ ID NO:113.
  • E99 The modified nucleic acid of any one of E95-E98, wherein the increased uptake by a target cell or tissue is an increase of 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 120%, at least 140%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500% or more.
  • a NTPT, a CTPT or both comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NQ:40, SEQ ID NO:41 and SEQ ID NO:42.
  • E102 The NTPT, CTPT or both of E100 or E101 , wherein the NTPT, CTPT or both are operably linked to a polypeptide.
  • E103 The NTPT, CTPT or both of E102, wherein the polypeptide is a GAA polypeptide.
  • E104 The NTPT, CTPT or both of E103, wherein the GAA polypeptide comprises or consists of an amino acid of SEQ ID NO:57.
  • E105 The NTPT, CTP or both of E103, wherein the GAA polypeptide is encoded by a nucleic acid comprising or consisting of a nucleic acid selected from the group consisting of SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55.
  • E106 The NTPT, CTPT or both of any one of E100-E105, wherein the NTPT, CTPT or both bind a cellular receptor to increase uptake of an operably linked polypeptide by a cell or tissue as compared to uptake of a polypeptide without either an operably linked NTPT or CTPT by an otherwise identical cell or tissue.
  • E107 The NTPT, CTPT or both of E106, wherein the cell is a cell of skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord or a combination thereof.
  • E108 The NTPT, CTPT or both of E106, wherein the tissue is skeletal muscle, cardiac muscle, diaphragm, brain, spinal cord or a combination thereof
  • E109 The NTPT, CTPT or both of any one of E103-E105, wherein the NTPT, CTPT or both facilitate transfer of the operably linked GAA across the blood-brain-barrier.
  • E110 The NTPT, CTPT or both of E109, wherein the transfer of the operably linked polypeptide across the blood-brain-barrier results in increased GAA activity in the brain, spinal cord or both.
  • E111 The NTPT, CTPT or both of E110, wherein the increased GAA activity in the brain, spinal cord or both results in decreased glycogen levels in the brain, spinal cord or both.
  • E112. The NTPT, CTPT or both of E100-E105, wherein the NTPT, a CTPT or both bind a cellular receptor and activate an intracellular signaling pathway.
  • E113 The NTPT, CTPT or both of E112, wherein the cellular receptor is any one of an IGF2 receptor, an IGF1 receptor and an insulin receptor.
  • E114 The NTPT, CTPT or both of E112, wherein the cellular receptor is an IGF1 receptor and the intracellular signally pathway stimulates cellular proliferation, blood glucose depletion or both.
  • E115 The NTPT, CTPT or both of any one of E100-E105, wherein the NTPT, CTPT or both exhibit a decreased binding affinity to a cellular IGF1 receptor, an insulin receptor or both as compared to the binding affinity of an wild type IGF2 polypeptide to a cellular IGF1 receptor, an insulin receptor or both on an otherwise identical cell.
  • E116 The NTPT, CTPT or both of E115, wherein the NTPT, CTPT or both maintain their binding affinity to an IGF2 receptor as compared to a binding affinity of a wild type IGF2 polypeptide to a IGF2 receptor on an otherwise identical cell.
  • E117 The NTPT, CTPT or both of E115 or E116, wherein the NTPT, CTPT or both are operably linked to a GAA polypeptide and wherein the wild type IGF2 polypeptide is operably linked to GAA polypeptide.
  • a recombinant nucleic acid comprising a modified nucleic acid encoding a polypeptide comprising a signal peptide, a NTPT, a linker and an acid alpha-glucosidase (GAA) polypeptide, the nucleic acid comprising a nucleic acid sequence at least about 80%, about 85%, about 90%, about 91%, about, 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:
  • a recombinant nucleic acid comprising a modified nucleic acid encoding a polypeptide comprising a signal peptide, a NTPT, a linker and an acid alpha-glucosidase (GAA) polypeptide, the nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:119 and SEQ ID NQ:120.
  • GAA acid alpha-glucosidase
  • a recombinant nucleic acid comprising a modified nucleic acid encoding a polypeptide comprising a signal peptide, a NTPT, a linker and an acid alpha-glucosidase (GAA) polypeptide, the nucleic acid comprising a nucleic acid sequence at least about 80%, about 85%, about 90%, about 91%, about, 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:79 and SEQ ID NQ:120.
  • GAA acid alpha-glucosidase
  • a recombinant nucleic acid comprising a modified nucleic acid encoding a polypeptide comprising a signal peptide, a NTPT, a linker and an acid alpha-glucosidase (GAA) polypeptide, the nucleic acid comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:79 and SEQ ID NQ:120.
  • GAA acid alpha-glucosidase
  • E122 The recombinant nucleic acid of any one of E118-E121 , further comprising at least one element selected from the group consisting of an enhancer, a promoter, an intron, and a polyadenylation (polyA) signal sequence.
  • polyA polyadenylation
  • E123 The recombinant nucleic acid of E122, wherein the promoter comprises a nucleic acid sequence at least about 80%, about 85%, about 90%, about 91%, about, 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identical to the nucleic acid sequence of SEQ ID NQ:106.
  • E124 The recombinant nucleic acid of E122 or E123, wherein the promoter comprises or consists of the nucleic acid sequence of SEQ ID NQ:106.
  • E125 The recombinant nucleic acid of any one of E122-E124, wherein the enhancer comprises a nucleic acid sequence at least about 80%, about 85%, about 90%, about 91%, about, 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identical to the nucleic acid sequence of SEQ ID NQ:105.
  • E126 The recombinant nucleic acid of any one of E122-E125, wherein the enhancer comprises or consists of the nucleic acid sequence of SEQ ID NO:105.
  • E127 The recombinant nucleic acid of any one of E122-E126, wherein the intron comprises a nucleic acid sequence at least about 80%, about 85%, about 90%, about 91%, about, 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identical to the nucleic acid sequence of SEQ ID NQ:107.
  • E128 The recombinant nucleic acid of any one of E122-E127, wherein the intron comprises or consists of the nucleic acid sequence of SEQ ID NQ:107.
  • E129 The recombinant nucleic acid of any one of E122-E128, wherein the polyA sequence comprises a nucleic acid sequence at least about 80%, about 85%, about 90%, about 91%, about, 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identical to the nucleic acid sequence of SEQ ID NO:108.
  • E130 The recombinant nucleic acid of any one of E122-E129, wherein the polyA sequence comprises or consists of the nucleic acid sequence of SEQ ID NO:108.
  • E131 The recombinant nucleic acid of any one of E122-E130, wherein the promoter is operably linked to the modified nucleic acid.
  • E132 The recombinant nucleic acid of any one of E122-E131 , wherein the enhancer is operably linked to the modified nucleic acid.
  • E133 The recombinant nucleic of any one of E118-E132, further comprising at least one element selected from the group consisting of an ApoE enhancer, a hAAT promoter, a chimeric intron, and a bovine grown hormone (BGH) polyA.
  • an ApoE enhancer a hAAT promoter
  • a chimeric intron a bovine grown hormone (BGH) polyA.
  • BGH bovine grown hormone
  • E134 The recombinant nucleic of any one of E118-E133, further comprising a least one element selected from the group consisting of an ApoE enhancer comprising the nucleic acid sequence of SEQ ID NQ:105, a hAAT promoter comprising the nucleic acid sequence of SEQ ID NQ:106, a chimeric intron comprising the nucleic acid sequence of SEQ ID NQ:107 and a BGH polyA comprising the nucleic acid sequence of SEQ ID NQ:108.
  • an ApoE enhancer comprising the nucleic acid sequence of SEQ ID NQ:105
  • a hAAT promoter comprising the nucleic acid sequence of SEQ ID NQ:106
  • a chimeric intron comprising the nucleic acid sequence of SEQ ID NQ:107
  • BGH polyA comprising the nucleic acid sequence of SEQ ID NQ:108.
  • a vector genome comprising a modified nucleic acid of any one of E7-E41 or E44-E99, a recombinant nucleic acid of any one of E118-E134 wherein the vector genome further comprises at least one AAV ITR sequence comprising a nucleic acid sequence at least about 80%, about 85%, about 90%, about 91%, about, 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identical to the nucleic acid sequence of SEQ ID NQ:109 or SEQ ID NO:110.
  • E136 The vector genome of E135, wherein the at least one AAV ITR sequence comprises or consists of the nucleic acid sequence of SEQ ID NO:109 or SEQ I D : 110.
  • E137 The vector genome of E135 or E136, comprising two AAV2 ITR sequences flanking a nucleic acid sequence encoding a polypeptide comprising a signal peptide, a NTPT, a linker and an acid alpha-glucosidase (GAA) polypeptide with a hAAT promoter upstream of the sequence encoding the signal peptide.
  • GAA acid alpha-glucosidase
  • E138 The vector genome of E137, wherein the sequence encoding the polypeptide comprises the nucleic acid sequence of SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:79 or SEQ ID NQ:120.
  • E139 The vector genome of any one of E136-E138, wherein the at least one AAV2 ITR sequence comprises the nucleic acid sequence of SEQ ID NQ:109 or SEQ ID NO:110.
  • a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NO:109 or SEQ ID NQ:110; b) an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105; c) a promoter comprising the nucleic acid sequence of SEQ ID NO:106; d) an intron comprising the nucleic acid sequence of SEQ ID NQ:107; e) a signal peptide comprising the nucleic acid sequence of any one of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22 and SEQ ID NO:23; f) a NTPT comprising the nucleic acid sequence of any one of SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:
  • a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NO:109 or SEQ ID NQ:110; b) an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105; c) a promoter comprising the nucleic acid sequence of SEQ ID NQ:106; d) an intron comprising the nucleic acid sequence of SEQ ID NQ:107; e) a signal peptide comprising the nucleic acid sequence of SEQ ID NO:18; f) a NTPT comprising the nucleic acid sequence of any one of SEQ ID NO:31 , SEQ ID NO:34 and SEQ ID NO:35; g) a linker comprising the nucleic acid sequence of L1 or L3; h) a modified nucleic acid encoding acid alpha-glucosidase (GAA) comprising the nucleic acid
  • a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NO:109 or SEQ ID NQ:110; b) an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105; c) a promoter comprising the nucleic acid sequence of SEQ ID NQ:106; d) an intron comprising the nucleic acid sequence of SEQ ID NQ:107; e) a signal peptide comprising the nucleic acid sequence of any one of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22 and SEQ ID NO:23; f) a NTPT modified GAA polypeptide comprising the nucleic acid sequence of any one of SEQ ID NO:59, SEQ ID NQ:60, SEQ ID NO:61 , SEQ ID NO
  • a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NO:109 or SEQ ID NO:110; b) an enhancer comprising the nucleic acid sequence of SEQ ID NO:105; c) a promoter comprising the nucleic acid sequence of SEQ ID NO:106; d) an intron comprising the nucleic acid sequence of SEQ ID NQ:107; e) a signal peptide comprising the nucleic acid sequence of SEQ ID NO:18; f) a NTPT modified GAA polypeptide comprising the nucleic acid sequence of any one of SEQ ID NO:59, SEQ ID NO:62 and SEQ ID NO:63; g) a polyA comprising the nucleic acid sequence of SEQ ID NO:108; and h) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NQ:109 or SEQ ID NO
  • a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NO:109 or SEQ ID NQ:110; b) an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105; c) a promoter comprising the nucleic acid sequence of SEQ ID NQ:106; d) an intron comp comprising the nucleic acid sequence of SEQ ID NQ:107; e) a signal peptide-NTPT modified GAA polypeptide comprising the nucleic acid sequence of any one of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85
  • a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NO:109 or SEQ ID NQ:110; b) an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105; c) a promoter comprising the nucleic acid sequence of SEQ ID NO:106; d) an intron comp comprising the nucleic acid sequence of SEQ ID NQ:107; e) a signal peptide-NTPT modified GAA polypeptide comprising the nucleic acid sequence of any one of SEQ ID NO:74, SEQ ID NO:77 and QEQ ID NO:79; f) a polyA comprising the nucleic acid sequence of SEQ ID NQ:108; and g) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NQ:109 or SEQ ID NO:110.
  • a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) a spacer comprising the nucleic acid sequence of any one of SEQ ID NO:99, SEQ ID NQ:100, SEQ ID NO:101 , SEQ ID NQ:102, SEQ ID NQ:103 and SEQ ID NQ:104; b) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NQ:109 or SEQ ID NQ:110; c) an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105; d) a promoter comprising the nucleic acid sequence of SEQ ID NO:106; e) an intron comprising the nucleic acid sequence of SEQ ID NQ:107; f) a signal peptide comprising the nucleic acid sequence of any one of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21
  • a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) a spacer comprising the nucleic acid sequence of any one of SEQ ID NO:99, SEQ ID NQ:100, SEQ ID NQ:102 and SEQ ID NQ:103; b) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NQ:109 or SEQ ID NQ:110; c) an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105; d) a promoter comprising the nucleic acid sequence of SEQ ID NO:106; e) an intron comprising the nucleic acid sequence of SEQ ID NQ:107; f) a signal peptide comprising the nucleic acid sequence of SEQ ID NO:18; g) a NTPT comprising the nucleic acid sequence of any one of SEQ ID NO:31 , SEQ ID NO:34 and SEQ ID NO:35; h) a link
  • a vector genome comprising a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:116 and SEQ ID NO:117.
  • a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) a spacer comprising the nucleic acid sequence of any one of SEQ ID NO:99, SEQ ID NQ:100, SEQ ID NQ:102 and SEQ ID NQ:103; b) a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:116 and SEQ ID NO:117; and c) a spacer comprising the nucleic acid sequence of any one of SEQ ID NO:99, SEQ ID NQ:100, SEQ ID NO:
  • a recombinant adeno-associated virus (rAAV) vector comprising the vector genome of any one of E135-E150 and a capsid.
  • the rAAV vector of E151 comprising a capsid selected from the group consisting of a capsid of AAV1 , AAV2, AAV3 (including AAV3A and AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV12, AAVrh8, AAVrhI O, AAVrh39, AAVrh43, AAVrh74, AAVrh32.22, AAV1.1 , AAV2.5, AAV6.1 , AAV6.2, AAV6.3.1 , AAV9.45, AAVShHIO, HSC15/17, RHM4-1 , RHM15-1 , RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6, AAVhu.26, AAV2i8, AAV29G, AAV2,8G9, AAV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312
  • a rAAV vector comprising a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NO:109 or SEQ ID NQ:110; b) an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105; c) a promoter comprising the nucleic acid sequence of SEQ ID NQ:106; d) an intron comprising the nucleic acid sequence of SEQ ID NQ:107; e) a signal peptide comprising the nucleic acid sequence of any one of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22 and SEQ ID NO:23; f) a NTPT comprising the nucleic acid sequence of any one of SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33,
  • a rAAV vector comprising a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NQ:109 or SEQ ID NQ:110; b) an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105; c) a promoter comprising the nucleic acid sequence of SEQ ID NO:106; d) an intron comprising the nucleic acid sequence of SEQ ID NQ:107; e) a signal peptide comprising the nucleic acid sequence of SEQ ID NO:18; f) a NTPT comprising the nucleic acid sequence of any one of SEQ ID NO:31 , SEQ ID NO:34 and SEQ ID NO:35; g) a linker comprising the nucleic acid sequence of L1 or L3; h) a modified nucleic acid encoding acid alpha-glucosidase (GA
  • a rAAV vector comprising a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NQ:109 or SEQ ID NQ:110; b) an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105; c) a promoter comprising the nucleic acid sequence of SEQ ID NQ:106; d) an intron comprising the nucleic acid sequence of SEQ ID NQ:107; e) a signal peptide comprising the nucleic acid sequence of any one of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:21 , SEQ ID NO:22 and SEQ ID NO:23; f) a NTPT modified GAA polypeptide comprising the nucleic acid sequence of any one of SEQ ID NO:59, SEQ ID NQ:60,
  • a rAAV vector comprising a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NQ:109 or SEQ ID NQ:110; b) an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105; c) a promoter comprising the nucleic acid sequence of SEQ ID NQ:106; d) an intron comprising the nucleic acid sequence of SEQ ID NQ:107; e) a signal peptide comprising the nucleic acid sequence of SEQ ID NO:18; f) a NTPT modified GAA polypeptide comprising the nucleic acid sequence of any one of SEQ ID NO:59, SEQ ID NO:62 and SEQ ID NO:63; g) a polyA comprising the nucleic acid sequence of SEQ ID NO:108; and h) an AAV2 ITR comprising the nucleic acid sequence
  • a rAAV vector comprising a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NQ:109 or SEQ ID NQ:110; b) an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105; c) a promoter comprising the nucleic acid sequence of SEQ ID NQ:106; d) an intron comp comprising the nucleic acid sequence of SEQ ID NQ:107; e) a signal peptide-NTPT modified GAA polypeptide comprising the nucleic acid sequence of any one of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:
  • a rAAV vector comprising a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NQ:109 or SEQ ID NQ:110; b) an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105; c) a promoter comprising the nucleic acid sequence of SEQ ID NQ:106; d) an intron comp comprising the nucleic acid sequence of SEQ ID NQ:107; e) a signal peptide-NTPT modified GAA polypeptide comprising the nucleic acid sequence of any one of SEQ ID NO:74, SEQ ID NO:77 and QEQ ID NO:79; f) a polyA comprising the nucleic acid sequence of SEQ ID NQ:108; and g) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NQ:109 or SEQ ID NO:110.
  • a rAAV vector comprising a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) a spacer comprising the nucleic acid sequence of any one of SEQ ID NO:99, SEQ ID NQ:100, SEQ ID NO:101 , SEQ ID NQ:102, SEQ ID NQ:103 and SEQ ID NQ:104; b) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NQ:109 or SEQ ID NQ:110; c) an enhancer comprising the nucleic acid sequence of SEQ ID NQ:105; d) a promoter comprising the nucleic acid sequence of SEQ ID NO:106; e) an intron comprising the nucleic acid sequence of SEQ ID NQ:107; f) a signal peptide comprising the nucleic acid sequence of any one of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NQ:
  • a rAAV vector comprising a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) a spacer comprising the nucleic acid sequence of any one of SEQ ID NO:99, SEQ ID NQ:100, SEQ ID NQ:102 and SEQ ID NQ:103; b) an AAV2 ITR comprising the nucleic acid sequence of SEQ ID NQ:109 or SEQ ID NO:110; c) an enhancer comprising the nucleic acid sequence of SEQ ID NO:105; d) a promoter comprising the nucleic acid sequence of SEQ ID NO:106; e) an intron comprising the nucleic acid sequence of SEQ ID NQ:107; f) a signal peptide comprising the nucleic acid sequence of SEQ ID NO:18; g) a NTPT comprising the nucleic acid sequence of any one of SEQ ID NO:31 , SEQ ID NO:34 and SEQ ID NO:35
  • a rAAV vector comprising a vector genome comprising a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:116 and SEQ ID NO:117.
  • a rAAV vector comprising a vector genome comprising a nucleic acid wherein the nucleic acid comprises from 5’ to 3’: a) a spacer comprising the nucleic acid sequence of any one of SEQ ID NO:99, SEQ ID NQ:100, SEQ ID NQ:102 and SEQ ID NQ:103; b) a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:116 and SEQ ID NO:117; and c) a spacer comprising the nucleic acid sequence of any one of SEQ ID NO:99, SEQ ID N
  • E164 The rAAV vector of E163, wherein the capsid is of AAV serotype AAV9 or rh74.
  • E165 The rAAV vector of any one of E151-E164, wherein the vector, when introduced into a cell, expresses a NTPT modified GAA polypeptide.
  • E166 The rAAV vector of E165, wherein the cell is a hepatocyte.
  • E167 The rAAV vector of any one of E151-E166, wherein administration of the vector to a subject with an GAA gene mutation increases a serum GAA activity level as compared to the serum GAA activity level in the subject before administration of the vector.
  • E168 The rAAV vector of any one of E151-E167, wherein administration of the vector to a subject with an GAA gene mutation increases tissue GAA activity level as compared to tissue GAA activity level in the subject before administration of the vector.
  • E169 The rAAV vector of E168, wherein the tissue is quadricep tissue, diaphragm tissue, heart tissue, brain tissue, spinal cord tissue or a combination thereof.
  • E170 The rAAV vector of any one of E151-E169, wherein administration of the vector to a subject with an GAA gene mutation decreases glycogen tissue levels in the subject as compared to glycogen levels in the subject before administration of the vector.
  • E171. The rAAV vector of E170, wherein the tissue is quadricep tissue, diaphragm tissue, heart tissue, brain tissue, spinal cord tissue or a combination thereof.
  • E172 The rAAV vector of any one of E151-E171 , wherein administration of the vector to a subject with an GAA gene mutation increases latency to fall in the subject as compared to latency to fall in the subject before administration of the vector.
  • E173 The rAAV vector of any one of E151-E172, wherein administration of the vector to a subject with an GAA gene mutation increases muscle tetanic force in the subject as compared to muscle tetanic force in the subject before administration of the vector
  • E174 The rAAV vector of any one of E167-E173, wherein the subject is a human patient.
  • E176 The rAAV vector of any one of E167-E175, wherein the subject has at least one GAA gene mutation.
  • a pharmaceutical composition comprising the modified nucleic acid of any one of E7- E41 or E44-E99, the recombinant nucleic acid of any one of E118-E134, the vector genome of any one of E135-E150 or the rAAV vector of any one of E151-E176.
  • a pharmaceutical composition comprising the modified nucleic acid of any one of E7- E41 or E44-E99, the recombinant nucleic acid of any one of E118-E134, the vector genome of any one of E135-E150 or the rAAV vector of any one of E151-E176 and a pharmaceutically acceptable carrier.
  • E179 A method of treating and/or preventing a disease, disorder or condition associated with deficiency or dysfunction of GAA, the method comprising administering a therapeutically effective amount of comprising a modified nucleic acid of any one of E7-E41 or E44-E99, a recombinant nucleic acid of any one of E118-E134, a vector genome of any one of E135-E150, a rAAV vector of any one of E151 -E176 or a pharmaceutical composition of E177 or E178 to a subject in need of treatment.
  • E180 The method of E179, wherein the disease, disorder or condition associated with deficiency or dysfunction of GAA is Pompe disease.
  • E181 The method of E179 or E180, wherein the modified nucleic acid, recombinant nucleic acid, vector genome, rAAV vector or pharmaceutical composition is administered intravenously to a subject in need of treatment.
  • E182 The method of any one of E179-E181 , wherein the subject is a human patient.
  • E183 The method of any one of E179-E182, wherein the subject is a human patient with Pompe disease or at-risk for developing Pompe disease.
  • E184 The method of any one of E179-E183, wherein the subject has at least one mutation in the GAA gene.
  • a method of treating or preventing Pompe disease comprising the steps of: i) assessing whether a subject comprises at least one GAA gene mutation and ii) administering to the subject a therapeutically effective amount of a modified nucleic acid of any one of E7-E41 or E44-E99, a recombinant nucleic acid of any one of E118-E134, a vector genome of any one of E135-E150, a rAAV vector of any one of E151-E176 or a pharmaceutical composition of E177 or E178, thereby treating or preventing Pompe disease in the subject.
  • E186 The method of E185, wherein the subject is diagnosed with Pompe disease or diagnosed as at-risk for developing Pompe disease.
  • a method of treating or preventing a disease associated with GAA deficiency in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a modified nucleic acid encoding a NTPT modified GAA wherein the modified nucleic acid has been codon-optimized.
  • E188 The method of E187, wherein the modified nucleic acid encoding the NTPT modified GAA comprises the nucleic acid sequence of SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:63 or SEQ ID NO:118.
  • E189 The method of E187 or E188, wherein the modified nucleic acid encoding the NTPT modified GAA encodes an polypeptide having the amino acid sequence of SEQ ID NO:69 or SEQ ID NO:72.
  • E190 The method of any one of E187-E189, wherein the modified nucleic acid encoding the NTPT modified GAA is expressed in a target cell and wherein the target cell is an hepatocyte.
  • E191 The method of any one of E187-E190, wherein the modified nucleic acid encoding the NTPT modified GAA is delivered in a vector to the target cell.
  • E192 The method of E191 , wherein the vector is a viral vector or a non-viral vector.
  • E193 The method of any one of E187-E192, wherein the vector is administered to the subject by a systemic injection.
  • the method of E194, wherein the modified nucleic acid encoding the signal peptide- NTPT modified GAA comprises the nucleic acid sequence of SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:79 or SEQ ID NO:120.
  • E196 The method of E194-E195, wherein the modified nucleic acid encoding the signal peptide-NTPT modified GAA encodes an polypeptide comprising the amino acid sequence of SEQ ID NQ:90 or SEQ ID NO:93.
  • E197 The method of any one of E194-E196, wherein the modified nucleic acid encoding the signal peptide-NTPT modified GAA is expressed in a target cell and wherein the target cell is a hepatocyte.
  • E198 The method of any one of E194-E197, wherein the modified nucleic acid encoding the signal peptide-NTPT modified GAA is delivered in a vector to the target cell.
  • E199 The method of E198, wherein the vector is a viral vector or a non-viral vector.
  • E200 The method of any one of E194-E199, wherein the vector is administered to the subject by a systemic injection.
  • a host cell comprising a modified nucleic acid of any one of E7-E41 or E44-E99, a recombinant nucleic acid of any one of E1 18-E134, a vector genome of any one of E135-E150 or a rAAV vector of any one of E151 -E176.
  • E202 The host cell of E201 , wherein the cell is selected from the group consisting of VERO, WI38, MRC5, A549, HEK293, B-50 or any other HeLa cell, HepG2, Saos-2, HuH7, and HT1080.
  • E203 The host cell of E201 -E202, wherein the cell is a HEK293 cell adapted to growth in suspension culture.
  • E204 The host cell of any one of E201 -E203, wherein the cell is a HEK293 cell having American Type Culture Collection (ATCC) No. PTA 13274.
  • ATCC American Type Culture Collection
  • E205 The host cell of any one of E202-E204, wherein the cell comprises at least one nucleic acid encoding at least one protein selected from the group consisting of an AAV Rep protein, an AAV capsid (Cap) protein, a adenovirus early region 1A (E1 a) protein, a E1 b protein, an E2a protein, an E4 protein and a viral associated (VA) RNA.
  • a kit for the treatment of Pompe disease comprising a therapeutically effective amount of i) a rAAV vector of any one of E151 -E176 or ii) a pharmaceutical composition of E177 or E178.
  • kit of E206 wherein the kit further comprises a label or insert including instructions for using one or more of the kit components.
  • E209 The modified nucleic acid, the recombinant nucleic acid, the vector genome, the rAAV vector, or the pharmaceutical composition for use of E208, wherein the disease, disorder or condition is Pompe disease.
  • E210 Use of a modified nucleic acid of any one of E7-E41 or E44-E99, a recombinant nucleic acid of any one of E118-E134, a vector genome of any one of E135-E150 or a rAAV vector of any one of E151 -E176 in the manufacture of a medicament for treating and/ or preventing a disease, disorder of condition associated with deficiency or dysfunction of GAA.
  • E211 The use of E210 wherein the disease, disorder or condition is Pompe disease.
  • E212 An intron comprising a nucleic acid sequence at least about 80%, about 85%, about 90%, about 91%, about, 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identical to the nucleic acid sequence of SEQ ID NOU 07.
  • E213. The recombinant nucleic acid of E212, wherein the intron comprises or consists of the nucleic acid sequence of SEQ ID NOU 07.
  • the correction included any one of an increase in GAA activity in serum, muscle tissue (e.g., quadricep, diaphragm, cardiac) and nervous system tissue (e.g., brain, spinal cord) and a decrease in glycogen storage in muscle tissue (e.g., quadricep, diaphragm, cardiac) and nervous system tissue (e.g., brain, spinal cord).
  • muscle tissue e.g., quadricep, diaphragm, cardiac
  • nervous system tissue e.g., brain, spinal cord
  • the nucleic acid encoding GAA was optimized by one or more of the following modifications: 1) codon optimization of the GAA coding sequence thereby increasing expression of the protein, 2) CpG modification of the GAA coding sequence to improve transgene expression, 3) operable linkage of the nucleic acid sequence encoding GAA to a nucleic acid sequence encoding a N- terminal peptide tag (NTPT) thereby increasing uptake of the expressed and secreted GAA by target cells (e.g., quadricep, heart, diaphragm, brain and spinal cord) and 4) operable linkage of the nucleic acid sequence encoding the NTPT modified GAA to a nucleic acid encoding a signal peptide thereby increasing GAA protein secretion from the cell in which it was expressed (e.g., a hepatocyte).
  • target cells e.g., quadricep, heart, diaphragm, brain and spinal cord
  • the N-terminal peptide tag was also modified to advantageously reduce binding to insulin-like growth factor receptor 1 (IGF1 R) and insulin (INSR) receptor to limit signaling through these receptors and to limit the potential for adverse proliferation and hypoglycemia, while maintaining binding to the IGF2 receptor.
  • IGF1 R insulin-like growth factor receptor 1
  • INSR insulin-like growth factor receptor 1
  • Example 1 Efficiency of in vitro uptake of modified GAA in rat muscle cells
  • the conditioned media was obtained by transfecting 1 E6 HEK293 cells with 5 pg of plasmid DNA comprising an expression cassette for the expression of wtGAA (SEQ ID NO:58) or NTPT-GAA (SEQ ID NO:69) using Lipofectamine® 2000 (ThermoFisher).
  • DMEM serum free media
  • serum free media high glucose, supplemented with 0.5 g/L bovine serum albumin (Sigma), 4mM L-glutamine (Gibco) and 20mM HEPES, pH 7.2.
  • Conditioned media was harvested after 72 hours of transfection.
  • the GAA activity of the culture media was measured in order to add specific amounts of wtGAA peptide or NTPT-GAA peptide to the L6 myoblast cultures.
  • L6 rat myoblasts (ATCC #CRL- 1458) were seeded at 3E4 cells per well in 96-well plate in 2% fetal bovine serum culture media. The cells were grown for 24 hours to approximately 90% confluency then varying amounts of conditioned media containing wtGAA or NTPT-GAA were added to the wells. Following an 18 hour incubation period, cells were washed twice with D-PBS and then lysed in 50 pL CelLytic M reagent (Sigma #C3228) by shaking for 20 minutes at room temperature.
  • Lysate debris was removed by centrifugation at 2,000 x g for five minutes. Lysate wtGAA activity and NPTP-GAA activity in triplicate wells was measured using the GAA 4- methylumbelliferone-based (4MU) assay. Briefly, the 4MU assay was performed by incubating cell lysate with 3 mM of the fluorescent substrate 4-methylumbelliferyl a-D-glucopyranoside for 1 hour at 37 °C. The reaction was stopped after 1 hour with sodium bicarbonate buffer (pH 11) and compared to a standard curve generated by diluting 4-methylumbelliferone (4-MU) in the stop buffer. The plate was read at /excitation 368 nm; emission 460 nm.
  • Results GAA activity from L6 cultured cells treated with increasing amounts of wtGAA and NTPT-GAA peptide in conditioned media was measured (FIG. 1). Each data point represents the mean of three wells. Error bars represent the standard deviation. The cells treated with NTPT-GAA containing media demonstrated significantly increased intracellular GAA activity as compared to cells treated with wtGAA containing media. It was observed that the higher amount of NTPT-GAA or wtGAA added to the L6 cell culture (shown on the x-axis as GAA activity), the larger the difference in GAA activity that was measured in the L6 cell lysates.
  • Example 2 Efficiency of in vivo uptake of modified GAA in mouse muscle cells
  • GAA KO mice were transduced with rAAV9 vectors comprising a nucleic acid encoding wtGAA (SEQ ID NO:58) or a nucleic acid encoding NTPT-GAA (SEQ ID NO:69) at three doses: 2.5E12 vg/kg, 7.5E12 vg/kg and 2.25E13 vg/kg.
  • Six to eight animals were transduced at each dose. The doses were chosen to generate a range of serum GAA activity.
  • the vectors targeted the liver for liver production and secretion of the wtGAA peptide and the NTPT-GAA peptide. Animals were euthanized 3 months after transduction. Serum and quadricep muscle was harvested for measurement of GAA activity.
  • mice were first pulverized in liquid nitrogen using the Covaris Automated Dry Pulverizer. 15-25 mg of tissue powder was weighed, and CelLytic MT (Sigma #C3228) was added to a concentration of 0.05mg/ul in a 2 mL tube. The tissue was homogenized with a 5mm steel bead in the Qiagen TissueLyser II for 2.5min at 2.5rpm. The samples were centrifuged at 14,000 rpm for 10 minutes, and the supernatant was collected. The supernatant was then incubated with 3 mM of fluorescent substrate 4-methylumbelliferyl a- D-glucopyranoside for 1 hour at 37 °C.
  • the reaction was stopped after 1 hour with sodium bicarbonate buffer (pH 11 ) and compared to a standard curve generated by diluting 4- methylumbelliferone (4-MU) in the stop buffer.
  • the plate was read at /excitation 368 nm; emission 460 nm.
  • Example 3 Modified GAA specific activity, N-terminal peptide tag (NTPT) integrity and proteolytic process
  • modified GAA e.g., NTPT-GAA
  • rhGAA R&D Systems #8329-GH
  • wtGAA wtGAA polypeptide
  • Flag tagged NTPT-GAA protein was produced and purified by first transiently transfecting HEK293 cells with a Flag tagged NTPT-GAA plasmid (SEQ ID NO:111). Conditioned medium was collected after 96 hours of expression. 0.1 L of conditioned medium was incubated with 1 mL FLAG M2 resin and incubated at 4°C overnight. Target protein was eluted with ProA buffer (150 mM Glycine Ph 3.5, 50 mM NaCI) and neutralized with 1 M Tris 8.0 immediately after elution.
  • ProA buffer 150 mM Glycine Ph 3.5, 50 mM NaCI
  • the factions of eluate were combined, concentrated, and subsequently loaded onto a Superdex200 10/300 column with a running buffer of PBS.
  • GAA activity of the purified NTPT-GAA polypeptide was tested using a 4-methylumbelliferone-based (4-MU) assay as described above.
  • GAA activity was used as the endpoint readout for several studies, we evaluated the correlation between enzyme activity and modified GAA (e.g., NTPT-GAA) expression level or enzyme activity and wtGAA expression level.
  • modified GAA e.g., NTPT-GAA
  • GAA knock out (KO) mice were intravenously dosed with 7.5E12 vg/kg of a rAAV9 vector comprising a nucleic acid encoding wtGAA (animals #1-3) (SEQ ID NO:16) or a rAAV9 vector comprising a nucleic acid encoding NTPT-GAA (animals #4-6) (SEQ ID NO:1) to express the enzymes.
  • the vectors targeted liver cells for production and secretion of the wtGAA polypeptide and the NTPT-GAA polypeptide . Blood was collected 1 month after the dosing.
  • Serum GAA protein level was measured using an anti-human GAA antibody (Invitrogen #PA5-96756) by WesTM (automated capillary-based immunoassay) (ProteinSimple). Varying amounts of rhGAA (1 .2 ng, 0.4 ng, 0.133 ng and 0.04 ng) (R&D Systems #8329-GH) were used to generate a standard curve for calculating the serum wtGAA or serum NTPT-GAA protein concentration. The GAA activity was tested using a 4-methylumbelliferone-based (4-MU) assay as described above.
  • 4-MU 4-methylumbelliferone-based
  • GAA KO mice were intravenously dosed with rAAV vector comprising a nucleic acid (SEQ ID NO:16) encoding wtGAA polypeptide (SEQ ID NO:58) or a nucleic acid (SEQ ID NO:1) encoding NTPT-GAA polypeptide (SEQ ID NO:69) at a dose level of 7.5E12 vg/kg.
  • Blood was collected 3 months after dosing.
  • Two sets of serum samples from the same animal were probed with anti-human GAA (Invitrogen #PA5-96756) and anti-human IGF2 (Abeam #ab9574) antibodies in parallel by WesTM analysis.
  • rhGAA (R&D #8329-GH) and rhlGF2 (R&D #292-G3) were used as positive controls for the enzyme and the NTPT.
  • the NTPT-GAA fusion protein was comprised of a mature IGF2 peptide (the NTPT), a linker and a GAA protein (SEQ ID NO:69).
  • FIG. 4 shows that both GAA and IGF2 were detected at the same molecular weight of 128-132 kDa in serum from mice dosed with the rAAV vector comprising a nucleic acid encoding NTPT-GAA.
  • only GAA was detected in the serum samples from mice dosed with the rAAV comprising a nucleic acid encoding wtGAA.
  • the rhlGF2 control was detected at 7.5-16 kDa.
  • GAA undergoes several proteolytic processing steps to convert the 110 kDa precursor protein to the mature 70 kDa lysosomal form after it is synthesized in the cell or taken up intracellularly.
  • HEK293 cells were transiently transfected with a plasmid encoding wtGAA (SEQ ID NO:58) or a plasmid encoding NTPT-GAA (SEQ ID NO:69).
  • Cell lysates and conditioned media were probed with anti-human GAA antibody (Invitrogen #PA5-96756) to visualize the GAA forms.
  • the conditioned media was also probed with anti-human IGF2 (Abeam #ab9574) to visualize the NTPT.
  • FIG. 5 shows that the intracellular precursor GAA, intermediate GAA and mature GAA forms were presented in the cell lysates from cells transfected with NTP-GAA plasmid and cells transfected with wtGAA plasmid.
  • the precursor forms of GAA were present in the conditioned media from cells transfected with NTP-GAA plasmid and cells transfected with wtGAA plasmid.
  • the IGF2 N-terminal peptide tag in the precursor protein was only identified in the conditioned media from the NTPT-GAA transfected cells (FIG. 5). This study demonstrates that modified GAA polypeptides were processed intercellu larly through similar proteolytic processing steps as wtGAA polypeptides.
  • a feature of Pompe disease is the abnormal buildup of glycogen in many tissues including cardiac muscle and skeletal muscle. Measurement of muscle glycogen level in disease animal models has been used as a biochemical functional assay for efficacy studies when testing potential therapeutic approaches. This study was performed to determine whether mice treated with a rAAV vector comprising a nucleic acid encoding a modified GAA (NTPT- GAA; SEQ ID NO:69) demonstrated better glycogen clearance from muscle than mice treated with a rAAV vector comprising a nucleic acid encoding wtAAV (wtGAA; SEQ ID NO:58).
  • the rAAV vector comprises an AAV9 serotype capsid.
  • FIG. 6A, FIG. 8A and FIG. 8B represent data only from the first study.
  • FIG. 7A shows quadricep tissue GAA activity levels from GAA KO mice treated with rAAV NTPT-GAA of 32% (2.5E12 vg/kg), 176% (7.5E12 vg/kg) and 204% (2.25E13 vg/kg) relative to the GAA activity levels in WT Untx mice.
  • rAAV wtGAA treated GAA KO mice treated with rAAV wtGAA showed lower quadricep tissue GAA activity level relative to WT Untx mice at 23% (2.5E12 vg/kg), 57% (7.5E12 vg/kg) and 50% (2.25E13 vg/kg.
  • FIG. 7B shows quadricep tissue glycogen levels from rAAV NTPT-GAA treated GAA KO mice as 116% (2.5E1 vg/kg), 45% (7.5E1 vg/kg) and 26% (2.25E13 vg/kg) relative io the glycogen levels in KO Untx mice.
  • rAAV wtGAA treated GAA KO mice showed quadricep tissue glycogen level relative to KO Untx as 1 2% (2.5E1 vg/kg), 53% (7.5E1 vg/kg) and 32% (2.25E13 vg/kg).
  • GAA activity in brain tissue was 0.13 nmol.hr.mg tissue from GAA KO mice treated with 7.5E12 vg/kg of rAAV NTPT-GAA vector, and 0.99 nmol.hr.mg tissue from untreated wild type mice. This demonstrated that the level of GAA entering the brain of treated mice was about 13% of the level of GAA entering the brains of wild type mice, and that this level was sufficient to reduce the glycogen in brain tissue.
  • the GAA activity in the brain tissue from GAA KO mice treated with rAAV NPTP-GAA vector was significantly greater than the GAA activity in the brains of untreated GAA knock out mice, and was significantly greater than the GAA activity in the brains of GAA KO mice that were treated with the rAAV wtGAA vector.
  • liver secreted modified GAA (NTPT-GAA) was taken up by cardiac muscle, skeletal muscle and the CNS and corrected the glycogen levels in all tissues.
  • Example 5 Alternative signal peptide in vitro screening and in vivo validation
  • a panel of alternative signal peptides were designed and tested in the GAA expression cassette in order to identify a signal peptide that would confer a higher level of protein secretion from liver cells than the endogenous GAA signal peptide (referred to as SP17) (SEQ ID NO:30). These variants were referred to as “signal peptide variants” or “SP variants”.
  • Methods Huh7 cells were seeded at 1 E4 cells per well in a 96-well plate. After overnight culture, cells were transiently transfected with 0.1 pg plasmid DNA using Lipofectamine® 2000 (ThermoFisher). GAA activity in conditioned media was measured post 48-hour transfection.
  • Lead signal peptides (SP1-SP16) were substituted for the endogenous IGF2 signal peptide (referred to as SP18; amino acids 1-24 of GenBank Ref. No. NP 000603.1 ; SEQ ID NO:25) in the modified GAA cassettes and subcloned in a vector backbone for vectorization.
  • a nucleic acid encoding the IGF2 signal peptide was codon optimized (e.g., SEQ ID NO:17, SEQ ID NO:19 and SEQ ID NO:22).
  • GAA KO male mice were administrated rAAV modified GAA vector at a dose of 1 .5E13 vg/kg. Serum was collected 3 weeks post injection and the GAA activity measured. Three mice were used in each of the untreated wild type and GAA KO groups. Six mice were used in each of the treatment groups.
  • GAA activity was highest in serum from mice administered 1 .5E13 vg/kg of a rAAV vector comprising a nucleic acid (SP13-NTPT-GAA; SEQ ID NO:2) encoding a modified GAA with an amino terminal SP13 SEQ ID NO:26) (FIG 10B).
  • This GAA activity level was 1.5-fold higher than the GAA activity in serum from mice administered 1 .5E13 vg/kg of a rAAV vector comprising a nucleic acid (NTPT-GAA; SEQ ID NO:1) encoding a modified GAA with the endogenouslGF2 signal peptide (SEQ ID NO:25).
  • GAA activity was lowest in serum from mice administered 1 .5E13 vg/kg of a rAAV vector comprising a nucleic acid (IgVH-NTPT-GAA; SEQ ID NO:15) encoding a modified GAA with an amino terminal IgVH signal peptide (ISEQ ID NO:29) (FIG 10B).
  • IgVH-NTPT-GAA nucleic acid
  • ISEQ ID NO:29 amino terminal IgVH signal peptide
  • Example 6 CpG modification variants in vitro screening and in vivo validation
  • CpG modification variants of the GAA transgene were designed and synthesized. These variants were first tested in vitro for expression.
  • the variant transgenes were referred to as CpGdl (SEQ ID NO:50), CpGd2 (SEQ ID NO:51) CpGd3 (SEQ ID NO:52) CpGd4 (SEQ ID NO:53), CpGd5 (SEQ ID NO:54) and CpGd6 (SEQ ID NO:55). All included the NTPT (SEQ ID NO:39).
  • the lead transgene, CpGdl was then vectorized and tested in vivo.
  • Huh7 cells were seeded at 3E4 cell per well in a 96- well plate. After overnight culture, cells were transiently transfected with 0.2 pg plasmid DNA using Lipofectamine® 2000 (ThermoFisher). GAA activity in conditioned media was measured post 72-hour transfection.
  • the lead CpG modification variant, CpGdl was subcloned in a vector backbone for vectorization.
  • GAA KO male mice were administrated 1 .5E13 vg/kg of a rAAV vector (SEQ ID NO:6) with the CpG modified GAA (NTPT-GAACpGd1).
  • Serum GAA was collected 3 weeks post injection and the GAA activity level was measured.
  • Example 7 In vivo validation of lead CpG modification variant combined with lead signal peptide.
  • the purpose of this study was to combine the optimized elements of the signal peptide, NTPT and CpG modified GAA transgene and achieve more a efficacious rAAV vector and a higher serum GAA level in mice.
  • the IGF2 signal peptide (SEQ ID NO:25) that was used in the parental NTPT-GAA construct (SEQ ID NO:1) was replaced with the signal peptide SP1 (SEQ ID NO:27), SP7 (SEQ ID NO:28) or SP13 (SEQ ID NO:26).
  • mice administered the rAAV vector with the SP13 signal peptide and the CpG modified GAA (SP13-NTPT-GAACpGd1).
  • the serum GAA activity in mice administered this rAAV vector was significantly higher than the serum GAA activity in mice administered vectors which used SP1 or SP7 and the CpG modified GAA (SP1 - NTPT-GAACpGd1 and SP7-NTPT-GAACpGd1 , respectively) and mice administered the vector which used SP13 but the non-codon-optimized GAA (SP13-NTPT-GAA).
  • Example 8 Binding profiles for GAA peptides with N-terminal peptide tags (NTPT) or C- terminal peptide tags (CTPT).
  • NTPT N-terminal peptide tags
  • CPT C- terminal peptide tags
  • IGF2 peptide as the N-terminal peptide tag (NTPT ; SEQ ID NO:39) or C-terminal peptide tag (CTPT), in addition to binding the “on-target” IGF- 11 receptor (IGF2R), also binds to insulin receptor (INSR) and IGF1 receptor (IGF1 R), which may trigger downstream pathways for proliferation and blood glucose depletion.
  • IGF2R insulin receptor
  • IGF1 R insulin receptor
  • an NTPT-GAA variant library was generated and the binding profiles of NTPT-GAA variants to IGF2R, INSR and IGF1 R were measured by Biacore SPR.
  • Mutations were identified for inclusion in the rational library based on computational alanine scanning, computational AAG predictions of the IGF-II/IGF-2R interface, and potential for salt bridge disruption and/or steric disruption of the IGF- 1 R and/or INS-R binding sites, resulting in a NTPT-GAA library size of approximately 6x10 A 4. Select point mutant constituents of the library were independently synthesized and tested.
  • Library fragments were synthesized and cloned into a custom-built SSI-compatible dropout vector containing the GAA transgene, and the resulting DNA was scaled up.
  • the libraries were transfected into CHO2.0 cells, and viability curves were generated to determine the optimal point to hand-off samples for screening.
  • the CHO cell library expressing Flag-tagged NTPT-GAA variants was seeded in 96- well plates at density of 3 cells per well. After 2-week culture, supernatants were collected and screened in 384-well plates with IGF-2R, IGF-1 R, INS-R, and anti-Flag ELISA Delfia direct binding assays and IGF- 11 ligand competition assay on Ni-NTA or Maxisorp plate to identify hits with increased binding to IGF-2R and reduced binding to IGF-1 R and INS-R. Selected hits were sequenced with PCR amplification of genomic DNA and further subcloned into expression vectors for expression, purification, and binding evaluation by Biacore SPR.
  • NTPTSeqO2-GAA SEQ ID NO:70
  • NTPTSeq43- GAA SEQ ID NO:71
  • NTPTSeq44-GAA SEQ ID NO:72
  • Table 1 summarizes Biacore SPR binding profiles of three lead NTPT-GAA variants relative to the parental NTPT-GAA. All were tested at concentrations up to 1000 nM. All 3 lead constructs completely ablate binding (below threshold of detection) to both receptors (IGF1 R & INSR) with only marginal drops in on-target (IGF2R) receptor binding. NTPTSeqO2-GAA has a larger reduction in IGF2R binding than either NTPTSeq43-GAA or NTPTSeq44-GAA.
  • Example 9 New NTPT-GAA variants correct both muscle and CNS phenotype in GAA KO mice.
  • rAAV vectors comprising a nucleic acid encoding a GAA transgene with a signal peptide and N-terminal peptide tag were evaluated in GAA KO mice for a number of parameters including serum GAA activity, latency to fall off a rotarod, muscle tetanic force, quadricep tissue GAA activity, quadricep tissue glycogen level, heart tissue glycogen level, diaphragm tissue glycogen level, brain GAA activity and brain tissue glycogen level.
  • GAA KO male mice were administrated a rAAV vector comprising a transgene cassette encoding SP13-NTPT-GAA (SEQ ID NO:2), SP13-NTPTseqO2-GAA (SEQ ID NO:3), SP13-NTPTseq43-GAA (SEQ ID NO:4), SP13-NTPTseq44-GAA (SEQ ID NO:5) or NTPT-GAACpGd1 (SEQ ID NO:6) at a dose of 1 .5E13 vg/kg. Details of the vector constructs are provided in Tables 2 and 3. Blood was collected biweekly to monitor serum GAA activity levels. Rotarod and muscle force behavior tests were run 11 weeks post injection. Mice were euthanized and tissues were harvested 12 weeks post injection for GAA activity (serum and tissue) and tissue glycogen measurements.
  • mice administered the rAAV vector comprising a nucleic acid encoding NTPT- GAACpGdl had the highest GAA activity level at both 3 weeks post injection (FIG. 13A) and 12 weeks post injection (FIG. 13B).
  • Untreated GAA KO (KO Untx) mice, untreated wildtype (WT Untx) mice and GAA KO mice administered a rAAV vector comprising a nucleic acid encoding a GAA transgene with a signal peptide and N-terminal peptide tag were evaluated by performance on a rotarod and by muscle tetanic force 11 weeks after treatment.
  • Untreated GAA KO mice showed significantly decreased latency to fall (in seconds) as compared to the untreated wildtype mice.
  • GAA KO mice administer the rAAV SP13-NTPT-GAA and rAAV NTPT-GAACpGd1 vectors suggested improvements of 20% and 42%, respectively, in the latency to fall as compared to GAA KO mice that were not treated (FIG. 14A).
  • mice administered rAAV SP13-NTPTseq44-GAA and rAAV NTPT-GAACpGd1 demonstrated significantly restored muscle strength as compared to muscle strength in untreated GAA KO mice and full muscle strength as compared to wild type mice.
  • Mice administered rAAV SP13-NTPT-GAA suggested a 11 .6% improvement in muscle strength as comparted to muscle strength in untreated GAA KO mice (FIG. 14B).
  • Quadricep tissue GAA activity and glycogen levels were measured in untreated GAA KO mice, untreated wildtype mice and GAA KO mice administered a rAAV vector comprising a nucleic acid encoding a GAA transgene with a signal peptide and N-terminal peptide tag 12 weeks after treatment.
  • GAA KO mice administered rAAV SP13-NTPT-GAA, rAAV SP13-NTPTseqO2-GAA, rAAV SP13-NTPTseq43-GAA, and rAAV SP13-NTPTseq44GAA vectors restored quadricep tissue GAA activity to levels that were comparable to quadricep tissue GAA activity levels in untreated wildtype mice.
  • GAA KO mice administered rAAV NTPT- GAACpGdl vector showed a 10.7-fold higher GAA activity level as compared to GAA activity levels in untreated wildtype mice (FIG. 15A). All of the treatment groups showed a significant correction of quadricep tissue glycogen levels as compared to untreated GAA KO mice.
  • the treatment groups administered rAAV SP13-NTPTseqO2-GAA, rAAV SP13-NTPTseq43-GAA, rAAV SP13-NTPTseq44GAA, and rAAV NTPT-GAACpGd1 vectors had mean quadricep tissue glycogen levels that were not significantly different from mean quadricep tissue glycogen levels in untreated wild type mice (FIG. 15B).
  • Diaphragm and heart tissue glycogen levels were measured in untreated GAA KO mice, untreated wildtype mice and GAA KO mice administered a rAAV vector comprising a nucleic acid encoding a GAA transgene with a signal peptide and N-terminal peptide tag 12 weeks after treatment.
  • GAA KO mice administered rAAV SP13-NTPT-GAA, rAAV SP13- NTPTseqO2-GAA, rAAV SP13-NTPTseq43-GAA, rAAV SP13-NTPTseq44GAA and rAAV NTPT-GAACpGd1 vectors reduced diaphragm and heart tissue glycogen to levels that were not significantly different from diaphragm and heart tissue glycogen in untreated wildtype mice and were significantly different from diaphragm and heart tissue glycogen in untreated GAA KO mice. All treated mice had diaphragm and heart tissue glycogen levels that were significantly lower than levels in untreated GAA KO mice (FIG. 16A and FIG. 16B).
  • GAA KO mice administered rAAV SP13-NTPT-GAA, rAAV SP13- NTPTseqO2-GAA, rAAV SP13-NTPTseq43-GAA, rAAV SP13-NTPTseq44GAA and rAAV NTPT-GAACpGd1 vectors had GAA activity levels in spinal cord tissue that was significantly increased as compared to GAA activity levels in spinal cord tissue from untreated GAA KO mice.
  • the rAAV NTPT-GAACpGd1 vector restored GAA activity in spinal cord tissue to GAA activity levels in untreated wildtype mice (FIG. 17A). All treated mice also demonstrated a significant reduction in spinal tissue glycogen as compared to untreated GAA KO mice.
  • the rAAV SP13-NTPT-GAA and rAAV NTPT-GAACpGd1 vectors reduced spinal cord tissue glycogen to levels that were not significantly different from glycogen levels in spinal cord tissue from untreated wildtype mice (FIG. 17B).
  • Brain GAA activity and glycogen levels were measured in untreated GAA KO mice, untreated wild type mice and GAA KO mice administered a rAAV vector comprising a nucleic acid encoding a GAA transgene with a signal peptide and N-terminal peptide tag 12 weeks after treatment.
  • GAA KO mice administered rAAV NTPT-GAACpGd1 vector restored brain tissue GAA activity to GAA activity levels in brains of untreated wildtype mice (FIG. 18A).
  • GAA KO mice administered rAAV SP13-NTPT-GAA, rAAV SP13-NTPTseqO2-GAA, rAAV SP13-NTPTseq43-GAA, rAAV SP13-NTPTseq44-GAA and rAAV NTPT-GAACpGd1 vectors had glycogen levels in brain tissue that were significantly decreased as compared to glycogen levels in brain tissue from untreated GAA KO mice.
  • the rAAV SP13-NTPT-GAA and rAAV NTPT-GAACpGd1 vectors reduced brain tissue glycogen levels to levels that were not significantly different from glycogen levels in brain tissue of untreated wild type mice (FIG.
  • rAAV9 vectors comprising an alpha-glucosidase (GAA) transgene modified with a signal peptide that improves secretion of the protein from hepatocytes, and a N-terminal peptide tag that improves uptake of the expressed protein by cardiac muscle, skeletal muscle, spinal cord and brain, provides full rescue of Pompe disease in GAA knockout mice including improved serum and tissue GAA activity levels, decreased tissue glycogen storage and improved muscle function.
  • GAA alpha-glucosidase
  • the present study evaluates the efficacy of rAAV SP13-NTPT-GAACpGd1 (SEQ ID NO:7) and SP13-NTPTseq44-GAACpGd1 (SEQ ID NO:117) in GAA KO mice, assessing several parameters including serum GAA activity, liver GAA to serum GAA ratio, quadricep tissue GAA activity, quadricep tissue GAA to serum GAA ratio, and tissue glycogen levels.
  • GAA KO male mice were administrated a rAAV vector comprising a transgene cassette encoding SP13-NTPT-GAACpGd1 (SEQ ID NO:7) and SP13-NTPTseq44- GAACpGdl (SEQ ID NO: i 17) at doses of 1 E12, 5E12 and 2.5E13 vg/kg, with blood collected biweekly to monitor serum GAA activity levels.
  • Mice were euthanized 12 weeks post injection, and tissues were harvested for GAA activity and glycogen measurements.
  • SP13-NTPTseq44-GAACpGd1 treated animals had the highest serum GAA activity to liver GAA activity ratio, suggesting a superior liver secretion efficiency compared to other constructs (FIG. 19B).
  • SP13-NTPT-GAACpGd1 treated animals had an increased serum GAA activity to liver GAA activity ratio compared to NTPT-GAA or wtGAA treated animals (FIG. 19B), indicating an improved liver secretion.
  • SP13-NTPT-GAACpGd1 at 5E12 vg/kg restored quadricep tissue GAA activity to wild-type levels
  • SP13-NTPT-GAACpGd1 and SP13-NTPTseq44-GAACpGd1 at 2.5E13 vg/kg showed 9.9-fold and 11 .5-fold higher GAA activity, respectively, compared to wildtype levels (FIG. 19C).
  • SP13-NTPT-GAACpGd1 treated animals had the highest quadricep GAA uptake efficiency, as indicated by quadricep GAA activity to serum GAA activity ratio (FIG.

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Abstract

La présente invention concerne des acides nucléiques recombinants et des vecteurs de thérapie génique comprenant un acide nucléique codant pour une alpha-glucosidase acide (GAA) modifiée, et des variants de ceux-ci, destinés à être utilisés dans le traitement de maladies et de troubles associés à une déficience ou un dysfonctionnement de GAA, et en particulier, la maladie de Pompe.
PCT/IB2023/056506 2022-06-28 2023-06-23 Acides nucléiques codant pour l'alpha-glucosidase acide (gaa) et vecteurs pour thérapie génique WO2024003687A1 (fr)

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