WO2024009280A1 - Integrated stress response inhibitors and methods of using the same - Google Patents

Integrated stress response inhibitors and methods of using the same Download PDF

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WO2024009280A1
WO2024009280A1 PCT/IB2023/057038 IB2023057038W WO2024009280A1 WO 2024009280 A1 WO2024009280 A1 WO 2024009280A1 IB 2023057038 W IB2023057038 W IB 2023057038W WO 2024009280 A1 WO2024009280 A1 WO 2024009280A1
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aav
construct
seq
isr
sequence
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PCT/IB2023/057038
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French (fr)
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Mauro Costa-Mattioli
Lucas REINEKE
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Baylor College Of Medicine
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This disclosure relates at least to the fields of aging biology, neurology, genetics, medicine, and gerontology.
  • proteostasis protein homeostasis
  • ISR integrated stress response
  • the ISR’s central regulatory hub lies in the Eukaryotic Initiation Factor 2 / Eukaryotic Initiation Factor 2B (eIF2/eIF2B) complex, which controls the formation of the eIF2»GTP»methionyl-initiator tRNA ternary complex (TC), a prerequisite for initiating new protein synthesis (see e.g., Hinnebusch etal., 2016; which is incorporated herein by reference for the purposes described herein).
  • eIF2/eIF2B Eukaryotic Initiation Factor 2B
  • eIF2B guanine nucleotide exchange factor termed eIF2B (see e.g., Bogorad et al., 2017; Kenner et al., 2019; Kashiwagi et al., 2019; Adomavicius et al., 2019; and Gordiyenko et al., 2019; each of which are incorporated herein by reference for the purposes described herein).
  • eIF2 protein kinase R-like endoplasmic reticulum kinase (PERK), eIF-2-alpha kinase (GCN2), protein kinase RNA- activated (PKR) and Heme-regulated eIF2a kinase (HRI)
  • PERK Protein kinase R-like endoplasmic reticulum kinase
  • GCN2 eIF-2-alpha kinase
  • PSR protein kinase RNA- activated
  • HRI Heme-regulated eIF2a kinase
  • eIF2-P also triggers the translation of specific mRNAs carrying short inhibitory upstream open reading frames (ORFs) in their 5 '-untranslated regions (5' UTRs), including key transcription factors, such as ATF4 (see e.g., FIG. 1).
  • ORFs inhibitory upstream open reading frames
  • ATF4 key transcription factors
  • ISR is a central regulator of longterm memory formation.
  • genetic or pharmacological inhibition of the ISR e.g., reduced eIF2-P
  • activation of ISR e.g., increased eIF2- P
  • the ISR is a central memory switch (see e.g., Batista et al., 2015; Ma et al., 2013; Sharma etal., 2018; Segev etal., 2015; Stem et al., 2013; Moreno et al., 2012; Kim etal., 2013; Wong et al., 2019; and reviewed in Costa-Mattioli & Walter, 2020; each of which are incorporated herein by reference for the purposes described herein).
  • ISR ISR
  • the ISR is activated in a variety of brain disorders that result from protein misfolding and aggregation problems, mitochondrial dysfunction, and/or oxidative stress.
  • activation of the ISR is a main causative mechanism underlying the memory deficits associated with, Down Syndrome (see e.g., Zhu et al., 2019), Alzheimer’s disease (AD) (see e.g., Ma et al., 2013; Segev et al., 2015; Tible et al., 2019; Hwang et al, 2017; and Lourenco et al., 2013; each of which are incorporated herein by reference for the purposes described herein), traumatic brain injury (TBI) (see e.g., Chou et al., 2017; and Sen et al., 2017; each of which are incorporated herein by reference for the purposes described herein), and aging (see e.g., Sharma et al., 2018, which is incorporated herein by reference for the
  • Some of these rare mutations map to the PPI binding site of CReP (encoded by PPP1R15B) and destabilize the CReP»PPl phosphatase complex, thereby increasing eIF2-P (see e.g., Abdulkarim et al., 2015; and Kemohan et al, 2015; each of which are incorporated herein by reference for the purposes described herein).
  • the present disclosure provides the recognition that diseases or conditions associated with Integrated Stress Response (ISR) activation (e.g., natural activation, hyperactivation, and/or aberrant activation) can be treated through the modulation (e.g., inhibition) of the ISR.
  • modulation of the ISR comprises the use of constructs, particles, polypeptides, polynucleotides, and/or compositions comprising said constructs, particles, proteins and/or nucleotides encoding said proteins, wherein the proteins comprise a PPI binding domain and an eIF2 binding domain.
  • a non-natural polynucleotide construct comprising a polynucleotide sequence encoding one or more inhibitor of the integrated stress response (ISR), wherein the one or more inhibitor comprises a protein phosphatase-1 (PPI) binding domain and an eukaryotic initiation factor 2 (eIF2) binding domain
  • the non-natural polynucleotide construct comprises: A) a heterologous promoter operably linked to the polynucleotide sequence encoding one or more inhibitors of the ISR, wherein the heterologous promoter is not a galactose inducible promoter; and/or B) a recombinant viral vector comprising a heterologous promoter operably linked to the polynucleotide sequence encoding one or more inhibitors of the ISR, wherein the heterologous promoter is not a galactose inducible promoter.
  • a non-natural polynucleotide construct comprising a polynucleotide sequence encoding one or more inhibitor of the ISR, wherein the one or more inhibitor comprises PPI binding domain and an eIF2 binding domain, wherein the polynucleotide construct further comprises a peptide linker sequence connecting the PPI binding domain and eIF2 binding domain, wherein the peptide linker sequence comprises a glutamic acid at the amino acid position represented by the E12 position of a DP71L protein (e.g., according to SEQ ID NO: 18), wherein the polynucleotide construct further comprises: A) a heterologous promoter operably linked to the polynucleotide sequence encoding one or more inhibitors of the ISR network; and/or B) a recombinant viral vector comprising a heterologous promoter operably linked to the polynucleotide sequence encoding one or more inhibitors of the ISR network
  • an ISR inhibitor binds PPla and/or PPly and/or eIF2 with greater affinity than other ISR inhibitors of similar primary amino acid sequence length. In some embodiments, an ISR inhibitor binds PPla and/or PPly and/or eIF2 with greater affinity than a ACREP protein. In some embodiments, binding affinity is assessed using immunoprecipitation assays. In some embodiments, binding affinity is assessed using immunoprecipitation assays performed on lysates from transgenic human cells (e.g., HEK293T cells).
  • transgenic human cells e.g., HEK293T cells
  • a construct that stably and/or transiently expresses a protein of interest e.g., an ISR inhibitor
  • a detectable marker e.g., a tag, e.g., a fluorescent marker, e.g., GFP (e.g., encoded by SEQ ID NO: 37).
  • a binding affinity is assayed using an antibody targeting a detectable marker and/or a protein of interest, and is detected (e.g., qualitatively and/or quantitatively) using any suitable means, such as but not limited to, ELISA assays, Western blot assays, immunoprecipitation mass spectrometry assays, etc. In some embodiments, binding affinity is assessed as described herein in FIG. 6.
  • a construct described herein encodes an inhibitor which comprises a PPI binding domain operable for recruiting a protein and/or protein complex comprising a serine/threonine-protein phosphatase PPI -alpha catalytic subunit (PPP1CA, aka PPla), a serine/threonine-protein phosphatase PPI -beta catalytic subunit (PPP1CB, akaPPip), and/or a serine/threonine-protein phosphatase PPI -gamma catalytic subunit (PPP ICC, aka PPly).
  • PPP1CA serine/threonine-protein phosphatase PPI -alpha catalytic subunit
  • PPP1CB serine/threonine-protein phosphatase PPI -beta catalytic subunit
  • PPP ICC serine/threonine-protein phosphatase PPI -gamma catalytic subunit
  • a construct described herein encodes an inhibitor which comprises an eIF2 binding domain operable for recruiting a protein and/or protein complex comprising an eIF2a, eIF2p, and/or eIF2y subunit.
  • a PPI binding domain comprises a RVxF PPI -binding motif and/or KGILK PPI binding motif.
  • a PPI binding domain comprises a KVRF, KVTF, RVRF, WVTF, IVRF, and/or HVRF PPI -binding motif.
  • an eIF2 binding domain comprises a RxGx- WxxxAxDRxRFxxRI eIF2 binding motif.
  • a construct described herein encodes an inhibitor which comprises a constitutive repressor of eIF2a phosphorylation (CReP) protein or a characteristic portion thereof.
  • a CReP protein comprises or consists of an amino acid sequence that is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6.
  • a CreP protein or a characteristic portion thereof further comprises a peptide linker sequence connecting the PPI binding domain and eIF2 binding domain.
  • the peptide linker sequence is derived from an African swine fever virus DP71L protein.
  • the peptide linker sequence is at least or exactly 73%, 82%, 91%, or 100% identical to SEQ ID NO: 21.
  • the CReP protein is a chimeric protein and comprises an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4.
  • the CReP protein is a chimeric protein and comprises an amino acid sequence according to SEQ ID NO: 4.
  • a construct described herein encodes an inhibitor which comprises a protein phosphatase 1 regulatory subunit 15A (GADD34) protein or a characteristic portion thereof. In some embodiments, a construct described herein encodes an inhibitor which comprises a Herpes simplex virus y34.5 protein or a characteristic portion thereof. In some embodiments, a construct described herein encodes an inhibitor which comprises a Canarypox virus CNPV231 protein or a characteristic portion thereof. In some embodiments, a construct described herein encodes an inhibitor which comprises a Macropoid herpes virus ICP34.5 protein or a characteristic portion thereof. In some embodiments, a construct described herein encodes an inhibitor which comprises an Amsacta moorei entom opoxvirus “L” AmEPV193 protein or a characteristic portion thereof.
  • a construct described herein encodes an inhibitor which comprises an African swine fever virus DP71L protein or a characteristic portion thereof.
  • the inhibitor comprises an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18.
  • the inhibitor comprises an amino acid sequence according to SEQ ID NO: 18.
  • the inhibitor is encoded by a polynucleotide comprising a coding sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 19.
  • the inhibitor is encoded by polynucleotide comprising a coding sequence according to SEQ ID NO: 19.
  • the inhibitor comprises a DP71L protein that does not comprise a V6E mutation and/or E12T mutation (e.g., relative to SEQ ID NO: 18).
  • a construct described herein encodes an inhibitor that comprises a peptide linker between the PPI binding domain and eIF2 binding domain.
  • the inhibitor comprises a peptide linker derived from a DP71L protein.
  • the peptide linker comprises a glutamic acid at the DP71L protein (SEQ ID NO: 18) E12 position.
  • the peptide linker comprises an amino acid sequence or a polynucleotide sequence encoding the same, that is at least or exactly 73%, 82%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20 or 21.
  • a construct described herein comprises more than one ISR inhibitor selected from the group of proteins consisting of CReP, GADD34, y34.5, CNPV231, ICP34.5, AmEPV193, and DP71L, and/or characteristic portions thereof.
  • a construct described herein comprises a heterologous promoter that is a CAG promoter.
  • a CAG promoter comprises a polynucleotide sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25 or 26.
  • a CAG promoter comprises a polynucleotide sequence according to SEQ ID NO: 25 or 26.
  • a construct described herein comprises an ISR inhibitor that does not comprise and/or is not fused to a Glutathione S-transferase (GST) polypeptide.
  • GST Glutathione S-transferase
  • a construct described herein is comprised in a retroviral capsid.
  • described herein is an AAV particle comprising any of the constructs disclosed herein, wherein the construct is comprised in an adeno associated virus (AAV) capsid.
  • AAV adeno associated virus
  • the AAV particle is of any one of the AAV-DJ/8, AAV9, AAV Php.B, and/or AAV Php.eB serotypes and/or pseudotypes.
  • the AAV particle is capable of retrograde infection.
  • the AAV particle comprises a nucleotide construct comprising a polynucleotide sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22.
  • the AAV particle comprises a polynucleotide sequence according to SEQ ID NO: 22.
  • the AAV capsid is an AAV-DJ/8, AAV9, AAV Php.B, and/or AAV Php.eB capsid.
  • polypeptides comprising an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, a polypeptide comprises an amino acid sequence according to SEQ ID NO: 4.
  • compositions comprising a non-natural construct, polypeptide, and/or AAV particle described herein.
  • cells comprising a non-natural construct, polypeptide, AAV particle, and/or composition disclosed herein.
  • Also disclosed herein are methods of delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing cognitive dysfunction in a subject comprising administering a non-natural construct, polypeptide, AAV particle, or composition disclosed herein to the subject (e.g., the mammalian subject, e.g., the human subject).
  • the administering is to the central nervous system.
  • the administering is to the peripheral nervous system.
  • the administering is to the cerebral spinal fluid (CSF).
  • the administering is to the hippocampus.
  • the administering is to the CAI, CA2, and/or CA3 region of the hippocampus. In some embodiments, the administering improves synaptic plasticity in the subject. In some embodiments, the administering improves long term memory formation in the subject. In some embodiments, the administering inhibits and/or counteracts the effects of one or more of Protein kinase R-like endoplasmic reticulum kinase (PERK), eIF-2-alpha kinase (GCN2), protein kinase RNA-activated (PKR), and/or Heme-regulated eIF2a kinase (HRI) in the subject.
  • PERK Protein kinase R-like endoplasmic reticulum kinase
  • GCN2 eIF-2-alpha kinase
  • PSR protein kinase RNA-activated
  • HRI Heme-regulated eIF2a kinase
  • the administering localizes a phosphorylase to an eIF2 protein.
  • the subject has been diagnosed with a disease and/or disorder associated with cognitive impairment or dysfunction.
  • the subject has a neurodevelopmental disorder.
  • the subject has a neurodegenerative disorder.
  • the subject has and/or is anticipated to develop Down Syndrome, Alzheimer’s disease, traumatic brain injury, vanishing white matter (VWM) disease, frontotemporal dementia, and/or aging-related cognitive decline.
  • VWM vanishing white matter
  • the disease is a cognitive disorder, neurodegeneration, cancer, diabetes, and/or a metabolic disorder.
  • a subject is less than 10 years of age.
  • a subject is older than 30 years of age.
  • a subject is older than 50 years of age.
  • a subject is older than 70 years of age.
  • a subject is a mammal. In some embodiments, a subject is a human.
  • methods of delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing cognitive dysfunction in a human subject comprising administering to the human subject: A) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence according to SEQ ID NO: 18; B) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence according to SEQ ID NO: 4; C) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence at least or exactly 85% , 90, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to SEQ ID NO: 18, wherein the inhibitor comprises a protein phosphatase-1 (PPI) binding domain and an eukaryotic initiation factor 2 (eIF2) binding domain; or D
  • PPI protein phosphatase-1
  • the polynucleotide comprises a polynucleotide sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22.
  • the polynucleotide comprises a sequence encoding a peptide linker sequence is at least or exactly 73%, 82%, 91%, or 100% identical to SEQ ID NO: 21.
  • the polynucleotide comprises a sequence encoding a CReP chimeric protein comprising an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4.
  • the polynucleotide is operably linked to a heterologous promoter.
  • the heterologous promoter is not a galactose-inducible promoter.
  • the disease is associated with activation, hyperactivation, and/or aberrant activation of the ISR in a human subject.
  • transgenic mice comprising a mutation in a Ppplrl5b gene.
  • the mutation is a R658C mutation.
  • the mutation is generated using an endonuclease.
  • the endonuclease is Cas9.
  • the generation comprises contacting the Ppplrl5b gene with a guide RNA comprising SEQ ID NO: 38.
  • Aspect 1 is a non-natural polynucleotide construct comprising a polynucleotide sequence encoding one or more inhibitor of the integrated stress response (ISR), wherein the one or more inhibitor comprises a protein phosphatase-1 (PPI) binding domain and an eukaryotic initiation factor 2 (eIF2) binding domain, wherein the non-natural polynucleotide construct comprises: A) a heterologous promoter operably linked to the polynucleotide sequence encoding one or more inhibitors of the ISR, wherein the heterologous promoter is not a galactose inducible promoter; and/or B) a recombinant viral vector comprising a heterologous promoter operably linked to the polynucleotide sequence encoding one or more inhibitors of the ISR, wherein the heterologous promoter is not a galactose inducible promoter.
  • ISR integrated stress response
  • PPI protein phosphatase-1
  • Aspect 2 is the construct of aspect 1, further comprising a peptide linker sequence connecting the PPI binding domain and eIF2 binding domain.
  • Aspect 3 is the construct of aspects 1 or 2, wherein the peptide linker sequence is at least or exactly 73%, 82%, 91%, or 100% identical to SEQ ID NO: 21.
  • Aspect 4 is the construct of any one of aspects 1-3, wherein the linker sequence comprises a glutamic acid at the DP71L protein E12 position according to SEQ ID NO: 18.
  • Aspect 5 is the construct of any one of aspects 1-4, wherein the inhibitor comprises an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4.
  • Aspect 6 is the construct of any one of aspects 1-5, wherein the inhibitor comprises an amino acid sequence according to SEQ ID NO: 4.
  • Aspect 7 is the construct of any one of aspects 1-4, wherein the inhibitor comprises an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18.
  • Aspect 8 is the construct of any one of aspects 1-5, wherein the inhibitor comprises an amino acid sequence according to SEQ ID NO: 18.
  • Aspect 9 is the construct of any one of aspects 1-8, wherein the heterologous promoter is a CAG promoter.
  • Aspect 10 is the construct of any one of aspects 1-9, wherein the non-natural construct is comprised in a retroviral capsid.
  • Aspect 11 is an AAV particle comprising the construct of any one of aspects 1-10 comprised in an adeno associated virus (AAV) capsid.
  • AAV adeno associated virus
  • Aspect 12 is the AAV particle of aspect 11, wherein the AAV particle is of any one of the AAV-DJ/8, AAV9, AAV Php.B, and/or AAV Php.eB serotypes and/or pseudotypes.
  • Aspect 13 is the AAV particle of aspects 11 or 12, wherein the AAV particle is capable of retrograde infection.
  • Aspect 14 is an AAV particle comprising a nucleotide construct comprising a polynucleotide sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22.
  • Aspect 15 is the AAV particle of aspect 14, comprising a polynucleotide sequence according to SEQ ID NO: 22.
  • Aspect 16 is an AAV particle of aspects 14 or 15, wherein the AAV capsid is an AAV-DJ/8, AAV9, AAV Php.B, and/or AAV Php.eB capsid.
  • Aspect 17 is a polypeptide comprising an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4.
  • Aspect 18 is the polypeptide of aspect 17, comprising an amino acid sequence according to SEQ ID NO: 4.
  • Aspect 19 is a pharmacologically acceptable composition comprising the nonnatural construct, polypeptide, or AAV particle of any one of aspects 1-18.
  • Aspect 20 is a human cell comprising the non-natural construct, polypeptide, AAV particle, or composition of any one of aspects 1-19.
  • Aspect 21 is a method of delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing cognitive dysfunction in a human subject, comprising administering the non-natural construct, polypeptide, AAV particle, or composition according to any one of aspects 1-20 to the human subject.
  • Aspect 22 is the method of aspect 21, wherein the administering is to the central nervous system.
  • Aspect 23 is the method of aspect 21, wherein the administering is to the peripheral nervous system.
  • Aspect 24 is the method of aspect 21, wherein the administering is to the cerebral spinal fluid (CSF).
  • CSF cerebral spinal fluid
  • Aspect 25 is the method of aspects 21 or 22, wherein the administering is to the hippocampus.
  • Aspect 26 is the method of aspect 25, wherein the administering is to the CAI, CA2, and/or CA3 region of the hippocampus.
  • Aspect 27 is the method of any one of aspects 21-26, wherein the human subject has been diagnosed with a disease and/or disorder associated with cognitive impairment or dysfunction.
  • Aspect 28 is the method of any one of aspects 21-27, wherein the subject has a neurodevelopmental disorder.
  • Aspect 29 is the method of any one of aspects 21-27, wherein the subject has a neurodegen erative disorder.
  • Aspect 30 is the method of any one of aspects 21-29, wherein the subject has and/or is anticipated to develop Down Syndrome, Alzheimer’s disease, traumatic brain injury, vanishing white matter (VWM) disease, frontotemporal dementia, and/or aging-related cognitive decline.
  • VWM vanishing white matter
  • Aspect 31 is a method of delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing a disease associated with activation, hyperactivation, and/or aberrant activation of the ISR in a human subject, comprising administering the non- natural construct, polypeptide, AAV particle, or composition according to any one of aspects 1-20 to the human subject.
  • Aspect 32 is the method of aspect 31, where the disease is a cognitive disorder, neurodegeneration, cancer, diabetes, and/or a metabolic disorder.
  • Aspect 33 is a method of delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing cognitive dysfunction in a human subject, comprising administering to the human subject: a) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence according to SEQ ID NO: 18; b) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence according to SEQ ID NO: 4; c) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence at least or exactly 85% , 90, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to SEQ ID NO: 18, wherein the inhibitor comprises a protein phosphatase-1 (PPI) binding domain and an eukaryotic initiation factor 2 (eIF2) binding domain; or d) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence according
  • Aspect 34 is the method of aspect 33, wherein the polynucleotide comprises a sequence encoding an inhibitor amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18.
  • Aspect 35 is the method of aspect 33, wherein the polynucleotide comprises a sequence encoding an inhibitor amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4.
  • Aspect 36 is the method of any one of aspects 33-35, wherein the polynucleotide comprises a sequence encoding a peptide linker sequence is at least or exactly 73%, 82%, 91%, or 100% identical to SEQ ID NO: 21.
  • Aspect 37 is the method of any one of aspects 33-36, wherein the polynucleotide is operably linked to a heterologous promoter.
  • Aspect 38 is the method of aspect 37, wherein the heterologous promoter is not a galactose-inducible promoter.
  • Aspect 39 is the method of any one of aspects 33-38, wherein the disease is associated with activation, hyperactivation, and/or aberrant activation of the ISR in a human subject.
  • Aspect 40 is a transgenic mouse comprising a mutation in a Ppplrl5b gene.
  • Aspect 41 is the transgenic mouse of aspect 40, wherein the mutation is a R658C mutation.
  • Aspect 42 is the transgenic mouse of aspects 40 or 41, wherein the mutation is generated using an endonuclease.
  • Aspect 43 is the transgenic mouse of aspect 42, wherein the endonuclease is Cas9.
  • Aspect 44 is the transgenic mouse of aspect 43, wherein the generation comprises contacting the Ppplrl5b gene with a guide RNA comprising SEQ ID NO: 38
  • FIG. 1 (PRIOR ART) The molecular wiring of the ISR.
  • the ISR kinases phosphorylate eIF2 in response to different cellular stresses. Phosphorylation of eIF2 leads to inhibition of eIF2B activity in the cell, thus reducing ternary complex (eIF2»GTP»methionyl- initiator tRNA) levels, which are controlled by GDP/GTP exchange on eIF2 by eIF2B.
  • the concentration of ternary complex determines the translational status of general protein synthesis (green) and translation of specific mRNAs, such as ATF4 (red).
  • Two phosphatase complexes antagonize the ISR, PP1CReP in a constitutive regime and PP 1 ⁇ GADD34 in a feedback regime in response to ISR activation.
  • FIGs. 2A-J The ISR was activated and translation was reduced in Ppp1r15b R658C mice.
  • FIGs. 3A-F Long-term fear and object recognition memory is impaired in Ppp1r15b R658C mice.
  • (3E) Novel object Discrimination Index (DI) was computed as DI (Novel Object Exploration Time - Familiar Object Exploration Time/Total Exploration Time) X 100 (see e.g., as described in Zhu, et al., 2019).
  • L-LTP Late Long-Term Potentiation
  • FIGs. 4A-D, DP71L was a potent pan-inhibitor of the ISR.
  • (4B-D) DP71L-GFP prevented the induction of the ISR by different stresses (oligomycin, poly(I:C), and thapsigargin, respectively (n 4 per group)).
  • 5A Schematic of DP71L and ACREP. Protein domains involved in the interaction of the eIF2a phosphatase cofactors with PPI and eIF2.
  • 5B Western blots revealed the activation of the ISR in cells transfected with either GRP, GFP-DP71L or GFP- ACREP.
  • 5C In vivo interaction between GFP-tagged DP71L or CREP with endogenous PPI or eIF2a. Levels of PPly, PPla and eIF2 in immunoprecipitates from GFP-DP71L or GFP-ACREP.
  • FIGs. 6A-D The interlink (also termed “linker” or “peptide linker”) region between PPI and eIF2a binding domains is necessary and sufficient for DP71L binding to PPL
  • linker also termed “linker” or “peptide linker”
  • 6A Schematic of different chimeric proteins and protein domains involved in PPI and eIF2 binding (e.g., DP71L, ACREP, and the linker mutant proteins (C8, C20, and D20 respectively)).
  • 6B Human HEK293T cells were transfected with the different GFP-constructs and subsequently immunoprecipitated with an anti-GFP antibody. Shown are levels of PPly, and PPla in immunoprecipitates from the different GFP-tagged chimeric proteins.
  • (6C) Schematic of DP71L, ACREP, and the linker chimera proteins “DP71L-linker” and “ACREP-linker” respectively.
  • (6D) HEK293T cells were transfected with the different GFP-constructs and proteins of interest were subsequently immunoprecipitated with an anti-GFP antibody. The results showed levels of in vivo interactions between GFP-tagged chimeric proteins and endogenous PPly, and PPla in immunoprecipitates from GFP-tagged DP71L, ACREP, and the linker swapped chimeric proteins (DP71L-linker and ACREP-linker respectively).
  • FIGs. 7A-C Mutation of the hydrogen bonding glutamic acid in the linker of DP71L impairs PPI binding.
  • (7 A) Alpha fold prediction of the complex of DP71L with eIF2 and PPL
  • (7B) Zoomed in Alpha fold prediction showing PPI (cyan) and the analogous regions of DP71L (magenta) and CreP (yellow). The glutamic acid involved in hydrogen bonding is highlighted next to the analogous threonine in ACREP.
  • 8A-E DP71L injection rescued the long-term memory deficits in mouse models of Down syndrome and Alzheimer’s disease.
  • 8D Freezing behavior in APP/PS1 mice (“humanized” model of Alzheimer’s disease) injected with either DP71L-GFP or GFP. Freezing behavior was assessed during a 2-min period prior to conditioning (naive) and then during a 5-min period 24 hr after training.
  • the work described herein provides at least new compositions and methods for ISR inhibition, including mitigation of ISR associated disorders.
  • the ISR and certain aspects of the systems association with disease has been reviewed in Mauro Costa-Mattioli and Peter Walter 2020 (see e.g., Mauro Costa-Mattioli and Peter Walter, The integrated stress response: From mechanism to disease. Science 2020 Apr 24; 368(6489): eaat5313, which is incorporated herein by reference for the purposes described herein).
  • constructs, particles, polypeptides, polynucleotides, and/or compositions described herein can be utilized in methods for inhibiting the ISR.
  • constructs, particles, polypeptides, polynucleotides, and/or compositions described herein can be utilized in methods of treatment for diseases associated with natural activation, hyperactivation, and/or aberrant activation of the ISR.
  • diseases associated with natural activation, hyperactivation, and/or aberrant activation of the ISR may include, but is not limited to, cognitive disorders, neurodegeneration, cancer, diabetes, muscle loss (e.g., cachexia), and/or metabolic disorders.
  • compositions e.g., including components comprised in a composition, such as constructs (e.g., polynucleotide constructs such RNA and/or DNA constructs), particles, polypeptides, etc.) may be employed based on methods described herein.
  • constructs e.g., polynucleotide constructs such RNA and/or DNA constructs
  • particles e.g., polypeptides, etc.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • “and/or” operates as an inclusive or.
  • compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.
  • the terms “individual, “subject,” and “patient” are used interchangeably and can refer to a human or non-human.
  • a “protein” or “polypeptide” refers to a molecule comprising at least five amino acid residues.
  • wild-type refers to the endogenous version of a molecule that occurs naturally in an organism.
  • wild-type versions of a protein or polypeptide are employed, however, in many embodiments of the disclosure, a modified and/or non-natural protein or polypeptide is employed to modulate (e.g., inhibit) ISR.
  • the terms described above may be used interchangeably.
  • a “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide.
  • a modified/variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified/variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity.
  • a “non-natural” occurring polypeptide and/or construct refers to an engineered polypeptide and/or construct that has been created by the hand of man and is not found in nature.
  • a non-natural polypeptide and/or construct is also understood to comprise non-natural amino acids and/or non-natural nucleotides.
  • a protein may be isolated directly from the organism of which it is native, produced by recombinant DNA/exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. In some embodiments, a protein is produced from an RNA construct.
  • RNA constructs encoding a protein can be produced synthetically and/or by in vitro methods.
  • the term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.
  • any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.
  • any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention.
  • any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.
  • Any embodiment discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa.
  • any step in a method described herein can apply to any other method.
  • any method described herein may have an exclusion of any step or combination of steps.
  • ISR Integrated Stress Response
  • technologies e.g., compositions and/or methods described herein are suitable for delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing a disease associated with activation, hyperactivation, and/or aberrant activation of the ISR in a human subject.
  • a disease is a disease associated with cognitive decline
  • technologies provided herein are suitable for delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing cognitive decline and/or other symptoms associated with the disease state.
  • cognitive decline is correlated with and/or caused by activation of the Integrated Stress Response (ISR) system.
  • ISR Integrated Stress Response
  • technologies described herein are suitable for the reversal, at least in part, of deficits in cognitive function (e.g., impaired cognitive function, such as but not limited to, impaired long term memory (LTM) formation, and/or retention).
  • technologies described herein are suitable for the reduction of risk of loss of LTM formation capacity.
  • technologies described herein are suitable for the treatment of loss of LTM formation capacity.
  • technologies described herein are suitable for the improvement of LTM formation capacity.
  • compositions and/or methods disclosed herein improve long-term potentiation (LTP) and/or synaptic plasticity.
  • compositions and/or methods disclosed herein improve long-lasting LTP.
  • compositions and/or methods disclosed herein improve late LTP.
  • technologies described herein are suitable to provide ISR inhibition in conditions of ISR hyperactivation, e.g., in conditions where traditional ISR inhibitory compositions (e.g., small molecules such as kinase inhibitors and/or ISRIB) are not sufficient for achievement of therapeutic disease state amelioration and/or prevention.
  • traditional ISR inhibitory compositions e.g., small molecules such as kinase inhibitors and/or ISRIB
  • Cognitive function may be assessed using methods (e.g. screening tests and/or questionnaires) well known to one of skill in the art, including but not limited to, e.g, assessment of attention, orientation, language, memory, visuospatial ability, social interaction, functional status and/or behavioral assessment.
  • methods e.g. screening tests and/or questionnaires
  • assessments including but not limited to, e.g, assessment of attention, orientation, language, memory, visuospatial ability, social interaction, functional status and/or behavioral assessment.
  • technologies described herein are suitable for prevention of, treatment of, and/or reduction of risk of various nervous system disorders (e.g., neurodegenerative disorders, neurodevelopmental disorders, etc.) associated with activation of ISR.
  • technologies described herein are suitable for improving brain function (e.g., brain cognitive function), including but not limited to, LTM formation and/or retention, associated with or not associated with activation of ISR.
  • a cognitive disorder is a neurological disorder, and is characterized in part by neurodegeneration and/or aberrant neurodevelopment (e.g., a neurodevelopmental disorder).
  • a neurological disorder may be but is not limited to Down syndrome (DS), Charcot-Marie-Tooth disease, major depressive disorder (MDD), schizophrenia, Alzheimer’s disease, Huntington disease, Parkinson’s disease, Amyotrophic lateral sclerosis (ALS), Multiple Sclerosis (MS), Prion disease, traumatic brain injury, Vanishing white matter (VWM) disease, frontotemporal dementia, and/or Aging (e.g., age-related cognitive decline).
  • technologies provided herein are suitable for delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing cognitive dysfunction associated with a cognitive disorder. In certain embodiments, technologies provided herein are suitable for delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing a neurological disorder.
  • Down syndrome is the most common genetic form of intellectual disability (ID) and occurs in ⁇ 10 of 10 000 live births (see e.g., Khoshnood et al., 2011). Individuals with DS show deficits in learning and memory, language and executive functions since early childhood, resulting from the presence of an extra copy of chromosome 21 (Hsa21). DS patients exhibit an age-dependent reduction in dendritic branching and spine density that likely involves impaired reorganization of the actin cytoskeleton in neurons (Marin-Padilla 1976; Takashima et al. 1981). In addition, DS patients show early onset Alzheimer-like neurodegeneration (reviewed in e.g., Lott and Dierssen 2010).
  • constructs and/or compositions described herein are suitable for the prevention, treatment, and/or alleviation of symptoms associated with Down syndrome.
  • Alzheimer’s disease is biologically defined by the presence of P-amyloid- containing plaques and tau-containing neurofibrillary tangles.
  • Alzheimer’s disease is a genetic and sporadic neurodegenerative disease that causes an amnestic cognitive impairment in its prototypical presentation and non-amnestic cognitive impairment in its less common variants.
  • Alzheimer’s disease is a common cause of cognitive impairment acquired in midlife and late- life but its clinical impact is modified by other neurodegenerative and cerebrovascular conditions.
  • Alzheimer’s disease biology can be thought of as a brain disorder that results from a complex interplay of loss of synaptic homeostasis and dysfunction in the highly interrelated endosomal/lysosomal clearance pathways in which the precursors, aggregated species and post-translationally modified products of Ap and tau play important roles.
  • Loss of synaptic homeostasis is known to promote the ISR.
  • Certain aspects of the ISR in Alzheimer’s disease are described in Tible et al., 2019; Hwang et al., 2017; and Lourenco et al., 2013; each of which are incorporated herein by reference for the purposes described herein.
  • constructs and/or compositions described herein are suitable for the prevention, treatment, and/or alleviation of symptoms associated with Alzheimer’s disease.
  • VWM Vanishing white matter
  • CACH central nervous system hypomyelination
  • the disease can get worse if the child has a fever, an infection, or a head injury.
  • an adult onset form of VWM may appear, and is often accompanied by behavioral abnormalities, severe headaches, and/or cognitive decline.
  • Certain roles of the ISR in VWM disease are described in Abbink et al., 2019 (see e.g., Abbink et al., Vanishing white matter: deregulated integrated stress response as therapy target. Ann Clin Transl Neurol. 2019 Aug; 6(8): 1407- 1422; which is incorporated herein by reference for the purposes described herein).
  • constructs and/or compositions described herein are suitable for the prevention, treatment, and/or alleviation of symptoms associated with VWM disease.
  • Frontotemporal dementia is an array of disorders that affect the frontal and temporal lobes of the brain. In certain cases, personality, emotions, behavior, memory formation, and speech are all controlled by these portions of the brain, and are lost as the associated cells lose their function. Symptoms of frontotemporal dementia are dependent upon the areas of the brain affected, but most symptoms can be categorized as either behavioral or language altering. In some embodiments, behavioral symptoms can include inappropriate actions, apathy, lack of interest and/or enthusiasm, lack of inhibition or restraint, neglect of person hygiene and/or care, and/or compulsive behavior. In some embodiments, language symptoms can include difficulty speaking and/or understanding speech, difficulty recalling appropriate language, loss of reading and/or writing skills, and/or difficulty with social interactions.
  • frontotemporal dementia may develop abnormal protein structures known as pick bodies.
  • the most common genetic cause of frontotemporal dementia are repeat expansions in the C9orf72 gene.
  • Protein accumulation associated with frontotemporal dementia activates the ISR response via the protein kinase PERK.
  • Certain roles of the ISR in frontotemporal dementia are described in Radford et al., 2015 (see e.g., Radford et al., PERK inhibition prevents tau-mediated neurodegeneration in a mouse model of frontotemporal dementia. Acta Neuropathol 130, 633-642 (2015); and Costa-Mattioli & Walter, 2020; each of which are incorporated herein by reference for the purposes described herein).
  • constructs and/or compositions described herein are suitable for the prevention, treatment, and/or alleviation of symptoms associated with frontotemporal dementia.
  • constructs and/or compositions described herein are suitable for the prevention, treatment, and/or alleviation of symptoms associated with aging. In certain embodiments, constructs and/or compositions described herein are suitable for the prevention, treatment, and/or alleviation of symptoms associated with aging related cognitive decline.
  • constructs, particles, polypeptides, polynucleotides, and/or compositions described herein may be used in a method of preventing, treating, reducing the progression of, and/or reducing the risk of a disease or disorder associated with ISR activation (e.g., normal activation, hyperactive, and/or aberrant activation) in a human subject.
  • ISR activation e.g., normal activation, hyperactive, and/or aberrant activation
  • constructs, particles, polypeptides, polynucleotides, and/or compositions described herein may be used in treating a disease or disorder, wherein the disease or disorder is a neurodegenerative disease, an inflammatory disease, an autoimmune disease, a metabolic syndrome, a cancer, a vascular disease, a fibrotic disease, a viral infection, a musculoskeletal disease (such as a myopathy), an ocular disease, or a genetic disorder.
  • the disease or disorder is a neurodegenerative disease, an inflammatory disease, an autoimmune disease, a metabolic syndrome, a cancer, a vascular disease, a fibrotic disease, a viral infection, a musculoskeletal disease (such as a myopathy), an ocular disease, or a genetic disorder.
  • the disease or disorder is a neurodegenerative disease.
  • the neurodegenerative disease is vanishing white matter disease, childhood ataxia with CNS hypomyelination, intellectual disability syndrome, Alzheimer’s disease, prion disease, Creutzfeldt- Jakob disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS) disease, Pelizaeus-Merzbacher disease, a cognitive impairment, a traumatic brain injury, a postoperative cognitive dysfunction (PCD), a neuro-otological syndrome, hearing loss, Huntington’s disease, stroke, chronic traumatic encephalopathy, spinal cord injury, dementia, frontotemporal dementia (FTD), depression, or a social behavior impairment.
  • ALS amyotrophic lateral sclerosis
  • PCD postoperative cognitive dysfunction
  • Huntington’s disease stroke, chronic traumatic encephalopathy, spinal cord injury, dementia, frontotemporal dementia (FTD), depression, or a social behavior impairment.
  • the cognitive impairment is triggered by ageing, radiation, sepsis, seizure, heart attack, heart surgery, liver failure, hepatic encephalopathy, anesthesia, brain injury, brain surgery, ischemia, chemotherapy, cancer treatment, critical illness, concussion, fibromyalgia, or depression.
  • the neurodegenerative disease is Alzheimer’s disease.
  • the neurodegenerative disease is ageing-related cognitive impairment.
  • the neurodegenerative disease is a traumatic brain injury.
  • constructs, particles, polypeptides, polynucleotides, and/or compositions described herein may be used in a method of treating Alzheimer’s disease.
  • neurodegeneration, and/or cognitive impairment is decreased.
  • the disease or disorder is an inflammatory disease.
  • the inflammatory disease is arthritis, psoriatic arthritis, psoriasis, juvenile idiopathic arthritis, asthma, allergic asthma, bronchial asthma, tuberculosis, chronic airway disorder, cystic fibrosis, glomerulonephritis, membranous nephropathy, sarcoidosis, vasculitis, ichthyosis, transplant rejection, interstitial cystitis, atopic dermatitis, or inflammatory bowel disease.
  • the inflammatory bowel disease is Crohn’ disease, ulcerative colitis, or celiac disease.
  • the disease or disorder is an autoimmune disease.
  • the autoimmune disease is systemic lupus erythematosus, type 1 diabetes, multiple sclerosis, or rheumatoid arthritis.
  • the disease or disorder is a metabolic syndrome.
  • the metabolic syndrome is acute pancreatitis, chronic pancreatitis, alcoholic liver steatosis, obesity, glucose intolerance, insulin resistance, hyperglycemia, fatty liver, dyslipidemia, hyperlipidemia, hyperhomocysteinemia, or type 2 diabetes.
  • the metabolic syndrome is alcoholic liver steatosis, obesity, glucose intolerance, insulin resistance, hyperglycemia, fatty liver, dyslipidemia, hyperlipidemia, hyperhomocysteinemia, or type 2 diabetes.
  • the disease or disorder is a cancer.
  • the cancer is pancreatic cancer, breast cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, urothelial cancer, endometrial cancer, ovarian cancer, cervical cancer, renal cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), multiple myeloma, cancer of secretory cells, thyroid cancer, gastrointestinal carcinoma, chronic myeloid leukemia, hepatocellular carcinoma, colon cancer, melanoma, malignant glioma, glioblastoma, glioblastoma multiforme, astrocytoma, dysplastic gangliocytoma of the cerebellum, Ewing’s sarcoma, rhabdomyosarcoma, ependymoma, medulloblastoma, ductal adenocarcinoma, adenosquamous carcinoma, nephroblastoma
  • the cancer of secretory cells is non-Hodgkin’s lymphoma, Burkitt’s lymphoma, chronic lymphocytic leukemia, monoclonal gammopathy of undetermined significance (MGUS), plasmacytoma, lymphoplasmacytic lymphoma or acute lymphoblastic leukemia.
  • the disease or disorder is a musculoskeletal disease (such as a myopathy).
  • the musculoskeletal disease is a myopathy, a muscular dystrophy, a muscular atrophy, a muscular wasting, or sarcopenia.
  • the muscular dystrophy is Duchenne muscular dystrophy (DMD), Becker’s disease, myotonic dystrophy, X-linked dilated cardiomyopathy, spinal muscular atrophy (SMA), or metaphyseal chondrodysplasia, Schmid type (MCDS).
  • the myopathy is a skeletal muscle atrophy.
  • the musculoskeletal disease (such as the skeletal muscle atrophy) is triggered by ageing, chronic diseases, stroke, malnutrition, bedrest, orthopedic injury, bone fracture, cachexia, starvation, heart failure, obstructive lung disease, renal failure, Acquired Immunodeficiency Syndrome (AIDS), sepsis, an immune disorder, a cancer, ALS, a burn injury, denervation, diabetes, muscle disuse, limb immobilization, mechanical unload, myositis, or a dystrophy.
  • ageing chronic diseases, stroke, malnutrition, bedrest, orthopedic injury, bone fracture, cachexia, starvation, heart failure, obstructive lung disease, renal failure, Acquired Immunodeficiency Syndrome (AIDS), sepsis, an immune disorder, a cancer, ALS, a burn injury, denervation, diabetes, muscle disuse, limb immobilization, mechanical unload, myositis, or a dystrophy.
  • constructs, particles, polypeptides, polynucleotides, and/or compositions described herein may be used in a method of treating musculoskeletal disease.
  • skeletal muscle mass, quality and/or strength are increased.
  • synthesis of muscle proteins is increased.
  • skeletal muscle fiber atrophy is inhibited.
  • the disease or disorder is a vascular disease.
  • the vascular disease is atherosclerosis, abdominal aortic aneurism, carotid artery disease, deep vein thrombosis, Buerger’s disease, chronic venous hypertension, vascular calcification, telangiectasia or lymphoedema.
  • the disease or disorder is an ocular disease.
  • the ocular disease is glaucoma, age-related macular degeneration, inflammatory retinal disease, retinal vascular disease, diabetic retinopathy, uveitis, rosacea, Sjogren's syndrome, or neovascularization in proliferative retinopathy.
  • a method of modulating an ISR pathway comprises modulating the ISR pathway in a cell by administering or delivering to the cell a construct and/or compound described herein.
  • the method of modulating an ISR pathway comprises modulating the ISR pathway in an individual by administering to the individual a construct and/or compound described herein.
  • modulating of the ISR pathway can be determined by methods known in the art, such as western blot, immunohistochemistry, or reporter cell line assays.
  • constructs e.g., polynucleotide constructs, e.g., linear and/or circular polynucleotide constructs, e.g., DNA and/or RNA constructs
  • polypeptides and/or compositions and/or methods of using the same, that consist of, consist essentially of, and/or comprise an inhibitor of the Integrated Stress Response (ISR) or a characteristic portion thereof and/or encode the same.
  • ISR Integrated Stress Response
  • the size of a protein or polypeptide may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,
  • polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.).
  • domain refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art (e.g., a PPI binding domain, an eIF2 binding domain, a peptide linker, etc. .
  • polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids or nucleic acid substitutions or be at least or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any de
  • a protein, polypeptide, or nucleic acid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
  • the polypeptide, protein, or nucleic acid may consist of, consist essentially of, or comprise at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
  • nucleotide as well as the protein, polypeptide, and peptide sequences for various genes have been previously disclosed, and may be found in the recognized computerized databases.
  • Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov/) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org).
  • Genbank and GenPept databases on the World Wide Web at ncbi.nlm.nih.gov/
  • the Universal Protein Resource UniProt; on the World Wide Web at uniprot.org.
  • the coding regions for these genes may be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.
  • an inhibitor of ISR according to the present disclosure consists of, consists essentially of, or comprises an amino acid sequence as described in Table 1.
  • a dash (-) as denoted in Table 1 is quantitative and stands for any amino acid.
  • an inhibitor of ISR consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, having at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NOs: 39 to 447.
  • an inhibitor of ISR according to the present disclosure consists of, consists essentially of, or comprises a constitutive repressor of eIF2a phosphorylation (CReP) protein (also known as Protein phosphatase 1 regulatory subunit 15B; PPP1R15B).
  • an inhibitor of ISR according to the present disclosure consists of, consists essentially of, or comprises a chimera of CReP and DP71L.
  • a chimera of CReP and DP71L comprises PPI and/or eIF2 binding domains derived from CReP, and an interjoining peptide linker sequence derived from DP71L.
  • an inhibitor of ISR consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, having at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NOs: 1 to 6.
  • SEQ ID NO: 3 Exemplary chimeric CReP polynucleotide sequence
  • SEQ ID NO: 4 Exemplary chimeric CReP protein amino acid sequence
  • SEQ ID NO: 5 Exemplary ACReP protein polynucleotide encoding sequence
  • an inhibitor of ISR consists of, consists essentially of, or comprises a protein phosphatase 1 regulatory subunit 15A (GADD34) protein.
  • an inhibitor of ISR consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, having at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NOs: 7 to 8.
  • an inhibitor of ISR consists of, consists essentially of, or comprises a Herpes simplex virus derived y34.5 protein.
  • an inhibitor of ISR consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, having at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NOs: 9 to 10.
  • an inhibitor of ISR consists of, consists essentially of, or comprises a Canarypox virus derived CNPV231 protein (MyD116- like protein; accession Q6VZB6).
  • an inhibitor of ISR consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, having at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NOs: 11 to 12.
  • an inhibitor of ISR consists of, consists essentially of, or comprises a Macropoid herpes virus derived ICP34.5 protein.
  • an inhibitor of ISR consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, having at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NOs: 13 to 14.
  • an inhibitor of ISR consists of, consists essentially of, or comprises an Amsacta moorei entomopoxvirus “L” derived AmEPV193 protein (accession Q9EML3).
  • an inhibitor of ISR consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, having at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NOs: 15 to 16.
  • African swine fever virus derived protein DP71L African swine fever virus derived protein DP71L
  • an inhibitor of ISR consists of, consists essentially of, or comprises an African swine fever virus derived DP71L protein.
  • a DP71L protein is a long form protein, while alternatively, in some embodiments, a DP71L protein is a short form protein.
  • DP71L is in reference to the short form protein (e.g., as represented by SEQ ID NO: 18), however, in some embodiments, slightly longer or shorter protein isoforms may be utilized (e.g., a protein isoform comprising an additional, or missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, amino acids relative to SEQ ID NO: 18).
  • an inhibitor of ISR is a chimeric protein (e.g., a synthetic protein comprised of domains derived from two or more proteins) that comprises a peptide linker sequence deposited between a PPI binding domain and an eIF2 binding domain that is derived from DP71L.
  • a peptide linker sequence derived from DP71L comprises 8, 9, 10, 11, or 12, amino acids.
  • a peptide linker sequence derived from DP71L enhances the ability of an ISR inhibitor to bind to PPI.
  • a peptide linker sequence derived from DP71L comprises one or more glutamic acids that may influence PPI binding domain inter- or intraprotein binding dynamics.
  • the one or more glutamic acid is glutamic acid E12 of the DP71L protein (e.g., the glutamic acid underlined in the sequence “MDVKHVRFAAAVEVWEADDI " (SEQ ID NO: 17), which is the E12 glutamic acid found in SEQ ID NO: 18 (e.g., the 12 th amino acid when not including the N-terminal methionine, or the 13 th amino acid when including the N-terminal methionine).
  • a linker sequence derived from DP71L consists of, consists essentially of, or comprises an polynucleotide sequence and/or amino acid sequence at least or exactly about 55%, 64%, 73%, 82%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NO: 20 to 21.
  • an inhibitor of ISR consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, having at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NOs: 18 to 19.
  • the amino acid subunits of a protein are modified to create an equivalent, or even improved, second-generation variant polypeptide or peptide.
  • certain amino acids may be substituted for other amino acids in a protein or polypeptide sequence with or without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein’s functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and nevertheless produce a protein with similar or desirable properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes which encode proteins without appreciable loss of their biological utility or activity.
  • the term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids.
  • Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants.
  • a variation in a polypeptide of the disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide, as compared to wild-type.
  • a variant can comprise an amino acid sequence that is at least or exactly about 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein.
  • a variant can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids.
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.
  • Deletion variants typically lack one or more residues of the native or wild type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein.
  • Insertional mutants typically involve the addition of amino acid residues at a nonterminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein or polypeptide, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar chemical properties.
  • Constant amino acid substitutions may involve exchange of a member of one amino acid class with another member of the same class.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to iso
  • substitutions may be “non-conservative”, such that a function or activity of the polypeptide is affected.
  • Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.
  • Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class.
  • inhibitors of ISR as described herein are encoded by a polynucleotide, such as a vector comprising a polynucleotide (e.g., a polynucleotide construct).
  • Vectors comprising polynucleotide constructs according to the present disclosure include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viral constructs (e.g., lentiviral, retroviral, adenoviral, and adeno associated viral constructs) that incorporate a polynucleotide comprising an inhibitor of ISR gene or characteristic portion thereof (e.g., as utilized herein, a “characteristic portion thereof’ refers to the portion of said protein required to perform the desired function, e.g., it comprises the ability to inhibit ISR, e.g., it comprises a functional PPI binding domain and an eIF2 binding domain).
  • a construct is a plasmid (i.e., a circular DNA molecule that can autonomously replicate inside a cell).
  • a construct can be a cosmid (e.g., pWE or sCos series).
  • a construct is a viral construct.
  • a viral construct is a lentivirus, retrovirus, adenovirus, or adeno-associated virus construct.
  • a construct is an adeno-associated virus (AAV) construct (see, e.g., Asokan et al., Mol. Then 20: 699-7080, 2012, which is incorporated herein by reference for the purposes described herein).
  • AAV adeno-associated virus
  • a viral construct is an adenovirus construct.
  • a viral construct may also be based on or derived from an alphavirus.
  • Alphaviruses include but are not limited to, Sindbis (and VEEV) virus, Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus, O’nyong-nyong virus, Pixuna virus, Rio Negro virus, Ross River virus, Salmon pancreas disease virus, Semliki Forest virus, Southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Whataroa virus.
  • viruses encode nonstructural (e.g., replicon) and structural proteins (e.g., capsid and envelope) that can be translated in the cytoplasm of the host cell.
  • Ross River virus, Sindbis virus, Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEEV) have all been used to develop viral constructs for coding sequence delivery.
  • Pseudotyped viruses may be formed by combining alphaviral envelope glycoproteins and retroviral capsids. Examples of alphaviral constructs can be found in U.S. Publication Nos. 20150050243, 20090305344, and 20060177819; constructs and methods of their making are incorporated herein by reference for the purposes described herein.
  • constructs provided herein can be of different sizes.
  • a construct is a plasmid and can include a total length of up to about 1 kb, up to about 2 kb, up to about 3 kb, up to about 4 kb, up to about 5 kb, up to about 6 kb, up to about 7 kb, up to about 8 kb, up to about 9 kb, up to about 10 kb, up to about 11 kb, up to about 12 kb, up to about 13 kb, up to about 14 kb, or up to about 15 kb.
  • a construct is a plasmid and can have a total length in a range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about
  • I I kb about 1 kb to about 12 kb, about 1 kb to about 13 kb, about 1 kb to about 14 kb, or about 1 kb to about 15 kb.
  • a construct is a viral construct and can have a total number of nucleotides of up to 10 kb.
  • a viral construct can have a total number of nucleotides in the range of about 1 kb to about 2 kb, 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 1 O kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 2 kb to about 9 kb,
  • a construct is a lentivirus construct and can have a total number of nucleotides of up to 8 kb.
  • a lentivirus construct can have a total number of nucleotides of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 4 kb, about 3 kb to about 4 kb, about 3 kb to about 5
  • a construct is an adenovirus construct and can have a total number of nucleotides of up to 8 kb.
  • an adenovirus construct can have a total number of nucleotides in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 4 kb, about 3 kb to about 4 kb, about 3 kb
  • any of the constructs described herein can further include a control sequence, e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly(A)) sequence, a Kozak consensus sequence, and/or additional untranslated regions which may house pre- or post-transcriptional regulatory and/or control elements.
  • a promoter can be a native promoter, a constitutive promoter, an inducible promoter, and/or a tissue-specific promoter.
  • control sequences are described herein.
  • AAV particles that comprise a polynucleotide construct encoding an inhibitor of ISR, and an AAV capsid.
  • AAV particles can be described as having a serotype, which is a description of the construct strain and the capsid strain.
  • an AAV particle may be described as AAV2, wherein the particle has an AAV2 capsid and a construct that comprises characteristic AAV2 Inverted Terminal Repeats (ITRs).
  • ITRs Inverted Terminal Repeats
  • an AAV particle may be described as a pseudotype, wherein the capsid and construct are derived from different AAV strains, for example, AAV2/9 would refer to an AAV particle that comprises a construct utilizing the AAV2 ITRs and an AAV9 capsid. Additional examples of pseudotyped AAV vectors include, but are not limited to, AAV2/1, AAV2/2, AAV2/3, AAV2/4, AAV2/5, AAV2/6, AAV2/7, AAV2/8 and AAV2/9.
  • AAV particles suitable for use according to the present disclosure may comprise or be derived from any natural or recombinant AAV serotype.
  • an AAV according to the present invention is selected from natural serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12; or pseudotypes, chimeras, and variants thereof.
  • chimera when referring to an AAV vector, or a “chimeric AAV vector”, refers to an AAV vector which comprises a capsid containing VP1, VP2 and VP3 proteins from at least two different AAV serotypes; or alternatively, which comprises VP1, VP2 and VP3 proteins, at least one of which comprises at least a portion from another AAV serotype.
  • chimeric AAV vectors include, but are not limited to, AAV-DJ, AAV-DJ/8, AAV2G9, AAV218, AAV218G9, AAV8G9, and AAV911.
  • an AAV serotype and/or pseudotype according to the present invention is selected from the group comprising or consisting of AAV1, AAV2, AAV3, AAV 4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV106.1/hu.37, AAV114.3/hu.4O, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.1/hu.43, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55, AAV16.12/hu.11 , AAV16.3, AAV16.8/hu.lO, AAV161.1O/hu.6O, AAV161.6/hu.61, AAVl-7/rh.48, AAVl-8/rh.49, AAV218, A
  • an AAV serotypes and/or pseudotype is AAV-DJ/8, AAV9, AAV Php.B, and/or AAV Php.eB.
  • an AAV serotype and/or pseudotype comprises AAV-DJ/8.
  • an AAV serotype and/or pseudotype comprises AAV9.
  • an AAV serotype and/or pseudotype comprises AAV Php.B.
  • an AAV serotype and/or pseudotype comprises AAV Php.eB.
  • an AAV is an AAV variant that has been genetically modified, e.g., by substitution, deletion or addition of one or several amino acid residues in one or more capsid proteins.
  • AAV2 with one or more of Y444F, Y500F, Y730F and/or S662V mutations
  • AAV3 with one or more of Y705F, Y731F and/or T492V mutations
  • AAV6 with one or more of S663V and/or T492V mutations.
  • an AAV capsid is modified to comprise at least one surfacebound saccharide or a derivative thereof.
  • surface-bound when referring to the at least one saccharide, means that said at least one saccharide is bound to and exposed at the outer surface of the AAV vector.
  • Suitable examples of saccharides include, but are not limited to, monosaccharides, oligosaccharides, polysaccharides, and derivatives thereof.
  • the present disclosure provides polynucleotide vectors (e.g., polynucleotide constructs) that comprise a polynucleotide sequence encoding an inhibitor of ISR or a characteristic portion thereof (e.g., comprising a PPI binding domain and an eIF2a binding domain).
  • a polynucleotide vector comprising an inhibitor of ISR is a polynucleotide construct, and can be comprised in an AAV capsid to produce an AAV particle (e.g., an AAV particle comprises an AAV construct comprised in an AAV capsid).
  • a polynucleotide construct comprises one or more components derived from or modified from a naturally occurring AAV genomic construct.
  • a sequence derived from an AAV construct is an AAV1 construct, an AAV2 construct, an AAV3 construct, an AAV4 construct, an AAV5 construct, an AAV6 construct, an AAV7 construct, an AAV8 construct, an AAV DJ/8 construct, an AAV9 construct, an AAV2.7m8 construct, an AAV8BP2 construct, an AAV293 construct, an AAVPhp.B construct, or AAVPhp.eB construct (see e.g., Chan et al., 2017).
  • Additional exemplary AAV constructs that can be used herein are known in the art. See, e.g., Kanaan etal., Mol. Ther. Nucleic Acids 8: 184- 197, 2017; Li et al., Mol. Ther. 16(7): 1252-1260, 2008; Adachi et al., Nat. Commun. 5: 3075, 2014; Isgrig et al., Nat. Commun. 10(1): 427, 2019; and Gao et al., J. Virol. 78(12): 6381-6388, 2004; each of which are incorporated herein by reference for the purposes described herein).
  • AAV derived sequences typically include the cis-acting 5' and 3' ITR sequences (see, e.g., B. J. Carter, in "Handbook of Parvoviruses,” ed., P. Tijsser, CRC Press, pp. 155 168, 1990, which is incorporated herein by reference for the purposes described herein).
  • Typical AAV2-derived ITR sequences are about 145 nucleotides in length.
  • At least or exactly 80% of a typical ITR sequence (e.g., at least or exactly 85%, at least or exactly 90%, at least or exactly 95%, or at least or exactly 100%, etc.) is incorporated into a construct provided herein.
  • the ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook etal., "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York, 1989; and K. Fisher et al., J Virol. 70:520 532, 1996, each of which is incorporated herein by reference for the purposes described herein).
  • any of the coding sequences and/or constructs described herein are flanked by 5' and 3' AAV ITR sequences.
  • the AAV ITR sequences may be obtained from any known AAV, including presently identified AAV types.
  • polynucleotide constructs described in accordance with this disclosure and in a pattern known to the art are typically comprised of, a coding sequence or a portion thereof, at least one and/or control sequence, and optionally 5' and 3' AAV inverted terminal repeats (ITRs).
  • provided constructs can be packaged into a capsid to create an AAV particle.
  • An AAV particle may be delivered to a selected target cell.
  • provided constructs comprise an additional optional coding sequence that is a nucleic acid sequence (e.g., inhibitory nucleic acid sequence), heterologous to the construct sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest.
  • a nucleic acid coding sequence is operatively linked to and/or control components in a manner that permits coding sequence transcription, translation, and/or expression in a cell of a target tissue.
  • an unmodified AAV endogenous genome includes two open reading frames, "cap” and "rep,” which are flanked by ITRs.
  • recombinant AAV constructs similarly comprise one or more open reading frames (e.g., a coding sequence comprising an ISR inhibitor encoding sequence) flanked by ITR sequences.
  • an AAV construct also comprises conventional control elements that are operably linked to the coding sequence in a manner that permits its transcription, translation and/or expression in a cell transfected with the polynucleotide construct or infected with a virus particle produced by the disclosure.
  • an AAV construct optionally comprises a promoter, an enhancer, an untranslated region (e.g., a 5' UTR, 3' UTR), a Kozak sequence, an internal ribosomal entry site (IRES), splicing sites (e.g., an acceptor site, a donor site), a polyadenylation site, or any combination thereof.
  • a construct is an AAV construct.
  • an AAV construct can include at least 500 bp, at least 1 kb, at least 1.5 kb, at least 2 kb, at least 2.5 kb, at least 3 kb, at least 3.5 kb, at least 4 kb, or at least 4.5 kb.
  • an AAV construct can include at most 7.5 kb, at most 7 kb, at most 6.5 kb, at most 6 kb, at most 5.5 kb, at most 5 kb, at most 4.5 kb, at most 4 kb, at most 3.5 kb, at most 3 kb, or at most 2.5 kb.
  • an AAV construct can include about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.
  • any of the constructs described herein can further include regulatory and/or control sequences, e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly(A)) sequence, a Kozak consensus sequence, and/or any combination thereof.
  • a promoter can be a native promoter, a constitutive promoter, an inducible promoter, and/or a tissue-specific promoter.
  • Non-limiting examples of control sequences are described herein and others are known in the art.
  • SEQ ID NO: 22 Exemplary AAV construct polynucleotide sequence
  • an AAV capsid is from or is derived from an AAV capsid of an AAV2, 3, 4, 5, 6, 7, 8, 9, 10, rh8, rhlO, rh39, rh43 or Ancestral serotype, or one or more hybrids thereof.
  • an AAV capsid is from an AAV ancestral serotype.
  • an AAV capsid is an ancestral (Anc) AAV capsid.
  • An Anc capsid is created from a construct sequence that is constructed using evolutionary probabilities and evolutionary modeling to determine a probable ancestral sequence.
  • an AAV capsid/construct sequence is not known to have existed in nature.
  • an AAV capsid is engineered and/or derived from an AAV9 capsid.
  • an AAV capsid is an AAV PHP.eB capsid.
  • an AAV capsid is an AAV PHP.B capsid (see e.g., Diptaman Chatterjee et al., Gene Therapy 29, 2290-387 (2022)).
  • any combination of AAV capsids and AAV constructs may be used in recombinant AAV particles of the present disclosure.
  • an AAV particle is wholly comprised of AAV2 and/or AAV9 components (e.g., capsid and ITRs are AAV2 and/or AAV9 serotype).
  • an AAV particle is an AAV2/6, AAV2/8 or AAV2/9 particle (e.g., an AAV6, AAV8 or AAV9 capsid with an AAV construct having AAV2 ITRs.
  • ITRs Inverted Terminal Repeat Sequences
  • AAV derived sequences of a construct typically comprises the cis-acting 5' and 3' ITRs (See, e.g., B. J. Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990), which is incorporated herein by reference for the purposes described herein).
  • ITRs are able to form a hairpin. The ability to form a hairpin can contribute to an ITRs ability to self-prime, allowing primase-independent synthesis of a second DNA strand. ITRs can also aid in efficient encapsidation of an AAV construct in an AAV particle.
  • An AAV particle of the present disclosure can comprise an AAV construct comprising a coding sequence (e.g., encoding an inhibitor of ISR) and associated elements flanked by a 5' and a 3' AAV ITR sequences.
  • a coding sequence e.g., encoding an inhibitor of ISR
  • an ITR is or comprises about 130 nucleic acids.
  • an ITR is or comprises about 145 nucleic acids.
  • all or substantially all of a sequence encoding an ITR is used.
  • an AAV ITR sequence may be obtained from any known AAV, including presently identified mammalian AAV types.
  • an ITR is an AAV2 ITR.
  • an ITR is an AAV9 ITR.
  • a non-limiting example of a polynucleotide construct of the present disclosure is a "cisacting" construct comprising a transgene, in which said transgene sequence and any associated regulatory elements are flanked by 5' or "left” and 3' or "right” AAV ITR sequences.
  • 5' and left designations refer to a position of an ITR sequence relative to an entire construct, read left to right, in a sense direction.
  • a 5' or left ITR is an ITR that is closest to a promoter (e.g., as opposed to a polyadenylation sequence) for a given construct, when a construct is depicted in a sense orientation, linearly.
  • 3' and right designations refer to a position of an ITR sequence relative to an entire construct, read left to right, in a sense direction.
  • a 3' or right ITR is an ITR that is closest to a polyadenylation sequence (e.g., as opposed to a promoter sequence) for a given construct, when a construct is depicted in a sense orientation, linearly.
  • ITRs as provided herein are depicted in 5' to 3' order in accordance with a sense strand. Accordingly, one of skill in the art will appreciate that a 5' or "left" orientation ITR can also be depicted as a 3' or "right” ITR when converting from sense to anti sense direction.
  • a given sense ITR sequence e.g., a 571 eft AAV ITR
  • an antisense sequence e.g., 37right ITR sequence
  • an ITR (e.g., a 5' ITR) can have a sequence according to SEQ ID NO: 23.
  • an ITR (e.g., a 3' ITR) can have a sequence according to SEQ ID NO: 24.
  • an ITR includes one or more modifications, e.g., truncations, deletions, substitutions or insertions, as is known in the art.
  • an ITR comprises fewer than 145 nucleotides, e.g., 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, or 141 nucleotides.
  • an ITR comprises 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143 144, or 145 nucleotides.
  • a 5' ITR sequence is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a 5' ITR sequence represented by SEQ ID NO: 23.
  • a 3' ITR sequence is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a 3' ITR sequence represented by SEQ ID NO: 24.
  • SEQ ID NO: 23 Exemplary 5' AAV ITR polynucleotide sequence
  • a construct (e.g., an AAV construct) comprises a promoter.
  • promoter refers to a DNA sequence recognized by enzymes/proteins that can promote and/or initiate transcription of an operably linked gene (e.g., encoding an inhibitor of ISR).
  • a promoter typically refers to, e.g., a polynucleotide sequence to which an RNA polymerase and/or any associated factor binds and from which it can initiate transcription.
  • a construct (e.g., an AAV construct) comprises a promoter operably linked to one of the non-limiting example promoters described herein.
  • a promoter is an inducible promoter, a constitutive promoter, a mammalian cell promoter, a viral promoter, a chimeric promoter, an engineered promoter, a tissuespecific promoter, or any other type of promoter known in the art.
  • a promoter is a RNA polymerase II promoter, such as a mammalian RNA polymerase II promoter.
  • a promoter is a RNA polymerase III promoter, including, but not limited to, a HI promoter, a human U6 promoter, a mouse U6 promoter, or a swine U6 promoter.
  • a promoter will generally be one that is able to promote transcription in a neurological cell.
  • promoters are known in the art, which in some embodiments, can be used herein.
  • Nonlimiting examples of promoters that can be used herein in some embodiments include: human EFla, human cytomegalovirus (CMV) (US Patent No.
  • human ubiquitin C UBC
  • mouse phosphoglycerate kinase 1 polyoma adenovirus
  • simian virus 40 SV40
  • P-globin P-actin
  • a- fetoprotein a-globin
  • P-interferon y-glutamyl transferase
  • mouse mammary tumor virus MMTV
  • Rous sarcoma virus rat insulin
  • glyceraldehyde-3-phosphate dehydrogenase metallothionein II (MT II)
  • amylase cathepsin
  • MI muscarinic receptor retroviral LTR (e.g., human T-cell leukemia virus HTLV), AAV ITR, interleukin-2, collagenase, platelet-derived growth factor, adenovirus 5 E2, stromelysin, murine MX gene, glucose regulated proteins (GRP78 and G
  • a promoter is the CMV immediate early promoter.
  • the promoter is a CAG promoter and/or a CAG/CBA promoter.
  • constitutive promoter refers to a polynucleotide and/or oligonucleotide sequence that, when operably linked with a nucleic acid encoding a protein (e.g., an inhibitor of ISR), causes RNA to be transcribed from the nucleic acid in a cell under most or all physiological conditions.
  • a protein e.g., an inhibitor of ISR
  • constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (see, e.g., Boshart et al., Cell 41 :521-530, 1985, which is incorporated herein by reference for the purposes described herein), the SV 40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFLalpha promoter (Invitrogen).
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only.
  • Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech, and Ariad. Additional examples of inducible promoters are known in the art.
  • inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex) inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (see e.g., WO 98/10088, which is incorporated herein by reference for the purposes described herein); the ecdysone insect promoter (see e.g., No et al., Proc. Natl. Acad Set.
  • MT zinc-inducible sheep metallothionein
  • Dex dexamethasone
  • MMTV mouse mammary tumor virus
  • T7 polymerase promoter system see e.g., WO 98/10088, which is incorporated herein by reference for the purposes described herein
  • ecdysone insect promoter see e.g., No et al., Proc. Natl. Acad Set.
  • tissue-specific promoter refers to a promoter that is active only in certain specific cell types and/or tissues (e.g., transcription of a specific gene occurs only within cells expressing transcription regulatory and/or control proteins that bind to the tissue-specific promoter).
  • regulatory and/or control sequences impart tissue-specific gene expression capabilities.
  • tissue-specific regulatory and/or control sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner.
  • a tissue-specific promoter is a neuron-specific promoter.
  • a tissue-specific promoter is glial cell-specific promoter.
  • a tissue-specific promoter is a hippocampal cell-specific promoter.
  • SEQ ID NO: 25 Exemplary CAG promoter polynucleotide sequence
  • SEQ ID NO: 26 Exemplary CAG promoter/enhancer polynucleotide sequence
  • a construct can include an enhancer sequence.
  • enhancer sequence refers to a polynucleotide and/or oligonucleotide sequence that can increase the level of transcription of a nucleic acid encoding a protein of interest (e.g., an inhibitor of ISR), and/or increase or modify the translational efficiency of a transcript following transcription.
  • enhancer sequences generally 50-1500 bp in length
  • an enhancer sequence is found within an intronic sequence.
  • an enhancer sequence is found in a 3' and/or 5' UTR.
  • an enhancer region is found downstream of a coding sequence comprising a transgene and proximal to a poly adenylation sequence.
  • enhancer sequences can act at much larger distance away from the transcription start site (e.g., as compared to a promoter).
  • Non-limiting examples of enhancers include a woodchuck hepatitis virus post- transcriptional regulatory element (WPRE), RSV enhancer, a CMV enhancer, and/or a SV40 enhancer.
  • any of the constructs described herein can include an untranslated region (UTR), such as a 5' UTR or a 3' UTR.
  • UTRs of a gene are transcribed but not translated.
  • a 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon.
  • a 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal.
  • the regulatory and/or control features of a UTR can be incorporated into any of the constructs, particles, polynucleotides, compositions, kits, or methods as described herein to enhance or otherwise modulate the expression of an inhibitor of ISR.
  • Natural 5' UTRs include a sequence that plays a role in translation initiation.
  • a 5' UTR can comprise sequences, like Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes.
  • Kozak sequences have the consensus sequence CCR(A/G)CCAUGG, where R is a purine (A or G) three bases upstream of the start codon (AUG), and the start codon is followed by another “G”.
  • 5' UTRs also form secondary structures that are involved in elongation factor binding.
  • a 5' UTR is included in any of the constructs described herein.
  • Non-limiting examples of 5' UTRs including those from the following genes: albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, and Factor VIII, can be used to enhance expression of a nucleic acid molecule, such as an mRNA.
  • AU-rich elements can be separated into three classes (see e.g., Chen et al., Mol. Cell. Biol. 15:5777-5788, 1995; Chen et al., Mol. Cell Biol. 15:2010-2018, 1995, each of which is incorporated herein by reference for the purposes described herein): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions.
  • c-Myc and MyoD mRNAs contain class I AREs.
  • Class II AREs possess two or more overlapping UUAUUUA(U/A) (U/A) nonamers.
  • GM-CSF and TNF-alpha mRNAs are examples that contain class II AREs.
  • Class III AREs are less well defined. These U-rich regions do not contain an AUUUA motif, two well-studied examples of this class are c-Jun and myogenin mRNAs.
  • AREs Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules may lead to HuR binding and thus, stabilization of the message in vivo. [0179] In some embodiments, the introduction, removal, or modification of 3' UTR AREs can be used to modulate the stability of an mRNA encoding an inhibitor of ISR protein. In other embodiments, AREs can be removed or mutated to increase the intracellular stability and thus increase translation and production of an inhibitor of ISR protein.
  • non-ARE sequences may be incorporated into the 5' or 3' UTRs.
  • introns or portions of intron sequences may be incorporated into the flanking regions of the polynucleotides in any of the constructs, particles, polynucleotides, compositions, kits, and methods provided herein. Incorporation of intronic sequences may increase protein production as well as mRNA levels.
  • IRS Internal Ribosome Entry Sites
  • a construct encoding an inhibitor of ISR protein can include an internal ribosome entry site (IRES).
  • IRES forms a complex secondary structure that allows translation initiation to occur from any position with an mRNA immediately downstream from where the IRES is located (see, e.g., Pelletier and Sonenberg, Mol. Cell. Biol. 8(3): 1103-1112, 1988, which is incorporated herein by reference for the purposes described herein).
  • IRES sequences known to those in skilled in the art, including those from, e.g., foot and mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), human rhinovirus (HRV), cricket paralysis virus, human immunodeficiency virus (HIV), hepatitis A virus (HA V), hepatitis C virus (HCV), and poliovirus (PV) (see e.g., Alberts, Molecular Biology of the Cell, Garland Science, 2002; and Hellen et al., Genes Dev. 15(13): 1593-612, 2001, each of which are incorporated herein by reference for the purposes described herein).
  • FMDV foot and mouth disease virus
  • EMCV encephalomyocarditis virus
  • HRV human rhinovirus
  • HCV hepatitis A virus
  • HCV hepatitis C virus
  • PV poliovirus
  • an IRES sequence that is incorporated into a construct that encodes an inhibitor of ISR protein is the foot and mouth disease virus (FMDV) 2A sequence.
  • the Foot and Mouth Disease Virus 2A sequence is a small peptide (approximately 18 amino acids in length) that has been shown to mediate the cleavage of polyproteins (see e.g., Ryan, MD et al., EMBO 4:928-933, 1994; Mattion et al., J Virology 70:8124-8127, 1996; Furler et al., Gene Therapy 8:864-873, 2001; and Halpin et al., Plant Journal 4:453-459, 1999, each of which is incorporated herein by reference for the purposes described herein).
  • the cleavage activity of the 2A sequence has previously been demonstrated in artificial systems including plasmids and gene therapy constructs (e.g., AAV and retroviruses) (see e.g., Ryan et al., EMBO 4:928-933, 1994; Mattion et al., J Virology 70:8124-8127, 1996; Furler et al., Gene Therapy 8:864-873, 2001; and Halpin et al., Plant Journal 4:453-459, 1999; de Felipe et al., Gene Therapy 6: 198-208, 1999; de Felipe et al., Human Gene Therapy II: 1921-1931, 2000; and Klump et al., Gene Therapy 8:811- 817, 2001, each of which is incorporated herein by reference for the purposes described herein).
  • gene therapy constructs e.g., AAV and retroviruses
  • an IRES can be utilized in an AAV construct.
  • a construct encoding an inhibitor of ISR protein can include a polynucleotide internal ribosome entry site (IRES).
  • IRES can be part of a composition comprising more than one construct.
  • an IRES is used to produce more than one polypeptide from a single gene transcript. f. Splice sites
  • any of the constructs provided herein can include splice donor and/or splice acceptor sequences, which are functional during RNA processing occurring during transcription.
  • splice sites are involved in trans-splicing.
  • a construct provided herein can include a polyadenylation (poly(A)) signal sequence.
  • poly(A) polyadenylation
  • a poly(A) tail confers mRNA stability and transferability (see e.g., Molecular Biology of the Cell, Third Edition by B. Alberts etal., Garland Publishing, 1994, which is incorporated herein by reference for the purposes described herein).
  • a poly(A) signal sequence is positioned 3' to a coding sequence.
  • polyadenylation refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule.
  • mRNA messenger RNA
  • a 3' poly(A) tail is a long sequence of adenine nucleotides (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase.
  • a poly(A) tail is added onto transcripts that contain a specific sequence, e.g., a poly(A) signal.
  • a poly(A) tail and associated proteins aid in protecting mRNA from degradation by exonucleases.
  • Polyadenylation also plays a role in transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation typically occurs in the nucleus immediately after transcription of DNA into RNA, but also can occur later in the cytoplasm. After transcription has been terminated, an mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. A cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3' end at the cleavage site.
  • a "poly(A) signal sequence” or “polyadenylation signal sequence” is a sequence that triggers the endonuclease cleavage of an mRNA and the addition of a series of adenosines to the 3' end of the cleaved mRNA.
  • poly(A) signal sequences that can be used in some embodiments, including those derived from bovine growth hormone (bGH) (Woychik et al., Proc. Natl. Acad Sci. U.S.A. 81(13):3944-3948, 1984; U.S. Patent No.
  • bGH bovine growth hormone
  • mouse-P-globin mouse-a-globin
  • human collagen human collagen
  • polyoma virus Bactet al., Mol. Cell Biol.
  • HSV TK Herpes simplex virus thymidine kinase gene
  • IgG heavy-chain gene polyadenylation signal US 2006/0040354, which is incorporated herein by reference for the purposes described herein
  • human growth hormone hGH
  • SV40 poly(A) site such as the SV40 late and early poly(A) site (see e.g., Schek et al., Mol Cell Biol. 12(12):5386-5393, 1992, which is incorporated herein by reference for the purposes described herein).
  • the poly(A) signal sequence can be AATAAA.
  • the AATAAA sequence may be substituted with other hexanucleotide sequences with homology to AATAAA and that are capable of signaling polyadenylation, including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, or AATAAG (see, e g., WO 06/12414, which is incorporated herein by reference for the purposes described herein).
  • a poly(A) signal sequence can be a synthetic polyadenylation site (see, e.g., the pCl- neo expression construct of Promega that is based on Levitt et al., Genes Dev. 3(7): 1019- 1025, 1989, which is incorporated herein by reference for the purposes described herein).
  • SEQ ID NO: 28 Exemplary SV40 poly A signal polynucleotide sequence
  • constructs of the present disclosure may comprise a 2A element or sequence.
  • constructs of the present disclosure may include one or more cloning sites.
  • cloning sites may not be fully removed prior to manufacturing for administration to a subject.
  • cloning sites may have functional roles including as linker sequences, or as portions of a Kozak site. As will be appreciated by those skilled in the art, cloning sites may vary significantly in primary sequence while retaining their desired function.
  • a 2A element is a T2A, P2A, E2A, and/or F2A element.
  • a 2A sequence may comprise an optional 5’ linker sequence, such as but not limited to GSG (e.g., Glycine, Serine, Glycine).
  • VKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 33 - Exemplary P2A oligonucleotide sequence
  • SEQ ID NO: 34 Exemplary transcriptional linker oligonucleotide sequence
  • SEQ ID NO: 35 Exemplary transcriptional linker oligonucleotide sequence
  • any of the constructs provided herein can optionally include a sequence encoding a destabilizing domain ("a destabilizing sequence") for temporal and/or spatial control of protein expression.
  • a destabilizing sequence include sequences encoding a FK506 sequence, a dihydrofolate reductase (DHFR) sequence, or other exemplary destabilizing sequences.
  • protein expression can be detected by conventional means, including enzymatic, radiographic, colorimetric, fluorescence, or other spectrographic assays, fluorescent activating cell sorting (FACS) assays, and/or immunological assays (e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).
  • FACS fluorescent activating cell sorting
  • destabilizing sequences are known in the art.
  • the destabilizing sequence is a FK506- and rapamycin-binding protein (FKBP12) sequence
  • the stabilizing ligand is Shield-I (Shldl) (see e.g., Banaszynski et al. (2012) Cell 126(5):995-1004, which is incorporated herein by reference for the purposes described herein).
  • a destabilizing sequence is a DHFR sequence
  • a stabilizing ligand is trimethoprim (TMP) (see e.g., Iwamoto etal., (2010) ChemBiol 17:981-988, which is incorporated herein by reference for the purposes described herein).
  • TMP trimethoprim
  • constructs provided herein can optionally include a sequence encoding a reporter polypeptide and/or protein ("a reporter sequence").
  • reporter sequences include DNA sequences encoding: a beta-lactamase, a betagalactosidase (LacZ), an alkaline phosphatase, a thymidine kinase, a green fluorescent protein (GFP), a red fluorescent protein, an mCherry fluorescent protein, a yellow fluorescent protein, a chloramphenicol acetyltransferase (CAT), and a luciferase. Additional examples of reporter sequences are known in the art.
  • the reporter sequence can provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence, or other spectrographic assays, fluorescent activating cell sorting (FACS) assays and/or immunological assays (e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).
  • FACS fluorescent activating cell sorting
  • immunological assays e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry.
  • a reporter sequence is the Lacz gene, and the presence of a construct carrying the Lacz gene in a cell is detected by assays for beta-galactosidase activity.
  • a reporter sequence is a fluorescent protein (e.g., green fluorescent protein (GFP)) or luciferase.
  • GFP green fluorescent protein
  • the presence of a construct carrying the fluorescent protein or luciferase in a cell may be measured by fluorescent imaging techniques (e.g., fluorescent microscopy or FACS) or light production in a luminometer (e.g., a spectrophotometer or an IVIS imaging instrument).
  • a reporter sequence can be used to verify tissue-specific targeting capabilities and/or tissue-specific promoter regulatory and/or control activity of any of the constructs described herein.
  • An exemplary GFP tag sequence is provided as SEQ ID NO: 37.
  • a reporter sequence is a FLAG tag (e.g., a 3xFLAG tag), and the presence of a construct carrying the FLAG tag in a cell is detected by protein binding or detection assays (e.g., Western blots, immunohistochemistry, radioimmunoassay (RIA), mass spectrometry).
  • protein binding or detection assays e.g., Western blots, immunohistochemistry, radioimmunoassay (RIA), mass spectrometry.
  • An exemplary 3xFLAGtag sequence is provided as SEQ ID NO: 36.
  • SEQ ID NO: 36 Exemplary 3xFLAG tag polynucleotide sequence
  • constructs, particles, polypeptides, polynucleotides, and/or compositions described herein may be comprised in a formulation with one or more additional therapeutic agents.
  • constructs, particles, polypeptides, polynucleotides, and/or compositions described herein may be comprised in a formulation wherein the formulation comprises pharmaceutically acceptable excipients.
  • constructs, particles, polypeptides, polynucleotides, and/or compositions described herein are administered to a subject in need thereof.
  • a subject may have, may be diagnosed with, or may be susceptible to developing Down syndrome (DS), Charcot-Marie-Tooth disease, major depressive disorder (MDD), schizophrenia, Alzheimer’s disease, Huntington disease, Parkinson’s disease, Amyotrophic lateral sclerosis (ALS), Multiple Sclerosis (MS), Prion disease, traumatic brain injury, Vanishing white matter (VWM) disease, frontotemporal dementia, and/or Aging (e.g., age-related cognitive decline).
  • DS Down syndrome
  • MDD major depressive disorder
  • ALS Amyotrophic lateral sclerosis
  • MS Multiple Sclerosis
  • Prion disease traumatic brain injury
  • VWM Vanishing white matter
  • frontotemporal dementia frontotemporal dementia
  • Aging e.g., age-related cognitive decline
  • a subject is a mammal. In some embodiments, a subject is a domestic animal. In some embodiments, a subject is a farm animal. In some embodiments, a subject is a zoo animal. In some embodiments, a subject is a dog or a cat. In some embodiments, a subject is a cow, a horse, a sheep, or a goat. In some embodiments, a subject can be but is not limited to, a dog, cat, ferret, rabbit, cow, duck, pig, goat, chicken, horse, llama, camel, ostrich, deer, turkey, dove, sheep, goose, oxen, and/or reindeer.
  • a subject is a human. In some embodiments, a subject is equal to, less than, or greater than 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
  • constructs, particles, polypeptides, polynucleotides, and/or compositions described herein according to the present invention may be administered by intraspinal and/or intracerebral administration.
  • intraspinal administration comprises or consists of intrathecal and epidural administration.
  • constructs, particles, polypeptides, polynucleotides, and/or compositions described herein are administered intracerebrally.
  • intracerebral administration is at a site selected from the group comprising or consisting of: hippocampus and/or hippocampal formation (such as, e.g., the cornu ammonis (e.g, CAI, CA2, and/or CA3), the subicular complex, the entorhinal cortex, and/or the dentate gyrus), striatum (such as, e.g., putamen, caudate nucleus, nucleus accumbens, olfactory tubercle, external globus pallidus and/or internal globus pallidus), thalamus, hypothalamus, epithalamus, subthalamus, parenchyma, cerebrum, medulla, deep cerebellar nuclei (such as, e.g., substanti
  • constructs, particles, polypeptides, polynucleotides, and/or compositions described herein are administered intrahippocampally (e.g., in the hippocampus, e.g., CAI, CA2, and/or CA3), intrastriataly (e.g., in the striatum, such as, e.g., in the putamen, caudate nucleus, nucleus accumbens, olfactory tubercle, external globus pallidus and/or internal globus pallidus), intrathalamicaly (e.g., in the thalamus), and/or intraci stemaly (e.g., in the subarachnoid cisterns, such as, e.g., in the cisterna magna, pontine cistem, interpeduncular cistern, chiasmatic cistern, cistern of lateral cerebral fossa, superior c
  • intrahippocampally
  • constructs, particles, polypeptides, polynucleotides, and/or compositions described herein are administered intracerebroventricularly, intrahippocampally, intraparenchymaly, intrastriataly, intrathalamicaly, intracisternaly, and/or intrathecally.
  • therapeutic regimens comprising constructs, particles, polypeptides, polynucleotides, and/or compositions described herein may comprise administration of a combination of therapeutic agents, such as a first therapy and a second therapy.
  • the therapies may be administered in any suitable manner known in the art.
  • the first and second therapy may be administered sequentially (at different times) or concurrently (at the same time).
  • the first and second therapies are administered in a separate composition.
  • the first and second therapies are in the same composition.
  • therapeutic regimens comprising constructs, particles, polypeptides, polynucleotides, and/or compositions described herein comprise administering of more than one composition, such as 2 compositions, 3 compositions, 4 compositions, or more than 4 compositions.
  • Various combinations of the agents may be employed.
  • therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration.
  • constructs, particles, polypeptides, polynucleotides, and/or compositions described herein and/or additional therapeutic agents are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
  • constructs, particles, polypeptides, polynucleotides, and/or compositions described herein and/or additional therapeutic agents are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
  • an appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.
  • treatments may include various “unit doses.”
  • Unit dose is defined as containing a predetermined- quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts.
  • a unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.
  • a unit dose comprises a single administrable dose.
  • the quantity to be administered depends on the treatment effect desired.
  • An effective dose is understood to refer to an amount necessary to achieve a particular effect.
  • doses in the range from 0.10 mg/kg to 200 mg/kg can affect the protective capability of these agents.
  • doses may comprise a composition comprising an AAV particle in a concentration of about 10 8 to about 10 14 viral genomes per ml.
  • such doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.
  • precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.
  • uptake is species and organ/tissue dependent.
  • the applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.
  • administrations of the composition e.g., 2, 3, 4, 5, 6 or more administrations.
  • the administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, 12 week, or more than 12 week intervals, including all ranges there between.
  • phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.
  • the active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes.
  • such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the proteinaceous compositions may be formulated into a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • a pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions described herein may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • formulations are administered in a variety of dosage forms, such as the type of injectable solutions described above.
  • the constructs, particles, polypeptides, polynucleotides, and/or compositions or agents for use in the methods are suitably contained in a pharmaceutically acceptable carrier.
  • the carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the agent.
  • the agents in some aspects of the disclosure may be formulated into preparations for local delivery (i.e.
  • compositions by coating medical devices and the like.
  • suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol.
  • sterile, fixed oils may be employed as a solvent or suspending medium.
  • any biocompatible oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.
  • the carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s).
  • a delivery vehicle may include, by way of non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.
  • the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration.
  • the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical compositions are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable or solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified.
  • a typical composition for such purpose comprises a pharmaceutically acceptable carrier.
  • the composition may contain 10 mg or less, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline.
  • Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.
  • non-limiting examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate.
  • non-limiting examples of aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.
  • intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial agents, antgifungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.
  • formulations comprising constructs described herein and/or co-administered formulations may be suitable for oral administration.
  • oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like.
  • the compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
  • unit dose or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen.
  • Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.
  • Mouse iPSCs were microinjected with specific gRNA (e.g., AGTGGTGATGAGGATCGCAA AGG (SEQ ID NO: 38); the PAM sequence is underlined and is not necessarily comprised within a gRNA sequence, (Synthego)), Cas9 mRNA (Sigma) and the donor DNA containing the R658C mutation.
  • the donor DNA was 500 bp in length and in addition to the point mutation of interest, silent mutations were introduced to prevent editing of the mutant back to WT.
  • the donor also contained approximately 250 bp flanking the mutated region to facilitate homologous recombination. Edited cells were implanted into surrogate females and the pups were genotyped by sequencing of the locus. These steps were performed at the core facility at Baylor College of Medicine (BCM). Pups carrying the desired mutation were bred and tested phenotypically.
  • BCM Baylor College of Medicine
  • AAVs Adeno-associated Viruses
  • Adeno associated viruses were generated by cloning the indicated sequences into an AAV2 plasmid backbone. DNA was then purified and sent to a core facility to produce AAVs serotyped with the neurotropic DJ/8. AAV was purified with commercially available kits (Cell Biolabs) and quantified by qPCR based assays. Typically, 0.5-1 pl of ⁇ 10 12 viral genomes per ml were injected into each hippocampus of the mouse in both the CAI and CA3 regions (cornu Ammonis subfields 1 and 3). AAVs were allowed to express the transgene for at least 14 days prior to the beginning of behavioral experiments. Subsequently the brains of the mice were dissected and samples were prepared for SDS-PAGE and western blotting.
  • mice were handled for 5 minutes three consecutive days to allow them to acclimatize to the experimenters. Subsequently the mice were placed in the conditioning chamber to allow them to habituate for 20 minutes each of two days. Mice were then trained with an aversive foot shock. Two training shocks were administered at 0.7 mA separated by 90 seconds each with freezing recorded in between and after the training shocks. Finally, 24 hours after training, mice were placed in the same chamber for 5 minutes and percent of time spent freezing was recorded using Freezeview software.
  • mice were handled for 5 days (5-10 min for each day) and then habituated to a black Plexiglas rectangular chamber (31 x 24 cm, height 27 cm) for 10 min under dim ambient light for 5 days.
  • Two identical objects were presented to mice to explore for 5 min, after which, mice were returned to the home cage. Twenty-four hours later, one object was replaced by one novel object and the mouse was again placed in the chamber for 5 min.
  • the novel object has the same height and volume but different shape and appearance. Exploration of the objects was defined as sniffing of the objects (with nose contact or head directed to the object) within at 2 cm radius of the objects.
  • DI Discrimination Index
  • Electrophysiological recordings were performed as previously described (see e.g., Zhu et al., 2019). Field recording were performed from CAI horizontal hippocampal slices (320 pm thick), which were cut from the brain of adult mice (3-6 months old) with a vibratome (Leica VT 1000S, Leica Microsystems, Buffalo Grove, IL) at 4 °C in artificial cerebrospinal fluid solution (ACSF; 95% 02 and 5% CO2) containing in mM: 124 NaCl, 2.0 KC1, 1.3 MgSO4, 2.5 CaC12, 1.2 KH2PO4, 25 NaHCO3, and 10 glucose (2-3 ml/min).
  • Tetanic LTP was induced by applying four trains of high- frequency stimulation (100 Hz, 1 s) separated by 5-min intervals.
  • Whole-cell recordings were performed using a MultiClamp 700B amplifier (Molecular Devices, Union City, CA) in a submerged chamber (2-3 ml/min) at 31-32 °C using conventional patch-clamp techniques.
  • CAI neurons were visually identified by infrared differential interference contrast video microscopy on the stage of an upright microscope (Axioskope FS2, Carl Zeiss, Oberkochen, Germany).
  • Patch pipettes (resistances 2-5 MW) were filled with (in mM): 140 CsCl, 10 HEPES, 10 Na2- phosphocreatine, 0.2 BAPTA, 2 Mg3-ATP, 0.2 Na3-GTP; pH was adjusted to 7.2 and osmolarity to 295-300 mOsm using a Wescor 5500 vapor pressure osmometer (Wescor, Logan, UT).
  • mIPSCs Miniature inhibitory postsynaptic currents
  • NBQX 5 pM, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline disodium salt
  • AP5 25 pM, DL-2-Amino-5-phosphonopentanoic acid
  • TTX 1 pM, tetrodotoxin
  • CsCl was replaced with 130 mM K-gluconate and 10 mM KC1.
  • EMCs excitatory postsynaptic currents
  • RNA sequencing was carried out as previously described (see e.g., Hinnebusch et al., 2016). Briefly, fresh 12 ml of 10-50% sucrose density gradients [10 mM HEPESKOH (pH 7.6), 5 mM MgC12, 150 mM KC1, 200 U/ml RNasin Rnase inhibitor (Promega, Madison, WI)] was prepared, as previously described (see e.g., Zhu et al., 2019; and Tible et al., 2019). Gradients were kept at 4 °C for at least 2 hours before use.
  • Mouse brain tissue was dissected in a cutting solution [1XHBSS, 2.5 mMHEPES-KOH (pH 7.6), 35 mM glucose, 4 mM NaHC03, 100 pg/ml cycloheximide (Sigma- Aldrich, St. Louis, MO)] and washed in ice-cold PBS containing 100 pg/ml cycloheximide by centrifugation at 3000 rpm for 10 min at 4 °C.
  • a cutting solution [1XHBSS, 2.5 mMHEPES-KOH (pH 7.6), 35 mM glucose, 4 mM NaHC03, 100 pg/ml cycloheximide (Sigma- Aldrich, St. Louis, MO)] and washed in ice-cold PBS containing 100 pg/ml cycloheximide by centrifugation at 3000 rpm for 10 min at 4 °C.
  • tissue was then lysed in polysome lysis buffer [10 mM HEPES-KOH (pH 7.4), 5 mM MgC12, 150 mM KC1, 0.5 mM DTT, 100 U/ml RNasin Rnase inhibitor (Promega, Madison, WI), 100 pg/ml cycloheximide, and EDTA-free protease inhibitors (Roche Indianapolis, IN)] and centrifuged at 2000 x g for 10 min at 4 °C. The supernatant was then transferred to a pre-chilled tube, supplemented with 0.5% NP-40, and kept on ice for 10 min.
  • polysome lysis buffer 10 mM HEPES-KOH (pH 7.4), 5 mM MgC12, 150 mM KC1, 0.5 mM DTT, 100 U/ml RNasin Rnase inhibitor (Promega, Madison, WI), 100 pg/ml cyclohex
  • RNA samples were centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was either layered onto sucrose gradient or reserved for total RNA isolation. Gradients were centrifuged in a SW-40Ti rotor at 35,000 rpm at 4 °C for 2 hours and then analyzed by piercing the tube with a Brandel tube piercer, passing 70% sucrose through the bottom of the tube and monitoring the absorbance of the material eluting from the tube using an ISCO UA-6 UV detector. Fractions were collected throughout and RNA was extracted with TRIzol following manufacturer’s instructions (Life Technologies, Carlsbad, CA). Experiments were performed in three biological replicates for each group.
  • Protein synthesis was measured using SUnSET, a non-radioactive labeling method to monitor protein synthesis, as previously described (see e.g., Zhu etal., 2019). Briefly, hippocampal slices were cut (300 pm) with a McIlwain Tissue Chopper (Mickle, UK) and incubated for 1 hour at room temperature in oxygenated (95% 02, 5% CO2) ACSF followed by incubation at 32 °C for 1 hour in oxygenated (95% 02, 5% C02) ACSF prior to treatment as we previously described (see e.g., Zhu et al., 2019).
  • Puromycin (10 pg/pl, dissolved in oxygenated ACSF) was bath applied to the slices for 20 min followed by a wash with untreated oxygenated ACSF. The slices were then snap-frozen on dry ice and stored at -80 °C until use. Frozen slices were lysed in homogenization buffer (in mM: 40 Tris HC1, pH 8.0, 150 NaCl, 25 b-glycerophosphate, 50 NaF, 2 Na3VO3, IX protease inhibitor cocktail, 10% glycerol, 1% Triton X-100).
  • homogenization buffer in mM: 40 Tris HC1, pH 8.0, 150 NaCl, 25 b-glycerophosphate, 50 NaF, 2 Na3VO3, IX protease inhibitor cocktail, 10% glycerol, 1% Triton X-100).
  • Puromycin incorporation was detected by Western blot using the 12D10 antibody to puromycin (Catalog # MABE343, 1:5000, EMD Millipore Corp, Darmstadt, Germany) as previously described (see e.g., Zhu et al., 2019). The density of the resulting bands was quantified using ImageJ and statistical significance assessed by Student’s t-test.
  • Example 1 Creation and characterization of Ppp1r15b R658C mice mice exhibited aberrant translational control and active ISR.
  • mice A new mouse model ( Ppp1r15b R658C mice) was generated, carrying a selective mutation (R658C) in PPP1R15B, which has been identified by whole-exome sequencing to be associated with intellectual disability in humans (see e.g., Abdulkarim et al., 2015; Kernohan et al., 2015; and Mohammad etal., 2016). Briefly, mice were generated with CRISPR using a guide RNA targeting a region of PPP1R15B in close proximity to the base position of encoding the R658C mutation.
  • donor DNA containing the R658C mutation, the Cas9 mRNA, and the guide RNA were co-injected into embryos that were subsequently implanted into surrogate mice.
  • Four 4 pups with homozygous targeting of the desired locus were identified (FIG. 2A).
  • Mice were bred and mutant and littermate controls were obtained. Consistent with the microcephaly observed in individuals carrying the homozygous mutation (see e.g., Abdulkarim etal., 2015; and Kernohan et al., 2015), Ppplrl5bR658C mice were also found to exhibit slightly smaller brains (FIG. 2B).
  • the R658C mutation inhibits the activity of the CReP»PPl phosphatase complex, leading to increased eIF2-P levels (see e.g., Abdulkarim et al., 2015; and Kernohan et al., 2015).
  • CReP levels were found to not be altered (FIG. 2C), but eIF2-P levels were found to be increased in the brains (FIG. 2D) and primary fibroblasts (FIG. 2E) of Ppp1r15b R658C mice.
  • the ISR was selectively perturbed in Ppp1r15b R658C mice.
  • mice as indicated by a decrease in polysomes and concomitant increase in monosomes (FIGs. 2G-H), consistent with increased eIF2-P levels (see e.g., Costa-Mattioli et al., 2007; and Harding et al., 2000).
  • LTP long-term potentiation
  • the ISR bidirectionally regulates L-LTP: that is, activation of the ISR impairs L-LTP, whereas inhibition of the ISR enhances it (see e.g., Costa-Mattioli et al., 2007; Zhu etal., 2011; Jiang etal., 2010; and Huang etal., 2016), and the immediate results showed that the ISR was selectively activated in Ppp1r15b R658C mice, the inventors investigated whether L-LTP was impaired in these mice. Indeed, in concordance with the field, four trains of 100 Hz induced a persistent L-LTP in WT slices (FIG. 3F).
  • eIF2-P phosphatase As modulation of eIF2-P phosphatase can prove to be a powerful mechanism to modulate the activity of the ISR, the inventors leveraged strategies employed by viruses to inhibit the ISR. Given that increased eIF2-P (that is, activated ISR) impair both cellular and viral protein synthesis, many viruses have evolved mechanisms to inactivate the ISR (see e.g., Garcia et al., 2007).
  • yeast genomes do not naturally encode eIF2-phosphatase cofactor orthologues (e.g., GADD34 or CreP-like proteins) that can inhibit the ISR, and B) the mechanism of recruitment of PPI to eIF2 in yeast is different than that in human cells (see e.g., Rojas et al., Protein phosphatase PP1/GLC7 interaction domain in yeast eIF2y bypasses targeting subunit requirement for eIF2a dephosphorylation.
  • eIF2-phosphatase cofactor orthologues e.g., GADD34 or CreP-like proteins
  • DP71L is the smallest protein (62 amino acids) of the family that contains both a PPI - and eIF2-binding domain (FIG. 4A).
  • the inventors first examined whether DP71L was able to inhibit the ISR activation induced by oligomycin (oligo), which is known to activate the ISR kinase HRI, in human cells.
  • oligo oligomycin
  • oligo induced the ISR, as determined by the increased eIF2-P.
  • expression of DP71L-GFP completely prevented oligo-induced ISR activation (FIG. 4B).
  • DP71L-GFP suppressed the activation of the ISR induced by Poly (I:C) and thapsigargin (thap), two well-known activators of the ISR kinases PKR and PERK, respectively.
  • DP71L is a potent pan-inhibitor of the ISR in human cells (FIGs. 4C-D), and can function to suppress ISR activation in human cells regardless of the ISR-branch that is activated.
  • DP71L is a scaffold protein that bridges the phosphatase PPI to the target eIF2, thus rendering the phosphatase specific to its target.
  • the inventors hypothesized that DP71L’s enhanced ISR suppression properties may be due to increased binding to PPI, eIF2, or both proteins.
  • the inventors compared head-to-head the binding of ACREP-GFP or DP71L-GFP to endogenous PPI and eIF2. While both ACREP-GFP and DP71L-GFP bound similarly to eIF2, DP71L-GFP showed a stronger binding to PPI than did ACREP-GFP (FIG. 5C), suggesting that DP71L may display increased PPI binding relative to other similarly sized PPI and eIF2 binding proteins.
  • ACREP-GFP that contains both the PPI binding domain and the linker region (e.g., region between PPI and eIF2 binding domains) was fused to the C terminus of DP71L (e.g., eIF2 binding domain).
  • DP71L e.g., eIF2 binding domain
  • the addition of the N-term of DP71L, including the PPI binding motif and the linker region, to ACREP-GFP was generated, D20, and this clone displayed improved binding to PPI when compared to ACREP-GFP (Fig. 6B).
  • the linker of DP71L contains 12 amino acids, whereas the linker in ACREP contains 13 amino acids.
  • Alpha-fold prediction of both protein structures together with PPI and the N- terminal domain (NTD) of eIF2a (FIGs. 7A-B) showed that DP71L contains a glutamic acid (tyrosine in ACREP) that was predicted to make a new hydrogen bond with PPI.
  • glutamic acid tyrosine in ACREP
  • DP71L a new gene therapy approach that reversed the cognitive deficits in mouse models of Down syndrome and Alzheimer’s disease.
  • ISRIB directly binds to eIF2B and enhances its activity by promoting its assembly (see e.g., Sidrauski et al., 2013; Sidrauski et al., 2015; Tsai et al., 2018; and Zyryanova et al., 2018).
  • ISRIB blocks the ISR only at intermediate activation levels (see e.g., Rabouw etal., 2019). That is, when the ISR is strongly activated, ISRIB fails to inhibit the ISR (see e.g., Halliday et al., 2015; and Rabouw etal., 2019).
  • Adeno-associated virus AAV are the leading platform for gene delivery for the targeted treatment of a variety of diseases. By optimizing the capsids of AAV, recent advances have contributed substantially to preclinical and clinical successes in AAV- mediated gene therapies.
  • AAV based therapies is the associated relatively small packaging capacity, e.g., it is limited to ⁇ 4.7 kb.
  • DP71L protein and/or constructs comprising polynucleotide sequences encoding the same, would be an ideal gene therapeutic approach to improve cognition and/or ameliorate and/or prevent symptoms of other diseases in which the ISR is activated.
  • the inventors cloned DP71L-GFP into an AAV based construct, formed an AAV particle, and delivered the AAV to the hippocampus, a brain structure that is required for long-term memory formation in mice (FIG. 8A).
  • Stereotactic injection of AAV expressing DP71L-GFP resulted in detectable protein expression in the hippocampus of mice (FIG.
  • the inventors and others have previously shown that inhibition of the ISR rescued the memory deficits associated with Down Syndrome (DS) (see e.g., Zhu et al., 2019) and Alzheimer’s disease (AD) (see e.g., Ma et al., 2013; Segev et al., 2015; Tible et al., 2019; Hwang et al., 2017; and Lourenco et al., 2013).
  • DS Down Syndrome
  • AD Alzheimer’s disease
  • DS remains the most common genetic form of intellectual disability.
  • Ts65Dn mice a well-characterized mouse model of DS, recapitulates the memory defects characteristic of DS patients, and exhibit deficits in long-term memory (see e.g., Zhu etal., 2019).
  • injection of DP71L-GFP but not GFP alone
  • the inventors then examined whether DP71L also restored the deficient strength of synaptic connections in the hippocampus of Ts65Dn mice. To this end, the inventors measured hippocampal L-LTP, a cellular model for memory formation.
  • APP/PS1 are double transgenic mice expressing a chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PSl-dE9), both directed to CNS neurons. Both mutations are associated with early-onset Alzheimer's disease.
  • This "humanized" APP/PS1 model has been used to study the pathology associated with AD.
  • the inventors found that APP/PS1 mice exhibited quite dramatic long-term memory and L-LTP deficits (FIG. 8D).
  • DP71L AAV therapy reversed both the long-term memory and L-LTP deficits found in APP/PS1 mice (FIG. 8E).
  • the inventors have identified a new and potent gene therapy (e.g., comprising an ISR inhibitor, e.g., comprising DP71L) that improved long-term memory formation in conditions in a wide range of cognitive disorders in which the ISR is activated.
  • an ISR inhibitor e.g., comprising DP71L
  • Kernohan, K. D. et al. Homozygous mutation in the eukaryotic translation initiation factor 2alpha phosphatase gene, PPP1R15B, is associated with severe microcephaly, short stature and intellectual disability.

Abstract

The present disclosure provides compositions comprising proteins and/or constructs comprising a coding sequence encoding an integrated stress response (ISR) inhibitory protein and/or a characteristic portion thereof. Exemplary constructs include AAV constructs. Also provided are methods of using disclosed constructs for the treatment and/or prevention of long term memory formation capacity diminishment and/or improvement of long term memory formation capacity.

Description

INTEGRATED STRESS RESPONSE INHIBITORS AND METHODS OF USING
THE SAME
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63/359,672 filed July 8, 2022, which is incorporated by reference herein in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on June 13, 2023, is named BAYM_P0373WO_Sequence_Listing.xml and is 441,028 bytes in size.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0003] This invention was made with government support under R01 NS076708-10 awarded by the National Institutes of Health. The government has certain rights in this invention.
BACKGROUND
I. Field
[0004] This disclosure relates at least to the fields of aging biology, neurology, genetics, medicine, and gerontology.
II. Background
[0005] To maintain cellular health, proteins need to be synthesized in proper amounts, folded with high fidelity and assembled, appropriately localized, and degraded. Specialized mechanisms respond to malfunction in these essential processes to maintain or reestablish protein homeostasis (proteostasis), when intracellular signaling networks are triggered by a variety of stress sensor molecules. One such network is the integrated stress response (ISR), a central and evolutionarily conserved signaling pathway that helps to restore the cell’s balance by reprogramming gene expression. The ISR’s central regulatory hub lies in the Eukaryotic Initiation Factor 2 / Eukaryotic Initiation Factor 2B (eIF2/eIF2B) complex, which controls the formation of the eIF2»GTP»methionyl-initiator tRNA ternary complex (TC), a prerequisite for initiating new protein synthesis (see e.g., Hinnebusch etal., 2016; which is incorporated herein by reference for the purposes described herein). Assembly of functional TC is inhibited by phosphorylation of a single serine in the alpha-subunit of the heterotrimeric translation initiation factor eIF2, which blocks the action of eIF2’s guanine nucleotide exchange factor termed eIF2B (see e.g., Bogorad et al., 2017; Kenner et al., 2019; Kashiwagi et al., 2019; Adomavicius et al., 2019; and Gordiyenko et al., 2019; each of which are incorporated herein by reference for the purposes described herein).
[0006] Different conditions, including proteostasis defects, nutrient deprivation, viral infection, and oxidative stress, are sensed by four specialized kinases (Protein kinase R-like endoplasmic reticulum kinase (PERK), eIF-2-alpha kinase (GCN2), protein kinase RNA- activated (PKR) and Heme-regulated eIF2a kinase (HRI)), which converge on the phosphorylation of eIF2. Phosphorylation of eIF2 (eIF2-P) results in a general reduction in protein synthesis. Paradoxically, eIF2-P also triggers the translation of specific mRNAs carrying short inhibitory upstream open reading frames (ORFs) in their 5 '-untranslated regions (5' UTRs), including key transcription factors, such as ATF4 (see e.g., FIG. 1). By turning down general translation and upregulating the synthesis of a few proteins that drive a new transcriptional program, the ISR aims to maintain homeostasis by reprogramming gene expression. However, if the stress cannot be mitigated, the ISR triggers apoptosis to eliminate the damaged cell.
[0007] The ISR and certain aspects of the systems association with disease states has been reviewed in Mauro Costa-Mattioli & Peter Walter, 2020 (see e.g., Mauro Costa-Mattioli and Peter Walter, The integrated stress response: From mechanism to disease. Science 2020 Apr 24; 368(6489): eaat5313, which is incorporated herein by reference for the purposes described herein). Natural activation, hyperactivation, and/or aberrant activation of the ISR has been implicated in various disease states including cognitive disorders, neurodegeneration, cancer, diabetes, muscle loss (e.g., cachexia), and metabolic disorders.
[0008] More than 15 years ago, it was discovered that the ISR is a central regulator of longterm memory formation. Briefly, genetic or pharmacological inhibition of the ISR (e.g., reduced eIF2-P) enhanced long-term memory, whereas activation of ISR (e.g., increased eIF2- P) impaired it (see e.g., Costa-Mattioli et al, 2007; Costa-Mattioli et al., 2005, and Zhu et al., 2011; each of which are incorporated herein by reference for the purposes described herein). Investigators around the world have reproduced, built upon and greatly expanded the notion that the ISR is a central memory switch (see e.g., Batista et al., 2015; Ma et al., 2013; Sharma etal., 2018; Segev etal., 2015; Stem et al., 2013; Moreno et al., 2012; Kim etal., 2013; Wong et al., 2019; and reviewed in Costa-Mattioli & Walter, 2020; each of which are incorporated herein by reference for the purposes described herein). Most notably, an inhibitor of the ISR (ISRIB) was discovered, which parallels the effect of reducing the ISR genetically: namely it enhances long-term memory formation (see e.g., Kenner etal., 2019; and Sidrauski etal., 2013; each of which are incorporated herein by reference for the purposes described herein).
[0009] The ISR is activated in a variety of brain disorders that result from protein misfolding and aggregation problems, mitochondrial dysfunction, and/or oxidative stress. For example, activation of the ISR is a main causative mechanism underlying the memory deficits associated with, Down Syndrome (see e.g., Zhu et al., 2019), Alzheimer’s disease (AD) (see e.g., Ma et al., 2013; Segev et al., 2015; Tible et al., 2019; Hwang et al, 2017; and Lourenco et al., 2013; each of which are incorporated herein by reference for the purposes described herein), traumatic brain injury (TBI) (see e.g., Chou et al., 2017; and Sen et al., 2017; each of which are incorporated herein by reference for the purposes described herein), and aging (see e.g., Sharma et al., 2018, which is incorporated herein by reference for the purposes described herein). Thus, the ISR is emerging as a promising avenue to reverse cognitive dysfunction in a wide range of memory disorders that result from disruption in protein homeostasis.
[0010] Finally, the importance of the ISR in cognitive dysfunction is highlighted by the identification of mutations that activate the ISR and are associated with intellectual disability in humans (see e.g., Abdulkarim et al., 2015; Kemohan et al., 2015; Borck et al., 2012; Skopkova et al, 2017; Gregory et al., 2019; Moortgat et al., 2015; and Costa-Mattioli et al., 2020; each of which are incorporated herein by reference for the purposes described herein). Some of these rare mutations map to the PPI binding site of CReP (encoded by PPP1R15B) and destabilize the CReP»PPl phosphatase complex, thereby increasing eIF2-P (see e.g., Abdulkarim et al., 2015; and Kemohan et al, 2015; each of which are incorporated herein by reference for the purposes described herein). Other mutations in the gene encoding CIF2Y reduce TC formation, and consequently induce the ISR, similarly leading to cognitive dysfunction in patients with MEHMO (mental deficiency, epilepsy, hypogenitalism, microcephaly, and obesity) syndrome, an X-linked ID syndrome (see e.g., Borck et al., 2012; Skopkova et al, 2017; Gregory et al., 2019; and Moortgat et al., 2016; each of which are incorporated herein by reference for the purposes described herein). Thus, activation of the ISR is associated with cognitive dysfunction in humans. [0011] The work described herein provides at least new compositions and methods for mitigation of ISR associated cognitive dysfunction.
SUMMARY
[0012] Among other things, the present disclosure provides the recognition that diseases or conditions associated with Integrated Stress Response (ISR) activation (e.g., natural activation, hyperactivation, and/or aberrant activation) can be treated through the modulation (e.g., inhibition) of the ISR. In certain embodiments, modulation of the ISR comprises the use of constructs, particles, polypeptides, polynucleotides, and/or compositions comprising said constructs, particles, proteins and/or nucleotides encoding said proteins, wherein the proteins comprise a PPI binding domain and an eIF2 binding domain. In some embodiments, disclosed herein is a non-natural polynucleotide construct comprising a polynucleotide sequence encoding one or more inhibitor of the integrated stress response (ISR), wherein the one or more inhibitor comprises a protein phosphatase-1 (PPI) binding domain and an eukaryotic initiation factor 2 (eIF2) binding domain, wherein the non-natural polynucleotide construct comprises: A) a heterologous promoter operably linked to the polynucleotide sequence encoding one or more inhibitors of the ISR, wherein the heterologous promoter is not a galactose inducible promoter; and/or B) a recombinant viral vector comprising a heterologous promoter operably linked to the polynucleotide sequence encoding one or more inhibitors of the ISR, wherein the heterologous promoter is not a galactose inducible promoter.
[0013] In some embodiments, disclosed herein is a non-natural polynucleotide construct comprising a polynucleotide sequence encoding one or more inhibitor of the ISR, wherein the one or more inhibitor comprises PPI binding domain and an eIF2 binding domain, wherein the polynucleotide construct further comprises a peptide linker sequence connecting the PPI binding domain and eIF2 binding domain, wherein the peptide linker sequence comprises a glutamic acid at the amino acid position represented by the E12 position of a DP71L protein (e.g., according to SEQ ID NO: 18), wherein the polynucleotide construct further comprises: A) a heterologous promoter operably linked to the polynucleotide sequence encoding one or more inhibitors of the ISR network; and/or B) a recombinant viral vector comprising a heterologous promoter operably linked to the polynucleotide sequence encoding one or more inhibitors of the ISR network. [0014] In some embodiments, an ISR inhibitor binds PPla and/or PPly and/or eIF2 with greater affinity than other ISR inhibitors of similar primary amino acid sequence length. In some embodiments, an ISR inhibitor binds PPla and/or PPly and/or eIF2 with greater affinity than a ACREP protein. In some embodiments, binding affinity is assessed using immunoprecipitation assays. In some embodiments, binding affinity is assessed using immunoprecipitation assays performed on lysates from transgenic human cells (e.g., HEK293T cells). In some embodiments, transgenic human cells (e.g., HEK293T cells) are transfected with a construct that stably and/or transiently expresses a protein of interest (e.g., an ISR inhibitor) and a detectable marker (e.g., a tag, e.g., a fluorescent marker, e.g., GFP (e.g., encoded by SEQ ID NO: 37). In some embodiments, a binding affinity is assayed using an antibody targeting a detectable marker and/or a protein of interest, and is detected (e.g., qualitatively and/or quantitatively) using any suitable means, such as but not limited to, ELISA assays, Western blot assays, immunoprecipitation mass spectrometry assays, etc. In some embodiments, binding affinity is assessed as described herein in FIG. 6.
[0015] In some embodiments, a construct described herein encodes an inhibitor which comprises a PPI binding domain operable for recruiting a protein and/or protein complex comprising a serine/threonine-protein phosphatase PPI -alpha catalytic subunit (PPP1CA, aka PPla), a serine/threonine-protein phosphatase PPI -beta catalytic subunit (PPP1CB, akaPPip), and/or a serine/threonine-protein phosphatase PPI -gamma catalytic subunit (PPP ICC, aka PPly). In some embodiments, a construct described herein encodes an inhibitor which comprises an eIF2 binding domain operable for recruiting a protein and/or protein complex comprising an eIF2a, eIF2p, and/or eIF2y subunit. In some embodiments, a PPI binding domain comprises a RVxF PPI -binding motif and/or KGILK PPI binding motif. In some embodiments, a PPI binding domain comprises a KVRF, KVTF, RVRF, WVTF, IVRF, and/or HVRF PPI -binding motif. In some embodiments, an eIF2 binding domain comprises a RxGx- WxxxAxDRxRFxxRI eIF2 binding motif.
[0016] In some embodiments, a construct described herein encodes an inhibitor which comprises a constitutive repressor of eIF2a phosphorylation (CReP) protein or a characteristic portion thereof. In some embodiments, a CReP protein comprises or consists of an amino acid sequence that is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6. In some embodiments, a CreP protein or a characteristic portion thereof further comprises a peptide linker sequence connecting the PPI binding domain and eIF2 binding domain. In some embodiments, the peptide linker sequence is derived from an African swine fever virus DP71L protein. In some embodiments, the peptide linker sequence is at least or exactly 73%, 82%, 91%, or 100% identical to SEQ ID NO: 21. In some embodiments, the CReP protein is a chimeric protein and comprises an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, the CReP protein is a chimeric protein and comprises an amino acid sequence according to SEQ ID NO: 4.
[0017] In some embodiments, a construct described herein encodes an inhibitor which comprises a protein phosphatase 1 regulatory subunit 15A (GADD34) protein or a characteristic portion thereof. In some embodiments, a construct described herein encodes an inhibitor which comprises a Herpes simplex virus y34.5 protein or a characteristic portion thereof. In some embodiments, a construct described herein encodes an inhibitor which comprises a Canarypox virus CNPV231 protein or a characteristic portion thereof. In some embodiments, a construct described herein encodes an inhibitor which comprises a Macropoid herpes virus ICP34.5 protein or a characteristic portion thereof. In some embodiments, a construct described herein encodes an inhibitor which comprises an Amsacta moorei entom opoxvirus “L” AmEPV193 protein or a characteristic portion thereof.
[0018] In some embodiments, a construct described herein encodes an inhibitor which comprises an African swine fever virus DP71L protein or a characteristic portion thereof. In some embodiments, the inhibitor comprises an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18. In some embodiments, the inhibitor comprises an amino acid sequence according to SEQ ID NO: 18. In some embodiments, the inhibitor is encoded by a polynucleotide comprising a coding sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 19. In some embodiments, the inhibitor is encoded by polynucleotide comprising a coding sequence according to SEQ ID NO: 19. In some embodiments, the inhibitor comprises a DP71L protein that does not comprise a V6E mutation and/or E12T mutation (e.g., relative to SEQ ID NO: 18).
[0019] In some embodiments, a construct described herein encodes an inhibitor that comprises a peptide linker between the PPI binding domain and eIF2 binding domain. In some embodiments, the inhibitor comprises a peptide linker derived from a DP71L protein. In some embodiments, the peptide linker comprises a glutamic acid at the DP71L protein (SEQ ID NO: 18) E12 position. In some embodiments, the peptide linker comprises an amino acid sequence or a polynucleotide sequence encoding the same, that is at least or exactly 73%, 82%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20 or 21. [0020] In some embodiments, a construct described herein comprises more than one ISR inhibitor selected from the group of proteins consisting of CReP, GADD34, y34.5, CNPV231, ICP34.5, AmEPV193, and DP71L, and/or characteristic portions thereof.
[0021] In some embodiments, a construct described herein comprises a heterologous promoter that is a CAG promoter. In some embodiments, a CAG promoter comprises a polynucleotide sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25 or 26. In some embodiments, a CAG promoter comprises a polynucleotide sequence according to SEQ ID NO: 25 or 26. In some embodiments, a construct described herein comprises an ISR inhibitor that does not comprise and/or is not fused to a Glutathione S-transferase (GST) polypeptide.
[0022] In some embodiments, a construct described herein is comprised in a retroviral capsid. In some embodiments, described herein is an AAV particle comprising any of the constructs disclosed herein, wherein the construct is comprised in an adeno associated virus (AAV) capsid. In some embodiments, the AAV particle is of any one of the AAV-DJ/8, AAV9, AAV Php.B, and/or AAV Php.eB serotypes and/or pseudotypes. In some embodiments, the AAV particle is capable of retrograde infection. In some embodiments, the AAV particle comprises a nucleotide construct comprising a polynucleotide sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22. In some embodiments, the AAV particle comprises a polynucleotide sequence according to SEQ ID NO: 22. In some embodiments, the AAV capsid is an AAV-DJ/8, AAV9, AAV Php.B, and/or AAV Php.eB capsid.
[0023] Also disclosed herein are polypeptides. In some embodiments, a polypeptide comprises an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, a polypeptide comprises an amino acid sequence according to SEQ ID NO: 4.
[0024] Also disclosed herein are pharmacologically acceptable compositions comprising a non-natural construct, polypeptide, and/or AAV particle described herein. Also disclosed herein are cells comprising a non-natural construct, polypeptide, AAV particle, and/or composition disclosed herein.
[0025] Also disclosed herein are methods of delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing cognitive dysfunction in a subject (e.g., a mammalian subject, e.g., a human subject), comprising administering a non-natural construct, polypeptide, AAV particle, or composition disclosed herein to the subject (e.g., the mammalian subject, e.g., the human subject). In some embodiments, the administering is to the central nervous system. In some embodiments, the administering is to the peripheral nervous system. In some embodiments, the administering is to the cerebral spinal fluid (CSF). In some embodiments, the administering is to the hippocampus. In some embodiments, the administering is to the CAI, CA2, and/or CA3 region of the hippocampus. In some embodiments, the administering improves synaptic plasticity in the subject. In some embodiments, the administering improves long term memory formation in the subject. In some embodiments, the administering inhibits and/or counteracts the effects of one or more of Protein kinase R-like endoplasmic reticulum kinase (PERK), eIF-2-alpha kinase (GCN2), protein kinase RNA-activated (PKR), and/or Heme-regulated eIF2a kinase (HRI) in the subject. In some embodiments, the administering localizes a phosphorylase to an eIF2 protein. In some embodiments, the subject has been diagnosed with a disease and/or disorder associated with cognitive impairment or dysfunction. In some embodiments, the subject has a neurodevelopmental disorder. In some embodiments, the subject has a neurodegenerative disorder. In some embodiments, the subject has and/or is anticipated to develop Down Syndrome, Alzheimer’s disease, traumatic brain injury, vanishing white matter (VWM) disease, frontotemporal dementia, and/or aging-related cognitive decline.
[0026] Also disclosed herein are methods of delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing a disease associated with activation, hyperactivation, and/or aberrant activation of the ISR in a human subject, comprising administering a non-natural construct, polypeptide, AAV particle, or composition described herein. In some embodiments, the disease is a cognitive disorder, neurodegeneration, cancer, diabetes, and/or a metabolic disorder. In some embodiments, a subject is less than 10 years of age. In some embodiments, a subject is older than 30 years of age. In some embodiments, a subject is older than 50 years of age. In some embodiments, a subject is older than 70 years of age. In some embodiments, a subject is a mammal. In some embodiments, a subject is a human. [0027] Also disclosed herein are methods of delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing cognitive dysfunction in a human subject, comprising administering to the human subject: A) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence according to SEQ ID NO: 18; B) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence according to SEQ ID NO: 4; C) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence at least or exactly 85% , 90, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to SEQ ID NO: 18, wherein the inhibitor comprises a protein phosphatase-1 (PPI) binding domain and an eukaryotic initiation factor 2 (eIF2) binding domain; or D) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence at least or exactly 85% , 90, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to SEQ ID NO: 4, wherein the inhibitor comprises a protein phosphatase-1 (PPI) binding domain and an eukaryotic initiation factor 2 (eIF2) binding domain. In some embodiments, the polynucleotide comprises a polynucleotide sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22. In some embodiments, the polynucleotide comprises a sequence encoding a peptide linker sequence is at least or exactly 73%, 82%, 91%, or 100% identical to SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a sequence encoding a CReP chimeric protein comprising an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, the polynucleotide is operably linked to a heterologous promoter. In some embodiments, the heterologous promoter is not a galactose-inducible promoter. In some embodiments, the disease is associated with activation, hyperactivation, and/or aberrant activation of the ISR in a human subject.
[0028] Also disclosed herein are transgenic mice comprising a mutation in a Ppplrl5b gene. In some embodiments, the mutation is a R658C mutation. In some embodiments, the mutation is generated using an endonuclease. In some embodiments, the endonuclease is Cas9. In some embodiments, the generation comprises contacting the Ppplrl5b gene with a guide RNA comprising SEQ ID NO: 38.
[0029] Certain embodiments of the present invention(s) are characterized through the following aspects.
[0030] Aspect 1 is a non-natural polynucleotide construct comprising a polynucleotide sequence encoding one or more inhibitor of the integrated stress response (ISR), wherein the one or more inhibitor comprises a protein phosphatase-1 (PPI) binding domain and an eukaryotic initiation factor 2 (eIF2) binding domain, wherein the non-natural polynucleotide construct comprises: A) a heterologous promoter operably linked to the polynucleotide sequence encoding one or more inhibitors of the ISR, wherein the heterologous promoter is not a galactose inducible promoter; and/or B) a recombinant viral vector comprising a heterologous promoter operably linked to the polynucleotide sequence encoding one or more inhibitors of the ISR, wherein the heterologous promoter is not a galactose inducible promoter.
[0031] Aspect 2 is the construct of aspect 1, further comprising a peptide linker sequence connecting the PPI binding domain and eIF2 binding domain.
[0032] Aspect 3 is the construct of aspects 1 or 2, wherein the peptide linker sequence is at least or exactly 73%, 82%, 91%, or 100% identical to SEQ ID NO: 21. [0033] Aspect 4 is the construct of any one of aspects 1-3, wherein the linker sequence comprises a glutamic acid at the DP71L protein E12 position according to SEQ ID NO: 18.
[0034] Aspect 5 is the construct of any one of aspects 1-4, wherein the inhibitor comprises an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4.
[0035] Aspect 6 is the construct of any one of aspects 1-5, wherein the inhibitor comprises an amino acid sequence according to SEQ ID NO: 4.
[0036] Aspect 7 is the construct of any one of aspects 1-4, wherein the inhibitor comprises an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18.
[0037] Aspect 8 is the construct of any one of aspects 1-5, wherein the inhibitor comprises an amino acid sequence according to SEQ ID NO: 18.
[0038] Aspect 9 is the construct of any one of aspects 1-8, wherein the heterologous promoter is a CAG promoter.
[0039] Aspect 10 is the construct of any one of aspects 1-9, wherein the non-natural construct is comprised in a retroviral capsid.
[0040] Aspect 11 is an AAV particle comprising the construct of any one of aspects 1-10 comprised in an adeno associated virus (AAV) capsid.
[0041] Aspect 12 is the AAV particle of aspect 11, wherein the AAV particle is of any one of the AAV-DJ/8, AAV9, AAV Php.B, and/or AAV Php.eB serotypes and/or pseudotypes.
[0042] Aspect 13 is the AAV particle of aspects 11 or 12, wherein the AAV particle is capable of retrograde infection.
[0043] Aspect 14 is an AAV particle comprising a nucleotide construct comprising a polynucleotide sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22.
[0044] Aspect 15 is the AAV particle of aspect 14, comprising a polynucleotide sequence according to SEQ ID NO: 22.
[0045] Aspect 16 is an AAV particle of aspects 14 or 15, wherein the AAV capsid is an AAV-DJ/8, AAV9, AAV Php.B, and/or AAV Php.eB capsid.
[0046] Aspect 17 is a polypeptide comprising an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4.
[0047] Aspect 18 is the polypeptide of aspect 17, comprising an amino acid sequence according to SEQ ID NO: 4. [0048] Aspect 19 is a pharmacologically acceptable composition comprising the nonnatural construct, polypeptide, or AAV particle of any one of aspects 1-18.
[0049] Aspect 20 is a human cell comprising the non-natural construct, polypeptide, AAV particle, or composition of any one of aspects 1-19.
[0050] Aspect 21 is a method of delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing cognitive dysfunction in a human subject, comprising administering the non-natural construct, polypeptide, AAV particle, or composition according to any one of aspects 1-20 to the human subject.
[0051] Aspect 22 is the method of aspect 21, wherein the administering is to the central nervous system.
[0052] Aspect 23 is the method of aspect 21, wherein the administering is to the peripheral nervous system.
[0053] Aspect 24 is the method of aspect 21, wherein the administering is to the cerebral spinal fluid (CSF).
[0054] Aspect 25 is the method of aspects 21 or 22, wherein the administering is to the hippocampus.
[0055] Aspect 26 is the method of aspect 25, wherein the administering is to the CAI, CA2, and/or CA3 region of the hippocampus.
[0056] Aspect 27 is the method of any one of aspects 21-26, wherein the human subject has been diagnosed with a disease and/or disorder associated with cognitive impairment or dysfunction.
[0057] Aspect 28 is the method of any one of aspects 21-27, wherein the subject has a neurodevelopmental disorder.
[0058] Aspect 29 is the method of any one of aspects 21-27, wherein the subject has a neurodegen erative disorder.
[0059] Aspect 30 is the method of any one of aspects 21-29, wherein the subject has and/or is anticipated to develop Down Syndrome, Alzheimer’s disease, traumatic brain injury, vanishing white matter (VWM) disease, frontotemporal dementia, and/or aging-related cognitive decline.
[0060] Aspect 31 is a method of delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing a disease associated with activation, hyperactivation, and/or aberrant activation of the ISR in a human subject, comprising administering the non- natural construct, polypeptide, AAV particle, or composition according to any one of aspects 1-20 to the human subject. [0061] Aspect 32 is the method of aspect 31, where the disease is a cognitive disorder, neurodegeneration, cancer, diabetes, and/or a metabolic disorder.
[0062] Aspect 33 is a method of delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing cognitive dysfunction in a human subject, comprising administering to the human subject: a) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence according to SEQ ID NO: 18; b) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence according to SEQ ID NO: 4; c) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence at least or exactly 85% , 90, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to SEQ ID NO: 18, wherein the inhibitor comprises a protein phosphatase-1 (PPI) binding domain and an eukaryotic initiation factor 2 (eIF2) binding domain; or d) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence at least or exactly 85% , 90, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to SEQ ID NO: 4, wherein the inhibitor comprises a protein phosphatase-1 (PPI) binding domain and an eukaryotic initiation factor 2 (eIF2) binding domain.
[0063] Aspect 34 is the method of aspect 33, wherein the polynucleotide comprises a sequence encoding an inhibitor amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18.
[0064] Aspect 35 is the method of aspect 33, wherein the polynucleotide comprises a sequence encoding an inhibitor amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4.
[0065] Aspect 36 is the method of any one of aspects 33-35, wherein the polynucleotide comprises a sequence encoding a peptide linker sequence is at least or exactly 73%, 82%, 91%, or 100% identical to SEQ ID NO: 21.
[0066] Aspect 37 is the method of any one of aspects 33-36, wherein the polynucleotide is operably linked to a heterologous promoter.
[0067] Aspect 38 is the method of aspect 37, wherein the heterologous promoter is not a galactose-inducible promoter.
[0068] Aspect 39 is the method of any one of aspects 33-38, wherein the disease is associated with activation, hyperactivation, and/or aberrant activation of the ISR in a human subject.
[0069] Aspect 40 is a transgenic mouse comprising a mutation in a Ppplrl5b gene.
[0070] Aspect 41 is the transgenic mouse of aspect 40, wherein the mutation is a R658C mutation. [0071] Aspect 42 is the transgenic mouse of aspects 40 or 41, wherein the mutation is generated using an endonuclease.
[0072] Aspect 43 is the transgenic mouse of aspect 42, wherein the endonuclease is Cas9. [0073] Aspect 44 is the transgenic mouse of aspect 43, wherein the generation comprises contacting the Ppplrl5b gene with a guide RNA comprising SEQ ID NO: 38
BRIEF DESCRIPTION OF THE DRAWINGS
[0074] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0075] FIG. 1, (PRIOR ART) The molecular wiring of the ISR. The ISR kinases phosphorylate eIF2 in response to different cellular stresses. Phosphorylation of eIF2 leads to inhibition of eIF2B activity in the cell, thus reducing ternary complex (eIF2»GTP»methionyl- initiator tRNA) levels, which are controlled by GDP/GTP exchange on eIF2 by eIF2B. The concentration of ternary complex determines the translational status of general protein synthesis (green) and translation of specific mRNAs, such as ATF4 (red). Two phosphatase complexes antagonize the ISR, PP1CReP in a constitutive regime and PP 1·GADD34 in a feedback regime in response to ISR activation.
[0076] FIGs. 2A-J, The ISR was activated and translation was reduced in Ppp1r15bR658C mice. (2A) Sanger sequencing indicating the mutation in Ppp1r15bR658C mice. (2B) Size of the brain in WT and Ppp1r15bR658C mice. (2C-D) Representative Western blot and quantification of CReP (2C) and eIF2-P levels (2D) in hippocampal extracts from WT and Ppp1r15bR658C mice. (2E) Representative Western blot and quantification of eIF2-P levels in primary fibroblast from WT and Ppp1r15bR658C. (2F) Schematic of polysome profiling sedimentation. Following ultracentrifugation, 40S, 60S, and 80S and polysomes were separated based on size. (2G-H) Polysome profile (2G) and quantification (2H) in the hippocampus of WT and Ppp1r15bR658C mice. (2I-J) Incorporation of puromycin into nascent peptides, using an anti- puromycin antibody. Data are means ± s.e.m. *P < 0.05, **P < 0.01.
[0077] FIGs. 3A-F, Long-term fear and object recognition memory is impaired in Ppp1r15bR658C mice. (3A) Schematic of fear conditioning. (3B) Freezing behavior was assessed during a 2-min period prior to conditioning (naive) and then during a 5-min period 24 hr after training in WT and Ppp1r15bR658C mice. (3C) Schematic of the object recognition task. (3D) Object exploration time during training. (3E) Novel object Discrimination Index (DI) was computed as DI = (Novel Object Exploration Time - Familiar Object Exploration Time/Total Exploration Time) X 100 (see e.g., as described in Zhu, et al., 2019). (3F) Late Long-Term Potentiation (L-LTP) was elicited by 4 trains at 100 Hz in WT and pplrlSh^'- ^- mice (at 220 min, p < 0.01). Data are means ± s.e.m. *P < 0.05, **P < 0.01.
[0078] FIGs. 4A-D, DP71L was a potent pan-inhibitor of the ISR. (4 A) Conserved PP1- and eIF2-binding motifs in CReP and viral phosphatases. (4B-D) DP71L-GFP prevented the induction of the ISR by different stresses (oligomycin, poly(I:C), and thapsigargin, respectively (n=4 per group)).
[0079] FIGs. 5A-C, DP71L bound strongly to PPI to attenuate the activation of the ISR. (5A) Schematic of DP71L and ACREP. Protein domains involved in the interaction of the eIF2a phosphatase cofactors with PPI and eIF2. (5B) Western blots revealed the activation of the ISR in cells transfected with either GRP, GFP-DP71L or GFP- ACREP. (5C) In vivo interaction between GFP-tagged DP71L or CREP with endogenous PPI or eIF2a. Levels of PPly, PPla and eIF2 in immunoprecipitates from GFP-DP71L or GFP-ACREP.
[0080] FIGs. 6A-D, The interlink (also termed “linker” or “peptide linker”) region between PPI and eIF2a binding domains is necessary and sufficient for DP71L binding to PPL (6A) Schematic of different chimeric proteins and protein domains involved in PPI and eIF2 binding (e.g., DP71L, ACREP, and the linker mutant proteins (C8, C20, and D20 respectively)). (6B) Human HEK293T cells were transfected with the different GFP-constructs and subsequently immunoprecipitated with an anti-GFP antibody. Shown are levels of PPly, and PPla in immunoprecipitates from the different GFP-tagged chimeric proteins. (6C) Schematic of DP71L, ACREP, and the linker chimera proteins “DP71L-linker” and “ACREP-linker” respectively. (6D) HEK293T cells were transfected with the different GFP-constructs and proteins of interest were subsequently immunoprecipitated with an anti-GFP antibody. The results showed levels of in vivo interactions between GFP-tagged chimeric proteins and endogenous PPly, and PPla in immunoprecipitates from GFP-tagged DP71L, ACREP, and the linker swapped chimeric proteins (DP71L-linker and ACREP-linker respectively).
[0081] FIGs. 7A-C, Mutation of the hydrogen bonding glutamic acid in the linker of DP71L impairs PPI binding. (7 A) Alpha fold prediction of the complex of DP71L with eIF2 and PPL (7B) Zoomed in Alpha fold prediction showing PPI (cyan) and the analogous regions of DP71L (magenta) and CreP (yellow). The glutamic acid involved in hydrogen bonding is highlighted next to the analogous threonine in ACREP. (7C) IP experiments with the glutamic acid mutant of DP71L (E12T) and a control tryptophan mutant (W14Y). [0082] FIGs. 8A-E, DP71L injection rescued the long-term memory deficits in mouse models of Down syndrome and Alzheimer’s disease. (8A) Representative pictures showing the expression of DP71L-GFP in the mouse brain. (8B) Freezing behavior in Ts65Dn mice (model of Down syndrome) injected with either DP71L-GFP or GFP. Freezing behavior was assessed during a 2-min period prior to conditioning (naive) and then during a 5-min period 24 hr after training. (8C) Long-term potentiation (LTP) in Ts65Dn mice injected with either DP71L-GFP or GFP. LTP was elicited by 4 trains at 100 Hz (at 220 min, p < 0.01; n=8-10 per group). (8D) Freezing behavior in APP/PS1 mice (“humanized” model of Alzheimer’s disease) injected with either DP71L-GFP or GFP. Freezing behavior was assessed during a 2-min period prior to conditioning (naive) and then during a 5-min period 24 hr after training. (8E) Long-term potentiation (LTP) in APP/PS1 mice injected with either DP71L-GFP or GFP. LTP was elicited by 4 trains at 100 Hz (at 220 min, p < 0.001, n=10 per group). Data are means ± s.e.m. *P < 0.05, ***P < 0.001.
DETAILED DESCRIPTION
[0083] The work described herein provides at least new compositions and methods for ISR inhibition, including mitigation of ISR associated disorders. The ISR and certain aspects of the systems association with disease has been reviewed in Mauro Costa-Mattioli and Peter Walter 2020 (see e.g., Mauro Costa-Mattioli and Peter Walter, The integrated stress response: From mechanism to disease. Science 2020 Apr 24; 368(6489): eaat5313, which is incorporated herein by reference for the purposes described herein). In certain embodiments, constructs, particles, polypeptides, polynucleotides, and/or compositions described herein can be utilized in methods for inhibiting the ISR. In certain embodiments, constructs, particles, polypeptides, polynucleotides, and/or compositions described herein can be utilized in methods of treatment for diseases associated with natural activation, hyperactivation, and/or aberrant activation of the ISR. In some embodiments, such a disease may include, but is not limited to, cognitive disorders, neurodegeneration, cancer, diabetes, muscle loss (e.g., cachexia), and/or metabolic disorders.
[0084] Use of the one or more compositions (e.g., including components comprised in a composition, such as constructs (e.g., polynucleotide constructs such RNA and/or DNA constructs), particles, polypeptides, etc.) may be employed based on methods described herein. Other embodiments are discussed throughout this application. Any embodiment discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. The embodiments in the Example section are understood to be embodiments that are applicable to all aspects of the technology described herein.
[0085] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.
[0086] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
[0087] The phrase “and/or” means “and” or “or”. To illustrate, A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and/or” operates as an inclusive or.
[0088] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
[0089] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.
[0090] The term “effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result.
[0091] The terms “individual, “subject,” and “patient” are used interchangeably and can refer to a human or non-human.
[0092] The terms “inhibiting” or “reducing” or “preventing” or “avoiding” or any variation of these terms, when used in the claims and/or the specification, includes any measurable decrease or complete inhibition to achieve a desired result.
[0093] As used herein, a “protein” or “polypeptide” refers to a molecule comprising at least five amino acid residues. As used herein, the term “wild-type” refers to the endogenous version of a molecule that occurs naturally in an organism. In some embodiments, wild-type versions of a protein or polypeptide are employed, however, in many embodiments of the disclosure, a modified and/or non-natural protein or polypeptide is employed to modulate (e.g., inhibit) ISR. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some embodiments, a modified/variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified/variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity. A “non-natural” occurring polypeptide and/or construct (e.g., a polynucleotide construct) refers to an engineered polypeptide and/or construct that has been created by the hand of man and is not found in nature. A non-natural polypeptide and/or construct is also understood to comprise non-natural amino acids and/or non-natural nucleotides. A protein may be isolated directly from the organism of which it is native, produced by recombinant DNA/exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. In some embodiments, a protein is produced from an RNA construct. In some embodiments, RNA constructs encoding a protein, (e.g., such as linear and/or circular constructs) can be produced synthetically and/or by in vitro methods. In particular embodiments, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide. The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.
[0094] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.
[0095] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Any embodiment discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. For example, any step in a method described herein can apply to any other method. Moreover, any method described herein may have an exclusion of any step or combination of steps. Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Detailed Description, Claims, and Brief Description of the Drawings.
[0096] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
I. Conditions associated with Integrated Stress Response (ISR) activation
A. Cognitive disorders
[0097] In some embodiments, technologies (e.g., compositions and/or methods) described herein are suitable for delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing a disease associated with activation, hyperactivation, and/or aberrant activation of the ISR in a human subject. In some embodiments, a disease is a disease associated with cognitive decline, and technologies provided herein are suitable for delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing cognitive decline and/or other symptoms associated with the disease state. As described herein, in certain embodiments, cognitive decline is correlated with and/or caused by activation of the Integrated Stress Response (ISR) system. In certain embodiments, technologies described herein are suitable for the reversal, at least in part, of deficits in cognitive function (e.g., impaired cognitive function, such as but not limited to, impaired long term memory (LTM) formation, and/or retention). In certain embodiments, technologies described herein are suitable for the reduction of risk of loss of LTM formation capacity. In certain embodiments, technologies described herein are suitable for the treatment of loss of LTM formation capacity. In certain embodiments, technologies described herein are suitable for the improvement of LTM formation capacity. In certain embodiments, compositions and/or methods disclosed herein improve long-term potentiation (LTP) and/or synaptic plasticity. In certain embodiments, compositions and/or methods disclosed herein improve long-lasting LTP. In certain embodiments, compositions and/or methods disclosed herein improve late LTP. In certain embodiments, technologies described herein are suitable to provide ISR inhibition in conditions of ISR hyperactivation, e.g., in conditions where traditional ISR inhibitory compositions (e.g., small molecules such as kinase inhibitors and/or ISRIB) are not sufficient for achievement of therapeutic disease state amelioration and/or prevention.
[0098] Cognitive function may be assessed using methods (e.g. screening tests and/or questionnaires) well known to one of skill in the art, including but not limited to, e.g, assessment of attention, orientation, language, memory, visuospatial ability, social interaction, functional status and/or behavioral assessment.
[0099] In certain embodiments, technologies described herein are suitable for prevention of, treatment of, and/or reduction of risk of various nervous system disorders (e.g., neurodegenerative disorders, neurodevelopmental disorders, etc.) associated with activation of ISR. In certain embodiments, technologies described herein are suitable for improving brain function (e.g., brain cognitive function), including but not limited to, LTM formation and/or retention, associated with or not associated with activation of ISR.
[0100] In some embodiments, a cognitive disorder is a neurological disorder, and is characterized in part by neurodegeneration and/or aberrant neurodevelopment (e.g., a neurodevelopmental disorder). In certain embodiments, a neurological disorder may be but is not limited to Down syndrome (DS), Charcot-Marie-Tooth disease, major depressive disorder (MDD), schizophrenia, Alzheimer’s disease, Huntington disease, Parkinson’s disease, Amyotrophic lateral sclerosis (ALS), Multiple Sclerosis (MS), Prion disease, traumatic brain injury, Vanishing white matter (VWM) disease, frontotemporal dementia, and/or Aging (e.g., age-related cognitive decline). In certain embodiments, technologies provided herein are suitable for delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing cognitive dysfunction associated with a cognitive disorder. In certain embodiments, technologies provided herein are suitable for delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing a neurological disorder.
[0101] Down syndrome (DS) is the most common genetic form of intellectual disability (ID) and occurs in ~ 10 of 10 000 live births (see e.g., Khoshnood et al., 2011). Individuals with DS show deficits in learning and memory, language and executive functions since early childhood, resulting from the presence of an extra copy of chromosome 21 (Hsa21). DS patients exhibit an age-dependent reduction in dendritic branching and spine density that likely involves impaired reorganization of the actin cytoskeleton in neurons (Marin-Padilla 1976; Takashima et al. 1981). In addition, DS patients show early onset Alzheimer-like neurodegeneration (reviewed in e.g., Lott and Dierssen 2010). The role of the ISR in DS symptom presentation and cognitive capacity is discussed in Zhu et al., 2019, which is incorporated herein by reference for the purposes described herein. In certain embodiments, constructs and/or compositions described herein are suitable for the prevention, treatment, and/or alleviation of symptoms associated with Down syndrome.
[0102] Alzheimer’s disease is biologically defined by the presence of P-amyloid- containing plaques and tau-containing neurofibrillary tangles. Alzheimer’s disease is a genetic and sporadic neurodegenerative disease that causes an amnestic cognitive impairment in its prototypical presentation and non-amnestic cognitive impairment in its less common variants. Alzheimer’s disease is a common cause of cognitive impairment acquired in midlife and late- life but its clinical impact is modified by other neurodegenerative and cerebrovascular conditions. Alzheimer’s disease biology can be thought of as a brain disorder that results from a complex interplay of loss of synaptic homeostasis and dysfunction in the highly interrelated endosomal/lysosomal clearance pathways in which the precursors, aggregated species and post-translationally modified products of Ap and tau play important roles. Loss of synaptic homeostasis is known to promote the ISR. Certain aspects of the ISR in Alzheimer’s disease are described in Tible et al., 2019; Hwang et al., 2017; and Lourenco et al., 2013; each of which are incorporated herein by reference for the purposes described herein. In certain embodiments, constructs and/or compositions described herein are suitable for the prevention, treatment, and/or alleviation of symptoms associated with Alzheimer’s disease.
[0103] Vanishing white matter (VWM) disease (also known as childhood ataxia with central nervous system hypomyelination (CACH) is a rare inherited neurological condition caused by a mutation in one or more of the EIF2B 1 , EIF2B2, EIF2B3, EIF2B4, and/or EIF2B5 genes. Children with VWM disease have one or more defective proteins that prevent the body from making enough myelin, a white, fatty substance that insulates nerve fibers, protecting them from damage. Nerve fibers covered by myelin are known as “white matter.” Without myelin, nerves throughout the body deteriorate and disappear. Symptoms, such as muscle stiffness and poor bodily coordination, often begin to appear between ages 2 and 6. The disease can get worse if the child has a fever, an infection, or a head injury. In certain conditions, an adult onset form of VWM may appear, and is often accompanied by behavioral abnormalities, severe headaches, and/or cognitive decline. Certain roles of the ISR in VWM disease are described in Abbink et al., 2019 (see e.g., Abbink et al., Vanishing white matter: deregulated integrated stress response as therapy target. Ann Clin Transl Neurol. 2019 Aug; 6(8): 1407- 1422; which is incorporated herein by reference for the purposes described herein). In certain embodiments, constructs and/or compositions described herein are suitable for the prevention, treatment, and/or alleviation of symptoms associated with VWM disease. [0104] Frontotemporal dementia is an array of disorders that affect the frontal and temporal lobes of the brain. In certain cases, personality, emotions, behavior, memory formation, and speech are all controlled by these portions of the brain, and are lost as the associated cells lose their function. Symptoms of frontotemporal dementia are dependent upon the areas of the brain affected, but most symptoms can be categorized as either behavioral or language altering. In some embodiments, behavioral symptoms can include inappropriate actions, apathy, lack of interest and/or enthusiasm, lack of inhibition or restraint, neglect of person hygiene and/or care, and/or compulsive behavior. In some embodiments, language symptoms can include difficulty speaking and/or understanding speech, difficulty recalling appropriate language, loss of reading and/or writing skills, and/or difficulty with social interactions. In certain situations, individuals with frontotemporal dementia may develop abnormal protein structures known as pick bodies. The most common genetic cause of frontotemporal dementia are repeat expansions in the C9orf72 gene. Protein accumulation associated with frontotemporal dementia activates the ISR response via the protein kinase PERK. Certain roles of the ISR in frontotemporal dementia are described in Radford et al., 2015 (see e.g., Radford et al., PERK inhibition prevents tau-mediated neurodegeneration in a mouse model of frontotemporal dementia. Acta Neuropathol 130, 633-642 (2015); and Costa-Mattioli & Walter, 2020; each of which are incorporated herein by reference for the purposes described herein). In certain embodiments, constructs and/or compositions described herein are suitable for the prevention, treatment, and/or alleviation of symptoms associated with frontotemporal dementia.
[0105] Healthy aging requires the coordination of numerous stress signaling pathways that converge on the protein homeostasis network. ISR activity increases with age, suggesting a potential link with the aging process. Although decreased protein biosynthesis, which occurs during ISR activation, has been linked to lifespan extension, recent data show that lifespan is limited by the ISR as its inhibition extends survival in nematodes and enhances cognitive function in aged mice. In certain embodiments, the ISR is modulated and also modulates the aging process. Certain roles of the ISR in aging are described in Derisbourg et al., 2021 (see e.g., Derisbourg et al., Perspective: Modulating the integrated stress response to slow aging and ameliorate age-related pathology. Nat Aging. 2021 Sep; l(9):760-768; which is incorporated herein by reference for the purposes described herein). In certain embodiments, constructs and/or compositions described herein are suitable for the prevention, treatment, and/or alleviation of symptoms associated with aging. In certain embodiments, constructs and/or compositions described herein are suitable for the prevention, treatment, and/or alleviation of symptoms associated with aging related cognitive decline. B. Additional disorders and diseases
[0106] In some embodiments, constructs, particles, polypeptides, polynucleotides, and/or compositions described herein may be used in a method of preventing, treating, reducing the progression of, and/or reducing the risk of a disease or disorder associated with ISR activation (e.g., normal activation, hyperactive, and/or aberrant activation) in a human subject. In some embodiments, constructs, particles, polypeptides, polynucleotides, and/or compositions described herein may be used in treating a disease or disorder, wherein the disease or disorder is a neurodegenerative disease, an inflammatory disease, an autoimmune disease, a metabolic syndrome, a cancer, a vascular disease, a fibrotic disease, a viral infection, a musculoskeletal disease (such as a myopathy), an ocular disease, or a genetic disorder.
[0107] In some embodiments, the disease or disorder is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is vanishing white matter disease, childhood ataxia with CNS hypomyelination, intellectual disability syndrome, Alzheimer’s disease, prion disease, Creutzfeldt- Jakob disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS) disease, Pelizaeus-Merzbacher disease, a cognitive impairment, a traumatic brain injury, a postoperative cognitive dysfunction (PCD), a neuro-otological syndrome, hearing loss, Huntington’s disease, stroke, chronic traumatic encephalopathy, spinal cord injury, dementia, frontotemporal dementia (FTD), depression, or a social behavior impairment. In some embodiments, the cognitive impairment is triggered by ageing, radiation, sepsis, seizure, heart attack, heart surgery, liver failure, hepatic encephalopathy, anesthesia, brain injury, brain surgery, ischemia, chemotherapy, cancer treatment, critical illness, concussion, fibromyalgia, or depression. In some embodiments, the neurodegenerative disease is Alzheimer’s disease. In some embodiments, the neurodegenerative disease is ageing-related cognitive impairment. In some embodiments, the neurodegenerative disease is a traumatic brain injury.
[0108] In some embodiments, constructs, particles, polypeptides, polynucleotides, and/or compositions described herein may be used in a method of treating Alzheimer’s disease. In some embodiments, neurodegeneration, and/or cognitive impairment is decreased.
[0109] In some embodiments, the disease or disorder is an inflammatory disease. In some embodiments, the inflammatory disease is arthritis, psoriatic arthritis, psoriasis, juvenile idiopathic arthritis, asthma, allergic asthma, bronchial asthma, tuberculosis, chronic airway disorder, cystic fibrosis, glomerulonephritis, membranous nephropathy, sarcoidosis, vasculitis, ichthyosis, transplant rejection, interstitial cystitis, atopic dermatitis, or inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is Crohn’ disease, ulcerative colitis, or celiac disease.
[0110] In some embodiments, the disease or disorder is an autoimmune disease. In some embodiments, the autoimmune disease is systemic lupus erythematosus, type 1 diabetes, multiple sclerosis, or rheumatoid arthritis.
[oni] In some embodiments, the disease or disorder is a metabolic syndrome. In some embodiments, the metabolic syndrome is acute pancreatitis, chronic pancreatitis, alcoholic liver steatosis, obesity, glucose intolerance, insulin resistance, hyperglycemia, fatty liver, dyslipidemia, hyperlipidemia, hyperhomocysteinemia, or type 2 diabetes. In some embodiments, the metabolic syndrome is alcoholic liver steatosis, obesity, glucose intolerance, insulin resistance, hyperglycemia, fatty liver, dyslipidemia, hyperlipidemia, hyperhomocysteinemia, or type 2 diabetes.
[0112] In some embodiments, the disease or disorder is a cancer. In some embodiments, the cancer is pancreatic cancer, breast cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, urothelial cancer, endometrial cancer, ovarian cancer, cervical cancer, renal cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), multiple myeloma, cancer of secretory cells, thyroid cancer, gastrointestinal carcinoma, chronic myeloid leukemia, hepatocellular carcinoma, colon cancer, melanoma, malignant glioma, glioblastoma, glioblastoma multiforme, astrocytoma, dysplastic gangliocytoma of the cerebellum, Ewing’s sarcoma, rhabdomyosarcoma, ependymoma, medulloblastoma, ductal adenocarcinoma, adenosquamous carcinoma, nephroblastoma, acinar cell carcinoma, neuroblastoma, or lung cancer. In some embodiments, the cancer of secretory cells is non-Hodgkin’s lymphoma, Burkitt’s lymphoma, chronic lymphocytic leukemia, monoclonal gammopathy of undetermined significance (MGUS), plasmacytoma, lymphoplasmacytic lymphoma or acute lymphoblastic leukemia.
[0113] In some embodiments, the disease or disorder is a musculoskeletal disease (such as a myopathy). In some embodiments, the musculoskeletal disease is a myopathy, a muscular dystrophy, a muscular atrophy, a muscular wasting, or sarcopenia. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD), Becker’s disease, myotonic dystrophy, X-linked dilated cardiomyopathy, spinal muscular atrophy (SMA), or metaphyseal chondrodysplasia, Schmid type (MCDS). In some embodiments, the myopathy is a skeletal muscle atrophy. In some embodiments, the musculoskeletal disease (such as the skeletal muscle atrophy) is triggered by ageing, chronic diseases, stroke, malnutrition, bedrest, orthopedic injury, bone fracture, cachexia, starvation, heart failure, obstructive lung disease, renal failure, Acquired Immunodeficiency Syndrome (AIDS), sepsis, an immune disorder, a cancer, ALS, a burn injury, denervation, diabetes, muscle disuse, limb immobilization, mechanical unload, myositis, or a dystrophy.
[0114] In some embodiments, constructs, particles, polypeptides, polynucleotides, and/or compositions described herein may be used in a method of treating musculoskeletal disease. In some embodiments, skeletal muscle mass, quality and/or strength are increased. In some embodiments, synthesis of muscle proteins is increased. In some embodiments, skeletal muscle fiber atrophy is inhibited.
[0115] In some embodiments, the disease or disorder is a vascular disease. In some embodiments, the vascular disease is atherosclerosis, abdominal aortic aneurism, carotid artery disease, deep vein thrombosis, Buerger’s disease, chronic venous hypertension, vascular calcification, telangiectasia or lymphoedema.
[0116] In some embodiments, the disease or disorder is an ocular disease. In some embodiments, the ocular disease is glaucoma, age-related macular degeneration, inflammatory retinal disease, retinal vascular disease, diabetic retinopathy, uveitis, rosacea, Sjogren's syndrome, or neovascularization in proliferative retinopathy.
[0117] In some embodiments, provided herein is a method of modulating an ISR pathway. The constructs, particles, polypeptides, polynucleotides, and/or compositions described herein are believed to be effective for modulating an ISR pathway. In some embodiments, the method of modulating an ISR pathway comprises modulating the ISR pathway in a cell by administering or delivering to the cell a construct and/or compound described herein. In some embodiments, the method of modulating an ISR pathway comprises modulating the ISR pathway in an individual by administering to the individual a construct and/or compound described herein. In some embodiments, modulating of the ISR pathway can be determined by methods known in the art, such as western blot, immunohistochemistry, or reporter cell line assays.
II. Proteins, Polypeptides, and Polynucleotides
[0118] In certain embodiments, provided herein are constructs (e.g., polynucleotide constructs, e.g., linear and/or circular polynucleotide constructs, e.g., DNA and/or RNA constructs), polypeptides and/or compositions, and/or methods of using the same, that consist of, consist essentially of, and/or comprise an inhibitor of the Integrated Stress Response (ISR) or a characteristic portion thereof and/or encode the same. In certain embodiments the size of a protein or polypeptide (wild-type or modified) may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 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, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or greater, and any range derivable therein, or derivative of a corresponding amino sequence described or referenced herein. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term “domain” refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art (e.g., a PPI binding domain, an eIF2 binding domain, a peptide linker, etc. .
[0119] The polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids or nucleic acid substitutions or be at least or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with at least, exactly, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,
119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,
138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,
157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,
195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,
214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,
233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300,
400, 500, 550, 1000 or more contiguous amino acids or nucleic acids, or any range derivable therein, of SEQ ID NOs: 1 to 447.
[0120] In some embodiments, a protein, polypeptide, or nucleic acid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104.
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560,
561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579,
580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598,
599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617,
618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636,
637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655,
656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674,
675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693,
694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712,
713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731,
732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750,
751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769,
770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788,
789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807,
808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826,
827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845,
846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864,
865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883,
884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902,
903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921,
922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940,
941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959,
960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978,
979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997,
998, 999, or 1000, (or any derivable range therein) or more contiguous amino acids or nucleic acids of SEQ ID NOs: 1 to 447.
[0121] In some embodiments, the polypeptide, protein, or nucleic acid may consist of, consist essentially of, or comprise at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,
110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, , 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318,, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337,, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375,, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413,, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432,, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470,, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489,, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508,, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527,, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546,, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565,, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584,, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603,, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622,, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641,, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660,, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679,, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698,, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717,, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736,, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755,, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774,, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812,
813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831,
832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850,
851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869,
870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888,
889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907,
908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926,
927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945,
946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964,
965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983,
984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any derivable range therein) contiguous amino acids or nucleic acids of SEQ ID NOs: 1 to 447 that are at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with one of SEQ ID NOs: 1 to 447. [0122] In some aspects there is a nucleic acid molecule or polypeptide starting at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,
103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,
122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,
141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,
160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,
179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,
198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,
236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,
255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,
274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292,
293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,
312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330,
331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, , 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368,, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387,, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406,, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425,, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444,, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463,, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501,, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539,, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558,, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577,, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596,, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615,, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634,, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653,, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672,, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691,, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710,, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729,, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748,, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767,, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786,, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805,, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824,, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843,, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862,, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881,, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900,, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919,, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938,, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957,, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976,, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 of any of SEQ ID NOs: 1 to 447 and comprising at least, at most or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101
102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671,
672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690,
691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709,
710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728,
729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747,
748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766,
767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785,
786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804,
805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823,
824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842,
843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861,
862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880,
881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899,
900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918,
919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937,
938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956,
957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975,
976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994,
995, 996, 997, 998, 999, or 1000 (or any derivable range therein) contiguous amino acids or nucleic acids of any of SEQ ID NOS: 1 to 447.
[0123] The nucleotide as well as the protein, polypeptide, and peptide sequences for various genes have been previously disclosed, and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov/) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org). The coding regions for these genes may be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.
[0124] In some embodiments, an inhibitor of ISR according to the present disclosure consists of, consists essentially of, or comprises an amino acid sequence as described in Table 1. In some embodiments, a dash (-) as denoted in Table 1 is quantitative and stands for any amino acid. In some embodiments, an inhibitor of ISR consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, having at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NOs: 39 to 447.
Table 1 - Protein variants comprising a PPI binding domain and eIF2 binding domain
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Constitutive repressor of eIF2a phosphorylation (CReP)
[0125] In some embodiments, an inhibitor of ISR according to the present disclosure consists of, consists essentially of, or comprises a constitutive repressor of eIF2a phosphorylation (CReP) protein (also known as Protein phosphatase 1 regulatory subunit 15B; PPP1R15B). In some embodiments, an inhibitor of ISR according to the present disclosure consists of, consists essentially of, or comprises a chimera of CReP and DP71L. In some embodiments, a chimera of CReP and DP71L comprises PPI and/or eIF2 binding domains derived from CReP, and an interjoining peptide linker sequence derived from DP71L. In some embodiments, an inhibitor of ISR consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, having at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NOs: 1 to 6.
SEQ ID NO: 1 - CReP amino acid sequence
MEPGTGGSRKRLGPRAGFRFWPPFFPRRSQAGSSKFPTPLGPENSGNPTLLSSAQPETRVSYWT KLLSQLLAPLPGLLQKVLIWSQLFGGMFPTRWLDFAGVYSALRALKGREKPAAPTAQKSLSSLQ LDSSDPSVTSPLDWLEEGIHWQYSPPDLKLELKAKGSALDPAAQAFLLEQQLWGVELLPSSLQS RLYSNRELGSSPSGPLNIQRIDNFSWSYLLNPSYLDCFPRLEVS YQNSDGNSEWGFQTLTPE SSCLREDHCHPQPLSAELI PASWQGCPPLSTEGLPEIHHLRMKRLEFLQQASKGQDLPTPDQDN GYHSLEEEHSLLRMDPKHCRDNPTQFVPAAGDI PGNTQESTEEKIELLTTEVPLALEEESPSEG CPSSEI PMEKEPGEGRI SWDYSYLEGDLPI SARPACSNKLIDYI LGGASSDLETSSDPEGEDW DEEAEDDGFDSDSSLSDSDLEQDPEGLHLWNSFCSVDPYNPQNFTATIQTAARIVPEEPSDSEK DLSGKSDLENSSQSGSLPETPEHSSGEEDDWESSADEAESLKLWNSFCNSDDPYNPLNFKAPFQ TSGENEKGCRDSKTPSES IVAI SECHTLLSCKVQLLGSQESECPDSVQRDVLSGGRHTHVKRKK VTFLEEVTEYYI SGDEDRKGPWEEFARDGCRFQKRIQETEDAIGYCLTFEHRERMFNRLQGTCF KGLNVLKQC
SEQ ID NO: 2 - CReP polynucleotide sequence
ATGGAGCCGGGGACAGGCGGATCGCGGAAACGGCTTGGCCCTCGGGCGGGCTTCCGGTTCTGGC CACCCTTTTTCCCTCGGCGATCGCAAGCAGGCTCTTCTAAGTTCCCGACGCCTCTTGGCCCGGA AAACTCCGGGAACCCCACACTGCTTTCCTCTGCCCAGCCCGAGACTCGGGTCAGTTACTGGACG AAACTGCTCTCCCAGCTCCTTGCGCCGCTCCCCGGATTGCTTCAGAAGGTGCTAATTTGGAGCC AACTTTTCGGTGGAATGTTTCCGACCAGATGGCTAGATTTTGCTGGAGTCTACAGCGCCCTGAG AGCCCTGAAGGGACGGGAGAAACCAGCCGCCCCCACAGCGCAGAAATCTTTGAGTTCGCTGCAG CTCGACTCCTCAGACCCCTCGGTCACCAGTCCCCTTGATTGGCTAGAGGAGGGGATCCACTGGC AATACTCGCCCCCAGACCTAAAATTGGAGCTTAAGGCCAAGGGAAGTGCTTTGGACCCTGCAGC ACAGGCTTTTCTCTTAGAGCAGCAGCTGTGGGGAGTGGAGCTGTTGCCCAGTAGCCTTCAATCC CGTCTGTACTCTAACCGGGAACTTGGCTCTTCGCCCTCTGGGCCTCTAAACATTCAACGCATAG ACAATTTCAGTGTGGTATCCTATTTGCTGAACCCTTCCTACCTGGACTGCTTTCCTAGGCTAGA AGTCAGCTATCAGAACAGTGATGGAAATAGCGAGGTAGTCGGCTTCCAGACACTAACCCCAGAG AGCAGCTGCCTGAGAGAGGACCATTGTCATCCCCAGCCGCTGAGTGCAGAACTCATTCCGGCCT CGTGGCAGGGATGTCCACCTCTTTCTACGGAAGGCCTACCAGAAATTCACCATCTTCGCATGAA ACGGCTGGAATTCCTTCAACAGGCTAGCAAGGGGCAAGATTTACCCACCCCTGACCAGGATAAT GGCTACCACAGCCTGGAGGAGGAACACAGCCTTCTCCGGATGGATCCAAAACACTGCAGAGATA ACCCAACACAGTTTGTTCCTGCTGCTGGAGACATTCCTGGAAACACCCAGGAATCCACTGAAGA AAAAATAGAATTATTAACTACAGAGGTTCCACTTGCTTTGGAAGAAGAGAGCCCTTCTGAGGGC TGTCCATCTAGTGAGATACCTATGGAAAAGGAGCCTGGAGAGGGCCGAATAAGTGTAGTTGATT ACTCATACCTAGAAGGTGACCTTCCCATTTCTGCCAGACCAGCTTGTAGTAACAAACTGATAGA TTATATTTTGGGAGGTGCATCCAGTGACCTGGAAACAAGTTCTGATCCAGAAGGTGAGGATTGG GATGAGGAAGCTGAGGATGATGGTTTTGATAGTGATAGCTCACTGTCAGACTCAGACCTTGAAC AAGACCCTGAAGGGCTTCACCTTTGGAACTCTTTCTGCAGTGTAGATCCTTATAATCCCCAGAA C T T T AC AG C AAC AAT T C AGAC T G C T G C C AGAAT T G T T C C T GAAGAG C C T T C T GAT T C AGAGAAG GATTTGTCTGGCAAGTCTGATCTAGAGAATTCCTCCCAGTCTGGAAGCCTTCCTGAGACCCCTG AGCATAGTTCTGGGGAGGAAGATGACTGGGAATCTAGTGCAGATGAAGCAGAGAGTCTCAAACT GTGGAACTCATTCTGTAATTCTGATGACCCCTACAACCCTTTAAATTTTAAGGCTCCTTTTCAA ACATCAGGGGAAAATGAGAAAGGCTGTCGTGACTCAAAGACCCCATCTGAGTCCATTGTGGCCA TTTCTGAGTGTCACACCTTACTTTCTTGTAAGGTGCAGCTGTTGGGGAGCCAAGAAAGTGAATG TCCAGACTCGGTACAGCGTGACGTTCTTTCTGGAGGAAGACACACACATGTCAAAAGAAAAAAG GTAACCTTCCTTGAAGAAGTTACTGAGTATTATATAAGTGGTGATGAGGATCGCAAAGGACCAT GGGAAGAAT T T GCAAGGGAT GGAT GCAGGT T CCAGAAACGAAT T CAAGAAACAGAAGAT GCTAT T GGATAT T GOT T GACAT T T GAACACAGAGAAAGAAT GT T TAATAGACT CCAGGGAACAT GOT T C AAAGGACTTAATGTTCTCAAGCAATGTTGA
SEQ ID NO: 3 - Exemplary chimeric CReP polynucleotide sequence
ATGAGGAAGAAGGTTACATTTGCAGCAGCAGTGGAAGTTTGGGAAGCAGATGACATCGATAGGA AGGGGCCCTGGGAGGAGTTCGCGCGTGACGGTTGCAGGTTTCAGAAGAGAATCCAGGAAACTGA GGACGCTATCGGATATTGCCTTACATTTGAGCACCGTGAAAGAATGTTTAACTGA
SEQ ID NO: 4 - Exemplary chimeric CReP protein amino acid sequence
MRKKVTFAAAVEVWEADDIDRKGPWEEFARDGCRFQKRIQETEDAIGYCLTFEHRERMFN
SEQ ID NO: 5 - Exemplary ACReP protein polynucleotide encoding sequence
ATGAGGAAGAAGGTTACATTTCTGGAGGAAGTGACGGAGTACTATATCTCCGGTGACGAAGATA GGAAGGGGCCCTGGGAGGAGTTCGCGCGTGACGGTTGCAGGTTTCAGAAGAGAATCCAGGAAAC TGAGGACGCTATCGGATATTGCCTTACATTTGAGCACCGTGAAAGAATGTTTAACTGA SEQ ID NO: 6 - Exemplary ACReP protein amino acid sequence
MRKKVTFLEEVTEYYI SGDEDRKGPWEEFARDGCRFQKRIQETEDAIGYCLTFEHRERMFN
Protein phosphatase 1 regulatory subunit 15A (GADD34)
[0126] In some embodiments, an inhibitor of ISR according to the present disclosure consists of, consists essentially of, or comprises a protein phosphatase 1 regulatory subunit 15A (GADD34) protein. In some embodiments, an inhibitor of ISR consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, having at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NOs: 7 to 8.
SEQ ID NO: 7 - GADD34 amino acid sequence
MAPGQAPHQATPWRDAHPFFLLSPVMGLLSRAWSRLRGLGPLEPWLVEAVKGAALVEAGLEGEA RTPLAI PHTPWGRRPEEEAEDSGGPGEDRETLGLKTSSSLPEAWGLLDDDDGMYGEREATSVPR GQGSQFADGQRAPLSPSLLIRTLQGSDKNPGEEKAEEEGVAEEEGVNKFSYPPSHRECCPAVEE EDDEEAVKKEAHRTSTSALSPGSKPSTWVSCPGEEENQATEDKRTERSKGARKTSVSPRSSGSD PRSWEYRSGEASEEKEEKAHKETGKGEAAPGPQSSAPAQRPQLKSWWCQPSDEEEGEVKALGAA EKDGEAECPPCI PPPSAFLKAWVYWPGEDTEEEEDEEEDEDSDSGSDEEEGEAEASSSTPATGV FLKSWVYQPGEDTEEEEDEDSDTGSAEDEREAETSASTPPASAFLKAWVYRPGEDTEEEEDEDV DSEDKEDDSEAALGEAESDPHPSHPDQRAHFRGWGYRPGKETEEEEAAEDWGEAEPCPFRVAIY VPGEKPPPPWAPPRLPLRLQRRLKRPETPTHDPDPETPLKARKVRFSEKVTVHFLAVWAGPAQA ARQGPWEQLARDRSRFARRITQAQEELSPCLTPAARARAWARLRNPPLAPI PALTQTLPSSSVP S S P VQT T P L S QAVAT P S RS S AAAAAAL DL S GRRG
SEQ ID NO: 8 - GADD34 polynucleotide sequence
ATGGCCCCAGGCCAAGCACCCCATCAGGCTACCCCGTGGAGGGATGCCCACCCTTTCTTCCTCC TGTCCCCAGTGATGGGCCTCCTCAGCCGCGCCTGGAGCCGCCTGAGGGGCCTGGGACCTCTAGA GCCCTGGCTGGTGGAAGCAGTAAAAGGAGCAGCTCTGGTAGAAGCTGGCCTGGAGGGAGAAGCT AGGACTCCTCTGGCAATCCCCCATACCCCTTGGGGCAGACGCCCTGAAGAGGAGGCTGAAGACA GTGGAGGCCCTGGAGAGGACAGAGAAACACTGGGGCTGAAAACCAGCAGTTCCCTTCCTGAAGC CTGGGGACTTTTGGATGATGATGATGGCATGTATGGTGAGCGAGAGGCAACCAGTGTCCCTAGA GGGCAGGGAAGTCAATTTGCAGATGGCCAGCGTGCTCCCCTGTCTCCCAGCCTTCTGATAAGGA CACTGCAAGGTTCTGATAAGAACCCAGGGGAGGAGAAAGCCGAGGAAGAGGGAGTTGCTGAAGA GGAGGGAGTTAACAAGTTCTCTTATCCACCATCACACCGGGAGTGTTGTCCAGCCGTGGAGGAG GAGGACGATGAAGAAGCTGTAAAGAAAGAAGCTCACAGAACCTCTACTTCTGCCTTGTCTCCAG GATCCAAGCCCAGCACTTGGGTGTCTTGCCCAGGGGAGGAAGAGAATCAAGCCACGGAGGATAA AAGAACAGAAAGAAGTAAAGGAGCCAGGAAGACCTCCGTGTCCCCCCGATCTTCAGGCTCCGAC CCCAGGTCCTGGGAGTATCGTTCAGGAGAGGCGTCCGAGGAGAAGGAGGAAAAGGCACACAAAG AAACTGGGAAAGGAGAAGCTGCCCCAGGGCCGCAATCCTCAGCCCCAGCCCAGAGGCCCCAGCT CAAGTCCTGGTGGTGCCAACCCAGTGATGAAGAGGAGGGTGAGGTCAAGGCTTTGGGGGCAGCT GAGAAGGATGGAGAAGCTGAGTGTCCTCCCTGCATCCCCCCACCAAGTGCCTTCCTGAAGGCCT GGGTGTATTGGCCAGGAGAGGACACAGAGGAAGAGGAAGATGAGGAAGAAGATGAGGACAGTGA CTCTGGATCAGATGAGGAAGAGGGAGAAGCTGAGGCTTCCTCTTCCACTCCTGCTACAGGTGTC TTCTTGAAGTCCTGGGTCTATCAGCCAGGAGAGGACACAGAGGAGGAGGAAGATGAGGACAGTG ATACAGGATCAGCCGAGGATGAAAGAGAAGCTGAGACTTCTGCTTCCACACCCCCTGCAAGTGC TTTCTTGAAGGCCTGGGTGTATCGGCCAGGAGAGGACACGGAGGAGGAGGAAGATGAGGATGTG GATAGTGAGGATAAGGAAGATGATTCAGAAGCAGCCTTGGGAGAAGCTGAGTCAGACCCACATC CCTCCCACCCGGACCAGAGGGCCCACTTCAGGGGCTGGGGATATCGACCTGGAAAAGAGACAGA GGAAGAGGAAGCTGCTGAGGACTGGGGAGAAGCTGAGCCCTGCCCCTTCCGAGTGGCCATCTAT GTACCTGGAGAGAAGCCACCGCCTCCCTGGGCTCCTCCTAGGCTGCCCCTCCGACTGCAAAGGC GGCTCAAGCGCCCAGAAACCCCTACTCATGATCCGGACCCTGAGACTCCCCTAAAGGCCAGAAA GGTGCGCTTCTCCGAGAAGGTCACTGTCCATTTCCTGGCTGTCTGGGCAGGGCCGGCCCAGGCC GCCCGCCAGGGCCCCTGGGAGCAGCTTGCTCGGGATCGCAGCCGCTTCGCACGCCGCATCACCC AGGCCCAGGAGGAGCTGAGCCCCTGCCTCACCCCTGCTGCCCGGGCCAGAGCCTGGGCACGCCT CAGGAACCCACCTTTAGCCCCCATCCCTGCCCTCACCCAGACCTTGCCTTCCTCCTCTGTCCCT TCGTCCCCAGTCCAGACCACGCCCTTGAGCCAAGCTGTGGCCACACCTTCCCGCTCGTCTGCTG CTGCAGCGGCTGCCCTGGACCTCAGTGGGAGGCGTGGCTGA
Herpes simplex virus derived protein y34.5
[0127] In some embodiments, an inhibitor of ISR according to the present disclosure consists of, consists essentially of, or comprises a Herpes simplex virus derived y34.5 protein. In some embodiments, an inhibitor of ISR consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, having at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NOs: 9 to 10.
SEQ ID NO: 9 - y34.5 amino acid sequence
MARRRRHRGPRRPRPPGPTGAVPTAQSQVTSTPNSEPAVRSAPAAAPPPPPAGGPPPSCSLLLR QWLHVPESASDDDDDDDWPDSPPPEPAPEARPTAAAPRPRPPPPGVGPGGGADPSHPPSRPFRL PPRLALRLRVTAEHLARLRLRRAGGE GAPE PPAT PAT PAT PAT PAT PARVRFS PHVRVRHLWW AS AARLARRG S WARE RADRARFRRRVAE AE AVI G P C L G PE ARARALARGAG PAN SV SEQ ID NO: 10 - y34.5 polynucleotide sequence
ATGGCCCGCCGCCGCCGCCATCGCGGCCCCCGCCGCCCCCGGCCGCCCGGGCCCACGGGCGCCG
TCCCAACCGCACAGTCCCAGGTAACCTCCACGCCCAACTCGGAACCCGCGGTCAGGAGCGCGCC
CGCGGCCGCCCCGCCGCCGCCCCCCGCCGGTGGGCCCCCGCCTTCTTGTTCGCTGCTGCTGCGC
CAGTGGCTCCACGTTCCCGAGTCCGCGTCCGACGACGACGATGACGACGACTGGCCGGACAGCC
CCCCGCCCGAGCCGGCGCCAGAGGCCCGGCCCACCGCCGCCGCCCCCCGGCCCCGGCCCCCACC
GCCCGGCGTGGGCCCGGGGGGCGGGGCTGACCCCTCCCACCCCCCCTCGCGCCCCTTCCGCCTT
CCGCCGCGCCTCGCCCTCCGCCTGCGCGTCACCGCGGAGCACCTGGCGCGCCTGCGCCTGCGAC
GCGCGGGCGGGGAGGGGGCGCCGGAGCCCCCCGCGACCCCCGCGACCCCCGCGACCCCCGCGAC
CCCCGCGACCCCCGCGCGGGTGCGCTTCTCGCCCCACGTCCGGGTGCGCCACCTGGTGGTCTGG
GCCTCGGCCGCCCGCCTGGCGCGCCGCGGCTCGTGGGCCCGCGAGCGGGCCGACCGGGCTCGGT
TCCGGCGCCGGGTGGCGGAGGCCGAGGCGGTCATCGGGCCGTGCCTGGGGCCCGAGGCCCGTGC
CCGGGCCCTGGCCCGCGGAGCCGGCCCGGCGAACTCGGTCTAA
Canarypox virus derived protein CNPV231
[0128] In some embodiments, an inhibitor of ISR according to the present disclosure consists of, consists essentially of, or comprises a Canarypox virus derived CNPV231 protein (MyD116- like protein; accession Q6VZB6). In some embodiments, an inhibitor of ISR consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, having at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NOs: 11 to 12.
SEQ ID NO: 11 - CNPV231 amino acid sequence
MEIQEKADVKKDMSLVDDNGVENKELSKLSETDSFQEWDESDEDS SNDTDTENMELWNMFCRSD DPYNLFTFTASVNKEWHSTSCHIDITKKSWTFSETI IEYHVPYEDRKGPWEEIARDRYRFEKR IKETAEI IEFCLSENHRRNIKTHLKYEDDK
SEQ ID NO: 12 - CNPV231 polynucleotide sequence
AT GGAAAT ACAAGAAAAAGCAGACGT AAAAAAAGATAT GT CT T TAGT GGAT GAT AACGGT GTAG
AAAATAAAGAACTATCGAAGCTAAGTGAAACGGACTCTTTCCAGGAATGGGACGAGTCTGATGA
AGACTCGTCCAACGATACTGATACTGAAAATATGGAATTATGGAATATGTTTTGTCGTTCTGAC GACCCTTATAATCTTTTTACGTTTACAGCATCGGTTAATAAAGAATGGCATAGCACTTCTTGCC ATATAGAT AT TACTAAGAAAT CT GT T GTAACGT T T AGT GAAACCATAATAGAAT AT CAT GT T C C T T AT GAAG AT AGAAAAG GAC C T T G G G AAGAGAT TGC T AGAGAT AG G T AT AGAT T T GAGAAAAG A AT T AAAGAAACAGCAGAAAT AAT AGAAT T C T GT T TAT C T GAAAAT CACAGAC GT AAT AT AAAAA C T CAT T T AAAAT AT GAG GAC GAT AAAT GA
Macropoid herpes virus derived protein ICP34.5
[0129] In some embodiments, an inhibitor of ISR according to the present disclosure consists of, consists essentially of, or comprises a Macropoid herpes virus derived ICP34.5 protein. In some embodiments, an inhibitor of ISR consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, having at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NOs: 13 to 14.
SEQ ID NO: 13 - ICP34.5 amino acid sequence
MSRRRGPRRRGPRRRPRPGAPAVPRPGAPAVPRPGALPTADSQMVPAYDSGTAVESAPAASSLL RRWLLVPQADDSDDADYAGNDDAEWANSPPSEGGGKAPEAPHAAPAAACPPPPPRKERGPQRPL PPHLALRLRTTTEYLARLSLRRRRPPASPPADAPRGKVCFSPRVQVRHLVAWETAARLARRGSW ARERADRDRFRRRVAAAEAVI GPCLE PEARARARARARAHEDGGPAEEEEAAAAARGS SAAAGP GRRAV
SEQ ID NO: 14 - ICP34.5 polynucleotide sequence
ATGTCCCGCCGCCGGGGTCCCCGCCGCCGGGGTCCCCGGCGCCGGCCGCGCCCCGGCGCTCCAG CCGTGCCGCGCCCCGGCGCTCCAGCCGTGCCGCGCCCCGGCGCGCTCCCAACCGCAGACTCCCA AATGGTCCCTGCGTACGACTCGGGAACCGCGGTCGAGAGCGCGCCGGCCGCGTCCTCGCTCCTG CGGCGCTGGCTGCTGGTGCCCCAGGCGGACGACAGCGACGACGCGGACTACGCCGGCAACGACG ACGCAGAGTGGGCGAACAGCCCCCCGAGCGAGGGCGGGGGGAAGGCGCCGGAGGCCCCGCACGC CGCGCCTGCCGCCGCCTGCCCCCCGCCGCCGCCGCGCAAGGAGCGCGGGCCGCAGCGCCCCCTT CCGCCCCACCTGGCGCTACGGCTGCGCACCACGACGGAGTACCTGGCGCGCCTGAGCCTGCGCC GGCGGCGGCCCCCCGCGTCCCCGCCCGCGGACGCGCCGTGCTTCTCGCCGCGCGTGCAGGTGCG CCATCTGGTGGCCTGGGAGACGGCCGCGCGCCTGGCCCGACGGGGGTCCTGGGCGCGCGAGCGG GCCGACCGCGACCGGTTCCGGCGCCGCGTGGCGGCGGCCGAGGCGGTCATCGGACCGTGCCTGG AGCCCGAGGCCCGAGCTCGGGCCCGAGCCCGAGCCCGGGCCCACGAAGACGGCGGACCCGCGGA GGAGGAGGAGGCGGCGGCGGCGGCGCGCGGGTCCTCCGCCGCCGCGGGCCCGGGCCGTCGGGCG GTCTAG Amsacta moorei entomopoxvirus “L” derived protein AmEPV193
[0130] In some embodiments, an inhibitor of ISR according to the present disclosure consists of, consists essentially of, or comprises an Amsacta moorei entomopoxvirus “L” derived AmEPV193 protein (accession Q9EML3). In some embodiments, an inhibitor of ISR consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, having at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NOs: 15 to 16.
SEQ ID NO: 15 - AmEPV193 amino acid sequence
MNVKI IEKYQHFKEDKYI SYYNI FIYILEEYI I ILYNYKLIYI INKNYIQYMYYNYLFKNNIYY NLKLYNNNKLLKHKPSKKVRFSSEPPKLHIMYVWLYAAKQTRKLYWDKFAIDRHRFKRRINDI D I S I SWVLT PHHRHKIMKHLKLI
SEQ ID NO: 16 - AmEPV193 polynucleotide sequence
AT GAAC GT AAAAAT T AT AGAAAAAT AT CAACAT T T T AAAGAAGAT AAAT AT AT AT CAT AT TATA AT AT AT T TAT AT AT AT AC T AGAAGAAT AT AT T AT AAT AT T AT AT AAT TAT AAAT T AAT AT AT AT AAT AAAT AAAAAT TAT AT AC AAT AT AT GT AT TAT AAT TAT T TAT T TAAAAATAATATATAT TAT AAT T T AAAAT TAT AT AAT AAT AAT AAAT TAT T AAAAC AT AAAC C G T C GAAAAAAG TACGCTTTT CATCCGAACCACCAAAACTCCACATTATGTATGTTTGGTTATATGCTGCAAAACAAACTCGAAA AT TATACT GGGATAAAT T T GCGAT T GATAGACATAGAT T CAAAAGAAGAAT TAAT GATATAGAT AT AT CAAT AT CTTGGGTTT T AAC T C C ACAT CACAGACAT AAAAT TAT GAAACAT C T T AAGT T AA TATAA
African swine fever virus derived protein DP71L
[0131] In some embodiments, an inhibitor of ISR according to the present disclosure consists of, consists essentially of, or comprises an African swine fever virus derived DP71L protein. In some embodiments, a DP71L protein is a long form protein, while alternatively, in some embodiments, a DP71L protein is a short form protein. In general, as utilized herein, DP71L is in reference to the short form protein (e.g., as represented by SEQ ID NO: 18), however, in some embodiments, slightly longer or shorter protein isoforms may be utilized (e.g., a protein isoform comprising an additional, or missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, amino acids relative to SEQ ID NO: 18). In some embodiments, an inhibitor of ISR according to the present disclosure is a chimeric protein (e.g., a synthetic protein comprised of domains derived from two or more proteins) that comprises a peptide linker sequence deposited between a PPI binding domain and an eIF2 binding domain that is derived from DP71L. In some embodiments, a peptide linker sequence derived from DP71L comprises 8, 9, 10, 11, or 12, amino acids. In some embodiments, a peptide linker sequence derived from DP71L enhances the ability of an ISR inhibitor to bind to PPI. In some embodiments, a peptide linker sequence derived from DP71L comprises one or more glutamic acids that may influence PPI binding domain inter- or intraprotein binding dynamics. In some embodiments, the one or more glutamic acid is glutamic acid E12 of the DP71L protein (e.g., the glutamic acid underlined in the sequence “MDVKHVRFAAAVEVWEADDI " (SEQ ID NO: 17), which is the E12 glutamic acid found in SEQ ID NO: 18 (e.g., the 12th amino acid when not including the N-terminal methionine, or the 13th amino acid when including the N-terminal methionine). In some embodiments, a linker sequence derived from DP71L consists of, consists essentially of, or comprises an polynucleotide sequence and/or amino acid sequence at least or exactly about 55%, 64%, 73%, 82%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NO: 20 to 21. In some embodiments, an inhibitor of ISR consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, having at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identity to SEQ ID NOs: 18 to 19.
SEQ ID NO: 18 - DP71L amino acid sequence
MDVKHVRFAAAVEVWEADDIERKGPWEQAAVDRFRFQRRIASVEELLSAVLLRQKKLLEQQ
SEQ ID NO: 19 - DP71L polynucleotide sequence
ATGGATGTAAAGCATGTTCGCTTTGCAGCAGCAGTGGAAGTTTGGGAAGCAGATGACATCGAGC GGAAGGGTCCTTGGGAGCAAGCTGCTGTGGACAGATTTCGATTTCAGAGACGAATAGCCTCCGT CGAGGAACTCCTTTCAGCCGTTCTGCTGCGACAAAAGAAACTTCTGGAGCAGCAGTGA
SEQ ID NO: 20 - DP71L derived peptide linker polynucleotide encoding sequence
GCAGCAGCAGTGGAAGTTTGGGAAGCAGATGACATC SEQ ID NO: 21 - DP71L derived peptide linker amino acid sequence
AAAVEVWEADDI
[0132] In some embodiments, the amino acid subunits of a protein are modified to create an equivalent, or even improved, second-generation variant polypeptide or peptide. For example, certain amino acids may be substituted for other amino acids in a protein or polypeptide sequence with or without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein’s functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and nevertheless produce a protein with similar or desirable properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes which encode proteins without appreciable loss of their biological utility or activity.
[0133] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids.
[0134] Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants. A variation in a polypeptide of the disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide, as compared to wild-type. A variant can comprise an amino acid sequence that is at least or exactly about 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein. A variant can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids.
[0135] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.
[0136] Deletion variants typically lack one or more residues of the native or wild type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein.
[0137] Insertional mutants typically involve the addition of amino acid residues at a nonterminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein. [0138] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein or polypeptide, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar chemical properties. “Conservative amino acid substitutions” may involve exchange of a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties.
[0139] Alternatively, substitutions may be “non-conservative”, such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class. III. Vectors
[0140] Among other things, the present disclosure provides that in some embodiments, inhibitors of ISR as described herein are encoded by a polynucleotide, such as a vector comprising a polynucleotide (e.g., a polynucleotide construct). Vectors comprising polynucleotide constructs according to the present disclosure include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viral constructs (e.g., lentiviral, retroviral, adenoviral, and adeno associated viral constructs) that incorporate a polynucleotide comprising an inhibitor of ISR gene or characteristic portion thereof (e.g., as utilized herein, a “characteristic portion thereof’ refers to the portion of said protein required to perform the desired function, e.g., it comprises the ability to inhibit ISR, e.g., it comprises a functional PPI binding domain and an eIF2 binding domain). Those of skill in the art will be capable of selecting suitable constructs, as well as cells, for making any of the polynucleotides described herein. In some embodiments, a construct is a plasmid (i.e., a circular DNA molecule that can autonomously replicate inside a cell). In some embodiments, a construct can be a cosmid (e.g., pWE or sCos series).
[0141] In some embodiments, a construct is a viral construct. In some embodiments, a viral construct is a lentivirus, retrovirus, adenovirus, or adeno-associated virus construct. In some embodiments, a construct is an adeno-associated virus (AAV) construct (see, e.g., Asokan et al., Mol. Then 20: 699-7080, 2012, which is incorporated herein by reference for the purposes described herein). In some embodiments, a viral construct is an adenovirus construct. In some embodiments, a viral construct may also be based on or derived from an alphavirus. Alphaviruses include but are not limited to, Sindbis (and VEEV) virus, Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus, O’nyong-nyong virus, Pixuna virus, Rio Negro virus, Ross River virus, Salmon pancreas disease virus, Semliki Forest virus, Southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Whataroa virus. Generally, the genome of such viruses encode nonstructural (e.g., replicon) and structural proteins (e.g., capsid and envelope) that can be translated in the cytoplasm of the host cell. Ross River virus, Sindbis virus, Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEEV) have all been used to develop viral constructs for coding sequence delivery. Pseudotyped viruses may be formed by combining alphaviral envelope glycoproteins and retroviral capsids. Examples of alphaviral constructs can be found in U.S. Publication Nos. 20150050243, 20090305344, and 20060177819; constructs and methods of their making are incorporated herein by reference for the purposes described herein.
[0142] In some embodiments, constructs provided herein can be of different sizes. In some embodiments, a construct is a plasmid and can include a total length of up to about 1 kb, up to about 2 kb, up to about 3 kb, up to about 4 kb, up to about 5 kb, up to about 6 kb, up to about 7 kb, up to about 8 kb, up to about 9 kb, up to about 10 kb, up to about 11 kb, up to about 12 kb, up to about 13 kb, up to about 14 kb, or up to about 15 kb. In some embodiments, a construct is a plasmid and can have a total length in a range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about
I kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 1 kb to about
I I kb, about 1 kb to about 12 kb, about 1 kb to about 13 kb, about 1 kb to about 14 kb, or about 1 kb to about 15 kb.
[0143] In some embodiments, a construct is a viral construct and can have a total number of nucleotides of up to 10 kb. In some embodiments, a viral construct can have a total number of nucleotides in the range of about 1 kb to about 2 kb, 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 1 O kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 2 kb to about 9 kb, about 2 kb to about 10 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 3 kb to about 9 kb, about 3 kb to about 10 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 4 kb to about 9 kb, about 4 kb to about 10 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 5 kb to about 9 kb, about 5 kb to about 10 kb, about 6 kb to about 7 kb, about 6 kb to about
8 kb, about 6 kb to about 9 kb, about 6 kb to about 10 kb, about 7 kb to about 8 kb, about 7 kb to about 9 kb, about 7 kb to about 10 kb, about 8 kb to about 9 kb, about 8 kb to about 10 kb, or about
9 kb to about 10 kb. [0144] In some embodiments, a construct is a lentivirus construct and can have a total number of nucleotides of up to 8 kb. In some examples, a lentivirus construct can have a total number of nucleotides of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, or about 7 kb to about 8 kb.
[0145] In some embodiments, a construct is an adenovirus construct and can have a total number of nucleotides of up to 8 kb. In some embodiments, an adenovirus construct can have a total number of nucleotides in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, or about 7 kb to about 8 kb.
[0146] Any of the constructs described herein can further include a control sequence, e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly(A)) sequence, a Kozak consensus sequence, and/or additional untranslated regions which may house pre- or post-transcriptional regulatory and/or control elements. In some embodiments, a promoter can be a native promoter, a constitutive promoter, an inducible promoter, and/or a tissue-specific promoter. Non-limiting examples of control sequences are described herein. A. AAV particles
[0147] Among other things, the present disclosure provides AAV particles that comprise a polynucleotide construct encoding an inhibitor of ISR, and an AAV capsid. In some embodiments, AAV particles can be described as having a serotype, which is a description of the construct strain and the capsid strain. For example, in some embodiments an AAV particle may be described as AAV2, wherein the particle has an AAV2 capsid and a construct that comprises characteristic AAV2 Inverted Terminal Repeats (ITRs). In some embodiments, an AAV particle may be described as a pseudotype, wherein the capsid and construct are derived from different AAV strains, for example, AAV2/9 would refer to an AAV particle that comprises a construct utilizing the AAV2 ITRs and an AAV9 capsid. Additional examples of pseudotyped AAV vectors include, but are not limited to, AAV2/1, AAV2/2, AAV2/3, AAV2/4, AAV2/5, AAV2/6, AAV2/7, AAV2/8 and AAV2/9.
[0148] In some embodiments, AAV particles suitable for use according to the present disclosure may comprise or be derived from any natural or recombinant AAV serotype. In some embodiments, an AAV according to the present invention is selected from natural serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12; or pseudotypes, chimeras, and variants thereof.
[0149] As used herein, the term “chimera” when referring to an AAV vector, or a “chimeric AAV vector”, refers to an AAV vector which comprises a capsid containing VP1, VP2 and VP3 proteins from at least two different AAV serotypes; or alternatively, which comprises VP1, VP2 and VP3 proteins, at least one of which comprises at least a portion from another AAV serotype. Examples of chimeric AAV vectors include, but are not limited to, AAV-DJ, AAV-DJ/8, AAV2G9, AAV218, AAV218G9, AAV8G9, and AAV911.
[0150] In some embodiments, an AAV serotype and/or pseudotype according to the present invention is selected from the group comprising or consisting of AAV1, AAV2, AAV3, AAV 4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV106.1/hu.37, AAV114.3/hu.4O, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.1/hu.43, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55, AAV16.12/hu.11 , AAV16.3, AAV16.8/hu.lO, AAV161.1O/hu.6O, AAV161.6/hu.61, AAVl-7/rh.48, AAVl-8/rh.49, AAV218, AAV218G9, AAV2-15/rh.62, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV2-3/rh.61, AAV24.1, AAV2-4/rh.5O, AAV2-5/rh.51, AAV2.5T, AAV27.3, AAV29.3/bb.l, AAV29.5/bb.2, AAV2G9, AAV3B, AAV3.1/hu.6, AAV3.1/hu.9, AAV3-l l/rh.53, AAV3-3, AAV33.12/hu.l7, AAV33.4/hu.l5, AAV33.8/hu. l6, AAV3-9/rh.52, AAV3a, AAV3b, AAV4-19/rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2, AAV44.5, AAV46.2/hu.28, AAV46.6/hu.29, AAV4-8/rh.64, AAV4-9/rh.54, AAV52.1/hu.20,AAV52/hu.l9, AAV5-22/rh.58, AAV5-3/rh.57, AAV54.1/hu.21, AAV54.2/hu.22,AAV54.4R/hu.27, AAV54.5/hu.23,
AAV54.7/hu.24, AAV58.2/hu.25, AAV6.1, AAV6.1.2, AAV6.2, AAV7m8, AAV7.2, AAV7.3/hu.7, AAV-8b, AAV8G9, AAV-8h, AAV911, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVcy.5Rl, AAVcy.5R2, AAVcy.5R3, AAVcy.5R4, AAVcy.6, AAVhu.l, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.8, AAVhu.9, AAVhu.10, AAVhu.l l, AAVhu.12, AAVhu.13, AAVhu.14/9, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.19, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.53, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVpi. l, AAVpi.2, AAVpi.3, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh8R R533A mutant, AAVrh8R A586R mutant, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh. 13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.44, AAVrh.45, AAVrh.46, AAVrh.47, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.50, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59, AAVrh.60, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.65, AAVrh.67, AAVrh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73, AAVrh.74, AAV-PHP.B, AAVPHP.A, AAV- G2B-26, AAV-G2B-13, AAV-TH1 .1-32, AAVTH1.1-35, AAV-PHP.B2, AAV-PHP.B3, AAV- PHP.N/PHP.B-DGT, AAV-PHP.B-EST, AAV-PHP.B-GGT, AAV-PHP.BATP, AAV-PHP.B- ATT-T, AAV-PHP.B-DGT-T, AAV-PHP.B-GGT-T, AAV-PHP.B-SGS, AAV-PHP.B-AQP, AAV-PHP.B-QQP, AAV-PHP.B-SNP(3), AAV-PHP.B-SNP, AAV-PHP.B-QGT, AAV-PHP.B- NQT, AAV-PHP.B-EGS, AAV-PHP.BSGN, AAV-PHP.B-EGT, AAV-PHP.B-DST, AAV- PHP.BDST, AAV-PHP.B-STP, AAV-PHP.B-PQP, AAV-PHP.BSQP, AAV-PHP.B-Q1P, AAV- PHP.B-TMP, AAV-PHP.BTTP, AAV-PHP.S/G2A12, AAV-G2A15/G2A3, AAV-G2B4, AAV- G2B5, PHP.S, AAAV, AAV A3.3, AAV A3.4, AAV A3.5, AAV A3.7, AAV CBr-7.3, AAV CBr- 7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-Pl, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-N4, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-Bl, AAV CKd-B2, AAV CKd-B3, AAV CKdB4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-Hl, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N9, AAV CLg-Fl, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg- F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-M9, AAV CLv-R6, AAV CLv-1, AAV CLvl-1, AAV CLvl-10, AAV CLvl-2, AAV CLv-12, AAV CLvl-3, AAV CLv-13, AAV CLvl-4, AAV CLvl-7, AAV CLvl-8, AAV CLvl-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-Dl, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El, AAV CLv-Kl, AAV CLv-K3, AAV CLv- K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-Ml, AAV CLv-Ml l, AAV CLv- M2, AAV CLv-M5, AAV CLv-M6, AAV CLvM7, AAV CLv-M8, AAV CLv-Rl, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-8.10, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp- 8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAVLK08, AAV-LK15, AAV Shuffle 100-1, AAV Shuffle 100-2, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV SM 100-10, AAV SM 100-3, AAV SM 10-1, AAV SM 10-2, AAV SM 10-8, AAV.VR-355, AAV-b, AAVC1, AAVC2, AAVC5, AAVCh.5, AAVCh.5Rl, AAV-DJ, AAV-DJ8, AAVF1/HSC1, AAVF11/HSC11, AAVF12/HSC12, AAVF13/HSC13, AAVF14/HSC14, AVF15/HSC15, AAVF16/HSC16, AAVF17/HSC17, AAVF2/HSC2, AAVF3, AAVF3/HSC3, AAVF4/HSC4, AAVF5, AAVF5/HSC5, AAVF6/HSC6, AAVF7/HSC7, AAVF8/HSC8, AAVF9/HSC9, AAV-h, AAVH- l/hu.l,AAVH2,AAVH-5/hu.3,AAVH6,AAVhEl.l, AAVhErl.14, AAVhErE16, AAVhErl.18, AAVhER1.23, AAVhErl.35, AAVhErl.36, AAVhErl.5, AAVhErl.7, AAVhErl.8, AAVhEr2.16, AAVhEr2.29, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhEr2.4, AAVhEr3.1, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39, AAVLG-9/hu.39, AAV- LK01, AAV-LK02, AAV-LK03, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAVLK07, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK16, AAV-LK17, AAVLK18, AAV-LK19, AAVN721-8/rh.43, AAV-PAEC, AAVPAEC12, AAV-PAEC11, AAV-PAEC2, AAV-PAEC4, AAVPAEC6, AAV-PAEC7, AAV- PAECS, Anc80, Anc80L65, Anc81, Anc82, Anc83, Anc84, Anc94, And 10, And 13, Ancl26, Ancl27, BAAV, BNP61 AAV, BNP62 AAV, BNP63 AAV, bovine AAV, caprine AAV, Japanese AAV10 serotype, UPENN AAV10, VOY101, and VOY201.
[0151] In certain embodiments, an AAV serotypes and/or pseudotype is AAV-DJ/8, AAV9, AAV Php.B, and/or AAV Php.eB. In certain embodiments, an AAV serotype and/or pseudotype comprises AAV-DJ/8. In certain embodiments, an AAV serotype and/or pseudotype comprises AAV9. In certain embodiments, an AAV serotype and/or pseudotype comprises AAV Php.B. In certain embodiments, an AAV serotype and/or pseudotype comprises AAV Php.eB.
[0152] In some embodiments, an AAV is an AAV variant that has been genetically modified, e.g., by substitution, deletion or addition of one or several amino acid residues in one or more capsid proteins. Examples of such variants include, but are not limited to, AAV2 with one or more of Y444F, Y500F, Y730F and/or S662V mutations; AAV3 with one or more of Y705F, Y731F and/or T492V mutations; and AAV6 with one or more of S663V and/or T492V mutations.
[0153] In some embodiments, an AAV capsid is modified to comprise at least one surfacebound saccharide or a derivative thereof. As used herein, the term “surface-bound”, when referring to the at least one saccharide, means that said at least one saccharide is bound to and exposed at the outer surface of the AAV vector. Suitable examples of saccharides include, but are not limited to, monosaccharides, oligosaccharides, polysaccharides, and derivatives thereof. 1. AAV constructs
[0154] In some embodiments, the present disclosure provides polynucleotide vectors (e.g., polynucleotide constructs) that comprise a polynucleotide sequence encoding an inhibitor of ISR or a characteristic portion thereof (e.g., comprising a PPI binding domain and an eIF2a binding domain). In some embodiments described herein, a polynucleotide vector comprising an inhibitor of ISR is a polynucleotide construct, and can be comprised in an AAV capsid to produce an AAV particle (e.g., an AAV particle comprises an AAV construct comprised in an AAV capsid).
[0155] In some embodiments, a polynucleotide construct comprises one or more components derived from or modified from a naturally occurring AAV genomic construct. In some embodiments, a sequence derived from an AAV construct is an AAV1 construct, an AAV2 construct, an AAV3 construct, an AAV4 construct, an AAV5 construct, an AAV6 construct, an AAV7 construct, an AAV8 construct, an AAV DJ/8 construct, an AAV9 construct, an AAV2.7m8 construct, an AAV8BP2 construct, an AAV293 construct, an AAVPhp.B construct, or AAVPhp.eB construct (see e.g., Chan et al., 2017). Additional exemplary AAV constructs that can be used herein are known in the art. See, e.g., Kanaan etal., Mol. Ther. Nucleic Acids 8: 184- 197, 2017; Li et al., Mol. Ther. 16(7): 1252-1260, 2008; Adachi et al., Nat. Commun. 5: 3075, 2014; Isgrig et al., Nat. Commun. 10(1): 427, 2019; and Gao et al., J. Virol. 78(12): 6381-6388, 2004; each of which are incorporated herein by reference for the purposes described herein).
[0156] In some embodiments, AAV derived sequences (e.g., which are comprised in a polynucleotide construct) typically include the cis-acting 5' and 3' ITR sequences (see, e.g., B. J. Carter, in "Handbook of Parvoviruses," ed., P. Tijsser, CRC Press, pp. 155 168, 1990, which is incorporated herein by reference for the purposes described herein). Typical AAV2-derived ITR sequences are about 145 nucleotides in length. In some embodiments, at least or exactly 80% of a typical ITR sequence (e.g., at least or exactly 85%, at least or exactly 90%, at least or exactly 95%, or at least or exactly 100%, etc.) is incorporated into a construct provided herein. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook etal., "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York, 1989; and K. Fisher et al., J Virol. 70:520 532, 1996, each of which is incorporated herein by reference for the purposes described herein). In some embodiments, any of the coding sequences and/or constructs described herein are flanked by 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including presently identified AAV types. [0157] In some embodiments, polynucleotide constructs described in accordance with this disclosure and in a pattern known to the art (see, e.g., Asokan et al., Mai. Ther. 20: 699- 7080, 2012, which is incorporated herein by reference for the purposes described herein) are typically comprised of, a coding sequence or a portion thereof, at least one and/or control sequence, and optionally 5' and 3' AAV inverted terminal repeats (ITRs). In some embodiments, provided constructs can be packaged into a capsid to create an AAV particle. An AAV particle may be delivered to a selected target cell. In some embodiments, provided constructs comprise an additional optional coding sequence that is a nucleic acid sequence (e.g., inhibitory nucleic acid sequence), heterologous to the construct sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest. In some embodiments, a nucleic acid coding sequence is operatively linked to and/or control components in a manner that permits coding sequence transcription, translation, and/or expression in a cell of a target tissue.
[0158] In some embodiments, an unmodified AAV endogenous genome includes two open reading frames, "cap" and "rep," which are flanked by ITRs. In some embodiments, recombinant AAV constructs similarly comprise one or more open reading frames (e.g., a coding sequence comprising an ISR inhibitor encoding sequence) flanked by ITR sequences. In some embodiments, an AAV construct also comprises conventional control elements that are operably linked to the coding sequence in a manner that permits its transcription, translation and/or expression in a cell transfected with the polynucleotide construct or infected with a virus particle produced by the disclosure. In some embodiments, an AAV construct optionally comprises a promoter, an enhancer, an untranslated region (e.g., a 5' UTR, 3' UTR), a Kozak sequence, an internal ribosomal entry site (IRES), splicing sites (e.g., an acceptor site, a donor site), a polyadenylation site, or any combination thereof.
[0159] In some embodiments, a construct is an AAV construct. In some embodiments, an AAV construct can include at least 500 bp, at least 1 kb, at least 1.5 kb, at least 2 kb, at least 2.5 kb, at least 3 kb, at least 3.5 kb, at least 4 kb, or at least 4.5 kb. In some embodiments, an AAV construct can include at most 7.5 kb, at most 7 kb, at most 6.5 kb, at most 6 kb, at most 5.5 kb, at most 5 kb, at most 4.5 kb, at most 4 kb, at most 3.5 kb, at most 3 kb, or at most 2.5 kb. In some embodiments, an AAV construct can include about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.
[0160] Any of the constructs described herein can further include regulatory and/or control sequences, e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly(A)) sequence, a Kozak consensus sequence, and/or any combination thereof. In some embodiments, a promoter can be a native promoter, a constitutive promoter, an inducible promoter, and/or a tissue-specific promoter. Non-limiting examples of control sequences are described herein and others are known in the art.
SEQ ID NO: 22 - Exemplary AAV construct polynucleotide sequence
CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTC GCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC CTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGGAAGAGTACC ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAA TGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTA CGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTT ATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTG AGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTAT TTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCGGGGGG GGGGGGGGGGGGGGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGC TCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCG GCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGC CCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTC CGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCT TGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGCTGTCCGCGGGGGGACGG CTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGA GCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTA TTGTGCTGTCTCATCATTTTGGCAAAGAATTGGATCCGCCACCATGGTGAGCAAGGGCGAGGAG CTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCA GCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATTTGCAC CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGC TTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCT ACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAA GTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGC AACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACA AGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCA GCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAAC CACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCC TGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGCTCGAGGC CACCAACTTCTCCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGGATGTA AAGCATGTTCGCTTTGCAGCAGCAGTGGAAGTTTGGGAAGCAGATGACATCGAGCGGAAGGGTC CTTGGGAGCAAGCTGCTGTGGACAGATTTCGATTTCAGAGACGAATAGCCTCCGTCGAGGAACT CCTTTCAGCCGTTCTGCTGCGACAAAAGAAACTTCTGGAGCAGCAGTGAGAATTCGATATCAAG C T T AT C GAT AAT C AAC C T C T G GAT T AC AAAAT T T G T GAAAGAT T G AC TGGTATTCT T AAC T AT G TTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCG TATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGG CCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGG GCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGC GGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAAT TCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGA TTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCG CGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATC TCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACCCGGGCGGCCGCTTCGAGCAGACATGA T AAGAT AC AT T GAT GAG T T T GGACAAAC CACAAC T AGAAT GCAGT GAAAAAAAT GC T T TAT T T G TGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAAC AACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGT AAAACCTCTACAAATGTGGTAAAATCGATAAGGATCTTCCTAGAGCATGGCTACGTAGATAAGT AGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGG GCGGCCTCAGTGAGCGAGCGAGCGCGCAG
2. AAV Capsids
[0161] In some embodiments, the present disclosure provides one or more polynucleotide constructs packaged into an AAV capsid. In some embodiments, an AAV capsid is from or is derived from an AAV capsid of an AAV2, 3, 4, 5, 6, 7, 8, 9, 10, rh8, rhlO, rh39, rh43 or Ancestral serotype, or one or more hybrids thereof. In some embodiments, an AAV capsid is from an AAV ancestral serotype. In some embodiments, an AAV capsid is an ancestral (Anc) AAV capsid. An Anc capsid is created from a construct sequence that is constructed using evolutionary probabilities and evolutionary modeling to determine a probable ancestral sequence. Thus, an Anc capsid/construct sequence is not known to have existed in nature. In some embodiments, an AAV capsid is engineered and/or derived from an AAV9 capsid. In some embodiments, an AAV capsid is an AAV PHP.eB capsid. In some embodiments, an AAV capsid is an AAV PHP.B capsid (see e.g., Diptaman Chatterjee et al., Gene Therapy 29, 2290-387 (2022)). [0162] As provided herein, in some embodiments, any combination of AAV capsids and AAV constructs (e.g., comprising AAV ITRs) may be used in recombinant AAV particles of the present disclosure. For example, wild type or variant AAV2 ITRs and AAV DJ/8, AAV9, AAV PHP.B, and/or AAV PHP.eB capsid, or variant AAV2 ITRs and AAV6 capsid, etc. In some embodiments of the present disclosure, an AAV particle is wholly comprised of AAV2 and/or AAV9 components (e.g., capsid and ITRs are AAV2 and/or AAV9 serotype). In some embodiments, an AAV particle is an AAV2/6, AAV2/8 or AAV2/9 particle (e.g., an AAV6, AAV8 or AAV9 capsid with an AAV construct having AAV2 ITRs.
3. Exemplary AAV construct components a. Inverted Terminal Repeat Sequences (ITRs)
[0163] AAV derived sequences of a construct typically comprises the cis-acting 5' and 3' ITRs (See, e.g., B. J. Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990), which is incorporated herein by reference for the purposes described herein). Generally, ITRs are able to form a hairpin. The ability to form a hairpin can contribute to an ITRs ability to self-prime, allowing primase-independent synthesis of a second DNA strand. ITRs can also aid in efficient encapsidation of an AAV construct in an AAV particle.
[0164] An AAV particle of the present disclosure can comprise an AAV construct comprising a coding sequence (e.g., encoding an inhibitor of ISR) and associated elements flanked by a 5' and a 3' AAV ITR sequences. In some embodiments, an ITR is or comprises about 130 nucleic acids. In some embodiments, an ITR is or comprises about 145 nucleic acids. In some embodiments, all or substantially all of a sequence encoding an ITR is used. In some embodiments, an AAV ITR sequence may be obtained from any known AAV, including presently identified mammalian AAV types. In some embodiments an ITR is an AAV2 ITR. In some embodiments, an ITR is an AAV9 ITR.
[0165] A non-limiting example of a polynucleotide construct of the present disclosure is a "cisacting" construct comprising a transgene, in which said transgene sequence and any associated regulatory elements are flanked by 5' or "left" and 3' or "right" AAV ITR sequences. 5' and left designations refer to a position of an ITR sequence relative to an entire construct, read left to right, in a sense direction. For example, in some embodiments, a 5' or left ITR is an ITR that is closest to a promoter (e.g., as opposed to a polyadenylation sequence) for a given construct, when a construct is depicted in a sense orientation, linearly. Concurrently, 3' and right designations refer to a position of an ITR sequence relative to an entire construct, read left to right, in a sense direction. For example, in some embodiments, a 3' or right ITR is an ITR that is closest to a polyadenylation sequence (e.g., as opposed to a promoter sequence) for a given construct, when a construct is depicted in a sense orientation, linearly. In general, ITRs as provided herein are depicted in 5' to 3' order in accordance with a sense strand. Accordingly, one of skill in the art will appreciate that a 5' or "left" orientation ITR can also be depicted as a 3' or "right" ITR when converting from sense to anti sense direction. Further, it is well within the ability of one of skill in the art to transform a given sense ITR sequence (e.g., a 571 eft AAV ITR) into an antisense sequence (e.g., 37right ITR sequence). One of ordinary skill in the art would understand how to modify a given ITR sequence for use as either a 571eft or 37right ITR, or an antisense version thereof.
[0166] In some embodiments, an ITR (e.g., a 5' ITR) can have a sequence according to SEQ ID NO: 23. In some embodiments, an ITR (e.g., a 3' ITR) can have a sequence according to SEQ ID NO: 24. In some embodiments, an ITR includes one or more modifications, e.g., truncations, deletions, substitutions or insertions, as is known in the art. In some embodiments, an ITR comprises fewer than 145 nucleotides, e.g., 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, or 141 nucleotides. For example, in some embodiments, an ITR comprises 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143 144, or 145 nucleotides. [0167] In some embodiments, a 5' ITR sequence is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a 5' ITR sequence represented by SEQ ID NO: 23. In some embodiments, a 3' ITR sequence is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a 3' ITR sequence represented by SEQ ID NO: 24.
SEQ ID NO: 23 - Exemplary 5' AAV ITR polynucleotide sequence
CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTC GCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC CT SEQ ID NO: 24 - Exemplary 3' AAV ITR polynucleotide sequence
AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGG
GCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC AG b. Promoters
[0168] In some embodiments, a construct (e.g., an AAV construct) comprises a promoter. The term "promoter" refers to a DNA sequence recognized by enzymes/proteins that can promote and/or initiate transcription of an operably linked gene (e.g., encoding an inhibitor of ISR). For example, a promoter typically refers to, e.g., a polynucleotide sequence to which an RNA polymerase and/or any associated factor binds and from which it can initiate transcription. Thus, in some embodiments, a construct (e.g., an AAV construct) comprises a promoter operably linked to one of the non-limiting example promoters described herein.
[0169] In some embodiments, a promoter is an inducible promoter, a constitutive promoter, a mammalian cell promoter, a viral promoter, a chimeric promoter, an engineered promoter, a tissuespecific promoter, or any other type of promoter known in the art. In some embodiments, a promoter is a RNA polymerase II promoter, such as a mammalian RNA polymerase II promoter. In some embodiments, a promoter is a RNA polymerase III promoter, including, but not limited to, a HI promoter, a human U6 promoter, a mouse U6 promoter, or a swine U6 promoter. A promoter will generally be one that is able to promote transcription in a neurological cell.
[0170] A variety of promoters are known in the art, which in some embodiments, can be used herein. Nonlimiting examples of promoters that can be used herein in some embodiments include: human EFla, human cytomegalovirus (CMV) (US Patent No. 5,168,062, which is incorporated herein by reference for the purposes described herein), human ubiquitin C (UBC), mouse phosphoglycerate kinase 1, polyoma adenovirus, simian virus 40 (SV40), P-globin, P-actin, a- fetoprotein, y-globin, P-interferon, y-glutamyl transferase, mouse mammary tumor virus (MMTV), Rous sarcoma virus, rat insulin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein II (MT II), amylase, cathepsin, MI muscarinic receptor, retroviral LTR (e.g., human T-cell leukemia virus HTLV), AAV ITR, interleukin-2, collagenase, platelet-derived growth factor, adenovirus 5 E2, stromelysin, murine MX gene, glucose regulated proteins (GRP78 and GRP94), a-2- macroglobulin, vimentin, MHC class I gene H-2K b, HSP70, proliferin, tumor necrosis factor, thyroid stimulating hormone a gene, immunoglobulin light chain, T-cell receptor, HL A DQa and DQ, interleukin-2 receptor, MHC class II, MHC class II HLA-DRa, muscle creatine kinase, prealbumin (transthyretin), elastase I, albumin gene, c-fos, c-HA-ras, neural cell adhesion molecule (NCAM), H2B (TH2B) histone, rat growth hormone, human serum amyloid (SAA), troponin I (TN I), duchenne muscular dystrophy, human immunodeficiency virus, and Gibbon Ape Leukemia Virus (GAL V) promoters. Additional examples of promoters are known in the art. See, e.g., Lodish, Molecular Cell Biology, Freeman and Company, New York 2007, each of which is incorporated herein by reference for the purposes described herein. In some embodiments, a promoter is the CMV immediate early promoter. In some embodiments, the promoter is a CAG promoter and/or a CAG/CBA promoter.
[0171] The term "constitutive" promoter refers to a polynucleotide and/or oligonucleotide sequence that, when operably linked with a nucleic acid encoding a protein (e.g., an inhibitor of ISR), causes RNA to be transcribed from the nucleic acid in a cell under most or all physiological conditions. Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (see, e.g., Boshart et al., Cell 41 :521-530, 1985, which is incorporated herein by reference for the purposes described herein), the SV 40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFLalpha promoter (Invitrogen).
[0172] Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech, and Ariad. Additional examples of inducible promoters are known in the art. Examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex) inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (see e.g., WO 98/10088, which is incorporated herein by reference for the purposes described herein); the ecdysone insect promoter (see e.g., No et al., Proc. Natl. Acad Set. U.S.A 93:3346-3351, 1996, which is incorporated herein by reference for the purposes described herein), the tetracycline-repressible system (see e.g., Gossen et al., Proc. Natl. Acad Set. U.S.A 89:5547-5551, 1992, which is incorporated herein by reference for the purposes described herein), the tetracycline-inducible system (see e.g., Gossen et al., Science 268: 1766- 1769, 1995, see also Harvey et al., Curr. Opin. Chem. Biol. 2:512-518, 1998, each of which is incorporated herein by reference for the purposes described herein), the RU486-inducible system (see e.g., Wang et al., Nat. Biotech. 15:239- 243, 1997, and Wang et al., Gene Then. 4:432-441, 1997, each of which is incorporated herein by reference for the purposes described herein), and the rapamycin-inducible system (see e.g., Magari et al., J Clin. Invest. 100:2865-2872, 1997, which is incorporated herein by reference for the purposes described herein).
[0173] The term "tissue-specific" promoter refers to a promoter that is active only in certain specific cell types and/or tissues (e.g., transcription of a specific gene occurs only within cells expressing transcription regulatory and/or control proteins that bind to the tissue-specific promoter). In some embodiments, regulatory and/or control sequences impart tissue-specific gene expression capabilities. In some cases, tissue-specific regulatory and/or control sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner. In some embodiments, a tissue-specific promoter is a neuron-specific promoter. In some embodiments, a tissue-specific promoter is glial cell-specific promoter. In some embodiments, a tissue-specific promoter is a hippocampal cell-specific promoter.
SEQ ID NO: 25 - Exemplary CAG promoter polynucleotide sequence
TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTT GTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCG CCGGGGGGGGGGGGGGGGGGGGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAG CGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCG
SEQ ID NO: 26 - Exemplary CAG promoter/enhancer polynucleotide sequence
TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTT GTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCG CCGGGGGGGGGGGGGGGGGGGGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAG CGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGA AGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCT CGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCC TTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGT GAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGCTGTCCGCGG GGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCG GCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGT GCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGGATCCGCCACC
Ill c. Enhancers
[0174] In some embodiments, a construct can include an enhancer sequence. The term "enhancer" as used herein refers to a polynucleotide and/or oligonucleotide sequence that can increase the level of transcription of a nucleic acid encoding a protein of interest (e.g., an inhibitor of ISR), and/or increase or modify the translational efficiency of a transcript following transcription. In some embodiments, enhancer sequences (generally 50-1500 bp in length) generally increase the level of transcription by providing additional binding sites for transcription- associated proteins (e.g., transcription factors), and/or stabilize or modify post-transcriptional regulatory machinery. In some embodiments, an enhancer sequence is found within an intronic sequence. In some embodiments, an enhancer sequence is found in a 3' and/or 5' UTR. In some embodiments, an enhancer region is found downstream of a coding sequence comprising a transgene and proximal to a poly adenylation sequence. Unlike promoter sequences, enhancer sequences can act at much larger distance away from the transcription start site (e.g., as compared to a promoter). Non-limiting examples of enhancers include a woodchuck hepatitis virus post- transcriptional regulatory element (WPRE), RSV enhancer, a CMV enhancer, and/or a SV40 enhancer.
SEQ ID NO: 27 - Exemplary WPRE polynucleotide sequence
AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTT TTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTT CATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTC AGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCA CCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCAT CGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTG TTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCG GGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCT GCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGG GCCGCCTCCCCGC d. Flanking untranslated regions, 5' UTR and 3' UTR
[0175] In some embodiments, any of the constructs described herein can include an untranslated region (UTR), such as a 5' UTR or a 3' UTR. UTRs of a gene are transcribed but not translated. A 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon. A 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. The regulatory and/or control features of a UTR can be incorporated into any of the constructs, particles, polynucleotides, compositions, kits, or methods as described herein to enhance or otherwise modulate the expression of an inhibitor of ISR.
[0176] Natural 5' UTRs include a sequence that plays a role in translation initiation. In some embodiments, a 5' UTR can comprise sequences, like Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus sequence CCR(A/G)CCAUGG, where R is a purine (A or G) three bases upstream of the start codon (AUG), and the start codon is followed by another “G”. In some embodiments, 5' UTRs also form secondary structures that are involved in elongation factor binding. In some embodiments, a 5' UTR is included in any of the constructs described herein. Non-limiting examples of 5' UTRs, including those from the following genes: albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, and Factor VIII, can be used to enhance expression of a nucleic acid molecule, such as an mRNA.
[0177] 3' UTRs are known to have stretches of adenosines and uridines (in the RNA form) or thymidines (in the DNA form) embedded in them. These AU-rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU-rich elements (AREs) can be separated into three classes (see e.g., Chen et al., Mol. Cell. Biol. 15:5777-5788, 1995; Chen et al., Mol. Cell Biol. 15:2010-2018, 1995, each of which is incorporated herein by reference for the purposes described herein): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. For example, c-Myc and MyoD mRNAs contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A) (U/A) nonamers. GM-CSF and TNF-alpha mRNAs are examples that contain class II AREs. Class III AREs are less well defined. These U-rich regions do not contain an AUUUA motif, two well-studied examples of this class are c-Jun and myogenin mRNAs.
[0178] Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules may lead to HuR binding and thus, stabilization of the message in vivo. [0179] In some embodiments, the introduction, removal, or modification of 3' UTR AREs can be used to modulate the stability of an mRNA encoding an inhibitor of ISR protein. In other embodiments, AREs can be removed or mutated to increase the intracellular stability and thus increase translation and production of an inhibitor of ISR protein.
[0180] In some embodiments, non-ARE sequences may be incorporated into the 5' or 3' UTRs. In some embodiments, introns or portions of intron sequences may be incorporated into the flanking regions of the polynucleotides in any of the constructs, particles, polynucleotides, compositions, kits, and methods provided herein. Incorporation of intronic sequences may increase protein production as well as mRNA levels. e. Internal Ribosome Entry Sites (IRES)
[0181] In some embodiments, a construct encoding an inhibitor of ISR protein can include an internal ribosome entry site (IRES). An IRES forms a complex secondary structure that allows translation initiation to occur from any position with an mRNA immediately downstream from where the IRES is located (see, e.g., Pelletier and Sonenberg, Mol. Cell. Biol. 8(3): 1103-1112, 1988, which is incorporated herein by reference for the purposes described herein). There are several IRES sequences known to those in skilled in the art, including those from, e.g., foot and mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), human rhinovirus (HRV), cricket paralysis virus, human immunodeficiency virus (HIV), hepatitis A virus (HA V), hepatitis C virus (HCV), and poliovirus (PV) (see e.g., Alberts, Molecular Biology of the Cell, Garland Science, 2002; and Hellen et al., Genes Dev. 15(13): 1593-612, 2001, each of which are incorporated herein by reference for the purposes described herein).
[0182] In some embodiments, an IRES sequence that is incorporated into a construct that encodes an inhibitor of ISR protein is the foot and mouth disease virus (FMDV) 2A sequence. The Foot and Mouth Disease Virus 2A sequence is a small peptide (approximately 18 amino acids in length) that has been shown to mediate the cleavage of polyproteins (see e.g., Ryan, MD et al., EMBO 4:928-933, 1994; Mattion et al., J Virology 70:8124-8127, 1996; Furler et al., Gene Therapy 8:864-873, 2001; and Halpin et al., Plant Journal 4:453-459, 1999, each of which is incorporated herein by reference for the purposes described herein). The cleavage activity of the 2A sequence has previously been demonstrated in artificial systems including plasmids and gene therapy constructs (e.g., AAV and retroviruses) (see e.g., Ryan et al., EMBO 4:928-933, 1994; Mattion et al., J Virology 70:8124-8127, 1996; Furler et al., Gene Therapy 8:864-873, 2001; and Halpin et al., Plant Journal 4:453-459, 1999; de Felipe et al., Gene Therapy 6: 198-208, 1999; de Felipe et al., Human Gene Therapy II: 1921-1931, 2000; and Klump et al., Gene Therapy 8:811- 817, 2001, each of which is incorporated herein by reference for the purposes described herein). [0183] In some embodiments, an IRES can be utilized in an AAV construct. In some embodiments, a construct encoding an inhibitor of ISR protein can include a polynucleotide internal ribosome entry site (IRES). In some embodiments, an IRES can be part of a composition comprising more than one construct. In some embodiments, an IRES is used to produce more than one polypeptide from a single gene transcript. f. Splice sites
[0184] In some embodiments, any of the constructs provided herein can include splice donor and/or splice acceptor sequences, which are functional during RNA processing occurring during transcription. In some embodiments, splice sites are involved in trans-splicing. g. Polyadenylation sequences
[0185] In some embodiments, a construct provided herein can include a polyadenylation (poly(A)) signal sequence. Most nascent eukaryotic mRNAs possess a poly (A) tail at their 3' end, which is added during a complex process that includes cleavage of the primary transcript and a coupled polyadenylation reaction driven by the poly(A) signal sequence (see, e.g., Proudfoot et al., Cell 108:501-512, 2002, which is incorporated herein by reference for the purposes described herein). A poly(A) tail confers mRNA stability and transferability (see e.g., Molecular Biology of the Cell, Third Edition by B. Alberts etal., Garland Publishing, 1994, which is incorporated herein by reference for the purposes described herein). In some embodiments, a poly(A) signal sequence is positioned 3' to a coding sequence.
[0186] As used herein, "polyadenylation" refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. A 3' poly(A) tail is a long sequence of adenine nucleotides (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase. In some embodiments, a poly(A) tail is added onto transcripts that contain a specific sequence, e.g., a poly(A) signal. A poly(A) tail and associated proteins aid in protecting mRNA from degradation by exonucleases. Polyadenylation also plays a role in transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation typically occurs in the nucleus immediately after transcription of DNA into RNA, but also can occur later in the cytoplasm. After transcription has been terminated, an mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. A cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3' end at the cleavage site.
[0187] As used herein, a "poly(A) signal sequence" or "polyadenylation signal sequence" is a sequence that triggers the endonuclease cleavage of an mRNA and the addition of a series of adenosines to the 3' end of the cleaved mRNA.
[0188] There are several poly(A) signal sequences that can be used in some embodiments, including those derived from bovine growth hormone (bGH) (Woychik et al., Proc. Natl. Acad Sci. U.S.A. 81(13):3944-3948, 1984; U.S. Patent No. 5,122,458, each of which is incorporated herein by reference for the purposes described herein), mouse-P-globin, mouse-a-globin (Orkin et al., EMBO J 4(2):453-456, 1985; Them et al., Blood 71 (2): 313-319, 1988, each of which is incorporated herein by reference for the purposes described herein), human collagen, polyoma virus (Batt et al., Mol. Cell Biol. 15(9):4783-4790, 1995, which is incorporated herein by reference for the purposes described herein), the Herpes simplex virus thymidine kinase gene (HSV TK), IgG heavy-chain gene polyadenylation signal (US 2006/0040354, which is incorporated herein by reference for the purposes described herein), human growth hormone (hGH) (Szymanski et al., Mol Therapy 15(7): 1340-1347, 2007, which is incorporated herein by reference for the purposes described herein), and/or the group consisting of SV40 poly(A) site, such as the SV40 late and early poly(A) site (see e.g., Schek et al., Mol Cell Biol. 12(12):5386-5393, 1992, which is incorporated herein by reference for the purposes described herein).
[0189] In some embodiments, the poly(A) signal sequence can be AATAAA. The AATAAA sequence may be substituted with other hexanucleotide sequences with homology to AATAAA and that are capable of signaling polyadenylation, including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, or AATAAG (see, e g., WO 06/12414, which is incorporated herein by reference for the purposes described herein). In some embodiments, a poly(A) signal sequence can be a synthetic polyadenylation site (see, e.g., the pCl- neo expression construct of Promega that is based on Levitt et al., Genes Dev. 3(7): 1019- 1025, 1989, which is incorporated herein by reference for the purposes described herein).
SEQ ID NO: 28 - Exemplary SV40 poly A signal polynucleotide sequence
T AAGAT AC AT T GAT GAG T T T GGACAAAC CACAAC T AGAAT GCAGT GAAAAAAAT GC T T TAT T T G TGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTT h. Additional sequences
[0190] In some embodiments, constructs of the present disclosure may comprise a 2A element or sequence. In some embodiments, constructs of the present disclosure may include one or more cloning sites. In some such embodiments, cloning sites may not be fully removed prior to manufacturing for administration to a subject. In some embodiments, cloning sites may have functional roles including as linker sequences, or as portions of a Kozak site. As will be appreciated by those skilled in the art, cloning sites may vary significantly in primary sequence while retaining their desired function.
[0191] In some embodiments, a 2A element is a T2A, P2A, E2A, and/or F2A element. In some embodiments, a 2A sequence may comprise an optional 5’ linker sequence, such as but not limited to GSG (e.g., Glycine, Serine, Glycine).
SEQ ID NO: 29 - Exemplary T2A amino acid sequence
EGRGSLLTCGDVEENPGP
SEQ ID NO: 30 - Exemplary P2A amino acid sequence
ATNFSLLKQAGDVEENPGP
SEQ ID NO: 31 - Exemplary E2A amino acid sequence
QCTNYALLKLAGDVESNPGP
SEQ ID NO: 32 - Exemplary F2A amino acid sequence
VKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 33 - Exemplary P2A oligonucleotide sequence
CTCGAGGCCACCAACTTCTCCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCC
SEQ ID NO: 34 - Exemplary transcriptional linker oligonucleotide sequence
GAAT T C GAT AT C AAG C T T AT C GAT
SEQ ID NO: 35 - Exemplary transcriptional linker oligonucleotide sequence
ATCGATACCGTCGACCCGGGCGGCCGCTTCGAGCAGACATGA i. Destabilization domains
[0192] In some embodiments, any of the constructs provided herein can optionally include a sequence encoding a destabilizing domain ("a destabilizing sequence") for temporal and/or spatial control of protein expression. Non-limiting examples of destabilizing sequences include sequences encoding a FK506 sequence, a dihydrofolate reductase (DHFR) sequence, or other exemplary destabilizing sequences.
[0193] In the absence of a stabilizing ligand, a protein sequence operatively linked to a destabilizing sequence is degraded by ubiquitination. In contrast, in the presence of a stabilizing ligand, protein degradation is inhibited, thereby allowing the protein sequence operatively linked to the destabilizing sequence to be actively expressed. As a positive control for stabilization of protein expression, protein expression can be detected by conventional means, including enzymatic, radiographic, colorimetric, fluorescence, or other spectrographic assays, fluorescent activating cell sorting (FACS) assays, and/or immunological assays (e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).
[0194] Additional examples of destabilizing sequences are known in the art. In some embodiments, the destabilizing sequence is a FK506- and rapamycin-binding protein (FKBP12) sequence, and the stabilizing ligand is Shield-I (Shldl) (see e.g., Banaszynski et al. (2012) Cell 126(5):995-1004, which is incorporated herein by reference for the purposes described herein). In some embodiments, a destabilizing sequence is a DHFR sequence, and a stabilizing ligand is trimethoprim (TMP) (see e.g., Iwamoto etal., (2010) ChemBiol 17:981-988, which is incorporated herein by reference for the purposes described herein). j. Reporter Sequences or Elements
[0195] In some embodiments, constructs provided herein can optionally include a sequence encoding a reporter polypeptide and/or protein ("a reporter sequence"). Non-limiting examples of reporter sequences include DNA sequences encoding: a beta-lactamase, a betagalactosidase (LacZ), an alkaline phosphatase, a thymidine kinase, a green fluorescent protein (GFP), a red fluorescent protein, an mCherry fluorescent protein, a yellow fluorescent protein, a chloramphenicol acetyltransferase (CAT), and a luciferase. Additional examples of reporter sequences are known in the art. When associated with control elements which drive their expression, the reporter sequence can provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence, or other spectrographic assays, fluorescent activating cell sorting (FACS) assays and/or immunological assays (e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).
[0196] In some embodiments, a reporter sequence is the Lacz gene, and the presence of a construct carrying the Lacz gene in a cell is detected by assays for beta-galactosidase activity. In some embodiments, a reporter sequence is a fluorescent protein (e.g., green fluorescent protein (GFP)) or luciferase. In embodiments where a reporter sequence is a fluorescent protein or luciferase, the presence of a construct carrying the fluorescent protein or luciferase in a cell may be measured by fluorescent imaging techniques (e.g., fluorescent microscopy or FACS) or light production in a luminometer (e.g., a spectrophotometer or an IVIS imaging instrument). In some embodiments, a reporter sequence can be used to verify tissue-specific targeting capabilities and/or tissue-specific promoter regulatory and/or control activity of any of the constructs described herein. An exemplary GFP tag sequence is provided as SEQ ID NO: 37.
[0197] In some embodiments, a reporter sequence is a FLAG tag (e.g., a 3xFLAG tag), and the presence of a construct carrying the FLAG tag in a cell is detected by protein binding or detection assays (e.g., Western blots, immunohistochemistry, radioimmunoassay (RIA), mass spectrometry). An exemplary 3xFLAGtag sequence is provided as SEQ ID NO: 36.
SEQ ID NO: 36 - Exemplary 3xFLAG tag polynucleotide sequence
GGATCCCGGGCTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGG AT GAC GAT GAC AAG SEQ ID NO: 37 - Exemplary GFP polynucleotide sequence
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCG ACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCT GACCCTGAAGTTCATTTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACC CTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCA AGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTA CAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGC ATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACA ACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAA CATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGC CCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACG AGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGA CGAGCTGTACAAG
IV. Administration of constructs and/or compositions
[0198] In some embodiments, constructs, particles, polypeptides, polynucleotides, and/or compositions described herein may be comprised in a formulation with one or more additional therapeutic agents.
[0199] In some embodiments, constructs, particles, polypeptides, polynucleotides, and/or compositions described herein may be comprised in a formulation wherein the formulation comprises pharmaceutically acceptable excipients.
[0200] In some embodiments, constructs, particles, polypeptides, polynucleotides, and/or compositions described herein are administered to a subject in need thereof. In some embodiments, a subject may have, may be diagnosed with, or may be susceptible to developing Down syndrome (DS), Charcot-Marie-Tooth disease, major depressive disorder (MDD), schizophrenia, Alzheimer’s disease, Huntington disease, Parkinson’s disease, Amyotrophic lateral sclerosis (ALS), Multiple Sclerosis (MS), Prion disease, traumatic brain injury, Vanishing white matter (VWM) disease, frontotemporal dementia, and/or Aging (e.g., age-related cognitive decline).
[0201] In some embodiments, a subject is a mammal. In some embodiments, a subject is a domestic animal. In some embodiments, a subject is a farm animal. In some embodiments, a subject is a zoo animal. In some embodiments, a subject is a dog or a cat. In some embodiments, a subject is a cow, a horse, a sheep, or a goat. In some embodiments, a subject can be but is not limited to, a dog, cat, ferret, rabbit, cow, duck, pig, goat, chicken, horse, llama, camel, ostrich, deer, turkey, dove, sheep, goose, oxen, and/or reindeer. In some embodiments, a subject is a human. In some embodiments, a subject is equal to, less than, or greater than 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 years of age.
[0202] In some embodiments, constructs, particles, polypeptides, polynucleotides, and/or compositions described herein according to the present invention may be administered by intraspinal and/or intracerebral administration. In some embodiments, intraspinal administration comprises or consists of intrathecal and epidural administration.
[0203] In some embodiments, constructs, particles, polypeptides, polynucleotides, and/or compositions described herein are administered intracerebrally. In some embodiments, intracerebral administration is at a site selected from the group comprising or consisting of: hippocampus and/or hippocampal formation (such as, e.g., the cornu ammonis (e.g, CAI, CA2, and/or CA3), the subicular complex, the entorhinal cortex, and/or the dentate gyrus), striatum (such as, e.g., putamen, caudate nucleus, nucleus accumbens, olfactory tubercle, external globus pallidus and/or internal globus pallidus), thalamus, hypothalamus, epithalamus, subthalamus, parenchyma, cerebrum, medulla, deep cerebellar nuclei (such as, e.g., substantia nigra, dentate, emboliform, globose and/or fastigii nucleus), cerebrospinal fluid (CSF), meninges, dura mater, arachnoid mater, pia mater, subarachnoid cisterns (such as, e.g., cisterna magna, pontine cistem, interpeduncular cistern, chiasmatic cistern, cistern of lateral cerebral fossa, superior cistern and/or cistern of lamina terminalis), subarachnoid space, cortex, septum, pons, and cerebellum.
[0204] In some embodiments, constructs, particles, polypeptides, polynucleotides, and/or compositions described herein are administered intrahippocampally (e.g., in the hippocampus, e.g., CAI, CA2, and/or CA3), intrastriataly (e.g., in the striatum, such as, e.g., in the putamen, caudate nucleus, nucleus accumbens, olfactory tubercle, external globus pallidus and/or internal globus pallidus), intrathalamicaly (e.g., in the thalamus), and/or intraci stemaly (e.g., in the subarachnoid cisterns, such as, e.g., in the cisterna magna, pontine cistem, interpeduncular cistern, chiasmatic cistern, cistern of lateral cerebral fossa, superior cistern and/or cistern of lamina terminalis). [0205] In some embodiments, constructs, particles, polypeptides, polynucleotides, and/or compositions described herein are administered intracerebroventricularly, intrahippocampally, intraparenchymaly, intrastriataly, intrathalamicaly, intracisternaly, and/or intrathecally.
[0206] In some embodiments, therapeutic regimens comprising constructs, particles, polypeptides, polynucleotides, and/or compositions described herein may comprise administration of a combination of therapeutic agents, such as a first therapy and a second therapy. The therapies may be administered in any suitable manner known in the art. For example, the first and second therapy may be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the first and second therapies are administered in a separate composition. In some embodiments, the first and second therapies are in the same composition.
[0207] In some embodiments, therapeutic regimens comprising constructs, particles, polypeptides, polynucleotides, and/or compositions described herein comprise administering of more than one composition, such as 2 compositions, 3 compositions, 4 compositions, or more than 4 compositions. Various combinations of the agents may be employed. In some embodiments, therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some embodiments, constructs, particles, polypeptides, polynucleotides, and/or compositions described herein and/or additional therapeutic agents are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, constructs, particles, polypeptides, polynucleotides, and/or compositions described herein and/or additional therapeutic agents are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, an appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.
[0208] In some embodiments, treatments may include various “unit doses.” Unit dose is defined as containing a predetermined- quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose.
[0209] In some embodiments, the quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 0.10 mg/kg to 200 mg/kg can affect the protective capability of these agents. In certain embodiments, it is contemplated that doses may comprise a composition comprising an AAV particle in a concentration of about 108 to about 1014 viral genomes per ml. Furthermore, such doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.
[0210] In certain embodiments, precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.
[0211] It is also understood that uptake is species and organ/tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.
[0212] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, 12 week, or more than 12 week intervals, including all ranges there between.
[0213] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.
[0214] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.
[0215] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
[0216] In some embodiments, wherein a composition is proteinaceous, the proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
[0217] In some embodiments, a pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
[0218] In some embodiments, sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[0219] In some embodiments, upon formulation, compositions described herein may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. In some embodiments, formulations are administered in a variety of dosage forms, such as the type of injectable solutions described above.
A. Pharmaceutical Compositions
[0220] In certain aspects, the constructs, particles, polypeptides, polynucleotides, and/or compositions or agents for use in the methods, such as methods of delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing cognitive dysfunction in a subject (e.g., a human subject), are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the agent. The agents in some aspects of the disclosure may be formulated into preparations for local delivery (i.e. to a specific location of the body, such as brain tissues, or other tissue) or systemic delivery, in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration. Certain aspects of the disclosure also contemplate local administration of the compositions by coating medical devices and the like.
[0221] In some embodiments, suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve. [0222] In certain embodiments, the carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.
[0223] In certain aspects, the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
[0224] In some embodiments, solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[0225] In certain aspects, the pharmaceutical compositions are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable or solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg or less, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.
[0226] In some embodiments, non-limiting examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. In some embodiments, non-limiting examples of aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. In some embodiments, intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antgifungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.
[0227] In certain embodiments, formulations comprising constructs described herein and/or co-administered formulations may be suitable for oral administration. In some embodiments, oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
[0228] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.
[0229] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.
EXAMPLES
[0230] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Materials and Methods -
[0231] Unless otherwise noted, the experiments and procedures described herein were conducted as follows.
Generation of Ppplrl5bR658C mice
[0232] Mouse iPSCs were microinjected with specific gRNA (e.g., AGTGGTGATGAGGATCGCAA AGG (SEQ ID NO: 38); the PAM sequence is underlined and is not necessarily comprised within a gRNA sequence, (Synthego)), Cas9 mRNA (Sigma) and the donor DNA containing the R658C mutation. The donor DNA was 500 bp in length and in addition to the point mutation of interest, silent mutations were introduced to prevent editing of the mutant back to WT. The donor also contained approximately 250 bp flanking the mutated region to facilitate homologous recombination. Edited cells were implanted into surrogate females and the pups were genotyped by sequencing of the locus. These steps were performed at the core facility at Baylor College of Medicine (BCM). Pups carrying the desired mutation were bred and tested phenotypically.
Cell based assays
[0233] For stress experiments, stable cells generated from lentiviral transduction were generated and selected with puromycin using standard transduction procedures. Cells were then treated with 1 pM oligomycin, 100 nM thapsigargin, or transfected with 1 pg poly(IC) using lipofectamine 2000 for three hours prior to harvesting cells. Lysates were prepared and standardized prior to analysis by SDS-PAGE and western blotting for the indicated proteins.
Immunoprecipitation
[0234] Cells were transfected using standard procedures with equivalent amounts of DNA plasmids expressing the indicated GFP-tagged genes and DP71L/ACREP chimera. 16 hours posttransfection, cells were washed and harvested in lysis buffer. Total protein was quantified and equivalent amounts of protein were used in immunoprecipitation reactions. Complexes were captured using magnetic GFP-trap resin (Proteintech Group) overnight at 4 °C with tumbling. The complexes were separated from the crude lysate using a magnet and washed thrice with ice-cold lysis buffer. Complexes were eluted from the resin with IX Laemmli buffer and analyzed by SDS- PAGE and western blotting.
Adeno-associated Viruses (AAVs)
[0235] Adeno associated viruses were generated by cloning the indicated sequences into an AAV2 plasmid backbone. DNA was then purified and sent to a core facility to produce AAVs serotyped with the neurotropic DJ/8. AAV was purified with commercially available kits (Cell Biolabs) and quantified by qPCR based assays. Typically, 0.5-1 pl of ~1012 viral genomes per ml were injected into each hippocampus of the mouse in both the CAI and CA3 regions (cornu Ammonis subfields 1 and 3). AAVs were allowed to express the transgene for at least 14 days prior to the beginning of behavioral experiments. Subsequently the brains of the mice were dissected and samples were prepared for SDS-PAGE and western blotting.
Behavioral tests
[0236] The specific procedures were performed as we previously published (see e.g., Zhu et al., 2019). Briefly, mice were handled for 5 minutes three consecutive days to allow them to acclimatize to the experimenters. Subsequently the mice were placed in the conditioning chamber to allow them to habituate for 20 minutes each of two days. Mice were then trained with an aversive foot shock. Two training shocks were administered at 0.7 mA separated by 90 seconds each with freezing recorded in between and after the training shocks. Finally, 24 hours after training, mice were placed in the same chamber for 5 minutes and percent of time spent freezing was recorded using Freezeview software. Object recognition was performed as we previously described (see e.g., Zhu et al., 2019) with only slight modifications. Briefly, mice were handled for 5 days (5-10 min for each day) and then habituated to a black Plexiglas rectangular chamber (31 x 24 cm, height 27 cm) for 10 min under dim ambient light for 5 days. Two identical objects were presented to mice to explore for 5 min, after which, mice were returned to the home cage. Twenty-four hours later, one object was replaced by one novel object and the mouse was again placed in the chamber for 5 min. The novel object has the same height and volume but different shape and appearance. Exploration of the objects was defined as sniffing of the objects (with nose contact or head directed to the object) within at 2 cm radius of the objects. Sitting or standing on the objects was not scored as exploration. Behavior was recorded from cameras positioned above the training chamber. Discrimination Index (DI) was computed as DI = (Novel Object Exploration Time - Familiar Object Exploration Time/Total Exploration Time) X 100. To control for odor cues, the open field arena and the objects were thoroughly cleaned with ethanol, dried, and ventilated between mice.
Electrophysiology
[0237] Electrophysiological recordings were performed as previously described (see e.g., Zhu et al., 2019). Field recording were performed from CAI horizontal hippocampal slices (320 pm thick), which were cut from the brain of adult mice (3-6 months old) with a vibratome (Leica VT 1000S, Leica Microsystems, Buffalo Grove, IL) at 4 °C in artificial cerebrospinal fluid solution (ACSF; 95% 02 and 5% CO2) containing in mM: 124 NaCl, 2.0 KC1, 1.3 MgSO4, 2.5 CaC12, 1.2 KH2PO4, 25 NaHCO3, and 10 glucose (2-3 ml/min). Slices were incubated for at least 60 min prior to recording in an interface chamber and continuously perfused with artificial cerebrospinal fluid (ACSF) at 28-29 °C at a flow rate of 2-3 ml/min. The recording electrodes were placed in the stratum radiatum. Field excitatory postsynaptic potentials (fEPSPs) were recorded with ACSF- filled micropipettes and were elicited by bipolar stimulating electrodes placed in the CAI stratum radiatum to excite Schaffer collateral and commissural fibers. The intensity of the 0.1 -ms pulses was adjusted to evoke 30-35% of maximal response. A stable baseline of responses at 0.033 Hz was established for at least 30 min. Tetanic LTP was induced by applying four trains of high- frequency stimulation (100 Hz, 1 s) separated by 5-min intervals. Whole-cell recordings were performed using a MultiClamp 700B amplifier (Molecular Devices, Union City, CA) in a submerged chamber (2-3 ml/min) at 31-32 °C using conventional patch-clamp techniques. CAI neurons were visually identified by infrared differential interference contrast video microscopy on the stage of an upright microscope (Axioskope FS2, Carl Zeiss, Oberkochen, Germany). Patch pipettes (resistances 2-5 MW) were filled with (in mM): 140 CsCl, 10 HEPES, 10 Na2- phosphocreatine, 0.2 BAPTA, 2 Mg3-ATP, 0.2 Na3-GTP; pH was adjusted to 7.2 and osmolarity to 295-300 mOsm using a Wescor 5500 vapor pressure osmometer (Wescor, Logan, UT). Miniature inhibitory postsynaptic currents (mIPSCs) were recorded at -60 mV in the presence of NBQX (5 pM, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline disodium salt), AP5 (25 pM, DL-2-Amino-5-phosphonopentanoic acid) and TTX (1 pM, tetrodotoxin). For recording excitatory currents, CsCl was replaced with 130 mM K-gluconate and 10 mM KC1. Excitatory postsynaptic currents (EPSCs) were recorded at -70 mV in the presence of 100 pM picrotoxin. All drugs were obtained from Tocris (Ellisville, MO).
Polysome profiling and RNA isolation
[0238] Polysome profiling followed by RNA sequencing was carried out as previously described (see e.g., Hinnebusch et al., 2016). Briefly, fresh 12 ml of 10-50% sucrose density gradients [10 mM HEPESKOH (pH 7.6), 5 mM MgC12, 150 mM KC1, 200 U/ml RNasin Rnase inhibitor (Promega, Madison, WI)] was prepared, as previously described (see e.g., Zhu et al., 2019; and Tible et al., 2019). Gradients were kept at 4 °C for at least 2 hours before use. Mouse brain tissue was dissected in a cutting solution [1XHBSS, 2.5 mMHEPES-KOH (pH 7.6), 35 mM glucose, 4 mM NaHC03, 100 pg/ml cycloheximide (Sigma- Aldrich, St. Louis, MO)] and washed in ice-cold PBS containing 100 pg/ml cycloheximide by centrifugation at 3000 rpm for 10 min at 4 °C. The tissue was then lysed in polysome lysis buffer [10 mM HEPES-KOH (pH 7.4), 5 mM MgC12, 150 mM KC1, 0.5 mM DTT, 100 U/ml RNasin Rnase inhibitor (Promega, Madison, WI), 100 pg/ml cycloheximide, and EDTA-free protease inhibitors (Roche Indianapolis, IN)] and centrifuged at 2000 x g for 10 min at 4 °C. The supernatant was then transferred to a pre-chilled tube, supplemented with 0.5% NP-40, and kept on ice for 10 min. Samples were centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was either layered onto sucrose gradient or reserved for total RNA isolation. Gradients were centrifuged in a SW-40Ti rotor at 35,000 rpm at 4 °C for 2 hours and then analyzed by piercing the tube with a Brandel tube piercer, passing 70% sucrose through the bottom of the tube and monitoring the absorbance of the material eluting from the tube using an ISCO UA-6 UV detector. Fractions were collected throughout and RNA was extracted with TRIzol following manufacturer’s instructions (Life Technologies, Carlsbad, CA). Experiments were performed in three biological replicates for each group.
Surface Sensing of Translation (SUnSET)
[0239] Protein synthesis was measured using SUnSET, a non-radioactive labeling method to monitor protein synthesis, as previously described (see e.g., Zhu etal., 2019). Briefly, hippocampal slices were cut (300 pm) with a McIlwain Tissue Chopper (Mickle, UK) and incubated for 1 hour at room temperature in oxygenated (95% 02, 5% CO2) ACSF followed by incubation at 32 °C for 1 hour in oxygenated (95% 02, 5% C02) ACSF prior to treatment as we previously described (see e.g., Zhu et al., 2019). Puromycin (10 pg/pl, dissolved in oxygenated ACSF) was bath applied to the slices for 20 min followed by a wash with untreated oxygenated ACSF. The slices were then snap-frozen on dry ice and stored at -80 °C until use. Frozen slices were lysed in homogenization buffer (in mM: 40 Tris HC1, pH 8.0, 150 NaCl, 25 b-glycerophosphate, 50 NaF, 2 Na3VO3, IX protease inhibitor cocktail, 10% glycerol, 1% Triton X-100). Puromycin incorporation was detected by Western blot using the 12D10 antibody to puromycin (Catalog # MABE343, 1:5000, EMD Millipore Corp, Darmstadt, Germany) as previously described (see e.g., Zhu et al., 2019). The density of the resulting bands was quantified using ImageJ and statistical significance assessed by Student’s t-test.
Example 1 - Creation and characterization of Ppp1r15bR658C mice mice exhibited aberrant translational control and active ISR.
Figure imgf000134_0001
[0240] A new mouse model ( Ppp1r15bR658C mice) was generated, carrying a selective mutation (R658C) in PPP1R15B, which has been identified by whole-exome sequencing to be associated with intellectual disability in humans (see e.g., Abdulkarim et al., 2015; Kernohan et al., 2015; and Mohammad etal., 2016). Briefly, mice were generated with CRISPR using a guide RNA targeting a region of PPP1R15B in close proximity to the base position of encoding the R658C mutation. Briefly, donor DNA containing the R658C mutation, the Cas9 mRNA, and the guide RNA were co-injected into embryos that were subsequently implanted into surrogate mice. Four 4 pups with homozygous targeting of the desired locus were identified (FIG. 2A). Mice were bred and mutant and littermate controls were obtained. Consistent with the microcephaly observed in individuals carrying the homozygous mutation (see e.g., Abdulkarim etal., 2015; and Kernohan et al., 2015), Ppplrl5bR658C mice were also found to exhibit slightly smaller brains (FIG. 2B). In humans, the R658C mutation inhibits the activity of the CReP»PPl phosphatase complex, leading to increased eIF2-P levels (see e.g., Abdulkarim et al., 2015; and Kernohan et al., 2015). In concordance with the human data, CReP levels were found to not be altered (FIG. 2C), but eIF2-P levels were found to be increased in the brains (FIG. 2D) and primary fibroblasts (FIG. 2E) of Ppp1r15bR658C mice. Thus, the ISR was selectively perturbed in Ppp1r15bR658C mice.
[0241] Because activation of the ISR inhibits general translation (see e.g., Costa-Mattioli et al., 2009), it was then investigated whether overall protein synthesis rates were reduced in Ppp1r15bR658C mice by polysome sedimentation in sucrose gradients, as recently described (see e.g., Zhu et al., 2019). In this technique, the position of a given mRNA in the sucrose gradient was determined by the number of associated ribosomes. mRNAs that were poorly or not translated accumulated near the top, whereas translationally active mRNAs were associated with multiple ribosomes (polysomes) and sediment to the bottom of the gradient (FIG. 2F). The results showed that general translation was inhibited in the brain of Pppl rl 5bw'^(: mice, as indicated by a decrease in polysomes and concomitant increase in monosomes (FIGs. 2G-H), consistent with increased eIF2-P levels (see e.g., Costa-Mattioli et al., 2007; and Harding et al., 2000). Moreover, when the inventors measured protein synthesis in vivo by assessing puromycin incorporation into nascent polypeptide chains (see e.g., Zhu et al., 2019; and Di Prisco et al., 2014), it was found that in fibroblasts from P pp 1 r 15 b^'-^ mice, puromycin incorporation was reduced (FIGs. 2I-J). Thus, when the ISR was activated in the brain of Pppl rl 5bw'^(: mice, protein synthesis rates were reduced.
Long-term memory was impaired in Ppplrl5bR658C mice.
[0242] The inventors and others have shown that activation of the ISR impairs long-term memory (see e.g., Costa-Mattioli et al., 2007; Batista et al., 2016; Jiang et al., 2015; Jian et al., 2014; and Shrestha et al., 2020). Given that I’pplrl5bw'5W mice had a selective activation of the ISR, the inventors then determined whether these mice had long-term memory impairment. To this end, hippocampus-dependent contextual fear memory was examined, where a context (conditioned stimulus; CS) was paired with a foot shock (the unconditioned stimulus; US). Twenty-four hours after training, mice were exposed to the CS and fear response (“freezing behavior”) was measured as an index of the strength of their long-term memory (FIG. 3A). The data showed that freezing prior to training was similar in Pppl rl 5/>R658C mice and naive control littermates, but PpplrlSb^65^ mice exhibited a significant reduction in freezing 24 hours after training, indicating that their long-term memory was impaired (FIG. 3B).
[0243] Additionally, long-term object recognition memory was also assessed, which also depends on the hippocampus (see e.g., Vann et al., 2011). In a long-term object recognition memory task, animals need to differentiate between a familiar and a novel object. Wild type (WT) mice are known to preferentially explore a novel object. During training, two identical objects were placed in a box and mice were allowed to explore the objects (FIG. 3C). The data showed that Ppp1r15bR658C and WT littermates spent, on average, an equal amount of time investigating the objects during training (FIG. 3D). However, when a new object was replaced 24 hours later, Ppp1r15bR658C mice exhibited reduced object discrimination, an indication that their long-term object recognition memory was deficient (FIG. 3E). Thus, long-term memory was impaired Ppplrl5b^c mice.
Long-term potentiation was impaired in mice.
Figure imgf000136_0001
[0244] The inventors measured long-term potentiation (LTP), a cellular model of memory formation (see e.g., Neves et al., 2008) in Ppp1r15bR658C mice. In hippocampal slices, four trains of 100 Hz stimulation induce a long lasting L-LTP that depends on new protein synthesis (see e.g., Costa-Mattioli etal., 2009; Kandel etal., 2001; and Kelleher etal., 2004). Given that the inventors and others have shown that the ISR bidirectionally regulates L-LTP: that is, activation of the ISR impairs L-LTP, whereas inhibition of the ISR enhances it (see e.g., Costa-Mattioli et al., 2007; Zhu etal., 2011; Jiang etal., 2010; and Huang etal., 2016), and the immediate results showed that the ISR was selectively activated in Ppp1r15bR658C mice, the inventors wondered whether L-LTP was impaired in these mice. Indeed, in concordance with the field, four trains of 100 Hz induced a persistent L-LTP in WT slices (FIG. 3F). However, the same stimulation protocol failed to do so in Ppp1r15bR658C slices (FIG. 3F). It was noted that basal synaptic transmission, as determined by input-output plots of field excitatory potentials as a function of presynaptic fiber volley and paired pulse facilitation, was similar in WT and Ppp1r15bR658C slices (data not shown). Thus, longterm potentiation was impaired in Ppp1r15bR658C mice.
Example 2 - Inhibition of the Integrated Stress Response (ISR)
Viral strategy to inhibit the ISR.
[0245] As modulation of eIF2-P phosphatase can prove to be a powerful mechanism to modulate the activity of the ISR, the inventors leveraged strategies employed by viruses to inhibit the ISR. Given that increased eIF2-P (that is, activated ISR) impair both cellular and viral protein synthesis, many viruses have evolved mechanisms to inactivate the ISR (see e.g., Garcia et al., 2007). Indeed, like the mammalian homologues GADD34 and CReP, the γ34.5 protein from herpes simplex virus, the DP71L protein from the African swine fever virus, the CNPV231 protein Canarypox virus (CNPV), the ICP34.5 protein from Macropoid herpes virus (MaHV), and the AmEPV193 protein from Amsacta moorei entomopoxvirus “L” (AmEPV), all are believed to recruit PPI to dephosphorylate eIF2 (see e.g., Rojas et al., 2015) (FIG. 4 A). In yeast, these proteins inhibit the ISR by reducing eIF2-P (see e.g., Rojas et al., 2015). However, A) to the inventors knowledge, yeast genomes do not naturally encode eIF2-phosphatase cofactor orthologues (e.g., GADD34 or CreP-like proteins) that can inhibit the ISR, and B) the mechanism of recruitment of PPI to eIF2 in yeast is different than that in human cells (see e.g., Rojas et al., Protein phosphatase PP1/GLC7 interaction domain in yeast eIF2y bypasses targeting subunit requirement for eIF2a dephosphorylation. PNAS, 2014, 111(14) E1344-E1353; which is incorporated herein by reference for the purposes described herein). Until now it was not clear whether these proteins would function in a similar manner in human cells. Moreover, until now, it was not clear that viral proteins (e. g. , DP71 L) could inhibit the ISR, whereby cognitive dysfunction and/or other diseases and disorders associated with ISR activation were reduced, treated, prevented, and/or reversed.
[0246] Herein, the inventors first focused on DP71L as it is the smallest protein (62 amino acids) of the family that contains both a PPI - and eIF2-binding domain (FIG. 4A). The inventors first examined whether DP71L was able to inhibit the ISR activation induced by oligomycin (oligo), which is known to activate the ISR kinase HRI, in human cells. As expected, in GFP transfected HEK293 cells, oligo induced the ISR, as determined by the increased eIF2-P. In contrast, expression of DP71L-GFP completely prevented oligo-induced ISR activation (FIG. 4B). Similarly, DP71L-GFP suppressed the activation of the ISR induced by Poly (I:C) and thapsigargin (thap), two well-known activators of the ISR kinases PKR and PERK, respectively. Thus, DP71L is a potent pan-inhibitor of the ISR in human cells (FIGs. 4C-D), and can function to suppress ISR activation in human cells regardless of the ISR-branch that is activated.
Molecular characterization of DP71L function.
[0247] To elucidate the mechanism underlying the DP71L potency with respect to inhibition of the ISR, the inventors first compared the effect of DP71L compared to a truncated version of CReP that is the same length of DP71L and contains the PPI and eIF2 binding motifs (ACREP, FIG. 5A; e.g., SEQ ID NO: 6). Upon induction of the ISR by thap, the inventors found that DP71L- GFP is a more potent inhibitor of the ISR when compared to ACREP-GFP (FIG. 5B). DP71L is a scaffold protein that bridges the phosphatase PPI to the target eIF2, thus rendering the phosphatase specific to its target. Based on this, the inventors hypothesized that DP71L’s enhanced ISR suppression properties may be due to increased binding to PPI, eIF2, or both proteins. To test this hypothesis, the inventors compared head-to-head the binding of ACREP-GFP or DP71L-GFP to endogenous PPI and eIF2. While both ACREP-GFP and DP71L-GFP bound similarly to eIF2, DP71L-GFP showed a stronger binding to PPI than did ACREP-GFP (FIG. 5C), suggesting that DP71L may display increased PPI binding relative to other similarly sized PPI and eIF2 binding proteins.
[0248] To identify the protein region(s) that differentiated ACREP-GFP from DP71L-GFP, the inventors generated disparate chimeric proteins comprising various portions of CREP and DP71L (FIG. 6A). First, the middle and C-term portions of DP71L were swapped in ACREP-GFP [C8 only contains the N-term (PPI binding) motif of ACREP], As noted above, stronger binding of full length DP71L-GFP to PPI compared to ACREP-GFP was observed (FIG. 6B). Interestingly, C8 showed strong binding to PPI, which was similar to full length DP71L. Next, the N-terminus of ACREP-GFP that contains both the PPI binding domain and the linker region (e.g., region between PPI and eIF2 binding domains) was fused to the C terminus of DP71L (e.g., eIF2 binding domain). This construct, C20, displayed reduced PPI binding compared to DP71L (FIG. 6B). Finally, the addition of the N-term of DP71L, including the PPI binding motif and the linker region, to ACREP-GFP was generated, D20, and this clone displayed improved binding to PPI when compared to ACREP-GFP (Fig. 6B). Thus, the data indicated that the linker regions between PPI - and eIF2-binding domains could quantitatively contribute to the binding efficiency of DP71L or ACREP-GFP to PPI. To further test this observation, the inventors swapped only the linker regions of DP71L and ACREP-GFP (FIG. 6C). In accordance with the earlier observed results, ACREP-GFP containing the linker of DP71 (e.g., “DP71 -linker”) binds more strongly to PPI when compared to ACREP-GFP. In contrast, adding the linker of ACREP-GFP to DP71L (e.g., “ACREP-linker”) reduced the binding of the chimeric protein to PPI when compared to WT DP71L (FIG. 6D). These results were surprising and unexpected, as it was not previously known that the linker region could contribute to binding to PPI.
[0249] The linker of DP71L contains 12 amino acids, whereas the linker in ACREP contains 13 amino acids. Alpha-fold prediction of both protein structures together with PPI and the N- terminal domain (NTD) of eIF2a (FIGs. 7A-B) showed that DP71L contains a glutamic acid (tyrosine in ACREP) that was predicted to make a new hydrogen bond with PPI. Given that hydrogen bonds are known to stabilize protein- protein interactions/structures and molecular recognition, the inventors hypothesized that mutation of the glutamic acid on DP71L would disrupt PPI binding. Consistent with this hypothesis, mutating the glutamic acid (El 2) and replacing it with tyrosine dramatically reduced the binding of DP71L to PPI (FIG. 7C). Finally, computational analyses revealed that there are > 440 proteins of different sizes in the animal kingdom (e.g., including viruses directed to said animals) containing both PPI and eIF2 binding domains (e.g., a subset of which is presented herein in Table 1). It is noteworthy that DP71L seems to be the shortest protein identified, and the only one in this list that contains a glutamic acid in this position.
Example 3 - Reversing cognitive deficits associated with ISR
DP71L: a new gene therapy approach that reversed the cognitive deficits in mouse models of Down syndrome and Alzheimer’s disease.
[0250] Tuning down the ISR is emerging as a promising avenue to reverse the cognitive dysfunction in a variety of memory disorders. The development of small molecule inhibitors for some of the ISR kinases (see e.g., Halliday etal., 2015; Nakamura etal., 2018; Axten etal., 2012; Rosen et al., 2009; Huang et al., 1990; Bryk et al., 2011; Robert et al., 2009; Yefidoff-Freedman et al. , 2017; and Hong et al. , 2016) has provided invaluable tools to understand the cellular function of these kinases and their associated targets. Unfortunately, however, the therapeutic promise of these small molecule inhibitors remains largely unobtained and hampered by their relative lack of specificity (see e.g., Rojas-Rivera et al., 2017) and significant toxic effects, such as pancreatic toxicity associated with use of a PERK inhibitor (see e.g., Yu et al., 2015). By far, ISRIB is the best characterized ISR inhibitor. Biochemical, genetic, and structural results show that ISRIB directly binds to eIF2B and enhances its activity by promoting its assembly (see e.g., Sidrauski et al., 2013; Sidrauski et al., 2015; Tsai et al., 2018; and Zyryanova et al., 2018). However, ISRIB blocks the ISR only at intermediate activation levels (see e.g., Rabouw etal., 2019). That is, when the ISR is strongly activated, ISRIB fails to inhibit the ISR (see e.g., Halliday et al., 2015; and Rabouw etal., 2019). In addition, ISRIB solubility is quite poor, which has significantly hampered its ability to penetrate to the brain through the blood-brain barrier and thus its development as medication. As such, new, more specific, and/or more potent ISR inhibitors are much needed. Given this need, the inventors have developed new and efficient and therapy-based approaches that silence and/or inhibit the ISR. Adeno-associated virus (AAV) are the leading platform for gene delivery for the targeted treatment of a variety of diseases. By optimizing the capsids of AAV, recent advances have contributed substantially to preclinical and clinical successes in AAV- mediated gene therapies. However, one major limitation of AAV based therapies is the associated relatively small packaging capacity, e.g., it is limited to ~4.7 kb.
[0251] Because of the small size of DP71L and its potent ISR suppression abilities, the inventors determined that DP71L protein and/or constructs comprising polynucleotide sequences encoding the same, would be an ideal gene therapeutic approach to improve cognition and/or ameliorate and/or prevent symptoms of other diseases in which the ISR is activated. To this end, the inventors cloned DP71L-GFP into an AAV based construct, formed an AAV particle, and delivered the AAV to the hippocampus, a brain structure that is required for long-term memory formation in mice (FIG. 8A). Stereotactic injection of AAV expressing DP71L-GFP resulted in detectable protein expression in the hippocampus of mice (FIG. 8A). The inventors and others have previously shown that inhibition of the ISR rescued the memory deficits associated with Down Syndrome (DS) (see e.g., Zhu et al., 2019) and Alzheimer’s disease (AD) (see e.g., Ma et al., 2013; Segev et al., 2015; Tible et al., 2019; Hwang et al., 2017; and Lourenco et al., 2013). Thus, the inventors tested whether injection of DP71L AAV rescued the long-term memory in these models of cognitive dysfunction.
[0252] DS remains the most common genetic form of intellectual disability. Ts65Dn mice, a well-characterized mouse model of DS, recapitulates the memory defects characteristic of DS patients, and exhibit deficits in long-term memory (see e.g., Zhu etal., 2019). Strikingly, injection of DP71L-GFP (but not GFP alone) into the hippocampus of Ts65Dn mice was sufficient to fully reverse the long-term memory deficits in this model (FIG. 8B). The inventors then examined whether DP71L also restored the deficient strength of synaptic connections in the hippocampus of Ts65Dn mice. To this end, the inventors measured hippocampal L-LTP, a cellular model for memory formation. In hippocampal slices, four trains of 100 Hz induce a long-lasting L-LTP. It was previously shown that L-LTP was impaired in slices from Ts65Dn mice due to activation of the ISR (see e.g., Zhu et al., 2019). In line with this observation, the inventors found that when compared to GFP-injected control animals, DP71L-GFP AAV injection rescued the deficits in L- LTP found in DS mice (FIG. 8C). These data showed that DP71L AAV therapy reversed the cognitive decline as well as the underlying long-lasting deficits in synaptic function associated with Down syndrome. [0253] Alzheimer’s disease is the most common form of dementia. While in in 2020, 5.8 million Americans were living with Alzheimer’s disease, this number is projected to triple to 14 million people by 2060. Currently there are no effective treatments that can alleviate and/or cure the memory decline associated with Alzheimer’s disease (AD). APP/PS1 are double transgenic mice expressing a chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PSl-dE9), both directed to CNS neurons. Both mutations are associated with early-onset Alzheimer's disease. This "humanized" APP/PS1 model has been used to study the pathology associated with AD. As previously reported, the inventors found that APP/PS1 mice exhibited quite dramatic long-term memory and L-LTP deficits (FIG. 8D). Remarkably, DP71L AAV therapy reversed both the long-term memory and L-LTP deficits found in APP/PS1 mice (FIG. 8E).
[0254] In conclusion, the inventors have identified a new and potent gene therapy (e.g., comprising an ISR inhibitor, e.g., comprising DP71L) that improved long-term memory formation in conditions in a wide range of cognitive disorders in which the ISR is activated.
* * *
[0255] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCES
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Claims

WHAT IS CLAIMED IS:
1) A non-natural polynucleotide construct comprising a polynucleotide sequence encoding one or more inhibitor of the integrated stress response (ISR), wherein the one or more inhibitor comprises a protein phosphatase- 1 (PPI) binding domain and an eukaryotic initiation factor 2 (eIF2) binding domain, wherein the non-natural polynucleotide construct comprises: A) a heterologous promoter operably linked to the polynucleotide sequence encoding one or more inhibitors of the ISR, wherein the heterologous promoter is not a galactose inducible promoter; and/or B) a recombinant viral vector comprising a heterologous promoter operably linked to the polynucleotide sequence encoding one or more inhibitors of the ISR, wherein the heterologous promoter is not a galactose inducible promoter.
2) The construct of claim 1 , further comprising a peptide linker sequence connecting the PPI binding domain and eIF2 binding domain.
3) The construct of claims 1 or 2, wherein the peptide linker sequence is at least or exactly 73%, 82%, 91%, or 100% identical to SEQ ID NO: 21.
4) The construct of any one of claims 1-3, wherein the linker sequence comprises a glutamic acid at the DP71L protein E12 position according to SEQ ID NO: 18.
5) The construct of any one of claims 1-4, wherein the inhibitor comprises an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4.
6) The construct of any one of claims 1-5, wherein the inhibitor comprises an amino acid sequence according to SEQ ID NO: 4.
7) The construct of any one of claims 1-4, wherein the inhibitor comprises an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18. 8) The construct of any one of claims 1-5, wherein the inhibitor comprises an amino acid sequence according to SEQ ID NO: 18.
9) The construct of any one of claims 1-8, wherein the heterologous promoter is a CAG promoter.
10) The construct of any one of claims 1-9, wherein the non- natural construct is comprised in a retroviral capsid.
11) An AAV particle comprising the construct of any one of claims 1-10 comprised in an adeno associated virus (AAV) capsid.
12) The AAV particle of claim 11, wherein the AAV particle is of any one of the AAV-DJ/8, AAV9, AAV Php.B, and/or AAV Php.eB serotypes and/or pseudotypes.
13) The AAV particle of claims 11 or 12, wherein the AAV particle is capable of retrograde infection.
14) An AAV particle comprising a nucleotide construct comprising a polynucleotide sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22.
15) The AAV particle of claim 14, comprising a polynucleotide sequence according to SEQ ID NO: 22.
16) An AAV particle of claims 14 or 15, wherein the AAV capsid is an AAV-DJ/8, AAV9, AAV Php.B, and/or AAV Php.eB capsid.
17) A polypeptide comprising an amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. 18) The polypeptide of claim 17, comprising an amino acid sequence according to SEQ ID NO: 4.
19) A pharmacologically acceptable composition comprising the non-natural construct, polypeptide, or AAV particle of any one of claims 1-18.
20) A human cell comprising the non-natural construct, polypeptide, AAV particle, or composition of any one of claims 1-19.
21) A method of delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing cognitive dysfunction in a human subject, comprising administering the non- natural construct, polypeptide, AAV particle, or composition according to any one of claims 1-20 to the human subject.
22) The method of claim 21, wherein the administering is to the central nervous system.
23) The method of claim 21, wherein the administering is to the peripheral nervous system.
24) The method of claim 21, wherein the administering is to the cerebral spinal fluid (CSF).
25) The method of claims 21 or 22, wherein the administering is to the hippocampus.
26) The method of claim 25, wherein the administering is to the CAI, CA2, and/or CA3 region of the hippocampus.
27) The method of any one of claims 21-26, wherein the human subject has been diagnosed with a disease and/or disorder associated with cognitive impairment or dysfunction.
28) The method of any one of claims 21-27, wherein the subject has a neurodevelopmental disorder. 29) The method of any one of claims 21-27, wherein the subject has a neurodegenerative disorder.
30) The method of any one of claims 21-29, wherein the subject has and/or is anticipated to develop Down Syndrome, Alzheimer’s disease, traumatic brain injury, vanishing white matter (VWM) disease, frontotemporal dementia, and/or aging-related cognitive decline.
31) A method of delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing a disease associated with activation, hyperactivation, and/or aberrant activation of the ISR in a human subject, comprising administering the non- natural construct, polypeptide, AAV particle, or composition according to any one of claims 1-20 to the human subject.
32) The method of claim 31, where the disease is a cognitive disorder, neurodegeneration, cancer, diabetes, and/or a metabolic disorder.
33) A method of delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing cognitive dysfunction in a human subject, comprising administering to the human subject:
A) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence according to SEQ ID NO: 18;
B) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence according to SEQ ID NO: 4;
C) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence at least or exactly 85% , 90, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to SEQ ID NO: 18, wherein the inhibitor comprises a protein phosphatase- 1 (PPI) binding domain and an eukaryotic initiation factor 2 (eIF2) binding domain; or
D) a polynucleotide encoding an inhibitor of the ISR comprising an amino acid sequence at least or exactly 85% , 90, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence according to SEQ ID NO: 4, wherein the inhibitor comprises a protein phosphatase-1 (PPI) binding domain and an eukaryotic initiation factor 2 (eIF2) binding domain. 34) The method of claim 33, wherein the polynucleotide comprises a sequence encoding an inhibitor amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18.
35) The method of claims 33, wherein the polynucleotide comprises a sequence encoding an inhibitor amino acid sequence at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4.
36) The method of any one of claims 33-35, wherein the polynucleotide comprises a sequence encoding a peptide linker sequence is at least or exactly 73%, 82%, 91%, or 100% identical to SEQ ID NO: 21.
37) The method of any one of claims 33-36, wherein the polynucleotide is operably linked to a heterologous promoter.
38) The method of claim 37, wherein the heterologous promoter is not a galactose-inducible promoter.
39) The method of any one of claims 33-38, wherein the disease is associated with activation, hyperactivation, and/or aberrant activation of the ISR in a human subject.
40) A transgenic mouse comprising a mutation in a Ppplrl5b gene.
41) The transgenic mouse of claim 40, wherein the mutation is a R658C mutation.
42) The transgenic mouse of claims 40 or 41, wherein the mutation is generated using an endonuclease.
43) The transgenic mouse of claim 42, wherein the endonuclease is Cas9. 44) The transgenic mouse of claim 43, wherein the generation comprises contacting the Ppplrl5b gene with a guide RNA comprising SEQ ID NO: 38.
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