TW202408545A - Integrated stress response inhibitors and methods of using the same - Google Patents

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 use

The present invention relates at least to the fields of aging biology, neurology, genetics, medicine and gerontology.

To maintain cellular health, proteins need to be synthesized in appropriate amounts, folded and assembled with high fidelity, properly localized, and degraded. When a variety of stress sensor molecules trigger intracellular signaling networks, specialized mechanisms respond to malfunctions in these essential processes to maintain or re-establish protein homeostasis (proteostasis). One such network is the integrated stress response (ISR), a central and evolutionarily conserved signaling pathway that helps restore cellular homeostasis by reprogramming gene expression. The central regulatory center of the ISR is located in the eukaryotic initiation factor 2/eukaryotic initiation factor 2B (eIF2/eIF2B) complex, which controls the formation of the eIF2•GTP•methionyl-initiation tRNA ternary complex (TC), which is a prerequisite for the initiation of new protein synthesis (see, e.g., Hinnebusch et al., 2016; which is incorporated herein by reference for the purposes described herein). Phosphorylation of a single serine in the α subunit of the heterotrimeric translation initiation factor eIF2 inhibits the assembly of functional TCs, thereby blocking the action of the guanine nucleotide exchange factor of eIF2, called 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 is incorporated herein by reference for the purposes described herein).

Through four specialized kinases that focus on the phosphorylation of eIF2 (Protein kinase R-like endoplasmic reticulum kinase (PERK)), eIF-2-α kinase (GCN2), and RNA-activated protein kinase (PKR) and proheme-regulated eIF2α kinase (HRI)) sense different disease conditions, including protein constant defects, nutritional deficiencies, viral infections, and oxidative stress. Phosphorylation of eIF2 (eIF2-P) results in a general reduction in protein synthesis. Paradoxically, eIF2-P also triggers the translation of specific mRNAs (including key transcription factors such as ATF4) carrying short repressive upstream open reading frames (ORFs) in their 5' untranslated regions (5' UTRs) (see e.g. Figure 1 ). ISR aims to maintain homeostasis by reprogramming gene expression by reducing general translation and upregulating the synthesis of a few proteins that drive new transcriptional programs. However, if the stress cannot be relieved, the ISR triggers apoptosis to eliminate damaged cells.

Certain aspects of the ISR and systems associated with disease conditions have 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, overactivation, and/or abnormal activation of the ISR has been implicated in a variety of disease conditions, including cognitive disorders, neurodegeneration, cancer, diabetes, muscle loss (e.g., cachexia), and metabolic disorders.

More than 15 years ago, ISR was discovered to be a central regulator of long-term memory formation. Briefly, genetic or pharmacological inhibition of the ISR (e.g., reduced eIF2-P) enhances long-term memory, whereas activation of the ISR (e.g., increased eIF2-P) attenuates long-term memory (see, e.g., Costa-Mattioli et al., 2007; Costa-Mattioli et al., 2005 and Zhu et al., 2011; each of which is incorporated herein by reference for the purposes described herein). Researchers around the world have reproduced, established, and greatly expanded the concept of the ISR as a central memory switch (see, e.g., Batista et al., 2015; Ma et al., 2013; Sharma et al., 2018; Segev et al., 2015; Stern et al. , 2013; Moreno et al., 2012; Kim et al., 2013; Wong et al., 2019; and Costa-Mattioli & Walter, 2020; each of which is incorporated by reference for the purposes described herein. middle). Most notably, an ISR inhibitor (ISRIB) was discovered that acts similarly to genetically lowering the ISR: i.e., it enhances long-term memory formation (see e.g. Kenner et al., 2019; and Sidrauski et al., 2013; their Each is incorporated herein by reference for the purposes described herein).

The ISR is activated in a variety of brain pathologies caused by protein misfolding and aggregation problems, mitochondrial dysfunction, and/or oxidative stress. For example, activation of the ISR is a major pathogenic mechanism implicated in 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 is incorporated by reference for the purposes described herein. incorporated herein), traumatic brain injury (TBI) (see, e.g., Chou et al., 2017; and Sen et al., 2017; each of which is incorporated 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). Therefore, ISR is emerging as a promising approach to reversing cognitive dysfunction in various memory pathologies caused by disruption of proteostasis.

Finally, the importance of the ISR in cognitive dysfunction has been 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; Kernohan 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 is incorporated herein by reference for the purposes described herein). Some of these rare mutations map to the PP1 binding site of CReP (encoded by PPP1R15B) and destabilize the CReP·PP1 phosphatase complex, thereby increasing eIF2-P (see e.g. Abdulkarim et al., 2015; and Kernohan et al. People, 2015; each of which is incorporated herein by reference for the purposes described herein). Other mutations in the gene encoding eIF2γ reduce TC formation and thus induce ISR, similarly causing mental deficiency, epilepsy, hypogenitalism, microcephaly, and obesity. obesity; MEHMO) 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 their are incorporated herein by reference for the purposes described herein). Therefore, activation of ISR is associated with cognitive dysfunction in humans.

The research described herein provides at least new compositions and methods for alleviating ISR-related cognitive dysfunction.

The present invention provides, among other things, the understanding that diseases or conditions associated with integrated stress response (ISR) activation (eg, natural activation, hyperactivation, and/or aberrant activation) can be treated by modulating (eg, inhibiting) the ISR. In certain embodiments, modulation of ISR includes the use of constructs, particles, polypeptides, polynucleotides, and/or compositions comprising such constructs, particles, proteins, and/or nucleotides encoding such proteins, The protein contains PP1 binding domain and eIF2 binding domain. In some embodiments, disclosed herein is a non-natural polynucleotide construct comprising a polynucleotide sequence encoding one or more inhibitors of the integrated stress response (ISR), wherein the one or more inhibitors comprise a protein phosphate Enzyme-1 (PP1) binding domain and eukaryotic initiation factor 2 (eIF2) binding domain, wherein the non-natural polynucleotide construct includes: A) a heterologous promoter operably linked to one of the encoding ISRs or Polynucleotide sequences for multiple inhibitors, wherein the heterologous promoter is not a galactose-inducible promoter; and/or B) a recombinant viral vector comprising a heterologous promoter operably linked to an encoding ISR Polynucleotide sequences for one or more inhibitors, wherein the heterologous promoter is not a galactose-inducible promoter.

In some embodiments, disclosed herein is a non-natural polynucleotide construct comprising a polynucleotide sequence encoding one or more inhibitors of an ISR, wherein the one or more inhibitors comprise a PP1 binding domain and an eIF2 binding domain, Wherein the polynucleotide construct further comprises a peptide linker sequence connecting the PP1 binding domain and the eIF2 binding domain, wherein the peptide linker sequence is included at the amino acid position represented by the E12 position of the DP71L protein (e.g. according to SEQ ID NO: 18) Glutamic acid, wherein the polynucleotide construct further comprises: A) a heterologous promoter operably linked to a 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 a polynucleotide sequence encoding one or more inhibitors of the ISR network.

In some embodiments, the ISR inhibitor binds to PP1α and/or PP1γ and/or eIF2 with higher affinity than other ISR inhibitors with similar primary amino acid sequence lengths. In some embodiments, the binding affinity of the ISR inhibitor to PP1α and/or PP1γ and/or eIF2 is higher than that of the ΔCREP protein. In some embodiments, binding affinity is assessed using an immunoprecipitation assay. In some embodiments, binding affinity is assessed using immunoprecipitation assays on lysates from transgenic human cells (eg, HEK293T cells). In some embodiments, transgenic human cells (e.g., HEK293T cells) are transfected with constructs that stably and/or transiently express a protein of interest (e.g., an ISR inhibitor) and a detectable marker (e.g., A tag, such as a fluorescent marker such as GFP (e.g. encoded by SEQ ID NO: 37)). In some embodiments, binding affinity is analyzed using antibodies targeting a detectable label and/or protein of interest, and using any suitable method, such as (but not limited to) ELISA analysis, Western blot ) assay, immunoprecipitation mass spectrometry, etc.) to detect binding affinity (e.g., qualitatively and/or quantitatively). In some embodiments, binding affinity is assessed as described in Figure 6 herein.

In some embodiments, the constructs described herein encode inhibitors, the inhibitors comprising a PP1 binding domain operable to recruit proteins and/or protein complexes comprising serine/threonine-protein phosphatase PP1-α catalytic subunit (PPP1CA, also known as PP1α), serine/threonine-protein phosphatase PP1-β catalytic subunit (PPP1CB, also known as PP1β) and/or serine/threonine-protein phosphatase PP1-γ catalytic subunit (PPP1CC, also known as PP1γ). In some embodiments, the constructs described herein encode inhibitors, the inhibitors comprising an eIF2 binding domain operable to recruit proteins and/or protein complexes comprising eIF2α, eIF2β and/or eIF2γ subunits. In some embodiments, the PP1 binding domain comprises a RVxF PP1 binding motif and/or a KGILK PP1 binding motif. In some embodiments, the PP1 binding domain comprises a KVRF, KVTF, RVRF, WVTF, IVRF, and/or HVRF PP1 binding motif. In some embodiments, the eIF2 binding domain comprises a RxGx-WxxxAxDRxRFxxRI eIF2 binding motif.

In some embodiments, the constructs described herein encode an inhibitor comprising a constitutive inhibitor of eIF2α phosphorylation (CReP) protein or a characteristic portion thereof. In some embodiments, the 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, the CreP protein or a characteristic portion thereof further comprises a peptide linker sequence that connects the PP1 binding domain to the eIF2 binding domain. In some embodiments, the peptide linker sequence is derived from the 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 that is 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.

In some embodiments, the construct encoding inhibitor described herein comprises a protein phosphatase 1 regulatory subunit 15A (GADD34) protein or a characteristic portion thereof. In some embodiments, the construct encoding inhibitor described herein comprises a Herpes simplex virus γ34.5 protein or a characteristic portion thereof. In some embodiments, the construct encoding inhibitor described herein comprises a Canarypox virus CNPV231 protein or a characteristic portion thereof. In some embodiments, the construct encoding inhibitor described herein comprises a kangaroo herpes virus ICP34.5 protein or a characteristic portion thereof. In some embodiments, the constructs described herein encode an inhibitor comprising the Amsacta moorei entomopoxvirus "L" AmEPV193 protein or a characteristic portion thereof.

In some embodiments, the constructs described herein encode an inhibitor comprising the African swine fever virus DP71L protein or a characteristic portion thereof. In some embodiments, the inhibitor comprises an amino acid sequence that is 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 that is 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 a 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 an E12T mutation (eg, relative to SEQ ID NO: 18).

In some embodiments, the constructs described herein encode an inhibitor comprising a peptide linker between the PP1 binding domain and the eIF2 binding domain. In some embodiments, the inhibitor comprises a peptide linker derived from a DP71L protein. In some embodiments, the peptide linker comprises glutamine at position E12 of the DP71L protein (SEQ ID NO: 18). In some embodiments, the peptide linker comprises an amino acid sequence or polynucleotide sequence encoding it 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.

In some embodiments, the constructs described herein comprise more than one ISR inhibitor selected from the group of proteins consisting of CReP, GADD34, γ34.5, CNPV231, ICP34.5, AmEPV193, and DP71L, and/or characteristic portions thereof.

In some embodiments, the constructs described herein comprise a heterologous promoter that is a CAG promoter. In some embodiments, the CAG promoter comprises a polynucleotide sequence that is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 25 or 26. In some embodiments, the CAG promoter comprises a polynucleotide sequence according to SEQ ID NO: 25 or 26. In some embodiments, the constructs described herein comprise an ISR inhibitor that does not comprise and/or is not fused to glutamine sulfhydryl S-transferase (GST).

In some embodiments, constructs described herein are comprised in retroviral capsids. In some embodiments, described herein is an AAV particle comprising any of the constructs disclosed herein, wherein the construct is contained within an adeno-associated virus (AAV) capsid. In some embodiments, the AAV particles have any of AAV-DJ/8, AAV9, AAV Php.B and/or AAV Php.eB serotypes and/or pseudotypes. In some embodiments, AAV particles are capable of retrograde infection. In some embodiments, the AAV particle comprises a nucleotide construct comprising at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99% of SEQ ID NO: 22 % or 100% identical polynucleotide sequence. In some embodiments, the AAV particle comprises a polynucleotide sequence according to SEQ ID NO: 22. In some embodiments, the AAV capsid is AAV-DJ/8, AAV9, AAV Php.B and/or AAV Php.eB capsid.

Polypeptides are also disclosed herein. In some embodiments, the polypeptide comprises an amino acid sequence that is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, the polypeptide comprises the amino acid sequence according to SEQ ID NO: 4.

Also disclosed herein are pharmacologically acceptable compositions comprising the non-natural constructs, polypeptides and/or AAV particles described herein. Also disclosed herein are cells comprising the non-natural constructs, polypeptides, AAV particles and/or compositions disclosed herein.

Also disclosed herein is delaying the onset of, treating the cognitive dysfunction, slowing the progression of the cognitive dysfunction, reducing the risk of the cognitive dysfunction, and/or preventing the cognitive dysfunction in an individual (eg, a mammalian individual, such as a human individual) Methods of dysfunction comprising administering to an individual (eg, a mammalian individual, such as a human individual) a non-natural construct, polypeptide, AAV particle, or composition disclosed herein. In some embodiments, administration is to the central nervous system. In some embodiments, the administration is to the peripheral nervous system. In some embodiments, the administration is to cerebrospinal fluid (CSF). In some embodiments, the administration is to the hippocampus. In some embodiments, the administration is to the CA1, CA2, and/or CA3 regions of the hippocampus. In some embodiments, administration improves synaptic plasticity in an individual. In some embodiments, administration improves long-term memory formation in an individual. In some embodiments, administering inhibits and/or counteracts protein kinase R-like endoplasmic reticulum kinase (PERK), eIF-2-alpha kinase (GCN2), RNA-activated protein kinase (PKR), and/or pro- The role of one or more of the heme-regulated eIF2α kinases (HRI). In some embodiments, administration localizes the phosphorylase to the eIF2 protein. In some embodiments, the individual has been diagnosed with a disease and/or condition associated with cognitive impairment or dysfunction. In some embodiments, the individual has a neurodevelopmental disorder. In some embodiments, the individual suffers from a neurodegenerative disorder. In some embodiments, the individual has and/or is expected to develop Down syndrome, Alzheimer's disease, traumatic brain injury, vanishing white matter (VWM) disorder, frontotemporal dementia, and /or aging-related cognitive decline.

Also disclosed herein are methods of delaying the onset of a disease associated with activation, overactivation, and/or aberrant activation of the ISR in a human subject, treating the disease, slowing the progression of the disease, reducing the risk of the disease, and/or preventing the disease, 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, the subject is less than 10 years old. In some embodiments, the subject is older than 30 years old. In some embodiments, the subject is older than 50 years old. In some embodiments, the subject is older than 70 years old. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

Also disclosed herein are methods of delaying the onset, treating the cognitive dysfunction, slowing the progression of the cognitive dysfunction, reducing the risk of the cognitive dysfunction, and/or preventing the cognitive dysfunction in a human subject, including providing Administration to an individual: A) a polynucleotide encoding an inhibitor of an ISR, comprising an amino acid sequence according to SEQ ID NO: 18; B) a polynucleotide encoding an inhibitor of an ISR, the ISR The inhibitor comprises the amino acid sequence according to SEQ ID NO: 4; C) a polynucleotide encoding an inhibitor of ISR, the inhibitor of the ISR comprises at least or exactly the amino acid sequence according to SEQ ID NO: 18 85%, 90%, 95%, 97%, 98%, 99% or 100% identical amino acid sequences, in which the inhibitor includes protein phosphatase-1 (PP1) binding domain and eukaryotic initiation factor 2 (eIF2) ) binding domain; or D) a polynucleotide encoding an inhibitor of an ISR that contains at least or exactly 85%, 90%, 95%, or 97% of the amino acid sequence according to SEQ ID NO: 4 , 98%, 99% or 100% identical amino acid sequences, in which the inhibitor includes protein phosphatase-1 (PP1) binding domain and eukaryotic initiation factor 2 (eIF2) binding domain. In some embodiments, the polynucleotide comprises a polynucleotide sequence that is 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 that 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 at least or exactly 85%, 90%, 95%, 96%, 97%, 98% of the same sequence as SEQ ID NO: 4 %, 99% or 100% identical amino acid sequence. 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, overactivation and/or abnormal activation of ISR in a human subject.

Also disclosed herein are transgenic mice containing mutations in the Ppp1r15b gene. In some embodiments, the mutation is the R658C mutation. In some embodiments, endonucleases are used to generate mutations. In some embodiments, the endonuclease is Cas9. In some embodiments, generating comprises contacting the Ppp1r15b gene with a guide RNA comprising SEQ ID NO: 38.

Certain embodiments of the invention are characterized by the following aspects.

Aspect 1 is a non-natural polynucleotide construct comprising a polynucleotide sequence encoding one or more inhibitors of the integrated stress response (ISR), wherein the one or more inhibitors comprise protein phosphatase-1 ( PP1) binding domain and eukaryotic initiation factor 2 (eIF2) binding domain, wherein the non-natural polynucleotide construct includes: A) a heterologous promoter operably linked to a protein encoding one or more inhibitors of the ISR a polynucleotide sequence, wherein the heterologous promoter is not a galactose-inducible promoter; and/or B) a recombinant viral vector comprising a heterologous promoter operably linked to one or more encoding ISRs Polynucleotide sequences of inhibitors in which the heterologous promoter is not a galactose-inducible promoter.

Aspect 2 is a construct as in aspect 1, further comprising a peptide linker sequence connecting the PP1 binding domain and the eIF2 binding domain.

Aspect 3 is a construct as in aspect 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 a construct as in any one of Aspects 1 to 3, wherein the linker sequence comprises glutamine at position E12 of the DP71L protein according to SEQ ID NO: 18.

Aspect 5 is the construct of any one of Aspects 1 to 4, wherein the inhibitor comprises an amino acid sequence that is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4.

Aspect 6 is a construct as in any one of aspects 1 to 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 to 4, wherein the inhibitor comprises an amino acid sequence that is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 18.

Aspect 8 is a construct as in any one of aspects 1 to 5, wherein the inhibitor comprises an amino acid sequence according to SEQ ID NO: 18.

Aspect 9 is a construct as in any one of aspects 1 to 8, wherein the heterologous promoter is a CAG promoter.

Aspect 10 is the construct of any one of aspects 1 to 9, wherein the non-native construct is contained in a retroviral capsid.

Aspect 11 is an AAV particle comprising the construct of any one of aspects 1 to 10 contained in an adeno-associated virus (AAV) capsid.

Aspect 12 is an AAV particle of aspect 11, wherein the AAV particle has any one of AAV-DJ/8, AAV9, AAV Php.B and/or AAV Php.eB serotypes and/or pseudotypes.

Aspect 13 is an AAV particle as in aspect 11 or 12, wherein the AAV particle is capable of retrograde infection.

Aspect 14 is an AAV particle comprising a nucleotide construct comprising at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, A polynucleotide sequence that is 99% or 100% identical.

Aspect 15 is an AAV particle of aspect 14, comprising a polynucleotide sequence according to SEQ ID NO: 22.

Aspect 16 is an AAV particle of aspect 14 or 15, wherein the AAV capsid is AAV-DJ/8, AAV9, AAV Php.B and/or AAV Php.eB capsid.

Aspect 17 is a polypeptide comprising an amino acid sequence that is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4.

Aspect 18 is a polypeptide as in aspect 17, comprising the amino acid sequence according to SEQ ID NO: 4.

Aspect 19 is a pharmacologically acceptable composition comprising the non-natural construct, polypeptide or AAV particle of any one of aspects 1 to 18.

Aspect 20 is a human cell comprising the non-natural construct, polypeptide, AAV particle or composition of any one of aspects 1 to 19.

Aspect 21 is a method of delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing a cognitive disorder in a human subject, comprising administering to the human subject a non-natural construct, polypeptide, AAV particle, or composition of any one of Aspects 1 to 20.

Aspect 22 is the method of aspect 21, wherein the administering is performed to the central nervous system.

Aspect 23 is the method of aspect 21, wherein the administration is performed to the peripheral nervous system.

Aspect 24 is the method of aspect 21, wherein the administering is performed into cerebrospinal fluid (CSF).

Aspect 25 is the method of Aspect 21 or 22, wherein the administration is to the hippocampus.

Aspect 26 is the method of aspect 25, wherein the CA1, CA2, and/or CA3 regions of the hippocampus are administered.

Aspect 27 is the method of any one of Aspects 21 to 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 to 27, wherein the individual suffers from a neurodevelopmental disorder.

Aspect 29 is the method of any one of aspects 21 to 27, wherein the subject has a neurodegenerative disorder.

Aspect 30 is the method of any one of aspects 21 to 29, wherein the individual suffers from and/or is expected to develop Down syndrome, Alzheimer's disease, traumatic brain injury, vanishing white matter (VWM) disease, frontotemporal dementia, and/or aging-related cognitive decline.

Aspect 31 is a method for delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing a disease associated with activation, overactivation, and/or aberrant activation of the ISR in a human subject, comprising administering to the human subject a non-natural construct, polypeptide, AAV particle, or composition of any one of Aspects 1 to 20.

Aspect 32 is the method of aspect 31, wherein 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 the cognitive dysfunction, slowing the progression of the cognitive dysfunction, reducing the risk of the cognitive dysfunction, and/or preventing the cognitive dysfunction in a human subject, comprising Administering to a human subject: a) a polynucleotide encoding an inhibitor of an ISR, the inhibitor of the ISR comprising an amino acid sequence according to SEQ ID NO: 18; b) a polynucleotide encoding an inhibitor of an ISR, The inhibitor of the ISR includes an amino acid sequence according to SEQ ID NO: 4; C) a polynucleotide encoding an inhibitor of the ISR, the inhibitor of the ISR includes at least an amino acid sequence according to SEQ ID NO: 18 or exactly 85%, 90%, 95%, 97%, 98%, 99% or 100% identical amino acid sequences, wherein the inhibitor includes protein phosphatase-1 (PP1) binding domain and eukaryotic initiation factor 2 (eIF2) binding domain; or d) a polynucleotide encoding an inhibitor of an ISR, the inhibitor of the ISR comprising at least or exactly 85%, 90%, 95%, 97%, 98%, 99% or 100% identical amino acid sequences, in which the inhibitor contains protein phosphatase-1 (PP1) binding domain and eukaryotic initiation factor 2 (eIF2) binding domain.

Aspect 34 is the method of aspect 33, wherein the polynucleotide comprises a sequence encoding an inhibitory amino acid sequence that is 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 that is at least or exactly 85%, 90%, or 95% identical to SEQ ID NO: 4 %, 96%, 97%, 98%, 99% or 100% agreement.

Aspect 36 is the method of any one of aspects 33 to 35, wherein the polynucleotide comprises a sequence encoding a peptide linker sequence that is at least or exactly 73%, 82%, 91% identical to SEQ ID NO: 21 % or 100% consistent.

Aspect 37 is the method of any one of Aspects 33 to 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 to 38, wherein the disease is associated with activation, overactivation, and/or abnormal activation of the ISR in the human subject.

Aspect 40 is a transgenic mouse containing a mutation in the Ppp1r15b gene.

Aspect 41 is a transgenic mouse as in aspect 40, wherein the mutation is a R658C mutation.

Aspect 42 is a transgenic mouse as described in aspect 40 or 41, wherein the mutation is generated using an endonuclease.

Aspect 43 is a transgenic mouse as in aspect 42, in which the endonuclease is Cas9.

Aspect 44 is a transgenic mouse as in aspect 43, wherein the generating comprises contacting the Ppp1r15b gene with a guide RNA comprising SEQ ID NO: 38.

Cross-reference to related applications

This application claims priority from U.S. Provisional Patent Application No. 63/359,672, filed on July 8, 2022, which is incorporated herein by reference in its entirety. sequence list

This application contains a sequence listing, which has been submitted in ST26 format and is incorporated herein by reference in its entirety. This ST26 copy was created on June 13, 2023, named BAYM_P0373WO_Sequence_Listing.xml and has a size of 441,028 bytes. Statement Regarding Federally Awarded Research or Development

This invention was made with government support under Grant R01 NS076708-10 from the National Institutes of Health. The government has certain rights in this invention.

The research described herein provides at least new compositions and methods for inhibiting the ISR, including alleviating ISR-related symptoms. Certain aspects of the ISR and disease-related systems have been summarized in Mauro Costa-Mattioli and Peter Walter 2020 (see, for example, 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 some embodiments, the constructs, particles, polypeptides, polynucleotides and/or compositions described herein can be used in methods for inhibiting the ISR. In some embodiments, the constructs, particles, polypeptides, polynucleotides and/or compositions described herein can be used in methods for treating diseases associated with natural activation, overactivation and/or abnormal activation of the ISR. In some embodiments, such diseases may include, but are not limited to, cognitive disorders, neurodegeneration, cancer, diabetes, muscle loss (eg, cachexia), and/or metabolic disorders.

One or more compositions may be used based on the methods described herein (e.g., including components included in the composition, such as constructs (e.g., polynucleotide constructs, such as RNA and/or DNA constructs), particles, peptides, etc.). Other embodiments are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies equally to other aspects of the invention, and vice versa. The examples in the Examples section are to be understood as applicable to all aspects of the technology described herein.

Throughout this application, the term "about" is used to indicate that a value includes the inherent variation in error used in a measurement or quantitative method.

When used in conjunction with the term "comprising", the use of the word "a" or "an" may mean "one", but it is also consistent with the meaning of "one or more", "at least one" and "one or more than one".

The phrase "and/or" means "and" or "or". For example, A, B and/or C includes: A alone, B alone, C alone, the combination of A and B, the combination of A and C, the combination of B and C, or the combination of A, B and C. In other words, "and/or" acts as an inclusive OR operator.

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 "contains" (containing)" (and any form of containing, such as "contains" and "containing") is inclusive or open and does not exclude additional unlisted elements or method steps.

Compositions and methods, when used, may "comprise", "consist essentially of" or "consist of" any of the ingredients or steps disclosed throughout this specification. Compositions and methods that "consist essentially of" any of the disclosed ingredients or steps limit the scope of the claim to specified materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

The term "effective," as the term is used in the specification and/or claims, means sufficient to achieve a desired, expected or intended result.

The terms "individual," "subject," and "patient" are used interchangeably and may refer to humans or non-humans.

As used in the claims and/or this specification, the terms "inhibit" or "reduce" or "prevent" or "avoid" or any variation of these terms include any measurable reduction or complete inhibition to achieve the desired result.

As used herein, "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, a wild type version of a protein or polypeptide is used, however, in many embodiments of the invention, a modified and/or non-natural protein or polypeptide is used to modulate (e.g., inhibit) ISR. The terms described above may be used interchangeably. "Modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, particularly amino acid sequence, is altered relative to a wild type protein or polypeptide. In some embodiments, a modified/variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). In particular, it is contemplated that a modified/variant protein or polypeptide may be altered with respect to one activity or function, but retain wild-type activity or function in other respects, such as immunogenicity. A "non-naturally occurring" polypeptide and/or construct (e.g., a polynucleotide construct) refers to an engineered polypeptide and/or construct that has been artificially produced and is not found in nature. Non-natural polypeptides and/or constructs are also understood to include non-natural amino acids and/or non-natural nucleotides. Proteins can be isolated directly from their native organisms, produced by recombinant DNA/exogenous expression methods, or produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In some embodiments, proteins are produced by RNA constructs. In some embodiments, RNA constructs encoding proteins (e.g., linear and/or cyclic constructs) can be produced synthetically and/or by in vitro methods. In certain embodiments, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding polypeptides. The term "recombinant" may be used in conjunction with the name of a polypeptide or specific polypeptide, and this generally refers to a polypeptide produced by a nucleic acid molecule that has been manipulated in vitro or is a replicate of such a molecule.

Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may also be described in "use" claim language, such as any compound, composition, or agent discussed herein for accomplishing or performing that purpose. The therapeutic, diagnostic, or physiological purpose or effect described.

It is particularly contemplated that any limitation discussed with respect to one embodiment of the present invention may apply to any other embodiment of the present invention. In addition, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to produce or utilize any composition of the present invention. Any embodiment discussed with respect to one aspect of the present invention is equally applicable to other aspects of the present invention, and vice versa. For example, any step in the method described herein may be applicable to any other method. In addition, any method described herein may exclude any step or combination of steps. The aspects of the embodiments described in the examples are also embodiments that may be implemented in the context of the embodiments discussed elsewhere in different examples or elsewhere in the present application (such as in the invention content, implementation method, application scope and simple description of the figures).

Other objects, features and advantages of the present invention will become apparent from the following embodiments. It is to be understood, however, that the embodiments and specific examples are intended to be indicative of specific embodiments of the invention, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from these embodiments. are provided for illustration only. I. Conditions associated with activation of the integrated stress response ( ISR ) A. Cognitive disorders

In some embodiments, the technology described herein (e.g., compositions and/or methods) is useful for delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing a disease associated with activation, overactivation, and/or aberrant activation of the ISR in a human subject. In some embodiments, the disease is a disease associated with cognitive decline, and the technology provided herein is applicable to delaying the onset of cognitive decline and/or other symptoms associated with the disease condition, treating cognitive decline and/or other symptoms associated with the disease condition, slowing the progression of cognitive decline and/or other symptoms associated with the disease condition, reducing the risk of cognitive decline and/or other symptoms associated with the disease condition, and/or preventing cognitive decline and/or other symptoms associated with the disease condition. As described herein, in certain embodiments, cognitive decline is associated with and/or caused by activation of the integrated stress response (ISR) system. In certain embodiments, the techniques described herein are applicable to at least partially reverse cognitive deficits (e.g., cognitive impairment, such as (but not limited to) impairment of long-term memory (LTM) formation, and/or retention). In certain embodiments, the techniques described herein are applicable to reducing the risk of loss of LTM formation ability. In certain embodiments, the techniques described herein are applicable to treating loss of LTM formation ability. In certain embodiments, the techniques described herein are applicable to improving LTM formation ability. In certain embodiments, the compositions and/or methods disclosed herein improve long-term potentiation (LTP) and/or synaptic plasticity. In certain embodiments, the compositions and/or methods disclosed herein improve long-term LTP. In certain embodiments, the compositions and/or methods disclosed herein improve late LTP. In certain embodiments, the technology described herein is useful for providing ISR inhibition under conditions of ISR overactivation, e.g., under conditions where traditional ISR inhibitory compositions (e.g., small molecules such as kinase inhibitors and/or ISRIBs) are insufficient to achieve amelioration and/or prevention of the therapeutic disease condition.

Cognitive functions, including (but not limited to) functions such as attention, orientation, language, memory, visuospatial abilities, social interaction, and functional status, may be assessed using methods well known to those skilled in the art (e.g., screening tests and/or questionnaires). Assessment and/or behavioral assessment.

In certain embodiments, the techniques described herein are suitable for preventing, treating, and/or reducing various neurological disorders associated with ISR activation (e.g., neurodegenerative disorders, neurodevelopmental disorders, etc.). risk of neurological disorders. In certain embodiments, the techniques described herein are suitable for improving brain function (eg, brain cognitive function), including (but not limited to) LTM formation and/or retention, with or without ISR activation.

In some embodiments, the cognitive disorder is a neurological disorder and is characterized in part by neurodegeneration and/or abnormal neurodevelopment (eg, neurodevelopmental disorders). In certain embodiments, the 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's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) ), Prion disease, traumatic brain injury, Vanishing White Matter (VWM) disorder, frontotemporal dementia, and/or aging (e.g., age-related cognitive decline). In certain embodiments, the techniques provided herein are suitable for delaying the onset of cognitive dysfunction associated with a cognitive disorder, treating the cognitive dysfunction, slowing the progression of the cognitive dysfunction, reducing the risk of the cognitive dysfunction, and/or or prevent the cognitive dysfunction. In certain embodiments, the techniques provided herein are suitable for delaying the onset of a neurological disorder, treating the neurological disorder, slowing the progression of the neurological disorder, reducing the risk of the neurological disorder, and/or preventing the neurological disorder.

Down syndrome (DS) is the most common genetic form of intellectual disability (ID) and affects approximately 10 in 10 000 live births (see, e.g., Khoshnood et al., 2011). Individuals with DS show deficits in learning and memory, language, and executive function from early childhood, caused by the presence of an extra copy of chromosome 21 (Hsa21). DS patients exhibit age-dependent reductions in dendritic branching and spine density, which may be related to impaired reorganization of the actin cytoskeleton in neurons (Marin-Padilla 1976; Takashima et al. 1981). Additionally, DS patients show early onset Alzheimer-like neurodegeneration (reviewed in, eg, Lott and Dierssen 2010). The role of ISR in DS symptom presentation and cognitive abilities is discussed in Zhu et al., 2019, which is incorporated herein by reference for the purposes described herein. In certain embodiments, the constructs and/or compositions described herein are suitable for preventing, treating, and/or alleviating symptoms associated with Down syndrome.

Alzheimer's disease is biologically defined by the presence of beta-amyloid-containing plaques and tau-containing neurofibrillary tangles. Alzheimer's disease is a hereditary and sporadic neurodegenerative disorder that causes 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 in middle and late life, but its clinical impact can be modified by other neurodegenerative conditions and cerebrovascular conditions. Alzheimer's disease biology can be viewed as a brain disorder caused by a complex interplay of synaptic homeostasis loss and dysfunction in highly interrelated endosomal/lysosomal clearance pathways, in which precursors, aggregated species, and post-translational modifications of Aβ and tau play important roles. Synaptic homeostasis loss 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 is incorporated herein by reference for the purposes described herein. In certain embodiments, the constructs and/or compositions described herein are suitable for preventing, treating, and/or alleviating symptoms associated with Alzheimer's disease.

Vanishing white matter (VWM) disease (also known as childhood ataxia with central nervous system hypomyelination (CACH)) is a disease caused by EIF2B1, EIF2B2, EIF2B3, EIF2B4 and/or A rare inherited neurological condition caused by mutations in one or more of the EIF2B5 genes. Children with VWM have one or more defective proteins that prevent the body from producing enough myelin, a white fatty substance that insulates nerve fibers and protects them from damage. The nerve fibers covered by myelin are called "white matter". Without myelin, nerves throughout the body deteriorate and disappear. Symptoms such as muscle stiffness and poor coordination usually begin between the ages of 2 and 6 years. The illness may worsen if your child has a fever, infection, or head injury. In some conditions, an adult-onset form of VWM may occur and is often accompanied by behavioral abnormalities, severe headaches, and/or cognitive decline. Some roles for 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 are incorporated herein by reference for the purposes described herein). In certain embodiments, the constructs and/or compositions described herein are suitable for preventing, treating, and/or alleviating symptoms associated with VWM disease.

Frontotemporal dementia is a group of conditions that affect the frontal and temporal lobes of the brain. In certain cases, personality, emotions, behavior, memory formation, and speech are controlled by these parts of the brain and are lost as the relevant cells lose their function. The symptoms of frontotemporal dementia depend on the area of the brain affected, but most symptoms can be classified as behavioral or language changes. In some embodiments, behavioral symptoms may include inappropriate actions, apathy, lack of interest and/or enthusiasm, lack of inhibition or restraint, neglect of personal hygiene and/or care, and/or compulsive behavior. In some embodiments, language symptoms may include difficulty speaking and/or understanding speech, difficulty using appropriate language, loss of reading and/or writing skills, and/or difficulty interacting socially. In certain instances, individuals with frontotemporal dementia may develop abnormal protein structures called pick bodies. The most common genetic cause of frontotemporal dementia is a repeat expansion of the C9orf72 gene. Protein accumulation associated with frontotemporal dementia activates the ISR response via the protein kinase PERK. Certain roles of 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 is incorporated herein by reference for the purposes described herein). In certain embodiments, the constructs and/or compositions described herein are suitable for preventing, treating, and/or alleviating symptoms associated with frontotemporal dementia.

Healthy aging requires the coordinated regulation of a large number of stress signaling pathways that converge on a network of protein homeostasis. ISR activity increases with age, suggesting a potential link to the aging process. Although the reduction in protein biosynthesis that occurs during ISR activation is associated with lifespan extension, recent data suggest that lifespan is limited by the ISR, as ISR inhibition extends survival in nematodes and enhances cognitive function in aged mice. In certain embodiments, the ISR is modulated and, at the same time, the aging process is also modulated. 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; 1(9):760-768; which is incorporated herein by reference for the purposes described herein). In certain embodiments, the constructs and/or compositions described herein are suitable for preventing, treating, and/or alleviating symptoms associated with aging. In certain embodiments, the constructs and/or compositions described herein are suitable for preventing, treating, and/or alleviating symptoms associated with aging-related cognitive decline. B. Other Conditions and Diseases

In some embodiments, the constructs, particles, polypeptides, polynucleotides, and/or compositions described herein can be used to prevent ISR activation (e.g., normal activation, hyperactivation, and/or aberrant activation) in human subjects. A disease or condition, a method of treating the disease or condition, reducing the progression of the disease or condition, and/or reducing the risk of the disease or condition. In some embodiments, the constructs, particles, polypeptides, polynucleotides, and/or compositions described herein can be used to treat a disease or disorder, wherein the disease or disorder is a neurodegenerative disease, an inflammatory disease, an autologous Immune diseases, metabolic syndromes, cancer, vascular diseases, fibrotic diseases, viral infections, musculoskeletal diseases (such as myopathies), eye diseases or genetic disorders.

In some embodiments, the disease or condition is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is leukoencephalopathy, childhood ataxia with CNS hypomyelination, mental retardation syndrome, Alzheimer's disease, Prion's disease, Creutzfeldt- Jakob disease, Parkinson's disease, ALS, Pelizaeus-Merzbacher disease, cognitive impairment, traumatic brain injury, post-surgical cognitive function disorder (PCD), neuro-otological syndrome, hearing loss, Huntington's disease, stroke, chronic traumatic encephalopathy, spinal cord injury, dementia, frontotemporal dementia (FTD), depression or social behavior obstacles. In some embodiments, cognitive impairment is caused by aging, radiation, sepsis, epilepsy, heart attack, cardiac surgery, liver failure, hepatic encephalopathy, anesthesia, brain injury, brain surgery, ischemia, chemotherapy, cancer treatment, Triggered by critical illness, concussion, fibromuscular pain, or depression. In some embodiments, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the neurodegenerative disease is aging-related cognitive impairment. In some embodiments, the neurodegenerative disease is traumatic brain injury.

In some embodiments, the constructs, particles, polypeptides, polynucleotides and/or compositions described herein can be used in methods for treating Alzheimer's disease. In some embodiments, neurodegeneration and/or cognitive impairment is reduced.

In some embodiments, the disease or condition 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 respiratory disease, cystic fibrosis, glomerulonephritis Nephritis, membranous nephropathy, sarcoidosis, vasculitis, ichthyosis, transplant rejection, interstitial cystitis, atopic dermatitis, or inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is Crohn's disease, ulcerative colitis, or celiac disease.

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.

In some embodiments, the disease or condition is metabolic syndrome. In some embodiments, the metabolic syndrome is acute pancreatitis, chronic pancreatitis, alcoholic steatosis, obesity, glucose intolerance, insulin resistance, hyperglycemia, fatty liver, dyslipidemia, hyperlipidemia, Cysteinoidemia or type 2 diabetes. In some embodiments, the metabolic syndrome is alcoholic hepatic steatosis, obesity, glucose intolerance, insulin resistance, hyperglycemia, fatty liver, dyslipidemia, hyperlipidemia, cystinoidemia, or type 2 diabetes.

In some embodiments, the disease or condition is 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, secretory cell carcinoma, thyroid cancer, gastrointestinal cancer, chronic myeloid leukemia, hepatocellular carcinoma, colon cancer, melanoma, malignant glioma, glioblastoma, glioblastoma multiforme Cell tumor, astrocytoma, cerebellar dysplastic ganglioneuroma, Ewing's sarcoma, rhabdomyosarcoma, ependymoma, medulloblastoma, ductal adenocarcinoma, adenosquamous carcinoma, nephroblastoma tumour, acinar cell carcinoma, neuroblastoma or lung cancer. In some embodiments, the secretory cell cancer is non-Hodgkin's lymphoma, Burkitt's lymphoma, chronic lymphocytic leukemia, monoclonal immunoglobulinemia of undetermined significance (monoclonal gammopathy of undetermined significance; MGUS), plasmacytoma, lymphoplasmacytic lymphoma, or acute lymphoblastic leukemia.

In some embodiments, the disease or disorder is a musculoskeletal disorder (such as myopathy). In some embodiments, the musculoskeletal disease is myopathy, muscular dystrophy, muscular atrophy, muscle wasting, or sarcopenia. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD), Becker's disease, myotonic dystrophy, X-linked dilated cardiomyopathy, spinal cord Muscular atrophy (spinal muscular atrophy; SMA), or metaphyseal chondrodysplasia, Schmid type; MCDS. In some embodiments, the myopathy is skeletal muscle atrophy. In some embodiments, musculoskeletal disorders (such as skeletal muscle atrophy) are caused by aging, chronic disease, stroke, malnutrition, bedrest, orthopedic injury, fractures, cachexia, starvation, heart failure, obstructive Lung disease, renal failure, acquired immunodeficiency syndrome (AIDS), sepsis, immune disorders, cancer, ALS, burns, denervation, diabetes, muscle disuse, immobility, mechanical unloading, myositis, or malnutrition trigger.

In some embodiments, the constructs, particles, polypeptides, polynucleotides and/or compositions described herein can be used in methods for treating musculoskeletal diseases. In some embodiments, skeletal muscle mass, quality and/or strength are increased. In some embodiments, muscle protein synthesis is increased. In some embodiments, skeletal muscle fiber atrophy is inhibited.

In some embodiments, the disease or disorder is a vascular disease. In some embodiments, the vascular disease is atherosclerosis, abdominal aortic aneurysm, carotid artery disease, deep venous embolism, Buerger's disease, chronic venous hypertension, vascular calcification, telangiectasia, or lymphedema.

In some embodiments, the disease or disorder is an eye disease. In some embodiments, the eye 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.

In some embodiments, provided herein is a method for modulating an ISR pathway. It is believed that the constructs, particles, polypeptides, polynucleotides and/or compositions described herein are effective in modulating an ISR pathway. In some embodiments, the method of modulating an ISR pathway comprises modulating an ISR pathway in a cell by administering or delivering a construct and/or compound described herein to the cell. In some embodiments, the method of modulating an ISR pathway comprises modulating an ISR pathway in an individual by administering a construct and/or compound described herein to an individual. In some embodiments, modulation of an ISR pathway can be determined by methods known in the art, such as Western blotting, immunohistochemistry, or reporter cell line assay. II. Proteins, polypeptides and polynucleotides

In certain embodiments, provided herein are constructs (e.g., polynucleotide constructs, e.g., linear and/or cyclic polynucleotide constructs, e.g., DNA and/or RNA constructs), polypeptides, and/or compositions , and/or methods of using the same, which consist of, consist essentially of and/or include an inhibitor of the integrated stress response (ISR) or a characteristic part thereof, and/or encodes the inhibitor or characteristic portion thereof. In certain embodiments, the size of the protein or polypeptide (wild-type or modified) may include, 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 more, and any derivable range therein, or as described or referenced herein Derivatives corresponding to the amine sequence. It is contemplated that a polypeptide may be mutated by truncation to make it shorter than its corresponding wild-type form, and may furthermore have a specific function by fusion or conjugation (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term "domain" refers to any unique functional or structural unit of a protein or polypeptide, and generally refers to an amino acid sequence having a structure or function recognized by one skilled in the art (e.g., a PP1 binding domain, eIF2 binding domain, peptide linker, etc.).

The polypeptides, proteins or polynucleotides encoding the polypeptides or proteins of the present invention 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 may be different from SEQ ID NO: 1 to 447 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 consecutive amino acids or nucleic acids (or any range derivable therein) 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 derivative thereof) are similar, identical or homologous.

In some embodiments, the protein, polypeptide or nucleic acid may comprise SEQ ID NO: 1 to 447 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 10, 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, 3 94, 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 5、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, 5 78, 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 9, 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 62, 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 3. 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, 946 990, 991, 992, 993, 994, 995, 996, 997, 998, 999 or 1000 (or any derivable range therein) or more consecutive amino acids or nucleic acids.

In some embodiments, a 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 of SEQ ID NOs: 1 to 447 ,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 thereof) consecutive amino acids Or a nucleic acid that is at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% with one of SEQ ID NO: 1 to 447 ,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 exportable range) similar, identical or homologous.

In some aspects, there is a nucleic acid molecule or polypeptide that begins with SEQ ID NO: 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, 2 69, 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 30, 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 1, 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 2, 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 58, 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, 8 19, 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, 88 0, 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, 941 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999 or 1000 and comprising SEQ ID NO: 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 6、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 9, 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, 3 92, 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 ,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,485,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500,501,502,503,504,505,506,507,508,509,510,511,512,513,514,5 15, 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 6、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 9. 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 22, 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, 945 949, 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) consecutive amino acids or nucleic acids.

The nucleotide and protein, polypeptide and peptide sequences of various genes have been previously disclosed and can be found in recognized computerized databases. Two commonly used databases are the Genbank and GenPept databases of the National Center for Biotechnology Information (ncbi.nlm.nih.gov/ on the World Wide Web) and the Universal Protein Resource (UniProt; uniprot.org on the World Wide Web). The coding regions of these genes can be expanded and/or expressed using techniques disclosed herein or known to those skilled in the art.

In some embodiments, the ISR inhibitor according to the present invention is obtained by surface 1Consists of, consists essentially of, or includes the amino acid sequence described in . In some embodiments, as surface 1The dash (-) indicated in is quantitative and represents any amino acid. In some embodiments, an inhibitor of an ISR consists of, consists essentially of, or includes an amino acid sequence, or is encoded by a polynucleotide sequence, which amino acid sequence or polynucleotide sequence The nucleotide sequence is 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 exportable range) consistency. surface 1 - Include PP1 Combined domain and eIF2 Binding domain protein variants Name genus door sequence SEQ ID NO tr|R7U6B5|R7U6B5_CAPTE Capitella teleta Annelida ------------------------------------ LCQA--YAEARKGEWEMFARDRSRFASRVQSLEKIISPILSEEHRQHIR-- 39 tr|T1EY96|T1EY96_HELRO Helobdella robusta Annelida ---DQPAKKNKKKVRFSDTVDCQIIDEGD----EKECRKGHWELFALDRARFRKRINEAEPVLAWLFYPNHRIKM--- 40 tr|F4WRG9|F4WRG9_ACREC Acromyrmex echinatior Arthropoda ------RNKQTKKVSFDPTPVVHV------A--YRAARRGPWEEIARDNERFRGRIKSIAIVLDPILKSKH------- 41 tr|Q17PS4|Q17PS4_AEDAE Aedes aegypti Arthropoda ----PDSGFEEKKVQFNLKPEVHVMRTWDFA--YRQARKGEWEIAARDRERFKNRIHETGKVLSTVFDKNLRDKVY-- 42 tr|A0A1S4EV95|A0A1S4EV95_AEDAE Aedes aegypti Arthropoda ----------EKKVQFNLKPEVH------FA--YRQARKGEWEIAARDRERFKNRIHETGKVLSTVFDKN-------- 43 tr|A0A023EUJ6|A0A023EUJ6_AEDAL Aedes albopictus Arthropoda ----PDSGFEEKKVQFNLNPEVHVIRAWDFA--YRQARKGEWEMAARDRERFKKRIQETENVLSSVFDKKLRDKVY-- 44 tr|A0A1W4X3S0|A0A1W4X3S0_AGRPL Agrilus planipennis Arthropoda -------ITNRKKVSFASDCKVHHMVKWSYA--YQAARKGPWEECARDRERFMLRIRNLEPELSKVFDPKLRKRIY-- 45 tr|A0A1E1XCI9|A0A1E1XCI9_9ACAR Amblyomma aureolatum Arthropods ------DVGKPPRAAKVNFPLL--VQVHS-A--DDLERKGPWEQIAVDRRRFQSRIASVESVLAPVLCPDHRQNVY-- 46 tr|A0A023FNM1|A0A023FNM1_9ACAR Amblyomma cajennense Arthropoda ---------------KVNFPGL--VQVHS-A--DDLERKGPWEQIALDRRRFQSRIASVECVLAPVLSAEHRQNVYQ- 47 tr|A0A023G0U4|A0A023G0U4_9ACAR Tiny flower tick ( Amblyomma parvum ) Arthropoda --------RAAKVNFPLL--VQVHS-A--DDFERKGPWEQIAVDRRRFQSRIASVESVLAPVLTAEHRQNVY-- 48 tr|A0A1E1XQC2|A0A1E1XQC2_9ACAR Sculptured flower tick ( Amblyomma sculptum ) Arthropoda ------DVGKPPRAAKVNFPLL--VQVHS-A--DDLERKGPWEQIALDRRRFQSRIASVECVLAPVLSAEHRQNVY-- 49 tr|A0A023GBB1|A0A023GBB1_9ACAR Trist flower tick ( Amblyomma triste ) Arthropoda ------DVGKPPRAAKVNFPLL--VQVHC-A--DDLERKGPWEQIAVDRRRFQSRIDSVERVLAPVLSPDHRRNVY-- 50 tr|A0A182ILC7|A0A182ILC7_9DIPT Anopheles atroparvus Arthropoda -------GFEEKKVRFNMKPVVHVMRAWDFA--YRQARKGDWEMAARDRERFRKRIDDLEPVLGPALQSNVRDKIY-- 51 tr|A0A2M3ZGE9|A0A2M3ZGE9_9DIPT Anopheles braziliensis Arthropods --------FEERKVRFNTKPVVH-----DFA--YRQARKGEWEMAARDRERFRKHIADLEPVLGPALQPALRERIYQ- 52 tr|A0A2M3Z3Q5|A0A2M3Z3Q5_9DIPT Anopheles brasiliensis Arthropoda -------GFEERKVRFNTKPVVHVMRAWDFA--YRQARKGEWEMAARDRERFRKHIADLEPVLGPALQPALRERIY-- 53 tr|A0A182JQ15|A0A182JQ15_9DIPT Anopheles christyi Arthropoda -------GFEEKKVRFNAKPVVHVMRAWDFA--YRQARKGDWEMAARDRERFRKRIADLEPVLGPALQPRIRDKVY-- 54 tr|A0A182MSM9|A0A182MSM9_9DIPT Anopheles culicifacies Arthropoda -------GFDEKKVRFNTKPVVHVMRVWDFA--YRQARKGDWETTARDSERFRKRIADLEPVLGPALQPALRDKIY-- 55 tr|W5JKI8|W5JKI8_ANODA Anopheles darlingi Arthropoda ----------ERKVRFNTKPVVHV-----FA--YRQARKGEWEMAARDRERFRKHVADLEPVLGPALQPALRERIYQ- 56 tr|A0A182NV32|A0A182NV32_9DIPT Anopheles dirus Arthropods ----------ERKVRFNLKPVVHV-----FA--YRQARKGDWEMAARDRERFRKRVADLEPVLGPALQSTLRDKIYT- 57 tr|A0A182P0K5|A0A182P0K5_9DIPT Anopheles epiroticus Arthropoda -------GFEEKKVRFNSKPVVHVMRAWDFA--YRQARKGDWETAARDRERFRKRIADLEPVLGAVLQPTLRDKIY-- 58 tr|A0A182QYU0|A0A182QYU0_9DIPT Anopheles farauti Arthropoda -------GYEEKKVRFNMKPVVHVMRAWDFA--YRQARKGDWEMAARDRERFRKRVADLEPVLGPALQSTLRDKIY-- 59 tr|A0A182RQF9|A0A182RQF9_ANOFN Anopheles funestus Arthropoda -------GFEEKKVRFNLKPVVHVMRAWDFA--YRQARKGDWEMAARDSERFRKRITELEPVLGPALQSALRDKIY-- 60 tr|Q7Q951|Q7Q951_ANOGA Anopheles gambiae Arthropods -------GFEEKKVRFNSKPVVHVMRAWDFA--YRQARKGEWEMAARDRERFRKRIAELEPLLGPALQPALRDKVY-- 61 tr|A0A2M4BJU3|A0A2M4BJU3_9DIPT Anopheles marajoara Arthropoda ----------ERKVRFNTKPVVHV------A--YRQARKGEWEMAARDRERFRKHIADLEPVLGPALQP--------- 62 tr|A0A182WMK0|A0A182WMK0_9DIPT Anopheles minimus Arthropoda -------GFDEKKVRFNSKPVVHIMRVWNFA--YRQARKGDWETTARDNERFQKRIAELEPVLTPALQPALRDKIY-- 63 tr|A0A182WS40|A0A182WS40_ANOQN Anopheles quadriannulatus Arthropoda --------FEEKKVRFNSKPVVH-----DFA--YRQARKGEWEMAARDRERFRKRIAELEPLLGPALQPALRDKVYS- 64 tr|A0A084VUD6|A0A084VUD6_ANOSI Anopheles sinensis Arthropoda -------DYRSYNVRFCQKPVIHVMRAWDFA--YRQARKGEWEMAARDRERFRKRIDDLEPVLGPALQPSVRDKIY-- 65 tr|A0A182YBW0|A0A182YBW0_ANOST Anopheles stephensi Arthropoda ----------EKKVRFNMQPVVHV-----FA--YRQARKGDWEMAARDRERFRKRVADLEPVLGPALQSTLRDKIYA- 66 tr|A0A2A3E3P4|A0A2A3E3P4_APICC Chinese honey bee ( Apis cerana cerana ) Arthropoda --------SPVQKVKFN------IMVQWDYA--YRAARKGPWEEMARDRERFRGRINCIERVLNPILTTQHR------ 67 tr|A0A088APR2|A0A088APR2_APIME Italian bee ( Apis mellifera ) Arthropoda -----RPLSPVQKVKFNLNPEIHIMVQWDYA--YRAARKGPWEEMARDRERFRGRINCIERVLNPILTTQHRTYIW-- 68 tr|A0A158NR84|A0A158NR84_ATTCE Atta cephalotes Arthropoda -------NTQTKKVSFDPTPVVH------YA--YRAARRGPWEEIARDNERFRGRIKSIAIVLDPILKSKH------- 69 tr|A0A195BFA1|A0A195BFA1_9HYME Colombian leafcutter ant ( Atta colombica ) Arthropoda ----------TKKVSFDPTPVVHVM-----A--YRAARRGPWEEIARDNERFRGRIKSIAIVLDPILKSKHRSQVWQ- 70 tr|A0A034WW97|A0A034WW97_BACDO Oriental fruit fly ( Bactrocera dorsalis ) Arthropoda -----KPPESSKRVRFNLNPEIHVMYAWSYA--YRSARKGHWESLARDRDRFRKRIENTSKFINPILTPEHRS----- 71 tr|A0A0K8V9E4|A0A0K8V9E4_BACLA Pepper fruit fly ( Bactrocera latifrons ) Arthropoda ---------SSKRVRFNLNPEIHVMYAWNFA--YRSARKGHWESLARDRDRFRKRIESTSKYINPILTPEHRS----- 72 tr|A0A0K8W450|A0A0K8W450_BACLA pepper fruit fly Arthropoda ---------SSKRVRFNLNPEIH------FA--YRSARKGHWESLARDRDRFRKRIESTSKYINPILTP--------- 73 tr|A0A2P8Z491|A0A2P8Z491_BLAGE German cockroach ( Blattella germanica ) Arthropoda ---------IPVKIGFSDEVEVHVMDKWVKE--YHEARIGIWMQLALDHARFQKRIKDLEPVLAPVLTQEHRD----- 74 tr|H9JNN9|H9JNN9_BOMMO House Silkworm ( Bombyx mori ) Arthropoda ------KDRPVKRVTFSDEPKVHVMRVWAFA--ARQARAGHWERYALDRERFKRRIADVEMAVSWVLKPQHRSRV--- 75 tr|E2A0E2|E2A0E2_CAMFO Florida Camponotus floridanus Arthropoda -------TTQTKKVSFAQTPIIHIMVTWNYA--YRAARKGQWEEMARDNERFKGRINSIAAVLDPILTR--------- 76 tr|W8BX39|W8BX39_CERCA Mediterranean fruit fly ( Ceratitis capitata ) Arthropoda -----SPIHKVKKVRFNLNPVVHTMYAWSFA--YKSARKGQWENFARDRDRFRRRIENTSKYLNPILTPEHRS----- 77 tr|A0A1B6DLP0|A0A1B6DLP0_9HEMI Clastoptera arizonana Arthropoda --------KINKKVTFAAEVKVHRMVVWDYA--YRMARTGPWEVYARDSARFMNRIGQIATVLNPILNSNHRQK---- 78 tr|U5EQS2|U5EQS2_9DIPT Appendicular midge ( Corethrella appendiculata ) Arthropoda --QQPDSGFEDKKVRFQLKPDVHVLRAWDFA--YRQARKGPWEEAARDRCRFSERIKRSSIILTPILNENHRDKIYS- 79 tr|A0A2J7QRK2|A0A2J7QRK2_9NEOP Drywood Termite ( Cryptotermes secundus ) Arthropoda --------KKPVKVHFACLMTVHPMVKWNHA--YHEARCGPWEQMARDRVRFMARIKQMEPVLAAVFSAEHRQCVWS- 80 tr|A0A1B6FV58|A0A1B6FV58_9HEMI Cuerna arida Arthropoda -------NPGAKKVRFAEGAQIHPMVTWDFA--YRSARKGPWETLALDSQRFKCRVARTEKILAPVLDPEHRAAVY-- 81 tr|A0A1B6FVF2|A0A1B6FVF2_9HEMI Field cicada Arthropoda -------NPGAKKVRFAEGAQIH-----DFA--YRSARKGPWETLALDSQRFKCRVARTEKILAPVLDPEHRAAVYR- 82 tr|B0WSX4|B0WSX4_CULQU Culex quinquefasciatus Arthropods --------YPTLTVRFNFKTQVHVMRVWDYA--YRQARKSEWEIAARDRAWFKRRIENTEPVLGPIFDRDLRERVY-- 83 tr|A0A1Q3F8E7|A0A1Q3F8E7_CULTA Culex tarsalis Arthropoda ------------TVRFNFKTQVHVMRVWDYA--YRQARSGSWQLAARDRAWFKRRIEKTEPILGPIFDRELRERVY-- 84 tr|A0A151IG91|A0A151IG91_9HYME Fungus Ant ( Cyphomyrmex costatus ) Arthropoda -------TTQTKKVSFDPTPVVHVMVTWNYA--YRAARRGPWEEIARDNERFKGRIKSIAIVLDPILKSKHRSQVW-- 85 tr|A0A212FG81|A0A212FG81_DANPL Black-veined Butterfly ( Danaus plexippus plexippus ) Arthropoda -------RQPKKRVRFSSQPKVHVMRVWAFA--ARQARAGHWERHALDRERFKRRIADVEMAISWVLKPQHRSRI--- 86 tr|A0A164LUI8|A0A164LUI8_9CRUS Daphnia magna Arthropoda -----RKKTPLKKVHFPQLIRV------RHA--HRAARRGDWERFARDADRFRERIDSTAAILTPILD---------- 87 tr|A0A0P5GPX7|A0A0P5GPX7_9CRUS Daphnia Arthropoda -----RKKTPLKKVHFPPLIRV------THA--HRAARRGDWERFARDADRFRERIDSTAAILTPILD---------- 88 tr|A0A0P5MHL2|A0A0P5MHL2_9CRUS Daphnia Arthropoda ------KKTPLKKVHFFPDIRVRPMLTWTHA--HRAARRGDWERFARDADRFRERIDSTAAILTPILDPLHRDRIR-- 89 tr|A0A0N8C389|A0A0N8C389_9CRUS Daphnia magna Arthropoda ---------PLKKVHFPPDHRV------THA--HRAARRGDWERFARDADRFREIDSTAAILTPILDPLHRDRIRQ- 90 tr|E9FSW1|E9FSW1_DAPPU Daphnia pulex Arthropoda --------SRSKQVHFPVDTRIRPMLTWTHA--HRAARKGDWERFARDADRFRERIDSTAAILAPILDPSHRDRM--- 91 tr|U4TY70|U4TY70_DENPD Central European mountain pine beetle ( Dendroctonus ponderosae ) Arthropoda -------VTPRK-VTFAPDCTVHRMIKWSFA--YEAARKGHWETYARDRCRFKDRILRVERNIKLVLDPKHRAKIYS- 92 tr|N6TSQ7|N6TSQ7_DENPD Dendroctonus serrata Arthropoda -------VTPRK-VTFANLCTVH-----YA--YEAARKGHWETYARDRCRFKDRILRVERNIKLVLDPK-------- 93 tr|B3MCP4|B3MCP4_DROAN Drosophila ananassae Arthropoda --------SAAKKVRFNLKPEVHVMHVWDFA--YRTARKSEWQLIARDRDRFEKRINRVAPILNPILSPNHRDSV--- 94 tr|A0A0M3QVM0|A0A0M3QVM0_DROBS Drosophila busckii Arthropoda --------PLQKKVRFNLKPEVHKMHTWDFA--YRAARKGMWQEVARDRERFKQRINRLEPILNIICSANHREKV--- 95 tr|B3NQ42|B3NQ42_DROER Drosophila erecta Arthropoda -------SIPVKKVRFNIKPEVH------YA--YRAARKSEWQVMARDRDRFQQRIRRISPILNAVLTSV-------- 96 tr|A0A1W4ULZ2|A0A1W4ULZ2_DROFC Drosophila ficusphila Arthropoda --------TTAKKVRFNMKPEVHVMLAWDYA--YRAARKSEWQVMARDRARFQQRIKRISPILNAVLTPIHRESV--- 97 tr|B4J663|B4J663_DROGR Drosophila grimshawi Arthropoda --------ELRKKVHFNLSPEVHVMHAWNFA--YRAARKGGWQQFARDRDRFSQRIKRVAPIIDIVLSPSHRDRV--- 98 tr|B4KLY0|B4KLY0_DROMO Drosophila mojavensis Arthropoda --------EHRKRVRFDLKPKVHVMHTWDYA--YRAARKGGWQQIALDRERFQQRINRIAPTLNIILTSNHRDKI--- 99 tr|B4GCG6|B4GCG6_DROPE Drosophila persimilis Arthropods --------TTNKKVRFNLKPEVHVMLAWDFA--YRAARKSEWQVMARDRFRFQQRIHRVAPILNPILTPNHREQV--- 100 tr|Q290B9|Q290B9_DROPS Drosophila pseudoobscura pseudoobscura Arthropoda ----------NKKVRFNLKPEVH-----DFA--YRAARKSEWQVMARDRFRFQQRIHRVAPILNPILTPNHREQVYK- 101 tr|B4QBG0|B4QBG0_DROSI Drosophila simulans Arthropoda -------SAPVKKVRFNMKPEVH------YA--YRAARKSEWQVMARDRDRFQQRIRRISPILNAVLTPV-------- 102 tr|B4LP12|B4LP12_DROVI Drosophila virilis Arthropoda --------EVQKRVRFNLKPKVHVMHAWDFA--YRAARKGDWQEVARDRDRFQQRINRIAPILNNVLSSNHREKV--- 103 tr|B4MRL4|B4MRL4_DROWI Drosophila willistoni Arthropoda --------KSPKKVRFNLVPDVHVMRTWNFA--YRAARKGDWQVYSRDRDRFQQRINRVAPILSTVLSSNHREKV--- 104 tr|B4PAB9|B4PAB9_DROYA Drosophila yakuba Arthropoda --------TPVKKVRFNMKPEVHVMLAWDYA--YRAARKSEWQVMARDRDRFQQRIRRISPILNAVLTPVHRERV--- 105 tr|A0A154PEC5|A0A154PEC5_9HYME Dufourea novaeangliae Arthropoda --------PPTQKVKFNLNPTVHIMVQWDYA--YRAARKGPWEEMARDRERFKGRINCIERVLNPILTTQHRTHMWQ- 106 tr|A0A0C9RD89|A0A0C9RD89_9HYME Alishan leafminer braconid wasp ( Fopius arisanus ) Arthropods ------PPESSKRVRFNLNPEIH------YA--YRSARKGHWESLARDRDRFRKRIENTSKFINPILTP--------- 107 tr|A0A0C9R4Y5|A0A0C9R4Y5_9HYME Alishan leafminer braconid wasp Arthropoda -----EEDTTEQKVRFNLEPTIHTMIQWNYA--YRAARRGPWEEMARDRERFRVRINCIGRVLEPILTAKHRECIW-- 108 tr|A0A1A9X5T0|A0A1A9X5T0_9MUSC Glossina brevipalpis Arthropoda ------SSDKPKKVRFDTKPVVHVMHTWNYA--YRAARKSEWGILINNRAHFKSRIQRVANELNPILSIEHRQKIY-- 109 tr|A0A1B0GC50|A0A1B0GC50_GLOMM Glossina morsitans morsitans Arthropoda ----------PKKVRFDTKPIVHVM-----A--YRAARKSEWQIILQDRAHFKSRIQRVANELNPILSIEHRQKIYE- 110 tr|A0A1A9ZZH5|A0A1A9ZZH5_GLOPL Glossina pallidipes Arthropoda ------SSEKPKKVRFDTKPIVHVMHAWDYA--YRAARKSEWQIILQDRAHFKSRIQRVANELPILSIEHRQKIY-- 111 tr|A0A1B6MLH5|A0A1B6MLH5_9HEMI Leafhopper ( Graphocephala atropunctata ) Arthropoda -------NPRTKKVRFAKGTQIHRMVTWDFA--YRSARKGPWETLALDSQRFKSRVAGTEEILAPILDPQHRAAVY-- 112 tr|A0A0L7QQH5|A0A0L7QQH5_9HYME Southeastern blueberry bee ( Habropoda laboriosa ) Arthropoda -----ESVPPVQKVKFNLNPVVHVMMHWDYA--YRAARKGPWEEMARDRERFRGRINCIERVLNPILTTQHRTHIW-- 113 tr|E2BRL2|E2BRL2_HARSA Indian springtail ( Harpegnathos saltator ) Arthropoda ---------------------VHIMVQWDYA--YRAARKGPWEEMARDRERFKRRINCIAAVLDPILASQHRAHVW-- 114 tr|A0A2W1BIE7|A0A2W1BIE7_HELAM Tomato armyworm ( Helicoverpa armigera ) Arthropoda ----------PKRVQFS--PKVH---VLAFA--ARQARAGHWERYALDRDRFKRRIADVEVAVSWVLRPQHRSRIM-- 115 tr|A0A2A4JAH5|A0A2A4JAH5_HELVI American tobacco leaf moth ( Heliothis virescens ) Arthropoda ------KGLPPKRVQFSSAPKVHVLRVWAFA--ARQARAGHWERYALDRDRFKRRIADVEVAVSWVLRPQHRSRIM-- 116 tr|A0A1B6IL55|A0A1B6IL55_9HEMI Homalodisca liturata Arthropoda ----------TKKVRFAEGAQIH-----DFA--YRSARKGPWETLALDSERFKCRVARTEKILAPVLDPEHRATVY-- 117 tr|A0A131XW59|A0A131XW59_IXORI Ixodes ricinus Arthropoda ------DEQAQHRISKVNFGTF--LEVYD-A--DDVDRRGPWEQMAIDRKRFQVRVVSTEAVLAPVFSAEHRQRTF-- 118 tr|B7P0Y0|B7P0Y0_IXOSC Ixodes scapularis Arthropoda ------DEQAQHRISKVNFGTF--MQVHD-A--DDVDRRGPWEQMAIDRKRFQTRVASTEAVLAPVFSAEHRQRTF-- 119 tr|A0A0L0BQF9|A0A0L0BQF9_LUCCU Lucilia cuprina Arthropoda ---------KKKKVRFNTKPTVHVMHTWNYA--YRAARKGEWEMYARDRERFKLRIQRAASTLNPILEREHRQK---- 120 tr|A0A1B0CCY5|A0A1B0CCY5_LUTLO Lutzomyia longipalpis Arthropods ------SGFEERKVRFNLKPEVHVIRAWEFA--YRQARKGNWDQVARDRDRFEKRILSLGRVLSPILEQNHREKVY-- 121 tr|A0A0K8SEF5|A0A0K8SEF5_LYGHE Lygus hesperus Arthropoda MPLVKGNGYETGVDPIVKIVGGFVGSKSQAGSSNHRGREFSLWDVIFDKIIESMEQPKQAARKWGDVGGRKVPRIEGFAMIGMRKLSVNDSGCFRPKWSSAPQEYGESECRNQFPELGHGQDCDIGKSRTTDDTPETRVNDFVVKSTQDCFTPDRTDCSSNSSDSTELTSPSEQPFELTPPPSVETCGTLHHPHDQSPVREVCPAEPTEKHPQ VVRSSSSSFQVPCQRLLSESEDDSIPSDVLSSSVESHIDTFPPHKRVQRSISECSADSDDSFIVFESTEDEPVIFDESDDDDDGSTDEDLDEIDNCVLIPHNCDFSAPQQYLSRLEEANLKWNKTYGSVPTKSKSNKKCASKKVKFAEGKDLAKVKRLVAWDFAHRAARVGPWEMYYRDSQRFKSRISSLSSVISPVLAQSHRKKIYTQRFQT 122 tr|A0A146LKA2|A0A146LKA2_LYGHE Lygus bug Arthropods ----SNKKCASKKVKFA------RLVAWDFA--HRAARVGPWEMYYRDSQRFKSRISSLSSVISPVLA--HRKKIYT- 123 tr|A0A146L9L8|A0A146L9L8_LYGHE Lygus bug Arthropoda --------CASKKVKFAEGAKVKRLVAWDFA--HRAARVGPWEMYYRDSQRFKSRISSLSSVISPVLAQSHRKKIYT- 124 tr|A0A0A9Z6A2|A0A0A9Z6A2_LYGHE Lygus bug Arthropoda ----------SKKVKFAEGKDL-----WDFA--HRAARVGPWEMYYRDSQRFKSRISSLSSVISPVLAQSHRKKIY-- 125 tr|A0A0M9A061|A0A0M9A061_9HYME Four-banded stingless bee ( Melipona quadrifasciata ) Arthropoda -----KLVAPIREVKFNLNPVVHLMVQWDYA--YRAARKGPWEEMARDRERFRGRINCIERVLNPILTVQHRIHIW-- 126 tr|A0A1I8MZQ9|A0A1I8MZQ9_MUSDO House fly ( Musca domestica ) Arthropoda -------CDKKKKVRFNTKPVVHVMHTWNYA--YRAARKGNWEMYARDRERFKMRIDRTAHVLNRILEPEHRQKIY-- 127 tr|T1PDC8|T1PDC8_MUSDO House Fly Arthropoda ----------KKKVRFNTKPVVH-----YA--YRAARKGNWEMYARDRERFKMRIDRTAHVLNRILE---------- 128 tr|K7JKC1|K7JKC1_NASVI Nasonia vitripennis Arthropoda -------NGNKKCVRFKEEPTIHLMYTWRFA--HHQVRRGKWEEAARDRERFRRRIVQTNEIIMPVLLKK-------- 129 tr|K7JVD2|K7JVD2_NASVI Blowfly pupa aggregator wasp Arthropoda ----QQQNGKEKCVRFKEEPTIHF------A--HHQARCGKWEEAARDRERFRRRIVQTNEIIMPVLLKKLK------ 130 tr|K7JQS0|K7JQS0_NASVI Chrysophora spp. Arthropoda ------QNGKEKCVRFKEEPTIHLMYTWRFA--HHQARCGKWEEAARDRERFRRRIVQTNEIIMPVLLKK-------- 131 tr|K7JET5|K7JET5_NASVI Chrysophora spp. Arthropoda ----------EKCVRFKEEPTIHLM-----A--HHQARCGKWEEAARDRERFRRRIVQTNEIIMPVLLK--------- 132 tr|K7JZ07|K7JZ07_NASVI Chrysophora spp. Arthropoda ----------KKRVHFQEKPTDHLMCGWTYA--HKNARCGEWEQVARDQERFKLRIERMGKIMVPTLL---------- 133 tr|K7JSI1|K7JSI1_NASVI Blowfly pupa aggregator wasp Arthropoda ---DNNSSKQNKNLRFSDKVLIHNMCTWTFA--YQSARRGEWMQAACDRERFRMRIERTGVIINPVLLK--------- 134 tr|K7JHK7|K7JHK7_NASVI Chrysophora spp. Arthropoda -----VHSIQPTKVKFDLNPTVHRMIKWNFA--YRAARPGPWEQIARDRERFNSRIKCIGRVLNPVLAKDHRDLVY-- 135 tr|A0A2R7W1J6|A0A2R7W1J6_ONCFA Oncopeltus fasciatus Arthropoda -----------KMVKWKELVKIHPLVVWDFA--SRAARIGPWEKYRVDRDRFRNRIQNASSTLNPILDPAYRLKVF-- 136 tr|A0A026W8Q1|A0A026W8Q1_OOCBI Ooceraea biroi Arthropoda -----EELMEARKVKFNPTPIVHVMIKWNYA--YRAARKGPWEEMARDNERFRGRINSIAAVLNPILTNQHRLQIW-- 137 tr|A0A1D2MWJ5|A0A1D2MWJ5_ORCCI Springtail ( Orchesella cincta ) Arthropoda ------KKRKSKKVSFDEENLVSVIEFEDDA--SRRARETYWEVFARDRARFQDRIFRTGDILEPILLGAHRTKVF-- 138 tr|A0A1D2MYI7|A0A1D2MYI7_ORCCI Springtail Arthropoda ------KEKKIKSVRFKEENEVIEFED-DIE--SKNNRKLYWESVAADRFRFKDRIERLQEILSPVLNSSHRNVVY-- 139 tr|A0A293L4Z5|A0A293L4Z5_ORNER Wandering Ornithodoros erraticus Arthropoda ----------DSKVHFPADDIL--VAVRT-A--DDIDRKGTWEQAANDRLRFQCRIDNAEKILSPVLSIEHRQKVL-- 140 tr|A0A1Z5KVX7|A0A1Z5KVX7_ORNMO African hairy blunt-edged tick ( Ornithodoros moubata ) Arthropoda ----------DAKVHFPTDDIL--VDVQK-A--DDIDRKGPWEQAANDRLRFQCRIANTEKILSPVLSAEHRQKVL-- 141 tr|A0A224XUG3|A0A224XUG3_9HEMI Striped cone bug ( Panstrongylus lignarius ) Arthropoda ----------PKKVSFANLVQ-----VWDFA--LRAARKGPWEMLALDRSRFKTKIQQLSNIISPILEENHRKQIFH- 142 tr|A0A0N0PEA1|A0A0N0PEA1_PAPMA Golden Butterfly ( Papilio machaon ) Arthropoda ----------RKTVRFS--PKVH---VWAFA--ARQARAGHWERHALDRERFKRRIADVEMAVSWVLKPQHRARVM-- 143 tr|A0A194PSV8|A0A194PSV8_PAPXU Citrus butterfly ( Papilio xuthus ) Arthropoda -------KQPCKTVRFSPNPKVHVMRVWAFA--ARQARAGHWERHALDRERFKRRIADVEMAVSWVLKPQHRARV--- 144 tr|S4P6Q9|S4P6Q9_9NEOP Pararge aegeria Arthropoda -------KLPPKKVTFSTQPKVHVMRVWAFA--ARQARAGHWERHARDRERFKRRIADVEMAISWVFKRQHRTRV--- 145 tr|A0A2L2Z1T4|A0A2L2Z1T4_PARTP Greenhouse Parasteatoda tepidariorum Arthropoda -----DKPRDPVKVTFAPLVTVHCFQ----K--PLEENQPSYDTSALDRIRFQNRIEELSRLLTPILTLEHRTNI--- 146 tr|A0A1E1W419|A0A1E1W419_PECGO Pink bollworm ( Pectinophora gossypiella ) Arthropods -------GQPPKRVSFSEKPKVHVMRVWTFA--ARQARAGHWERHALDRERFKRRIADVDMAVSWVLKPQHRSRV--- 147 tr|A0A1B0DBA5|A0A1B0DBA5_PHLPP Phlebotomus papatasi Arthropods ---------ETREVRFNLKPEVHVIRAWDFA--YRTARKGHWEQVARDRERFGKRILSLGHVLSPILEKSHREK---- 148 tr|A0A1Y1MCV4|A0A1Y1MCV4_PHOPY Fireflies ( Photinus pyralis ) Arthropoda ------DEVPAKKVRFASECEVHPMIQWSYA--YQKARKGPWEQFALDRMRFRNRVANVQPILDRILEPSHRSKIY-- 149 tr|A0A1Y1M8V8|A0A1Y1M8V8_PHOPY fireflies Arthropoda ----------AKKVRFASEEEVHPM-----A--YQKARKGPWEQFALDRMRFRNRVANVQPILDRILEPSHRSKIYK- 150 tr|A0A131YLQ0|A0A131YLQ0_RHIAP Rhipicephalus appendiculatus Arthropoda ---------------KVSFPQL--VEVHN-A--DDMERKGPWEQIAVDRRRFQSRIASVESVLAPVLSREHRQKVYQ- 151 tr|A0A224YPI8|A0A224YPI8_9ACAR Zambezi Rhipicephalus tick ( Rhipicephalus zambeziensis ) Arthropods ------DVSGAPRSAKVSFPVL--VEVHS-A--DDMERKGPWEQIAVDRRRFQSRIASVESVLAPVLSHEHRQKVY-- 152 tr|T1HNP2|T1HNP2_RHOPR Rhodnius prolixus Arthropoda --SDLTSPTSPKKVSFANGVQIRKMVVWDFA--LRAARKGPWEMIALDRSRFKTKIQQLSNIISPILEQKHRRRIF-- 153 tr|E9IRX6|E9IRX6_SOLIN Fire ant ( Solenopsis invicta ) Arthropods ------LTAQTKKVKFNLQPVVHVMIAWDYA--YRAARKGHWEQFARDNERFRGRINSISIVLDPILKNQHRSQVWQ- 154 tr|A0A2H1VH82|A0A2H1VH82_SPOFR Fall Armyworm ( Spodoptera frugiperda ) Arthropoda -------------VHFSE--KVH---VWSFA--ARQARAGHWERFALDRDRFKRRIADVDMAVSWVLKPQHRSRVM-- 155 tr|A0A2H1VER0|A0A2H1VER0_SPOFR Fall Armyworm Arthropoda -------------VHFSEQPKVHVMRVWSFA--ARQARAGHWERYALDRDRFKRRIADVDMAVSWVLKPQHRSRV--- 156 tr|A0A087UMN6|A0A087UMN6_9ARAC Bump-headed spider ( Stegodyphus mimosarum ) Arthropoda -----DTPKLPSKVSFAPEADLVQIVPVEYC-----ERKGEWEMYASERLRFKRKIDDLEKVLSPCFSSSHRAKVF-- 157 tr|A0A1I8NP93|A0A1I8NP93_STOCA Fly ( Stomoxys calcitrans ) Arthropoda -------CDKKKKVRFNTKPEVHVMHAWNYA--YRAARKGHWEMYARDRERFKMRINRVANILNPILEPEHRLKVY-- 158 tr|T1JNK2|T1JNK2_STRMM Sea centipede ( Strigamia maritima ) Arthropoda ------SCSEKKKVHFASLVEVHSLAIED----DEDSRKGPWEEYARDRERFTKKIEDLDTIISPVLSATHRAKIY-- 159 tr|A0A0K8TR91|A0A0K8TR91_TABBR Polyphonic Fly ( Tabanus bromius ) Arthropoda --------KKEKKVRFNLKPDIHVMHSWDFA--YRAARKGPWEVIARDRCRFKQRIEKLGEIIGPILSDNHRKK---- 160 tr|A0A151IU56|A0A151IU56_9HYME Trachymyrmex cornetzi Arthropoda ---------QTKKVSFDPTPVVHV------A--YRAARRGPWEEIARDNERFRGRIKSIAIVLDPILKSKH------- 161 tr|A0A195FJL5|A0A195FJL5_9HYME Northern leafcutter ant ( Trachymyrmex septentrionalis ) Arthropods -------NTQSKKVSFDPTPVVHVMVTWNYA--YRAARRGPWEEIARDNERFKKRIKSIAIVLDPILKSKHRSQIW-- 162 tr|A0A151X539|A0A151X539_9HYME Trachymyrmex zeteki Arthropoda -------NTQIKKVSFDPTPVVHVMVTWNYA--YRAARRGPWEELARDNERFRGRIKSIAIVLDPILKSKHRSRVW-- 163 tr|A0A170YYB9|A0A170YYB9_TRIIF Triatoma infestans Arthropods -----TSPKSPKKVSFANLVQVRTMVVWDFA--LRAARKGPWEMLALDRSRFKTKIQQLSNIISPILEQNHRKQIF-- 164 tr|D2A5U3|D2A5U3_TRICA Tribolium castaneum Arthropoda -------LVPDKKVRFASECEVHPMIMWSFA--YQAARKGPWEEYARDRDRFNKRVRNTEAIIGHVFSDEHRSKIY-- 165 tr|D7GYC2|D7GYC2_TRICA Chimiao Grain Thief Arthropoda ----------LKQVRFNEDVTLYVLNV-------NEDRKGYW---VRDRMWFQKRIRDVEMILKPFLEL--------- 166 tr|A0A232F6X1|A0A232F6X1_9HYME Trichomalopsis sarcophagae Arthropoda ----------PTKVKFDLNPTVHRM-----A--YRAARPGPWEQIARDRERFNSRIKCIGRVLNPVLAKEHRDLVYN- 167 tr|A0A0A1XII6|A0A0A1XII6_ZEUCU Zeugodacus cucurbitae Arthropoda ---------NCKRVRF--NPEVH----WSYA--YKSARKGHWESLARDRDRFRKRIENTSKYINPILTPEHRS----- 168 tr|A0A1Q3DL60|A0A1Q3DL60_9VIRU Chionoecetes opilio bacilliform virus Retrovirus phylum (Artverviricota) -------GTNTQRITFGGVTATHHIIAWSFA--YQEARRGHWEQLARDRTRLRNRVTRVYNI---------------- 169 tr|A0A1S3IHP0|A0A1S3IHP0_LINUN Lingula unguis Brachiopoda ------SDTTPKRVHFPDEETVYEIM--QMD--ENECRKGPWEAYAVDRCRFQKRIAEVNDAIGYCFDPQHRQHIL-- 170 tr|A0A091MPX3|A0A091MPX3_9PASS Spurred Ibis ( Acanthisitta chloris ) Chordata --------PKRKKVTFLEQVTEYYIS-------SEEDRKGPWEEMARDSCRFQKRIQETEEAIGYCFTTEHRQRVF-- 171 tr|D2H8S4|D2H8S4_AILME Giant Panda ( Ailuropoda melanoleuca ) Chordata -----RAHTKRKKVTFLEEVTEYYIS-------DDEDRKGPWEEFARDGCRFQKRIQETEVAIGYCLTREHRERMF-- 172 tr|D2H7Y3|D2H7Y3_AILME giant panda Chordata WHPDPETPLKPRKVRFSEKVSVHLLVVWAGP--AQAARRGPWEQLARDRSRFARRIAQAQELLGPCLTPAARARAWA- 173 tr|A0A151NK26|A0A151NK26_ALLMI American alligator ( Alligator mississippiensis ) Chordata ---------AAKKVHFCPVVTVH-----DFA--SRTARCGPWEELARDRCRFRQRIEQAEVVLGPCLEPGHRAKAW-- 174 tr|A0A151MAW2|A0A151MAW2_ALLMI American alligator Chordata --------TKKKKVTFLDEVTEYYVS-------GEEDRKGPWEEFARDGCRFQKRIQETEDAIGHTSHSC-------- 175 tr|A0A1U7RBG9|A0A1U7RBG9_ALLSI Yangtze River Alligator ( Alligator sinensis ) Chordata ---DQEGNRAAKKVRFCPVVTVHPLIVWDFA--SRAARCGPWEELARDRCRFRQRIEQAEVVLGPCLEPGHRAKAW-- 176 tr|A0A1U7S3B9|A0A1U7S3B9_ALLSI Yangzijiang Crocodile Chordata -----HTHTKKKKVTFLDEVTEYYVS-------SEEDRKGPWEEFARDGCRFQKRIQETEDAIGYCLTVEHRQKI--- 177 tr|A0A0Q3TCT0|A0A0Q3TCT0_AMAAE Amazona aestiva Chordata -------PLTNWGGTFLDKVTEYYVS-------SEEDRKGPWEEIARDGCRFQKRIQETEEAIGYCFTADHRQRVF-- 178 tr|R0L938|R0L938_ANAPL Mallard Duck ( Anas platyrhynchos ) Chordata --------TTRKKVTFLEKVTEYYVS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIGYCFTTEHRQRVL-- 179 tr|A0A0E9TVB7|A0A0E9TVB7_ANGAN European eel ( Anguilla anguilla ) Chordata --------------------------------------------------MARDRDRFRRRVQTASDIISPFLLANHRARVWE- 180 tr|G1KGT3|G1KGT3_ANOCA Anolis carolinensis Chordata ---------TKKKVTFLEEVVEYYIS-------SEEDRKGPWEELARDGCRFLKRIQETENAIGYCLTMEHRQQIF-- 181 tr|A0A2K5CYS5|A0A2K5CYS5_AOTNA Night monkey ( Aotus nancymaae ) Chordata ----------ARKVHFSEKVTVHL------P--AHAARRGPWEQFARDRSRFARRIAQAQELLSPCLTP--------- 182 tr|A0A091NH62|A0A091NH62_APAVI Spotted-tailed African dart ( Apaloderma vittatum ) Chordata --------TKRKKVTFLEKVTEYYIS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIGYCFSTEHRQRVF-- 183 tr|A0A087QPR2|A0A087QPR2_APTFO Emperor Penguin ( Aptenodytes forsteri ) Chordata ---EKCRSTKRKKVTFLEKVT-YYIS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIGYCFTTE-------- 184 tr|W5JYE7|W5JYE7_ASTMX Mexican Fat Carp ( Astyanax mexicanus ) Chordata ---------LVKKVKFSPVVQVHKMRAWSFA--FQASRKGPWEEHARDRDRFQRRIMKTEQAIGYCFSLSHRQSLQH- 185 tr|W5KS24|W5KS24_ASTMX Mexican Fat Carp Chordata -------CVRLKKVTFVEEVEEFYAS-------SDEDRHGPWEEFARDRCRFQRRVQDVEETISYCLAPTFRLVIF-- 186 tr|W5K4R1|W5K4R1_ASTMX Mexican carp Chordata ---------GTKKVRFSEEVKVHNLVAWSFA--SREARDGSWMQLARDRERFKRRVERTEEVLSPCLTSQHRARVW-- 187 tr|A0A2I4BUP4|A0A2I4BUP4_9TELE Austrofundulus limnaeus Chordata ---------VQKKVQFSPVVQVHVMRTWPFA--RQATRKGHWEEMARDRDRFRRRIEETEQAVGRCLTPAHRQKMWA- 188 tr|A0A2I4C690|A0A2I4C690_9TELE Linnaeus pupfish Chordata -------CKNLKKVRFCDQVDEFFASCG--E--EEEDRRGPWEELARDRCRFQRRCEEVEQSIGFCLQPQHRHRVL-- 189 tr|A0A087V878|A0A087V878_BALRE Balearica regulorum gibbericeps Chordata ----------RKKVTFLEKVTEYYVS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIGYCFTAEHRQRVFN- 190 tr|A0A2P4T271|A0A2P4T271_BAMTH Grey-breasted Bamboo Partridge ( Bambusicola thoracicus ) Chordata -------HLTVCRVTFLEKVTEYYVS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIDDIDWLD--LRIFFI 191 tr|L8IKH8|L8IKH8_9CETA Wild Yak ( Bos mutus ) Chordata RHQDLEPLLKTRKVRFSEKVSIHPLVVWAGP--AQAARRGPWEQFARDRSRFARRIAQQADATPPFPK---------- 192 tr|E1BKX2|E1BKX2_BOVIN European cattle ( Bos taurus ) Chordata -SGETHTHIRRKKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCRFQKRIQETEEAIGYCLTFEHREKMFN- 193 tr|G3X6A2|G3X6A2_BOVIN european cattle Chordata RHQDLERLLKTRKVRFSEKVSIHPLVVWAGP--AQAARRGPWEQFARDRSRFARRIAQVQEELGPYLTPAARARAWA- 194 sp|Q2KI51|PR15A_BOVIN European Beef Chordata -----------RKVRFSEKVSIHPLVV-------QAARRGPWEQFARDRSRFARRIAQVQEELGPYLTPAARARA--- 195 tr|A0A226NIW5|A0A226NIW5_CALSU Scaled Quail ( Calipepla squamata ) Chordata ----------VCRVTFLEKVTEYYVS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIGYCFTPEHRQRVLN- 196 tr|A0A091IAC0|A0A091IAC0_CALAN Ann's Hummingbird ( Calypte anna ) Chordata ----------RKKVTFLEKVTEYYIS-------GEEDRKGPWEELARDGCRFQKRIQETEEAIGYCFTTEHRQKVFN- 197 tr|T0M6R5|T0M6R5_CAMFR Wild Camel ( Camelus ferus ) Chordata ---------KVQLVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCRFQKRIQETEDAIGD------------- 198 tr|S9XM19|S9XM19_CAMFR wild bactrian camel Chordata ----------ETPVRFSEKVSVHLLAVWAGP--ARAARRGPWEQFARDRSRFARRVARAQEELGPYLTPDARARAWA- 199 tr|E2RII3|E2RII3_CANLF Domestic dog ( Canis lupus familiaris ) Chordata ------ARIKRKKVTFLEEVTEYYIS-------EDEDRRGPWEQFARDACRFQKRIQETEDAIGYCLTFEHRERMF-- 200 tr|J9P332|J9P332_CANLF Domestic dog Chordata RHPDPETPLTARKVRFSEKVSVHLLAVWAGP--AQAARRGPWEQLARDRSRFARRIAQAQEQLGPCLTPAARARAWA- 201 tr|A0A091M8U5|A0A091M8U5_CARIC Red-legged Crane ( Cariama cristata ) Chordata ----------QKKVTFLEKVTEYYIS-------SEEDRKGPWEEIARDGCRFQKRIQETEEAIGYCFTTEHRQRVFN- 202 tr|H0V6Q0|H0V6Q0_CAVPO Guinea pig ( Cavia porcellus ) Chordata -----HTSIKRKKVTFVEEVTEYYIS-------GDEDRKGPWEEFARDACRFRKRIQETEDAIGYCLTFEYRERIF-- 203 tr|A0A286XWE4|A0A286XWE4_CAVPO guinea pig Chordata ----------RKKVTFVEEVTEYYIS-------GDEDRKGPWEEFARDACRFRKRIQETEDAIGYCLTFEYRERIFN- 204 tr|A0A2K5QP44|A0A2K5QP44_CEBCA Panamanian white-faced capuchin monkey ( Cebus capucinus imitator ) Chordata RDLDPETPLKARKVHFSEKVTVHLLAVWAGP--AHAARQGPWEQLARDRSRFARRIAQAQELLSPCLTPAARARAWA- 205 tr|A0A2K5LT72|A0A2K5LT72_CERAT White-capped sooty mangabey ( Cercocebus atys ) Chordata ----------RRKVTFLEEVTE-YIS-------GDEDRKGPWEEFARDGCRFQKRIQETEDAIGYCLTF--------- 206 tr|A0A212CRD6|A0A212CRD6_CEREH European red deer ( Cervus elaphus hippelaphus ) Chordata ----------RKKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCSFQKRIQETEEAIGYCLTFEHREKMFN- 207 tr|A0A093BFU8|A0A093BFU8_CHAPE Chimney Swift ( Chaetura pelagica ) Chordata --------HKRKKVTFLEKVTEYYIS-------GEEDRKGPWEELARDGCRFQKRIQETEEAIGYCFTPEHRQRVF-- 208 tr|A0A0A0AQW6|A0A0A0AQW6_CHAVO Two-collared Plover ( Charadrius vociferus ) Chordata ----KCRSTKRKKVTFLEKV-EYYIS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIGHCFTAE-------- 209 tr|M7BFJ5|M7BFJ5_CHEMY Green sea turtle ( Chelonia mydas ) Chordata -----HTSTKRKKVTFLEEVTEYYVS-------SEEDRKGPWEELARDGCRFQKRIQETEDAIGYCLTIEHRQRI--- 210 tr|A0A0D9S311|A0A0D9S311_CHLSB West African Green Monkey ( Chlorocebus sabaeus ) Chordata ----------ARKVHFSEKVTVHFPA--------QAARQGPWEQLARDRSRFARRIAQAQEELSPCLTP--------- 211 tr|A0A1W2WF74|A0A1W2WF74_CIOIN Ciona intestinalis Chordata --------KAPSKVRFMDKVVVHPMVAWSYA--YREARKGRWDSAARDRHRFGDRVRCVGDAISWCFNTSHRGKIYA- 212 tr|H2XQL9|H2XQL9_CIOIN Ciona Chordata -----------SKVRFMDKVVVHPM-----A--YREARKGRWDSAARDRHRFGDRVRCVGDAISWCFNTSHRGKIY-- 213 tr|A0A226PWT9|A0A226PWT9_COLVI Bobwhite Quail ( Colinus virginianus ) Chordata -------HLTVCRVTFLEKVTEYYVS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIGYCFTTEHRQRVL-- 214 tr|A0A2I0M025|A0A2I0M025_COLLI Wild pigeon ( Columba livia ) Chordata ----KCGSTKRKKVTFLEKVTE-YIS-------GEEDRKGPWEELARDGCRFQRRIQETEEAIGYCFTAE-------- 215 tr|A0A091EF33|A0A091EF33_CORBR Short-billed crow ( Corvus brachyrhynchos ) Chordata ----------RKKVTFLEKVTEYYIS-------SEEDRKGPWEEMARDGCRFQKRIQETEEAIGYCLTTEHRQRVFH- 216 tr|G3HQB0|G3HQB0_CRIGR Gray hamster ( Cricetulus griseus ) Chordata -SGETCTHNKRKKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCRFQKRIQETEDAIGYCLTFEHRERMFN- 217 tr|G3HZX4|G3HZX4_CRIGR Gray Hamster Chordata -----------RKVHFSENVTVHFLAV-------QAARPGPWEQLARDRSRFARRIAQAEEKLGPYLTPAFRARAW-- 218 sp|Q60465|PR15A_CRILO Long-tailed hamster ( Cricetulus longicaudatus ) Chordata QDQDPETPLRARKVHFSENVTVHFLAVWAGP--AQAARRGPWEQLARDRSRFARRIAQAEEKLGPYLTPAFRARAWA- 219 tr|A4IG88|A4IG88_DANRE Zebrafish ( Danio rerio ) Chordata ---------RLKKVKFSPVVQVHKMRAWSFA--LQASRKGPWEELARDRDRFRKRIIDTEKAIGYCFSLSHREKVWA- 220 tr|F1QPN1|F1QPN1_DANRE zebrafish Chordata ---------KLKKVKFSPVVQVHKMRAWSFA--LQASRKGPWEELARDRDRFRKRIGDTEKAIGYCFSLSNREKMRA- 221 tr|A0A2R8Q2M1|A0A2R8Q2M1_DANRE zebrafish Chordata ----------LKKVKFSPVVQVH-----SFA--LQASRKGPWEELARDRDRFRKRIGDTEKAIGYCFSLSNREKMR-- 222 tr|A3KNJ7|A3KNJ7_DANRE Zebrafish Chordata ---------SGKKVCFSERVCVRPLLVWSYA--SRSARDGSWLQMARDRERFRRRVQTLEPVLKPCLMPEHRARVW-- 223 tr|A0A2Y9PE36|A0A2Y9PE36_DELLE Beluga whale ( Delphinapterus leucas ) Chordata -----------RKVRFSEKVSVHLLAV-------QAARRGPWEQFARDRSRFARRIAQAQEELGPYLTPAARARA--- 224 tr|K9IMI7|K9IMI7_DESRO Vampire bat ( Desmodus rotundus ) Chordata ------THTKKKKVTFLEEVTEYYVS-------GDEDRKGPWEEFARDGCRFQKRIQETEDAIGYCLTFEHRERMF-- 225 tr|K9IKB2|K9IKB2_DESRO Vampire Bat Chordata ---------PSCEVTFLEEVTEYYVS-------GDEDRKGPWEEFARDGCRFQKRIQETEDAIGYCLTFEHRERMF-- 226 tr|K9IM54|K9IM54_DESRO vampire bat Chordata QHLDPETPQKTTKVRFSEKVSIHYLVVWAGP--AQAARQGPWEQFARDRSRFARRIAQVEEVLGPCFTLAARARAWA- 227 tr|A0A1S3F936|A0A1S3F936_DIPOR Dipodomys ordii Chordata -SGDRHTHLKRKKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCRFQKRIQETEVAIGYCLTFEHRERMFN- 228 tr|A0A1S3G2W4|A0A1S3G2W4_DIPOR Osborne's kangaroo Chordata QEPNDEIPLNTRKVRFSEKVTVHFLVVWAGA--AQAARQGPWEQFARDRSRFARRIARAQEELGPCLTPTWRARAWA- 229 tr|A0A093GY39|A0A093GY39_DRYPU Downy Woodpecker ( Dryobates pubescens ) Chordata --------TKRKKVTFQEQVTEYYIS-------SEEDRKGPWEELARDSCRFQKRIQETEEAIGHCLTPQHRHRVW-- 230 tr|A0A2Y9JYL5|A0A2Y9JYL5_ENHLU Alaska sea otter ( Enhydra lutris kenyoni ) Chordata ------TRIKRKKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCRFQKRIQETENVIGYCLTFEHRERMF-- 231 tr|A0A2Y9L316|A0A2Y9L316_ENHLU Alaska sea otter Chordata -----------RKVRFSENVSVHLLAV-------QAARRGPWEQLARDRSRFARRIAQAQEQLGPCLTPAARARA--- 232 tr|K7Z365|K7Z365_EPIAW Blue grouper ( Epinephelus awoara ) Chordata ---------SQKRVRFSPLVQVHVMRTWQFA--RQASRKGHWEEMARDDRDRFRRRVQETEQAIGHCFTQQHRER---- 233 tr|F6PQN6|F6PQN6_HORSE Domestic horse ( Equus caballus ) Chordata RDLEPETPLKARKVCFSEKVTIHFLVVWAGP--AQAARRGPWEQLARDRSRFAHRIARAQEELGPCLTPAARARAWA- 234 tr|A0A1S2ZR15|A0A1S2ZR15_ERIEU Western European hedgehog ( Erinaceus europaeus ) Chordata ------VHTKRKKVTFLEEITEYYIS-------DDEDRKGPWEEFARDACRFQKRIQETEDAIGYCLTFEHRERMF-- 235 tr|A0A1S3AJH2|A0A1S3AJH2_ERIEU Western European Hedgehog Chordata QLPDPGTPPQGRKVRFSEEVSVHILAVWAGP--AQAARRGPWEQMARDRSRFARRIAQAQEELGPCLTPEARARAWA- 236 tr|A0A2F0B7Y3|A0A2F0B7Y3_ESCRO Gray whale ( Eschrichtius robustus ) Chordata ------THIQRKKVCFFLLTCLIICIA-----------------------------SDVVKDISMTFLEA-------- 237 tr|A0A2F0AY78|A0A2F0AY78_ESCRO gray whale Chordata RHRDPEPPLKTRKVRFSEKVSVHLLAVWAGP--AQAARRGPWEQFARDRSRFARRIAQAQEELGPYLTPAARARAWA- 238 tr|A0A093JBH0|A0A093JBH0_EURHL Sun Bittern ( Eurypyga helias ) Chordata --------SKRKKVTFLEKVTEYYVS-------SEEDRKGPWEEFARDGCRFQKRIQETEEAIGHCFTAEHRQRVF-- 239 tr|M3WUP7|M3WUP7_FELCA Domestic cat ( Felis catus ) Chordata -SGQRHTRIKRKKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCRFQKRIQETEDAIGYCLTVEHRERMFN- 240 tr|M3XBR1|M3XBR1_FELCA House cat Chordata RHPDPEPPVKARKVRFSEKVSVHLLAVWAGP--ARAARRGPWEQLARDRSRFARRIAQAQEQLGPCLTPAARARAWA- 241 tr|U3JCE0|U3JCE0_FICAL White-collared Flycatcher ( Ficedula albicollis ) Chordata -----TANSKRKKVTFLEEVTEYLIS-------SEEDRRGPWEELARDGCRFQRRIQETEEAIGYCLRPEHRLRV--- 242 tr|A0A091CPQ2|A0A091CPQ2_FUKDA Damaran Mole ( Fukomys damarensis ) Chordata ----------RKKVTFLEEVTEYYVS-------GDEDRKGPWEEFARDACRFRKRIQETEDAIGHCLTFEHRERMFN- 243 tr|A0A147AAX9|A0A147AAX9_FUNHE Fundulus heteroclitus Chordata ---------RQKKVRFSPLVHVHVMRTWPFA--RQASRKGHWEELARDRDRFRRRIADTERAVGRCLSRPHRERMRA- 244 tr|A0A147AUZ6|A0A147AUZ6_FUNHE Bottom Chordata --------KLPKKVRFCDDVEEFFASGG--E--EEEDRRGPWEELARDRCRFLRRCQEVEQSIGFCLQPQHRRQVF-- 245 tr|F1NUR5|F1NUR5_CHICK Jungle fowl ( Gallus gallus ) Chordata -------RLTVCRVTFLEKVTEYYVS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIGYCFTTE---RVFHR 246 tr|G3NU49|G3NU49_GASAC Three-spined fish ( Gasterosteus aculeatus ) Chordata ---------TRKKVHFSPEVQVHVMRTWPFA--RQASRKGHWEEMARDRDRFRRRVWEAEQTIGRCLSPDHRDGMRA- 247 tr|G5BMM3|G5BMM3_HETGA Naked mole rat ( Heterocephalus glaber ) Chordata -----HTPIKGKKVTFLEEVTEYYVS-------GDEDRKGPWEEFARDACRFRKRIQETEDAIGYCLTFEHRERMF-- 248 tr|A0A0P6JG56|A0A0P6JG56_HETGA naked mole rat Chordata ----------GKKVTFLEEVTE-YVS------GDEDRKGPWEEFARDACRFRKRIQETEDAIGYCLTF--------- 249 sp|O75807|PR15A_HUMAN Homo sapiens Chordata HDPDPETPLKARKVRFSEKVTVHFLAVWAGP--AQAARQGPWEQLARDRSRFARRITQAQEELSPCLTPAARARAWA- 250 tr|C1JC98|C1JC98_HYPNO Squid ( Hypophthalmichthys nobilis ) Chordata ------------QVCFSDKVTVHPLVVWSFA--SRAARDGCWLQMARDRERFRRRVENIENVIKPCLTPEHRASVW-- 251 tr|A0A1A7YZB7|A0A1A7YZB7_9TELE Iconisemion striatum Chordata ---------TQKKVQFSPLVQVHVMRTWPFA--RQASRKGHWEELARDRDRFRRRVLDTERAVGHCFTQPHRERMRA- 252 tr|A0A2D0QSY1|A0A2D0QSY1_ICTPU Spotted Catfish ( Ictalurus punctatus ) Chordata ---------TVKRVKFSPVVQIHKMRAWSFA--LQACRKGPWEEHARDRDRFQRRILETEQAIGYCFMRSHRDKFFI- 253 tr|A0A2D0PHH9|A0A2D0PHH9_ICTPU channel catfish Chordata -------VVRLKKVTFIEEVEEFYAS-------SDEERHGPWEEYARDRCRFQRRVQEVEESISYCLSPSFRLNIF-- 254 tr|A0A2D0RC33|A0A2D0RC33_ICTPU Catfish Chordata -----STKERATKVRFSEEVTIHYIE-WDAA--NQAARDGSWMEMARDRDRFKRRVEQIGEIISPCLTAQHRAK---- 255 tr|I3NCX6|I3NCX6_ICTTR Thirteen-striped ground squirrel ( Ictidomys tridecemlineatus ) Chordata -----HMHIKRKKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCRFQKRIQETEDAIGYCLTFEHRERMF-- 256 tr|A0A0F8AW99|A0A0F8AW99_LARCR Large yellow croaker ( Larimichthys crocea ) Chordata ---------TQKKVRFSPLVQVHVMRTWPFA--RRASRKGHWEEMVRDRDRFRRRRIQETEQAICHCFTQEHRERIRA- 257 tr|A0A0F8AW45|A0A0F8AW45_LARCR Large yellow croaker Chordata -------CSTTKKVRFCDDVEEFFASGG--E--EDEDRHGPWEELARDRCRFLRRCQEVEQSIAFCLQPQHRRKVY-- 258 tr|M3XK44|M3XK44_LATCH Latimeria chalumnae Chordata ------RDVTPKKVLFLDEVTEYYLS-------DEEDRKGPWEAFARDRCRFKKRIQEIEEAVAYCLAPEHRRKV--- 259 tr|W5M566|W5M566_LEPOC Eye-spotted gar ( Lepisosteus oculatus ) Chordata ---------TEKKVSFSPVVQVHVMHAWSFA--LRAARRGPWEEMARDRARFRRRIEETEKAIGHCLSTAHREKILA- 260 tr|W5NND4|W5NND4_LEPOC Eye-spotted gar Chordata ------------QVRFSDTVVVRPLVVWAFA--SRAARSGCWQEMARDRDRFQRRVQQTSVAIEPCLQPEHRARVW-- 261 tr|A0A2U3XJE9|A0A2U3XJE9_LEPWE Weddell's seal ( Leptonychotes weddellii ) Chordata -SGERHAHIKRKKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCRFQKRIQETEDAIGYCLTFEHRERMFN- 262 tr|A0A2U3YVJ2|A0A2U3YVJ2_LEPWE Wedder seal Chordata -----------RKVHFSEKVSVHLLAV-------QAARRGPWEQLARDRSRFAWRIAQAQEQLGPCLTPAARARA--- 263 tr|A0A091PQ00|A0A091PQ00_LEPDC Cuckoo ( Leptosomus discolor ) Chordata --------TKQKKVTFLEEVTEYYVS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIGYCFTTEHRQRVF-- 264 tr|A0A2I0THG7|A0A2I0THG7_LIMLA Spot-tailed Godwit ( Limosa lapponica baueri ) Chordata ------------RVTFLEKVTE-YIS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIGYCFTRE-------- 265 tr|A0A218UHA0|A0A218UHA0_9PASE Ten-sister bird ( Lonchura striata domestica ) Chordata -----TASSKRKKVTFHEEVTEYYIS-------SEEDRKGPWEEMARDGCRFQKRIQETEEAIGYCLSPEHRLRV--- 266 tr|G3UEH1|G3UEH1_LOXAF African savanna elephant ( Loxodonta africana ) Chordata QNPDLGIPLKATKVHFSEKVSVHYLAV-------------PWEQLARDRSRFARRIAGVQEELEPCLTPAARARAWA- 267 tr|G3UNC1|G3UNC1_LOXAF African savanna elephant Chordata ---------KATKVHFSEKVSVHYLA--------------PWEQLARDRSRFARRIAGVQEELEPCLTPAARARAW-- 268 tr|A0A2K5VWQ4|A0A2K5VWQ4_MACFA Long-tailed macaque ( Macaca fascicularis ) Chordata ---------KARKVHFSEKVTVHF------P--AQAARQGPWEQLARDRSRFARRIALAQEELSPCLTP--------- 269 tr|A0A1D5R262|A0A1D5R262_MACMU Rhesus macaque ( Macaca mulatta ) Chordata RDLDPEIPLKARKVHFSEKVTVHFLAVWAGP--AQAARQGPWEQLARDRSRFARRIALAQEELSPCLTPAARARAWA- 270 tr|A0A093PIN3|A0A093PIN3_9PASS Golden-collared Flycatcher ( Manacus vitellinus ) Chordata --------SKRKKVTFLEKVTEYYIS-------SEEDRKGPWEEMARDGCRFQKRIQETEEAIGYCFTSEHRQRVF-- 271 tr|A0A2K5YBX8|A0A2K5YBX8_MANLE Drill Baboon ( Mandrillus leucophaeus ) Chordata -----QTHVKRRKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCRFQKRIQETEDAIGYCLTFEHRERMF-- 272 tr|A0A091QM69|A0A091QM69_MERNU Red Bee-eater ( Merops nubicus ) Chordata ---ERCRRTKRKKVTFLEKVT-YYIS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIGHCFTTE-------- 273 tr|A0A1U8CJL6|A0A1U8CJL6_MESAU Golden Hamster ( Mesocricetus auratus ) Chordata QDQDPDTPPRARKVRFSEKVAVHFLAVWAGP--AQAARRGPWEQLARDRSRFARRIAQAEEKLGPCLTPAFRARAWA- 274 tr|U3FZ67|U3FZ67_MICFL Coral snake ( Micrurus fulvius ) Chordata -----EKCKKKKKVTFLEEVTEYYVS-------IEEDRKGPWEELARDGCRFHKRIQETEDAIGYCLTVEHRQRI--- 275 tr|A0A2H6MW52|A0A2H6MW52_MICLE Micrurus lemniscatus carvalhoi Chordata ----REKCKKKKKVTFLEEVTE-YVS-------IEEDRKGPWEELARDGCRFHKRIQETEDAIGYCLTVE-------- 276 tr|A0A2D4HKZ6|A0A2D4HKZ6_MICLE South American Coral Snake ( Micrurus lemniscatus lemniscatus ) Chordata --------QVKKKVRFSPVVTVH-----DYA--SRAARRGPWEEMARDRCRFHRRITQMAAILEPCFAEDHRDKVWK- 277 tr|A0A2D4IKG3|A0A2D4IKG3_MICLE South American Coral Snake Chordata ---------KKKKVTFLEEVTE-YVS-------IEEDRKGPWEELARDGCRFHKRIQETEDAIGYCLTVE-------- 278 tr|A0A2D4Q362|A0A2D4Q362_MICSU Suriname coral snake ( Micrurus surinamensis ) Chordata ----------KMKVRFSPVVTVHSLVVWDYA--SRAARRGPWEEMARDRCRFHRRITQMAAILEPCFAEDHRDKVW-- 279 tr|A0A2D4NWJ1|A0A2D4NWJ1_MICSU Suriname Coral Snake Chordata -----EKCKKKKKVTFLEEVTY-YVS-------IEEDRKGPWEELARDGCRFHKRIQETEDAIGYCLTVE-------- 280 tr|F7BX77|F7BX77_MONDO Negative rat ( Monodelphis domestica ) Chordata -----HTATRRKKVTFLEEVTEYYIS-------SDEDRKGPWEELARDGCRFQKRIQETEDAIGYCLTFEHRQKIF-- 281 sp|Q8BFW3|PR15B_MOUSE House mouse ( Mus musculus ) Chordata ----------RKKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCRFQKRIQETEVAIGYCLAFEHREKMFN- 282 tr|A0A1B0GRX4|A0A1B0GRX4_MOUSE Mus musculus Chordata ---------GPRDSSKSSE----------GP--AQAARRGPWEQFARDRSRFARRIAQAEEKLGPYLTPDSRARAWA- 283 sp|P17564|PR15A_MOUSE House mouse Chordata ----------ARKVHFAEKVTVHFPA--------QAARRGPWEQFARDRSRFARRIAQAEEKLGPYLTP--------- 284 tr|B2RRL7|B2RRL7_MOUSE House mouse Chordata -----------RKVHFAEKVTVHFLAV-------QAARRGPWEQFARDRSRFARRIAQAEEKLGPYLTPDSRARA--- 285 tr|G9KI60|G9KI60_MUSPF Ferret ( Mustela putorius furo ) Chordata ------KRIKKKKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDAFRFQKRIRETENAIGYCLTFEHRERMF-- 286 tr|M3YJU7|M3YJU7_MUSPF Ferrets Chordata ----------KKKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDAFRFQKRIRETENAIGYCLTFEHRERMFN- 287 tr|S7Q597|S7Q597_MYOBR Myotis brandtii Chordata QHPDPETPEKARKVHFSEKVSIHFLIVWAGP--AQAARRGPWEQFARDRSRFARRIAQAEEKLGPCLTPAARARAWA- 288 tr|L5LGR6|L5LGR6_MYODS David's Myotis davidii Chordata -----------RKVHFSEKVSIHFLIV-------QAARRGPWEQFARDRSRFARRIAQAEEKLGPCLTPAARARA--- 289 tr|G1P3R3|G1P3R3_MYOLU Light-avoiding myotis bat ( Myotis lucifugus ) Chordata ----------ARKVHFSEKVSIHFPA--------QAARRGPWEQFARDRSRFARRIAQAEEKLGPCLTP--------- 290 tr|A0A1A6G2E1|A0A1A6G2E1_NEOLE Desert wood rat ( Neotoma lepida ) Chordata RDQDPETPLRARKVHFSEKVTVHFLAVWAGP--AQAARRGPWEQVARDRSRFARRIAQAEEELGPYLTPAFRARAWA- 291 tr|A0A091V327|A0A091V327_NIPNI Crested Ibis ( Nipponia nippon ) Chordata ----------VCRVTFLEKVTEYYIS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIGYCFTTEHRQRVFN- 292 tr|A0A2I3GRM3|A0A2I3GRM3_NOMLE White-cheeked Gibbon ( Nomascus leucogenys ) Chordata -----------RQVHFSEKVTVHFLAV-------QAARQGPWEQLARDRSRFARRIAQAQEELSPCLTPAARARA--- 293 tr|A0A1A8D4T8|A0A1A8D4T8_9TELE Nothobranchius kadleci Chordata -----KASSTQKKVHFSPLVQVH-----PFA--RQASRKGHWEELARDRDRFRRRVLDTERAIGNCFTQSHREKMR-- 294 tr|A0A1A8GYN8|A0A1A8GYN8_9TELE Nothobranchius korthausae Chordata -----KARSTQKKVQFSPRVQVH------FA--RQASRKGHWEELARDRDDRFRRRVLDTERAIGNCFTQ--------- 295 tr|A0A1A8G5F4|A0A1A8G5F4_9TELE False gillfish Chordata ----------SKKVQFCDDVEEFFAS-------EEEDRRGPWEELARDRCRFLRRCEEVEKNISYCLQPQHRRQVL-- 296 tr|A0A1A8HUB8|A0A1A8HUB8_NOTKU Nothobranchius kuhntae Chordata ---------TQKKVQFSPLVQVHVMRTWPFA--RQASRKGHWEELARDRDDRFRRRVLDTERAIGNCFTQSHREKMRA- 297 tr|A0A1A8MZ04|A0A1A8MZ04_9TELE Nothobranchius pienaari Chordata ----------QKKVHFSPLVQVH------VA--RQASRKGHWEELARDRDDRFRRRVLDTERAIGNCFTQS-------- 298 tr|A0A1A8RGY5|A0A1A8RGY5_9TELE Nothobranchius rachovii Chordata -----KASSTRKKVHFSPLVQVH------FA--RQASRKGHWEELARDRDRFRRRVLDTERAIGNCFTQ--------- 299 tr|A0A1A8RDN9|A0A1A8RDN9_9TELE Pseudoglossus lagomorpha Chordata -------GRCSKKVRFCDDVEEFFASCG--E--EEEDRRGPWEELARDRCRFLRRCEEVEKNISYCLQPHHRRQVL-- 300 tr|A0A2U3ZVT9|A0A2U3ZVT9_ODORO Pacific walrus ( Odobenus rosmarus divergens ) Chordata ----------ARKVHFSEKVSVHL------P--AQAARRGPWEQLARDRSRFARRIAQAQEQLGPCLTP--------- 301 tr|A0A060VZ96|A0A060VZ96_ONCMY Rainbow trout ( Oncorhynchus mykiss ) Chordata ----------LKKVRFSPLVQVH-----PFA--RQMSRKGPWEELARDRDRFRKRIQETEQAIGYCFSQSHRDTIR-- 302 tr|A0A060WIS8|A0A060WIS8_ONCMY rainbow trout Chordata ---------PLKKVRFSPLVQVHVMRTWPFA--RHVSRKGPWEELARDRGRFRRRIQEAEQTIDYCFSQSHREKIWA- 303 tr|A0A060XUB5|A0A060XUB5_ONCMY Rainbow Trout Chordata -------CVTVKKVCFCDTVEEFYAS----E--DDEDRCGPWEELARDRARFLRRVQDVEDGIGYVLSTTFRVAVY-- 304 tr|V8P8I2|V8P8I2_OPHHA Spectacled Kingsnake ( Ophiophagus hannah ) Chordata ----------KKKVRFSPVVTVH-----SLA--SRAARRGPWEEMARDRFRFHRRITQMAAILEPCFTEDHRDKVWK- 305 tr|F7FB61|F7FB61_ORNAN Platypus ( Ornithorhynchus anatinus ) Chordata -----QVHTRRKKVTFLEEVTEYYIS-------SDEDRKGPWEELARDGCRFQKRIQETEDAIG-------------- 306 tr|K7EHP9|K7EHP9_ORNAN platypus Chordata -----ERSMAGRKVQFSSQVSVHLMAVWAGP--ARAARRGPWEQLARDRSRFSRRIAQAERELAPCLSPAARAAAWA- 307 tr|G1TKF7|G1TKF7_RABIT Rabbit ( Oryctolagus cuniculus ) Chordata ------AHSKRKKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCRFQKRIQETEDAIGYCLTLEHRQRVF-- 308 tr|G1TEF9|G1TEF9_RABIT Rabbit Chordata RGPDPETPL-ARKVRFSDKVTVHLLAVWAGP--AQAARKGPWEQFARDRSRFARRIARAQEELGPCLSPAARARAWA- 309 tr|H2L6W6|H2L6W6_ORYLA Killifish ( Oryzias latipes ) Chordata ----------SKQVRFSPQVQVHVMRTWPFA--RQASRKGLWEEMARDRDRFRRRIVETEQAIGYCFSPPHRQK---- 310 tr|H2M4Z9|H2M4Z9_ORYLA Killifish Chordata --------RSSKKVRFCDTVEEFFASCE-EE--GEEDRQGPWEQMARDRCRFLRRCQEVEQQIAYCLQPQHRLRVY-- 311 tr|H0WWF1|H0WWF1_OTOGA Small-eared baby monkey ( Otolemur garnettii ) Chordata WDPDPETPLRGRKVRFSEKVTVHLLAVWAGP--AQAARRGPWEQFARDRSRFQRRITQAQEELGPCLTPAARARAWT- 312 tr|W5NWY5|W5NWY5_SHEEP Sheep( Ovis aries ) Chordata ------THIRRKKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCRFQKRIQETEEAIGYCLTFEHREKMF-- 313 tr|W5PS00|W5PS00_SHEEP Sheep Chordata RHLDLEPLPKTRKVRFSEKVSVHPLVVWAGP--AQAARRGPWEQFARD--RFPRRTARAQGELGPSLTPAARARAWA- 314 tr|A0A2R8ZMT3|A0A2R8ZMT3_PANPA Bonobo ( Pan paniscus ) Chordata ---------KARKVHFSEKVTVHFLAA-------QAARQGPWEQLARDRSRFARRIAQAQEELNPCLTPAARARAW-- 315 tr|A0A1V4KX71|A0A1V4KX71_PATFA Spot-tailed pigeon ( Patagioenas fasciata monilis ) Chordata -----CRSTKRKKVTFLEKVTEYYIS-------SEEDRKGPWEELARDGCRFQRRIQETEEAIGYCFTAEHRQRV--- 316 tr|K7FEU8|K7FEU8_PELSI Chinese soft-shell turtle ( Pelodiscus sinensis ) Chordata -----HAKTKRKKVTFLEEVTEYYIS-------SEEDRKGPWEELARDGCRFQKRIQETEDAIGYCLTIEHRQTI--- 317 tr|A0A091TM09|A0A091TM09_PHALP White-tailed grebe ( Phaethon lepturus ) Chordata ----KRRSTKRKKVTFLEKV-EYYIS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIGYCFTTE-------- 318 tr|A0A2Y9F179|A0A2Y9F179_PHYCD Sperm whale ( Physeter catodon ) Chordata ---------NTRKVRFSEKVSVHLPA--------QAARRGPWEQFARDRSRFARRIAQAQEELGPYLTP--------- 319 tr|A0A094KNW3|A0A094KNW3_9AVES Fengtou ( Podiceps cristatus ) Chordata ----------RKKVTFLDKVTEYYIS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIGYCFTREHRQRVFN- 320 tr|A0A096MBK4|A0A096MBK4_POEFO Poecilia formosa Chordata ---------RQKKVQFSPLVQVHVMRTWPFA--RQASRKGHWEELARDRDRFRRRIADTEQAVGYCLTQPHREKMRA- 321 tr|A0A096M760|A0A096M760_POEFO Beautiful medaka Chordata -------NRSTKKVRFCDEVEEFFASC------SEEDRRGPWEELARDRCRFRRRCQEVEHSIGFCLAPAHRWRVY-- 322 tr|A0A0S7MCR7|A0A0S7MCR7_9TELE Poeciliopsis prolifica Chordata ---------RQKKVQFSPLVQVHVMRTWPFA--RQASRKGHWEELARD--------------------PPWP------ 323 tr|A0A2J8U7R8|A0A2J8U7R8_PONAB Sumatran orangutan ( Pongo abelii ) Chordata HDPDPETPLKARKVHFSEKVTVHFLAVWAGP--AQAARQGPWEQLARDRSRFARRIARAQKELSPCLTPAARARAWA- 324 tr|H2NZI7|H2NZI7_PONAB Sumatran Orangutan Chordata -----------RKVHFSEKVTVHFLAV-------QAARQGPWEQLARDRSRFARRIARAQKELSPCLTPAARARA--- 325 tr|A0A2K6G4D3|A0A2K6G4D3_PROCO Propithecus coquereli Chordata QDPDPETPLKARKVRFSEKVTVHFLYVWAGP--AQAARRGPWEQLARDRSRFARRIAQAQEELGPCLTPAARARAWA- 326 tr|A0A093CBD4|A0A093CBD4_9AVES Yellow-throated sand grouse ( Pterocles gutturalis ) Chordata ----------RKKVTFLEKVTEYYIS-------SEEDRKGPWEEFARDGCRFQKRIQETEEAIGYCFTPEHRQRVFN- 327 tr|L5L4K0|L5L4K0_PTEAL Central flying fox ( Pteropus alecto ) Chordata TFSESDGQSSDRWVRFSEKVSIHLLAVWAGP--AQAARRGPWEQFARDRSRFARRIAQAQEVLGPCLTPAARARAWA- 328 tr|B5DF35|B5DF35_RAT Brown rat ( Rattus norvegicus ) Chordata --------RASDQVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCRFQKRIQETEVAIGYCLTFEHRERVF-- 329 tr|D3ZP67|D3ZP67_RAT Rattus norvegicus Chordata -----YTHIKRKKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCRFQKRIQETEVAIGYCLTFEHRERVF-- 330 tr|A0A0G2JSW4|A0A0G2JSW4_RAT Rattus norvegicus Chordata RNQDPEIPLKGRKVHFSEKVTVHFLAVWAGP--AQAARRGPWEQFARDRSRFARRIAQAEEQLGPYLTPAFRARAWT- 331 tr|A0A2K6MMD3|A0A2K6MMD3_RHIBE Yunnan golden monkey ( Rhinopithecus bieti ) Chordata -SGGRHTHVKRKKVTFLEEVTEYYIS-------DDEDRKGPWEEFARDGCRFQKRIQETEDAIGYCLTFEHRERMFN- 332 tr|A0A2K6UW65|A0A2K6UW65_SAIBB Amazon squirrel monkey ( Saimiri boliviensis boliviensis ) Chordata ----------ARKVHFSEKVTVHL------P--AHAARQGPWEQLARDRSRFARRIAQTQELLSPCLTP--------- 333 tr|A0A2K6UWC3|A0A2K6UWC3_SAIBB Amazon squirrel monkey Chordata ---------KARKVHFSEKVTVHL------P--AHAARQGPWEQLARDRSRFARRIAQTQELLSPCLTP--------- 334 tr|A0A1S3KNK9|A0A1S3KNK9_SALSA Atlantic salmon ( Salmo salar ) Chordata ---------PLKKVRFSPLVQVHVMHTWPFA--RQMSRKGPWEELARDRDRFRKRIQETEQAIGYCFSQSHRDTIRA- 335 tr|A0A1S3KPD5|A0A1S3KPD5_SALSA atlantic salmon Chordata -----KFNHPLKKVRFSPLVQVH-----PFA--RQMSRKGPWEELARDRDRFRKRIQETEQAIGYCFSQSHRDTIR-- 336 tr|A0A1S3PDA1|A0A1S3PDA1_SALSA Atlantic salmon Chordata ---------PLKKVRFSLLVQVHVMRTWPFA--RQVSRKGPWEELARDRGRFRRRIQEAEQTIGYFFSQSHREKIWA- 337 tr|A0A1S3P6L6|A0A1S3P6L6_SALSA Atlantic salmon Chordata -------CVTVKKVGFRDEVDEFYAS----E--DDEDRRGPWEELARDRCRFLRRVQEVEESIGYVFSSTFRLTVC-- 338 tr|A0A1S3L8J1|A0A1S3L8J1_SALSA Atlantic salmon Chordata --------VTVKRVCFCDTVEEFYAS--------DEDRSGPWEELARDRARFLRRVQDVEDGIGYVLSTTFR------ 339 tr|G3VPS2|G3VPS2_SARHA Tasmanian devil ( Sarcophilus harrisii ) Chordata ---------KEGEVRFSSTVGLRLLVVWAGP--ARAARRGPWEQLARDRDRFARRIAQAEALLGPCLSPATRTRAWA- 340 tr|A0A1W5AQX9|A0A1W5AQX9_9TELE Asian dragon fish ( Scleropages formosus ) Chordata ----------LPKVRFSPVVEVH------VA--LQASRRGPWEEMARDRDRFKRRIAETEQAIGYCLLPS-------- 341 tr|A0A1W5ARN7|A0A1W5ARN7_9TELE Asian arowana Chordata ---------ILPKVRFSPVVEVHVMRSWSFA--LQASRRGPWEEMARDRDRFKRRIAETEQAIGYCLLPSHREKILA- 342 tr|A0A0P7UI69|A0A0P7UI69_9TELE Asian Dragon Fish Chordata ----------LPKVRFSPVVEVH-----SFA--LQATRRGPWEEMARDRDRFKRRIAETEQAIGYCLLPSHREKIL-- 343 tr|A0A0P7USJ6|A0A0P7USJ6_9TELE Asian arowana Chordata -------CAKLKKVRFVEEVEEFYAS-------SDEDRRGPWEEFARDRCRFLRRVQETEEVISYCLAPTFRLLIF-- 344 tr|A0A1W5AE17|A0A1W5AE17_9TELE Asian arowana Chordata ---------KLKKVRFVEEVEEFYA--------SDEDRRGPWEEFARDRCRFLRRVQETEEVISYCLAPTFRLL---- 345 tr|A0A1W4YP86|A0A1W4YP86_9TELE Asian arowana Chordata ----------SRKVRFSNVVKVPP-----FA--SRAARDGCWQEMARDRARFKRRVDAVSEIISPCLLPEHRAMVWE- 346 tr|A0A0P7VNB0|A0A0P7VNB0_9TELE Asian Dragon Fish Chordata ------AVVSSRKVRFSNVVKVHPLVTWSFA--SRAARDGSWQEMARDRARFKRRVDAVSEIISPCLLPEHRAMVWE- 347 tr|A0A1W4YP94|A0A1W4YP94_9TELE Asian Dragon Fish Chordata ------MMVSSRKVRFSNVVKVHPLVTWSFA--SRAARDGSWQEIARDRARFKRRVDAVNEIISPCLLPEHRAMVWE- 348 tr|A0A1W5ANM8|A0A1W5ANM8_9TELE Asian Dragon Fish Chordata -------KQGVKKVRFSDAVKVHPMVVWQFA--SRVARDGSWLEMARDRERFRRRVRAASDVISPCLLPQHRARVWR- 349 tr|A0A1W5APZ6|A0A1W5APZ6_9TELE Asian arowana Chordata -RTRQNQKQGVKKVRFSDAVKVH------FA--SRVARDGSWLEMARDRERFRRRVRAASDVISPCLLPQH------- 350 tr|A0A2U9BVW2|A0A2U9BVW2_SCOMX Turbot ( Scophthalmus maximus ) Chordata ---------AQKKVRFSPVVQVHVMRTWPFA--RQASRKGHWEVMARDRDRFQRRIRETEPTIRHVLEQSHREKIQA- 351 tr|A0A2U9BEM8|A0A2U9BEM8_SCOMX Turbot Chordata ------RCGSTKKVRFCDHVEEFFASCGEEE--EEEDRRGPWEELARDRSRFLRRCLEVEQSIAYCLQPQHRSLVY-- 352 tr|A0A093HKD2|A0A093HKD2_STRCA African ostrich ( Struthio camelus australis ) Chordata ----------RKKVTFLEKVTEYYIS-------SEEDRKGPWEELARDGCRFQKRIQETEDAIGYCLTTEHRQKVFN- 353 tr|I3LIA2|I3LIA2_PIG Wild boar ( Sus scrofa ) Chordata ------THTQRKKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCRFQKRIQETEDAIGYCLTFEHRERIF-- 354 tr|F1RIN9|F1RIN9_PIG Wild Boar Chordata RHQDPEPPRKTRKVRFSEKVSVHLLAVWAGP--AQAARRGPWEQFARDRSRFARRIAQAEEVLGPYLTPAARARAWA- 355 tr|A0A2I7ZPD2|A0A2I7ZPD2_TACFU Yellowtail ( Tachysurus fulvidraco ) Chordata -NPDQSTKESAKKVRFSEEVTIHTLEAWGFA--SQAARDGSWMEMARDRDRFKKRVEKAVELISPCLTAQHRARV--- 356 tr|H1A4D9|H1A4D9_TAEGU Zebra Finch ( Taeniopygia guttata ) Chordata ---EKTASSKRKKVTFHEEVT-YYIS-------SEEDRKGPWEEMARDGCRFQKRIQETEEAIAYCLSPE-------- 357 tr|H2RNL4|H2RNL4_TAKRU Tiger fish ( Takifugu rubripes ) Chordata --------RTIKKVRFCEHVDEVVI-GA--E--EEEDRRGPWEELARDRCRFLRRCQEVEQSIAYCLQPQHRRAVY-- 358 tr|A0A1U7U7Z2|A0A1U7U7Z2_TARSY Philippine spectacled monkey ( Tarsius syrichta ) Chordata -----HALIKRKKVTFLEEITEYYIS-------DDEDRKGPWEEFARDACRFQKRIQETEDAIGYCLTFEHRERVF-- 359 tr|A0A1U7SRC3|A0A1U7SRC3_TARSY Philippine Tarsier Chordata RDPDPETPLKPRKVCFSEKVTVHFLAVWAGP--AQANRRGPWEQLARDRSRFARRIAQAQEVLGPCLTPAARARAWA- 360 tr|A0A093BVK0|A0A093BVK0_TAUER Tauraco erythrolophus Chordata ----------RKKVTFLEKVTEYYIS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIGYCFTTEHRQRVFD- 361 tr|Q4T9W6|Q4T9W6_TETNG Green river dolphin ( Tetraodon nigroviridis ) Chordata ---------GNSRVRFSPVVHVHVMRSWTFA--RQASRKGNWEEMARDRDRFQRRIREAEAQLGPCLSPAHRRKQDR- 362 tr|A0A099ZQY2|A0A099ZQY2_TINGU diphtheria ( Tinamus guttatus ) Chordata --------SKRKKVTFLEKVTEYYIS-------SEEDRKGPWEELARDGCRFQKRIQETEDAIGYCLTIEHRQKVF-- 363 tr|A0A2Y9DVH9|A0A2Y9DVH9_TRIMA West Indian manatee ( Trichechus manatus latirostris ) Chordata RDLDPGTPLKATKVRFSEKVSVHYLAVWAGP--ARAARRGPWEQLARDRNRFARRIACVQEELNPCLTPAARARAWA- 364 tr|L9JAZ8|L9JAZ8_TUPCH Northern tree shrew ( Tupaia chinensis ) Chordata ----------RKKVTFLEEVTEYYIS-------DDEDRKGPWEEFARDACRFQKRIQETEDAIGYCLTFEHREKMFN- 365 tr|L9J9G4|L9J9G4_TUPCH northern tree shrew Chordata WDPDPETPLKARKVRFSEKVTVHFLTVWAGP--AQAARRGPWEEFARDRSRFARRIAQAQEELGPYLTPAARARAWT- 366 tr|A0A2U3V9Z3|A0A2U3V9Z3_TURTR Bottlenose dolphin ( Tursiops truncatus ) Chordata -SGARHTHIQRKKVTFLEEVTEYYIS-------GDEDRKGPWEEFARDGCRFQKRIQETEDAIGYCLTFEHREKMFN- 367 tr|A0A093EKS1|A0A093EKS1_TYTAL Owl ( Tyto alba ) Chordata -SGEKCRSTKRKKVTFLEKVTEYYIS-------SEEDRKGPWEELARDGCRFQKRIQETEEAIGYCFTTEHRQRVFN- 368 tr|A0A1L8FH50|A0A1L8FH50_XENLA African clawed toad ( Xenopus laevis ) Chordata ----------IKRVRFSPTVKVHHMVAWSYA--YRTARKGPWEEYARDRCRFQRRIAETEAAIGFCLDPQHREKIRA- 369 tr|A0A1L8FH48|A0A1L8FH48_XENLA African Xenopus Chordata ----------IKKVHFSPTVKVHHMVVWSYA--YRMARKGPWEEYARDRCRFQRRIAETDGVIRNFLEPQHREKIWT- 370 tr|Q5U232|Q5U232_XENLA African Xenopus Chordata ----------IKRVHFSPTVKVHHMVVWSYA--YRMARKGPWEEYARDRCRFQRRIAEAEGVIRNFLQPQHREKIWT- 371 tr|Q5U4R3|Q5U4R3_XENLA African clawed frog Chordata -----------KKVHFSPTVKVHHM-----A--YRMARKGPWEEYARDRCRFQRRIAETDGVIRNFLEPQHREKIW-- 372 tr|A0A1L8FNH1|A0A1L8FNH1_XENLA African clawed frog Chordata ----------IKHVRFSSSVTVHHMVVWSYA--HRMARRGSWEEYARDRCRFQKRIAETEAAIGLCMEPQHREKIWA- 373 tr|F7DWG0|F7DWG0_XENTR Tropical claw toad ( Xenopus tropicalis ) Chordata ----------IKHVRFSPSVTVHHIVVWSYA--HRMARRGIWEEYARDRCRFQRRIAETEAAIGFCMEPPHRKKIWA- 374 tr|M4AD25|M4AD25_XIPMA Pied swordtail fish ( Xiphophorus maculatus ) Chordata ----------QKKVQFSPLVQVH-----PFA--RQASRKGHWEELARDRDRFRRRIADTERAVGYCLSQPHREKMR-- 375 tr|A7RH49|A7RH49_NEMVE Star-shaped sea anemone ( Nematostella vectensis ) Cnidaria ------AGTGTKKVKFCEEDDVHVMYTWDFA--YRAARRSEWCQCATDREHFKLRINQTEQLLKPVLLD--------- 376 tr|A0A2B4SRF3|A0A2B4SRF3_STYPI Stylophora pistillata Cnidaria ------GNKKKKRVCFKPDTVIHPMIVWSFA--YKQARKGTWEMEALDRLRFQKRIRDLDEILTPVLLA--------- 377 tr|A0A2V2PBT9|A0A2V2PBT9_ENTFC Enterococcus faecium Firmicutes ------QNIQQKKVRFSDEVTVCPLVDWPQA--SSDARDGSWMAMARDRDRFSRRVED-------------------- 378 tr|A0A0B7BHB3|A0A0B7BHB3_9EUPU Common slug ( Arion vulgaris ) Mollusca -----------IKVHFASEPTVH------IV--GTEERGGI-SHYALDRERFERRILESEQIISPVLDADHRAKILE- 379 tr|A0A2C9KDJ8|A0A2C9KDJ8_BIOGL Biomphalaria glabrata Molluscs ----------LIKVRFAAEPEVY------LV--GTEDRCGTWQHYALDRERFGRRIQEISNIVSPILEQKHRAKIC-- 380 tr|V4A4E2|V4A4E2_LOTGI Owl Limpet ( Lottia gigantea ) Molluscs ------KSKTQKKVRFEEGTTVYQMVAWSFA--YRASRKGPWEQYARDRERFQRKIRDLEEVLSQILIDSHRSKIF-- 381 tr|A0A210Q4Q9|A0A210Q4Q9_MIZYE Mizuhopecten yessoensis Mollusca -------KKSKSKVTFAEGDTVYDL--------DDEDRKSHWEECVRDRERFQRRITEVGEIMKPVLSMERRDRIY-- 382 tr|A0A210Q4Q8|A0A210Q4Q8_MIZYE Shrimp Platter with Scallops Molluscs -----QREKSKSKVTFAQLCTVYDL--------DDEDRKSHWEECVRDRERFQRRITEVGEIMKPVLSMER------- 383 tr|A0A0L8HX49|A0A0L8HX49_OCTBM California two-spot octopus ( Octopus bimaculoides ) Molluscs ------VHHKTSKVRFAADETKHFMLVWSFA--YQAARKGEWVQIHIDSERFRCRIENTEQVLKPILDPSHRLKILA- 384 sp|P0C754|DP71L_ASFK5 African swine fever virus (isolate Pig/Kenya/KEN-50/1950) NA ----------------------------EA--DDIDRKGPWEQAAVDRLRFQRRITDTEEILSTVFL---------- 385 tr|A9JM96|A9JM96_ASFPP African swine fever virus (isolate Pig/Portugal/OURT88/1988) NA ------------DTKHVRFAAA--VEVWE-A--DDIERKGPWEQVAVDRFRFQRRIASVEELLSTVLLRQKKLLEQQ- 386 sp|P0C755|DP71L_ASFM2 African swine fever virus (isolate Tick/Malawi/Lil 20-1/1983) NA ----------------------------------DDIDRKGPWEQAAVDRLRFQRRIADTEKILSAVLL---------- 387 sp|P0C756|DP71L_ASFP4 African swine fever virus (isolate Tick/South Africa/Pretoriuskop Pr4/1996) NA -MGGRRRKKRTNDVKHVRFAAA--VEVWE-A--DDIERKGPWEQAAVDRFRFQRRIASVEELLSAVLLRQKKLLEQQ- 388 sp|P0C753|DP71L_ASFWA African swine fever virus (isolate Warthog/Namibia/Wart80/1980) NA ------------DVKHVRFAAA--VEVWE-A--DDIERKGPWEQAAVDRFRFQRRIASVEELLSAVLLR--------- 389 tr|Q8B591|Q8B591_GVCF Choristoneura fumiferana granulovirus NA ---------DKKIVTFIENSKILVMRVWLFA--ARQSRVGHWQNYAIDRERFKTRIARVAKKISWIFESRHRIKIN-- 390 tr|A0A0K0WS63|A0A0K0WS63_9BBAC Clostera anastomosis granulovirus B NA ---------CRRRVTFSDKNQIFIMWVWLYA--ANKARSIYWEKCAADRVRFQKRIKEVGTKIEWVLSDSHRSSMY-- 391 tr|A0A097DAN4|A0A097DAN4_9BBAC Erinnyis ello granulovirus NA ---------KNKRVSFNPNTRTYIMWVWLYA--AKKARSNYWEQCAVDRARFHRRIKEVGLKISWVLSNEHRSKI--- 392 tr|A0A288WJ04|A0A288WJ04_9BBAC cassava hornworm granulosa virus NA ---------KNKRVSFNPNTRTY-----LYA--AKKARSNYWEQCAVDRARFQRRIKEVGLKISWVLSNEHRSKI--- 393 tr|B0YLU8|B0YLU8_GHVS Glossina hytrovirus (isolate Glossina pallidipes/Ethiopia/ Seibersdorf/-) NA ------KAENNKKVRFDPAPPVHKIIAWDFA--YRAARKGEWEIIVRDRARFKTRITRTENKIKHILDPEYRNKIY-- 394 tr|A0A2G9P657|A0A2G9P657_LITCT American bullfrog (Lithobates catesbeiana) NA ----------VKKVRFSSNVTVHRMVTWSYA--YRMSRKGPWEEYARDHCRFQKRIAEAEAAIGFCLQPDHREKIWA- 395 tr|E0VSW1|E0VSW1_PEDHC body lice subspecies NA ---DDVKSKSSKKVKFNNNVKVNKIIAWDHA--YRNARLGPWEQMARDRVRFHERISRLSDVISPILGDKHRRKI--- 396 tr|A0A2S1KM50|A0A2S1KM50_ASF African swine fever virus Nucleocytoviricota AKHVRFAAA--VEVWE-A--DDIERKGPWEQVAVDRFRFQRRIASVEELLSAVLLRQ 397 tr|A0A2S1KM30|A0A2S1KM30_ASF African swine fever virus Cytosporine Viruses AKHVHFATA--VEVWE-A--DDIERKGPWEQVAVDRFRFQRRIASVEELLTAVLLRQ 398 tr|Q76SC7|Q76SC7_ASF African swine fever virus Cytosporine Viruses ------------DTKHVRFAAA--VEVWE-A--DDIERKGPWEQVAVDRFRFQRRIASVEELLSTVLLR--------- 399 tr|A0A2S1KM36|A0A2S1KM36_ASF African swine fever virus Cytosporine Viruses ------------DAKHVHFATA--VEVWE-A--DDIERKGPWEQVAVDRFRFQRRIASVEELLSAVLLR--------- 400 tr|P90496|P90496_ASF African swine fever virus nucleovirus ------------DAKHVRFAAA--VEVWE-A--DDIERKGPWEQVAVDRFRFQRRIASVEELLSAMLLR--------- 401 tr|A0A1C6ZY93|A0A1C6ZY93_ASF African swine fever virus Cytosporine Viruses --------KRTNDTKHVRFAAA--VEVWE-A--DDIERKGPWEQVAVDRFRFQRRIASVEELLSTVLLRQ-------- 402 tr|A0A2S1KM38|A0A2S1KM38_ASF African swine fever virus nucleovirus ------------DMKHVRFAAV--VEVWE-A--DDIERKGPWEQAAVDRFRFQRRIASVEELLTAVLLR--------- 403 tr|A0A2S1KM42|A0A2S1KM42_ASF African swine fever virus nucleovirus -------------MKHVRFAAV--VEVWE-A--DDIERKGPWEQAAVDRFRFQRRIASVEELLSAVLLRQ-------- 404 tr|A0A0C5AZK7|A0A0C5AZK7_ASF African swine fever virus nucleovirus ----------------------------EA--DDIDRKGPWEQAAADRLRFQRRIANAEEILSVALL---------- 405 tr|Q89571|Q89571_ASF African swine fever virus nucleovirus ----------------------------EA--DDIDRKGPWEQAAVDRLRFQRRIADTEKILSAVLL---------- 406 tr|A0A2S1KM37|A0A2S1KM37_ASF African swine fever virus Cytosporine Viruses --------------------IL--VDVRE-A--DDIDRKGPWEQAAVDRLRFQRRITDTEKILSAVLLRKK------- 407 tr|A0A0C5AWH3|A0A0C5AWH3_ASF African swine fever virus Cytosporine Viruses ----------------------------EA--DDINRKGPWEQAAVDRLRFQRRIADAEEILSAALL---------- 408 tr|A0A2S1KM46|A0A2S1KM46_ASF African swine fever virus Cytosporine Viruses ----------------VHFAIL--VDLRE-A--DDIDRKGPWEQAAVDRLRFQRRITDTEEILSAVFLRKK------- 409 tr|A0A2S1KM59|A0A2S1KM59_ASF African swine fever virus Cytosporine Viruses ----------------------------EA--DDIDRKGPWEQAAVDRLRFQRRITDTEEILSAVLL---------- 410 tr|A0A2S1KM45|A0A2S1KM45_ASF African swine fever virus Cytosporine Viruses ---------------------------------DDIDRKSPWEQAAVDRLRFQRRITDTEEILSAVLL---------- 411 tr|Q9EML3|Q9EML3_AMEPV Entomopoxvirus nucleovirus ------KHKPSKKVRFSSEPKLHIMYVWLYA--AKQTRKLYWDKFAIDRHRFKRRINDIDISISWVLTPHHRHKIM-- 412 tr|W8QF34|W8QF34_9VIRU Anopheles minimus irodovirus nucleovirus ----------PRQKIRFSQKIIII------FV--DKEDRRGPWEMLARDRDRFHHRIQNINNEIGWIFSPTHRTKI--- 413 tr|Q6VZB6|Q6VZB6_CNPV Canarypox virus nucleovirus ------------VVTFSETIIEYHVP--------YEDRKGPWEEIARDRYRFEKRIKETAEIIEFCLSENHRRNI--- 414 tr|W8W1F7|W8W1F7_9VIRU Invertebrate iridovirus 22 Cytosporine Viruses ----------KHKVTFAPKPIIIFI--------EIEDRKGPWETYALDRYRFNCRIKDVESKIGWCLGSNHRKNILI- 415 tr|W8W278|W8W278_9VIRU Invertebrate Iridescent Virus 30 Cytosporine Viruses -------KNSKHKVTFASKPIIIFI--------EIEDRKGPWETYALDRYRFHRRIKDVESKIGWCLGSNHRKNIF-- 416 tr|S6DF93|S6DF93_9VIRU Invertebrate iridovirus 22 Cytosporine Viruses -------IKCKHKVTFAPKPIIIFI--------EIEDRKGPWETYALDRYRFNCRIKDVESKIGWCLGSDHRKNIL-- 417 tr|W8W243|W8W243_9VIRU Invertebrate iridovirus 25 nucleovirus --------KCFRKVTFALRPMII------IIE-DDEDRKGPWEILAVDRDRFNRRILNVESQIGWCFGEKHRENIF-- 418 tr|A0A1V0QGI8|A0A1V0QGI8_CNPV Shearwaterpox virus Cytosporine Viruses ---------KKSVVTFSETIIEYHVP--------YEDRKGPWEEIARDRYRFEKRIKETAEMIEFCLSENHRRNI--- 419 tr|A0A1V0S876|A0A1V0S876_CNPV Poxvirus Cytosporine Viruses ---------SSITVKFNEKVTVYYVP--------YEDRKGPWEEFARDRYRFKKRINETGEIIERCLTESHRQ----- 420 tr|A0A2N9QUY5|A0A2N9QUY5_9VIRU Trichoplusia ni ascovirus 6b Cytosporine Viruses ---------LLKRVKFSDQPHIHIMWVWSFA--ARQARVGEWDRYAINRDRFRRRIAEVDSAVSWILNPVHRSKIM-- 421 tr|G0T5C9|G0T5C9_IRV9 Wiseana iridescent virus Cytosporine Viruses -----------RKVKFTLMPTII------IIEDDDEDRKGPWEIFAVDRDRFNRRILNVESQIGWCFQPKHRENIF-- 422 tr|K9MHD6|K9MHD6_9ALPH Chimpanzee herpesvirus strain 105640 Peploviricota ---------PPRKVCFSPRVRVRHLVAWETA--ARLARRGTWARERADRDRFRRRVAAAEAVIGPCLEPEARAR-- 423 tr|A0A060Q1X2|A0A060Q1X2_9ALPH Fruit bat alphaherpesvirus 1 Capsid virus -----DSPASTARVRFSPSVR--ILVTWQVA--ARQARRGSWAHHQADRARFRRRVAEAEAALGPILCAEARERARA- 424 tr|G8H8Q0|G8H8Q0_HHV1 Human herpes virus type 1 Capsid virus -----AXXXXPGGVRFSPHVR--HLVVWAXX--XXXXXXXSWARERADRARFGRRVAEAEAVIGPCLGPEARARALA- 425 tr|A0A0F7GW96|A0A0F7GW96_HHV1 Human herpes virus type 1 Capsid virus -----ATPATPARVRFSPHVR--HLVVWASA--ARLXXXXXXXXXXADRARFRRRVAEAEAVTRPSLGPEARARALA- 426 tr|A0A0F7GU37|A0A0F7GU37_HHV1 Human herpes virus type 1 Capsid virus -----XTPATPARVRFSPHVR--HLVVWASA--ARXXXRGSWARERADRARVPRRGAEAEAVIGPCPGPEARARALA- 427 tr|A0A193GT91|A0A193GT91_HHV1 human herpesvirus type 1 capsid virus phylum -----ATPATPATPATPXXXXXXXXXXXXXX--XXXXXRGSWARERADRARFRRRVAEAEAVIGPCLGPEARARALA- 428 tr|A0A0F7GVQ8|A0A0F7GVQ8_HHV1 human herpesvirus type 1 capsid virus phylum -----------ARVRFSPHVR--------SA--ARLARRGSWARKRADRARFRRRVAEAEAVIGPGLGPEARARAL-- 429 tr|A0A0F7GTV6|A0A0F7GTV6_HHV1 Human herpes virus type 1 Capsid virus -----XTPATPARVRFSPHVR--HLVVWASA--ARLARRGSWARGRADRAPCRRRGAEAEAVIGPCLGPEARARALA- 430 tr|A0A0F7GUX7|A0A0F7GUX7_HHV1 human herpesvirus type 1 capsid virus phylum -----ATPATPARVASPRPAR--HLVVWASA--ARLARRGSWARERADRARFRRRVAEAEAVIGPCLGPEARARALA- 431 tr|A0A2U9AA93|A0A2U9AA93_HHV1 human herpesvirus type 1 capsid virus phylum -----------ARVRFSPHVRVRH------A--ARLARRGSWARERADRARFRRRVAEAEAVIGPCLGPE-------- 432 tr|A0A0F7GS11|A0A0F7GS11_HHV1 Human herpes virus type 1 Capsid virus ----------PARVRFSPLVV------WPPP--ARLARRGSWARERADRARFRRRVAEAEAVIGPCLGP--------- 433 tr|D3YPC3|D3YPC3_HHV1 Human herpes virus type 1 Capsid virus -----------ARVRFSPHVRVRHL-----A--ARLARRGSWARERADRARFRRRVAEAEAVIGPCLGPEARARAL-- 434 tr|A0A1C3J794|A0A1C3J794_HHV1 human herpesvirus type 1 capsid virus phylum -----------ARVRFSPHVV------WASA--ARLARRGSWARERADRARFRRRVAEAEAVIGPCLGP--------- 435 tr|A0A0X8EA43|A0A0X8EA43_HHV1 Human herpes virus type 1 Capsid virus ---GPATPATPARVRFSPHVRVRHLVVWASA--ARLARRGSWARERADRARFRRRVAEAEAVIGPCLGPEARARALA- 436 tr|H9E905|H9E905_HHV1 Human herpes virus type 1 Capsid virus ------------RVRFSPHVR------WASA--ARLARRGSWARERADRARFRRRVAEAEAVIGPCLGPE-------- 437 tr|A0A181ZG78|A0A181ZG78_HHV11 Human herpesvirus type 1 (strain 17) capsid virus phylum -----KPPKTPARVRFSPHVR--HLVVWASA--ARLARRGSWARERADRARFRRRVAEAEAVIGPCLGPQARARALA- 438 sp|P37319|ICP34_HHV1N Human herpesvirus type 1 (strain MGH-10) capsid virus phylum ----------PARVRFSPLVV------WASA--ARLARRGSWARERADRARFRRRVAEAEAVIGPCLGP--------- 439 tr|G9I220|G9I220_HHV2 human herpesvirus type 2 capsid virus phylum ----------RGKVCFSP--LVA----WETA--ARLARRGSWARERADRDRFRRRVAAAEAVIGPCLEP--------- 440 tr|A0A290Y4M3|A0A290Y4M3_HHV2 human herpesvirus type 2 capsid virus phylum -----------GKVCFSP--RVQ------TA--ARLARRGSWARERADRDRFRRRVAAAEAVIGPCLEPEARAR-- 441 sp|P28283|ICP34_HHV2H Human herpesvirus type 2 (strain HG52) capsid virus phylum -----AADAPRGKVCFSP--RVQHLVAWETA--ARLARRGSWARERADRDRFRRRVAAAEAVIGPCLEPEARARARA- 442 tr|A0A0Y0A6B1|A0A0Y0A6B1_9ALPH Kangaroo alphaherpesvirus type 1 capsid virus phylum -----------SIVRFATTVRVHPLVVWNTA--ARLARRGSWEHHLADRMRFDRRVRELEVLLGPSLQPHIRA----- 443 tr|Q91CH8|Q91CH8_9ALPH Kangaroo alphaherpesvirus type 1 capsid virus phylum ------------IVRFATTVRVHPL-----A--ARLARRGSWEPPLADRMRFDRRVRELEVLLGPSLQPHIRACV--- 444 tr|E2IUH1|E2IUH1_SHV1 Simian herpesvirus type 1 (strain MV-5-4-PSL) Capsid virus --PDIDPRGKHRVVRFSDTVRVHRLVVWESA--ARQARRGTWLQDSADRARFQRRITEAEATIGSCLTLEARAKAWA- 445 tr|B3SBK1|B3SBK1_TRIAD Trichoplax adhaerens Placozoa ---DSSSPSRPTRVHFKPGKEVHLMVTWNFA--YRAARKATWCQYRIDHIRFLNRIKKVEEAIRYVFQPQHREKML-- 446 tr|A0A1X7U8I0|A0A1X7U8I0_AMPQE Queensland Great Barrier Reef sponge (Amphimedon queenslandica) Sponges (Porifera) ------QPSSCKRVQFKPEPQVHHMVTWRYA--YRTARKGHWEQLGLDRDRFQKRIETIGKAIGPCLK--------- 447 eIF2α constitutive inhibitor of phosphorylation ( CReP )

In some embodiments, an ISR inhibitor according to the present invention consists of, consists essentially of, or includes the following: constitutive repressor of eIF2α phosphorylation (CReP) protein (also known as protein phosphatase 1 regulatory subunit 15B; PPP1R15B). In some embodiments, an ISR inhibitor according to the invention consists of, consists essentially of, or includes a chimera of CReP and DP71L. In some embodiments, a chimera of CReP and DP71L includes a PP1 and/or eIF2 binding domain derived from CReP, and an interconnecting peptide linker sequence derived from DP71L. In some embodiments, the ISR inhibitor consists of, consists essentially of, or includes an amino acid sequence, or is encoded by a polynucleotide sequence, the amino acid sequence or a polynucleoside Acid sequences that are at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86% identical to SEQ ID NOs: 1 to 6 , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any exportable range therein ) consistency. SEQ ID NO: 1 - CReP amino acid sequence SEQ ID NO: 2 - CReP polynucleotide sequence 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 ΔCReP protein polynucleotide coding sequence SEQ ID NO: 6 - Exemplary ΔCReP protein amino acid sequence Protein phosphatase 1 regulatory subunit 15A ( GADD34 )

In some embodiments, an ISR inhibitor according to the present invention consists of, consists essentially of, or includes a protein phosphatase 1 regulatory subunit 15A (GADD34) protein. 1 regulatory subunit 15A (GADD34) protein. In some embodiments, the ISR inhibitor consists of, consists essentially of, or includes an amino acid sequence, or is encoded by a polynucleotide sequence, the amino acid sequence or a polynucleoside Acid sequences that are at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86% identical to SEQ ID NOs: 7 to 8 , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any exportable range therein ) consistency. SEQ ID NO: 7 - GADD34 amino acid sequence SEQ ID NO: 8 - GADD34 polynucleotide sequence Herpes simplex virus-derived protein gamma 34.5

In some embodiments, an ISR inhibitor according to the present invention consists of, consists essentially of, or includes herpes simplex virus-derived γ34.5 protein. . In some embodiments, the ISR inhibitor consists of, consists essentially of, or includes an amino acid sequence, or is encoded by a polynucleotide sequence, the amino acid sequence or a polynucleoside Acid sequences that are at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86% identical to SEQ ID NO: 9 to 10 , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any exportable range therein ) consistency. SEQ ID NO: 9 - γ 34.5 amino acid sequence SEQ ID NO: 10 - γ34.5 polynucleotide sequence Canarypox virus-derived protein CNPV231

In some embodiments, an ISR inhibitor according to the present invention consists of, consists essentially of, or includes the following: canarypox virus-derived CNPV231 protein (MyD116-like protein; accession number Q6VZB6). In some embodiments, the ISR inhibitor consists of, consists essentially of, or includes an amino acid sequence, or is encoded by a polynucleotide sequence, the amino acid sequence or a polynucleoside Acid sequences that are at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86% identical to SEQ ID NO: 11 to 12 , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any exportable range therein ) consistency. SEQ ID NO: 11 - CNPV231 amino acid sequence SEQ ID NO: 12 - CNPV231 polynucleotide sequence Kangaroo herpesvirus -derived protein ICP34.5

In some embodiments, the ISR inhibitor according to the present invention consists of, consists essentially of, or comprises an ICP34.5 protein derived from a kangaroo herpesvirus. In some embodiments, the ISR inhibitor consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, which has 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% identity (or any range derivable therein) to SEQ ID NOs: 13-14. SEQ ID NO: 13 - ICP34.5 amino acid sequence SEQ ID NO: 14 - ICP34.5 polynucleotide sequence AmEPV193 protein derived from Entomopoxvirus ' L ' of the mulberry lantern moth

In some embodiments, an ISR inhibitor according to the present invention consists of, consists essentially of, or comprises AmEPV193 protein derived from Lanterna Insectpoxvirus "L" (Accession No. Q9EML3). In some embodiments, an ISR inhibitor consists of, consists essentially of, or comprises an amino acid sequence, or is encoded by a polynucleotide sequence, which has 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% identity (or any range derivable therein) to SEQ ID NOs: 15-16. SEQ ID NO: 15 - AmEPV193 amino acid sequence SEQ ID NO: 16 - AmEPV193 polynucleotide sequence African swine fever virus-derived protein DP71L

In some embodiments, an ISR inhibitor according to the present invention consists of, consists essentially of, or includes an African swine fever virus-derived DP71L protein. In some embodiments, the DP71L protein is a long form protein, and alternatively, in some embodiments, the DP71L protein is a short form protein. Generally, as used herein, DP71L refers to the short form of the protein (e.g., as represented by SEQ ID NO: 18), however, in some embodiments, slightly longer or shorter protein isoforms may be used. (For example, relative to SEQ ID NO: 18, any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids is additionally included or deleted. protein isoforms). In some embodiments, an ISR inhibitor according to the invention is a chimeric protein (e.g., a synthetic protein containing domains derived from two or more proteins) that contains a PP1 binding domain disposed to bind eIF2 Peptide linker sequence derived from DP71L between domains. In some embodiments, the peptide linker sequence derived from DP71L contains 8, 9, 10, 11, or 12 amino acids. In some embodiments, the peptide linker sequence derived from DP71L enhances the ability of the ISR inhibitor to bind to PP1. In some embodiments, the peptide linker sequence derived from DP71L contains one or more glutamic acids, which can affect inter- or intra-protein binding kinetics of the PP1 binding domain. In some embodiments, the one or more glutamines are glutamine E12 of the DP71L protein (e.g., the underlined glutamine in the sequence "MDVKHVRFAAAV E VWEADDI" (SEQ ID NO: 17), which is SEQ ID NO : E12 glutamic acid found in 18 (e.g., the 12th amino acid when N-terminal methionine is not included, or the 13th amino acid when N-terminal methionine is included)). In some embodiments, the linker sequence derived from DP71L consists of, consists essentially of, or includes at least or exactly about 55%, 64%, 73%, 82% of SEQ ID NOs: 20 to 21 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any derivable range thereof) identical polynucleotide sequences and/or Amino acid sequence. In some embodiments, the ISR inhibitor consists of, consists essentially of, or includes an amino acid sequence, or is encoded by a polynucleotide sequence, the amino acid sequence or a polynucleoside Acid sequences that are at least or exactly about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86% identical to SEQ ID NO: 18 to 19 , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any exportable range therein ) consistency. SEQ ID NO: 18 - DP71L amino acid sequence SEQ ID NO: 19 - DP71L polynucleotide sequence SEQ ID NO: 20 - DP71L derived peptide linker polynucleotide coding sequence SEQ ID NO: 21 -Amino acid sequence of DP71L-derived peptide linker

In some embodiments, the amino acid subunits of a protein are modified to produce equivalent or even improved second generation variant polypeptides or peptides. For example, certain amino acids may be substituted with other amino acids in the protein or polypeptide sequence with or without significant loss of interaction binding with a structure such as an antigen binding region of an antibody or binding site on a substrate molecule. Since the interaction ability and properties of a protein define the functional activity of the protein, certain amino acid substitutions may be made in the protein sequence and in its corresponding DNA coding sequence and still produce a protein with similar or desired properties. Therefore, the inventors contemplate that various changes may be made in the DNA sequence of the gene encoding the protein without significant loss of the biological utility or activity of the protein.

The term "functionally equivalent codons" is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also contemplated are "neutral substitutions" or "neutral mutations," which refer to changes in one or more codons that encode biologically equivalent amino acids.

The amino acid sequence variants of the present invention may be substitution, insertion or deletion variants. Compared with the wild type, the changes in the polypeptide of the present invention can affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 of the protein or polypeptide ,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-consecutive or continuous amino acids. Variants may comprise an amino acid sequence that is at least or exactly about 50%, 60%, 70%, 80% or 90% (including all values and ranges therebetween) identical to any sequence provided or referenced herein. ) are consistent. Variants may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substituted amino acids .

It will also be understood that amino acid and nucleic acid sequences, respectively, may include other residues, such as other N-terminal or C-terminal amino acids, or 5' or 3' sequences, and still be within one of the sequences disclosed herein. Substantially consistent as set forth, so long as the sequence meets the criteria set forth above, including maintaining biological protein activity related to protein expression. The addition of terminal sequences is particularly suitable for nucleic acid sequences that may, for example, include various non-coding sequences flanking either the 5' or 3' portion of the coding region.

Deletion variants generally lack one or more residues of the native or wild-type protein. Individual residues may be deleted or multiple consecutive amino acids may be deleted. A stop codon may be introduced (by substitution or insertion) into the coding nucleic acid sequence to produce a truncated protein.

Insertion mutants generally involve the addition of amino acid residues at non-terminal points in a polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be produced and may include fusion proteins, which are polymers or concatemers of one or more peptides or polypeptides described or referenced herein.

Substitution variants typically contain a replacement of one amino acid for another at one or more sites within a protein or polypeptide and can be designed to modulate one or more properties of a polypeptide with or without loss of other functions or properties. Substitutions can be conservative, meaning that one amino acid is replaced with an amino acid having similar chemical properties. "Conservative amino acid substitutions" can involve the replacement 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 following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamine; cysteine to serine; glutamine to asparagine; glutamine to aspartate; glycine to proline; histidine to asparagine or glutamine; isothiocyanate to oligonucleotides; Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. Such amino acid residues include peptide mimetics or other reversed or inverted forms of amino acid moieties.

Alternatively, a substitution may be "non-conservative," thereby affecting the function or activity of the polypeptide. Non-conservative changes generally involve the substitution of an amino acid residue with a chemically distinct amino acid residue, such as the substitution of a polar or charged amino acid for a non-polar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve replacing a member of one amino acid class with a member from another class. III. Carrier

Among other things, the present invention provides that, in some embodiments, the ISR inhibitors described herein are encoded by polynucleotides, such as vectors comprising polynucleotides (e.g., polynucleotide constructs). Vectors comprising polynucleotide constructs according to the present invention include all vectors 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) incorporating polynucleotides comprising an inhibitor of an ISR gene or a characteristic portion thereof (e.g., as used herein, "characteristic portion thereof" refers to a portion of the protein that is necessary to perform a desired function (e.g., it comprises the ability to inhibit the ISR), such as the portion comprising a functional PP1 binding domain and an eIF2 binding domain). Those skilled in the art will be able to select appropriate constructs and cells to produce any of the polynucleotides described herein. In some embodiments, the construct is a plasmid (i.e., a circular DNA molecule that can replicate autonomously inside a cell). In some embodiments, the construct can be a cosmid (e.g., pWE or sCos series).

In some embodiments, the construct is a viral construct. In some embodiments, the viral construct is a lentiviral, retroviral, adenoviral, or adeno-associated viral construct. In some embodiments, the construct is an adeno-associated viral (AAV) construct (see, e.g., Asokan et al., Mol. Ther. 20: 699-7080, 2012, which is incorporated herein by reference for the purposes described herein). In some embodiments, the viral construct is an adenoviral construct. In some embodiments, the viral construct may also be based on or derived from an alpha virus. 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, 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. Typically, the genomes of these viruses encode nonstructural proteins (e.g., replicon) and structural proteins (e.g., capsid and envelope) that are translated in the cytoplasm of host cells. Ross River virus, Sindby virus, Victory Forest virus (SFV), and Venezuelan equine encephalitis virus (VEEV) have all been used to develop viral constructs for coding sequence delivery. Pseudotyped viruses can be formed by combining alphavirus envelope glycoproteins with retroviral capsids. Examples of alphavirus constructs can be found in U.S. Publication Nos. 20150050243, 20090305344, and 20060177819; the constructs and methods for making them are incorporated herein by reference for the purposes described herein.

In some embodiments, the constructs provided herein can have different sizes. In some embodiments, the construct is a plastid and can include 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 in full length , 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, the construct is a plastid and can have a full length 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 1 kb to about 9 kb, about 1 kb to about 10 kb, about 1 kb to about 11 kb, about 1 kb to About 12 kb, about 1 kb to about 13 kb, about 1 kb to about 14 kb, or in the range of about 1 kb to about 15 kb.

In some embodiments, the construct is a viral construct and may have a total nucleotide number of up to 10 kb. In some embodiments, the viral construct can have a total nucleotide number 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 10 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 in the range of about 9 kb to about 10 kb.

In some embodiments, the construct is a lentiviral construct and can have a total nucleotide number of up to 8 kb. In some examples, the lentiviral construct can have a total nucleotide count 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.

In some embodiments, the construct is an adenoviral construct and can have a total nucleotide number of up to 8 kb. In some examples, the adenoviral construct can have a length between about 1 kb and about 2 kb, about 1 kb and about 3 kb, about 1 kb and about 4 kb, about 1 kb and about 5 kb, about 1 kb and about 6 kb, about 1 kb and about 7 kb, about 1 kb and about 8 kb, about 2 kb and about 3 kb, about 2 kb and about 4 kb, about 2 kb and about 5 kb, about 2 kb and about 6 kb, about 2 kb and about 7 kb, about 2 kb and about 8 kb, about 3 kb and about 4 kb, about 3 kb and about 5 kb, about 3 kb and about 6 kb, about 3 kb and about 7 kb, about 3 kb and about 8 kb, about 4 kb and about 5 kb, about 4 kb and about 6 kb, about 4 kb and about 7 kb, about 4 kb and about 8 kb, about 5 kb and about 6 kb, about 5 kb and about 7 kb 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.

Any of the constructs described herein may further include control sequences, for example, control sequences selected from the group consisting of transcription initiation sequences, transcription termination sequences, promoter sequences, enhancer sequences, RNA splicing sequences, polypeptide sequences, Adenylation (poly(A)) sequences, Kozak consensus sequences, and/or other non-translated regions that can accommodate pre- or post-transcriptional regulatory and/or control elements. In some embodiments, the promoter can be a native promoter, a constitutive promoter, an inducible promoter, and/or a tissue-specific promoter. This document describes non-limiting examples of control sequences. A. AAV particles

Among other things, the present invention provides AAV particles comprising a polynucleotide construct encoding an ISR inhibitor, and an AAV capsid. In some embodiments, an AAV particle may 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 comprising 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 comprising a construct utilizing the AAV2 ITRs and an AAV9 capsid. Other 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.

In some embodiments, AAV particles suitable for use according to the present invention may comprise or be derived from any natural or recombinant AAV serotype. In some embodiments, the 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.

As used herein, when referring to an AAV vector or a "chimeric AAV vector," the term "chimera" refers to an AAV vector that contains a coat containing VP1, VP2, and VP3 proteins from at least two different AAV serotypes. shell; or alternatively, it 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, AAV2i8, AAV2i8G9, AAV8G9, and AAV9i1.

In some embodiments, the AAV serotype and/or pseudotype according to the present invention is selected from the group consisting of: AAV1, AAV2, AAV3, AAV 4. AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV106.1/hu.37, AAV114.3/hu.40, 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.10, AAV161.1 0/hu.60, AAV161.6/hu.61, AAV1-7/rh.48, AAV1-8/rh.49, AAV2i8, AAV2i8G9, 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.50, AAV2-5/rh.51, AAV2.5T, AAV27.3, AAV29.3/bb.1, AAV29.5/bb.2, AAV2G9, AAV3B, AAV3. 1/hu.6, AAV3.1/hu.9, AAV3-11/rh.53, AAV3-3, AAV33.12/hu.17, AAV33.4/hu.15, AAV33.8/hu.16, AAV3-9/rh.52, AAV3a, AAV3b, AAV4-19/rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV 42-6b, 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.19, AAV5-22/rh.58, AAV5-3/rh.57, AAV54.1/hu.21, AAV5 4.2/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, AAV9i1, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAVcy.2, AAVcy.3, A AAVcy.4, AAVcy.5, AAVcy.5R1, AAVcy.5R2, AAVcy.5R3, AAVcy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.8, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.12, AAVhu.13, AAVhu.14/9, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.19, AAVhu.20, AAVh u.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.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.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu. Vhu.48, AAVhu.48R1, 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.1, 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, A AVrh.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.4 8.2, 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, AAV-PHP.B-SNP(3). -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 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-E1, 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 AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, 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-B1, AAV CKd-B2, AAV CKd-B3, AAV CKdB4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N9, AAV CLg-F1, 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 CLv1-1, AAV CLv1-10, AAV CLv1-2, AAV CLv-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV CLv1-7, AAV CLv1-8, AAV CLv1-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-R1, 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 shuffled 100-1, AAV shuffled 100-2, AAV shuffled 100-3, AAV shuffled 100-7, AAV shuffled 10-2, AAV shuffled 10-6, AAV shuffled 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.5R1, 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, AAV F5/HSC5, AAVF6/HSC6, AAVF7/HSC7, AAVF8/HSC8, AAVF9/HSC9, AAV-h, AAVH-1/hu.1, AAVH2, AAVH-5/hu.3, AAVH6, AAVhE1.1, AAVhEr1.14, AAVhEr1.16, AAVhEr1.18, AAVhER1.23, AAVhEr1.35, AAVhEr1.36, AAVhEr1.5, AAVhEr1.7, AAVhEr1.8, AAVhEr2.16, AAVhEr2.29, AA VhEr2.30, 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, Anc110, Anc113, Anc126, Anc127, BAAV, BNP61 AAV, BNP62 AAV, BNP63 AAV, bovine AAV, goat AAV, Japanese AAV10 serotype, UPENN AAV10, VOY101 and VOY201.

In some embodiments, the AAV serotype and/or pseudotype is AAV-DJ/8, AAV9, AAV Php.B and/or AAV Php.eB. In some embodiments, the AAV serotype and/or pseudotype comprises AAV-DJ/8. In some embodiments, the AAV serotype and/or pseudotype comprises AAV9. In some embodiments, the AAV serotype and/or pseudotype comprises AAV Php.B. In some embodiments, the AAV serotype and/or pseudotype comprises AAV Php.eB.

In some embodiments, AAV is an AAV variant that has been genetically modified, for example, by substitution, deletion, or addition of one or more amino acid residues in one or more shell proteins. Examples of such variants include (but are not limited to) AAV2 with one or more of the Y444F, Y500F, Y730F and/or S662V mutations; AAV3 with one or more of the Y705F, Y731F and/or T492V mutations; and AAV6 having one or more of the S663V and/or T492V mutations.

In some embodiments, AAV capsids are modified to include at least one surface-bound sugar or derivative thereof. As used herein, the term "surface bound" when referring to at least one sugar means that the at least one sugar is bound to and exposed to the outer surface of the AAV vector. Suitable examples of sugars include, but are not limited to, monosaccharides, oligosaccharides, polysaccharides and derivatives thereof. 1. AAV construct

In some embodiments, the invention provides polynucleotide vectors (e.g., polynucleotide constructs) comprising polynucleotides encoding ISR inhibitors or characteristic portions thereof (e.g., comprising a PP1 binding domain and an eIF2α binding domain) sequence. In some embodiments described herein, polynucleotide vectors including ISR inhibitors are polynucleotide constructs and can be included in AAV capsids to produce AAV particles (e.g., AAV particles include AAV capsids AAV constructs included in ).

In some embodiments, a polynucleotide construct includes one or more components derived from or modified from a naturally occurring AAV genome construct. In some embodiments, the sequence derived from an AAV construct is AAV1 construct, AAV2 construct, AAV3 construct, AAV4 construct, AAV5 construct, AAV6 construct, AAV7 construct, AAV8 construct, AAV DJ/8 construct, AAV9 construct, AAV2.7m8 construct, AAV8BP2 construct, AAV293 construct, AAVPhp.B construct or AAVPhp.eB construct (see e.g. Chan et al., 2017). Other exemplary AAV constructs that can be used herein are known in the art. (See, e.g., Kanaan et al., 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 is described herein. purposes are incorporated herein by reference).

In some embodiments, AAV-derived sequences (e.g., sequences contained in polynucleotide constructs) typically include 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 typical ITR sequences (e.g., at least or exactly 85%, at least or exactly 90%, at least or exactly 95%, or at least or exactly 100%, etc.) are incorporated into the constructs provided herein. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2nd 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. AAV ITR sequences can be obtained from any known AAV, including the currently identified AAV types.

In some embodiments, polynucleotide constructs according to the present invention and described in a manner known in the art (see, e.g., Asokan et al., Mal. Ther . 20: 699-7080, 2012, which is incorporated herein by reference for purposes described herein) generally comprise a coding sequence or portion thereof, at least one sequence and/or control sequence, and optionally 5' and 3' AAV inverted terminal repeats (ITRs). In some embodiments, the provided constructs can be encapsulated in capsids to produce AAV particles. The AAV particles can be delivered to selected target cells. In some embodiments, the provided constructs include other optional coding sequences, which are nucleic acid sequences (e.g., inhibitory nucleic acid sequences), heterologous to the construct sequences encoding polypeptides, proteins, functional RNA molecules (e.g., miRNA, miRNA inhibitors), or other gene products of interest. In some embodiments, the nucleic acid coding sequence is operably linked to a component and/or control component in a manner that permits transcription, translation, and/or expression of the coding sequence in cells of a target tissue.

In some embodiments, the unmodified AAV endogenous genome includes two open reading frames, "cap" and "rep", flanked by ITRs. In some embodiments, recombinant AAV constructs similarly comprise one or more open reading frames flanked by ITR sequences (eg, coding sequences comprising ISR inhibitor coding sequences). In some embodiments, AAV constructs also include conventional control elements that permit their transcription, translation and/or expression in cells transfected with the polynucleotide construct or infected with virions produced by the invention. means operably linked to the coding sequence. In some embodiments, the AAV construct optionally includes a promoter, enhancer, untranslated region (e.g., 5' UTR, 3' UTR), Kozak sequence, internal ribosomal entry site (IRES), splicing site (e.g., acceptor site, donor site), polyadenylation site, or any combination thereof.

In some embodiments, the construct is an AAV construct. In some embodiments, the AAV construct may 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, the AAV construct may 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, the AAV construct may comprise 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 may further comprise regulatory and/or control sequences, such as control sequences selected from the group consisting of: transcription initiation sequences, transcription termination sequences, promoter sequences, enhancer sequences, RNA Splice sequences, polyadenylation (poly(A)) sequences, Kozak consensus sequences, and/or any combination thereof. In some embodiments, the 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 2. AAV capsid

In some embodiments, the present invention provides one or more polynucleotide constructs encapsulated in an AAV capsid. In some embodiments, the AAV capsid is from or derived from an AAV capsid of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh8, AAVrhlO, AAVrh39, AAVrh43, or AAV ancestral serotype, or one or more hybrids thereof. In some embodiments, the AAV capsid is from an AAV ancestral serotype. In some embodiments, the AAV capsid is an ancestral (Anc) AAV capsid. Anc capsids are formed from construct sequences that were constructed using evolutionary probability and evolutionary modeling to determine possible ancestral sequences. Thus, it is known that Anc capsid/construct sequences do not exist in nature. In some embodiments, the AAV capsid is engineered and/or derived from an AAV9 capsid. In some embodiments, the AAV capsid is an AAV PHP.eB capsid. In some embodiments, the AAV capsid is an AAV PHP.B capsid (see, e.g., Diptaman Chatterjee et al., Gene Therapy 29, 2290-387 (2022)).

As provided herein, in some embodiments, any combination of AAV capsids and AAV constructs (eg, including AAV ITRs) may be used in the recombinant AAV particles of the invention. For example, wild-type or variant AAV2 ITR and AAV DJ/8, AAV9, AAV PHP.B and/or AAV PHP.eB capsids, or variant AAV2 ITR and AAV6 capsids, etc. In some embodiments of the invention, the AAV particles are composed entirely of AAV2 and/or AAV9 components (eg, the capsid and ITR are of AAV2 and/or AAV9 serotypes). In some embodiments, the AAV particle is an AAV2/6, AAV2/8, or AAV2/9 particle (eg, an AAV6, AAV8, or AAV9 capsid of an AAV construct with an AAV2 ITR). 3. Exemplary AAV construct components a. Inverted terminal repeats ( ITR )

AAV-derived sequences of constructs typically contain cis-acting 5' and 3' ITRs (see, e.g., B. J. Carter, "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155-168 (1990), ed. are incorporated herein by reference for the purposes described herein). Typically, ITRs are capable of forming hairpins. The ability to form hairpins may contribute to the ability of the ITR to self-prime, allowing primer enzyme-independent synthesis of the second DNA strand. ITR can also contribute to efficient encapsidation of AAV constructs in AAV particles.

The AAV particles of the present invention may comprise an AAV construct comprising a coding sequence (e.g., encoding an inhibitor of the ISR) and associated elements flanked by 5' and 3' AAV ITR sequences. In some embodiments, the ITR is or comprises about 130 nucleic acids. In some embodiments, the ITR is or comprises about 145 nucleic acids. In some embodiments, all or substantially all sequences encoding the ITRs are used. In some embodiments, the AAV ITR sequences can be obtained from any known AAV, including currently identified mammalian AAV types. In some embodiments, the ITR is an AAV2 ITR. In some embodiments, the ITR is an AAV9 ITR.

A non-limiting example of a polynucleotide construct of the invention is a "cis-acting" construct comprising a transgene, wherein the transgene sequence and any associated regulatory elements are linked by 5' or "left" and 3' Or "right" AAV ITR sequence flanking. The 5' and left designations refer to the position of the ITR sequence relative to the overall construct, which is read from left to right in the sense direction. For example, in some embodiments, when the construct is drawn in a linear fashion in a meaningful orientation, the 5' or left ITR is the ITR closest to the promoter of a given construct (e.g., opposite the polyadenylation sequence) . Meanwhile, the 3' and right designations refer to the position of the ITR sequence relative to the entire construct, which is read from left to right in the sense direction. For example, in some embodiments, when the construct is drawn in a linear fashion in a meaningful orientation, the 3' or right-hand ITR is the one closest to the polyadenylation sequence (e.g., as opposed to the promoter sequence) of a given construct ITR. Generally speaking, ITRs as provided herein are plotted in order from 5' to 3' based on the shares. Therefore, those skilled in the art should understand that a 5' or "left" directed ITR can also be depicted as a 3' or "right" ITR when switching from the sense direction to the antisense direction. Furthermore, converting a given sense ITR sequence (e.g., 5'/left AAV ITR) into an antisense sequence (e.g., 3'/right ITR sequence) is well within the capabilities of those skilled in the art. One of ordinary skill in the art will understand how a given ITR sequence can be modified to function as a 5'/left or 3'/right ITR or an antisense version thereof.

In some embodiments, the ITR (eg, 5' ITR) can have a sequence according to SEQ ID NO: 23. In some embodiments, the ITR (eg, 3' ITR) can have a sequence according to SEQ ID NO: 24. In some embodiments, an ITR includes one or more modifications as known in the art, such as truncation, deletion, substitution, or insertion. In some embodiments, the ITR contains less than 145 nucleotides, such as 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, or 141 nucleotides. For example, in some embodiments, the ITR includes 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.

In some embodiments, the 5' ITR sequence is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the 5' ITR sequence represented by SEQ ID NO: 23. In some embodiments, the 3' ITR sequence is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the 3' ITR sequence represented by SEQ ID NO: 24. SEQ ID NO: 23 - Exemplary 5' AAV ITR Polynucleotide Sequences SEQ ID NO: 24 - Exemplary 3' AAV ITR polynucleotide sequence b. Starter

In some embodiments, the construct (e.g., AAV construct) comprises a promoter. The term "promoter" refers to a DNA sequence recognized by an enzyme/protein that can promote and/or initiate transcription of an operably linked gene (e.g., encoding an inhibitor of the ISR). For example, a promoter generally refers to a polynucleotide sequence, such as one that binds to an RNA polymerase and/or any associated factors and from which transcription can be initiated. Thus, in some embodiments, the construct (e.g., AAV construct) comprises a promoter that is operably linked to one of the non-limiting exemplary promoters described herein.

In some embodiments, the promoter is an inducible promoter, a constitutive promoter, a mammalian cell promoter, a viral promoter, a chimeric promoter, an engineered promoter, a tissue-specific promoter, or the technology any other type of promoter known in . In some embodiments, the promoter is an RNA polymerase II promoter, such as a mammalian RNA polymerase II promoter. In some embodiments, the promoter is an RNA polymerase III promoter, including but not limited to the HI promoter, human U6 promoter, mouse U6 promoter, or porcine U6 promoter. The promoter will generally be one capable of promoting transcription in neural cells.

Various promoters are known in the art and may be used herein in some embodiments. In some embodiments, non-limiting examples of promoters that can be used herein include: human EF1α, human cytomegalovirus (CMV) (U.S. 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), β-globulin, β-actin, α-fetoprotein, γ-globulin, β-interferon, γ-glutamyl transferase, mouse mammary tumor virus (MMTV), Rous sarcoma virus, rat insulin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein II (MT II), amylase, histoplastin, MI muscarinic acid receptor, retrovirus LTR (e.g. human T-cell leukemia virus HTLV), AAV ITR, interleukin-2, collagenase, platelet-derived growth factor, adenovirus 5 E2, stromelysin, mouse MX gene, glucose-regulated proteins (GRP78 and GRP94), alpha-2-macroglobulin, vimentin, class I MHC gene H-2K b, HSP70, proliferative protein, tumor necrosis factor, thyrotropin alpha gene, immunoglobulin light chain, T cell receptor, HLA DQa and DQ, interleukin-2 receptor, class II MHC, class II MHC HLA-DRa, muscle creatine kinase, prealbumin (thyroxine transporter), elastase I, albumin gene, c-fos, c-HA-ras, neural cell adhesion molecule (NCAM), H2B (TH2B) histone, rat growth hormone, human serum amyloid protein (SAA), tyrosin I (TN I), Duchenne muscular dystrophy, human immunodeficiency virus and Gibbon Ape Leukemia Virus (GAL V) promoter. Other 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, the promoter is a CMV immediate early promoter. In some embodiments, the promoter is a CAG promoter and/or a CAG/CBA promoter.

The term "constitutive" promoter refers to a polynucleotide and/or oligonucleotide sequence that, when operably linked to a nucleic acid encoding a protein (e.g., an inhibitor of the ISR), allows RNA to be transcribed from the nucleic acid in a cell under most or all physiological conditions. Examples of constitutive promoters include, but are not limited to, 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 β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1-α promoter (Invitrogen).

Inducible promoters allow for regulation of gene expression and can be regulated by compounds provided exogenously; environmental factors such as temperature; or the presence of a specific physiological state (e.g., acute phase, a specific differentiation state of cells), or simply being present in replicating cells. Inducible promoters and inducible systems are available from a variety of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Other examples of inducible promoters are known in the art. Examples of inducible promoters regulated by exogenously provided 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 Sci. USA 93:3346-3351, 1996, which is incorporated herein by reference for the purposes described herein), the tetracycline-inhibitable system (see, e.g., Gossen et al., Proc. Natl. Acad Sci . USA 93:3346-3351, 1996, which is incorporated herein by reference for the purposes described herein), the tetracycline-inhibitable system (see, e.g., Gossen et al., Proc. Natl. Acad Sci. USA 89:5547-5551, 1992, which is incorporated herein by reference for the purposes described herein), tetracycline-inducible system (see, e.g., Gossen et al., Science 268: 1766-1769, 1995, and 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), RU486-inducible system (see, e.g., Wang et al., Nat. Biotech . 15:239-243, 1997 and Wang et al., Gene Ther . 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).

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 in cells expressing transcriptional regulatory and/or control proteins that and other proteins that bind to tissue-specific promoters). In some embodiments, regulatory and/or control sequences confer 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, the tissue-specific promoter is a neuron-specific promoter. In some embodiments, the tissue-specific promoter is a glial cell-specific promoter. In some embodiments, the 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 c. Enhancer

In some embodiments, a construct may include enhancer sequences. The term "enhancer" as used herein refers to polynucleotide and/or oligonucleotide sequences that increase the level of transcription of a nucleic acid encoding a protein of interest (e.g., an inhibitor of an ISR), and/or in Increase or alter the translation efficiency of the transcript after transcription. In some embodiments, enhancer sequences (typically 50 to 1500 bp in length) typically increase transcription levels by providing additional binding sites for transcription-related proteins (eg, transcription factors) and/or stabilizing or modifying post-transcriptional regulatory machinery. In some embodiments, enhancer sequences are found within intronic sequences. In some embodiments, enhancer sequences are found in the 3' and/or 5' UTR. In some embodiments, the enhancer region is found downstream of the coding sequence that contains the transgene and is proximate to the polyadenylation sequence. Unlike promoter sequences, enhancer sequences can act at a much greater distance from the start site of transcription (eg, compared to a promoter). Non-limiting examples of enhancers include woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), RSV enhancer, CMV enhancer and/or SV40 enhancer. SEQ ID NO: 27 - Exemplary WPRE polynucleotide sequence d.Flanking untranslated regions , 5' UTR and 3' UTR

In some embodiments, any of the constructs described herein may include an untranslated region (UTR), such as a 5' UTR or a 3' UTR. The UTR of a gene can be transcribed but not translated. The 5' UTR begins at the start of transcription and continues up to, but not including, the start codon. The 3' UTR begins immediately after the stop codon and continues until the transcription termination signal. Modulation and/or control features of the UTR may be incorporated into any of the constructs, particles, polynucleotides, compositions, kits, or methods described herein to enhance or otherwise modulate the ISR. Inhibitor performance.

The native 5' UTR includes sequences that function in translation initiation. In some embodiments, the 5' UTR may comprise sequences, such as Kozak sequences, which are generally known to be involved in the process by which ribosomes initiate translation of many genes. The Kozak sequence has the consensus sequence CCR(A/G)CCAUGG, where R is a purine (A or G) three bases upstream of the start codon (AUG), and there is another "G" after the start codon. In some embodiments, the 5' UTR also forms secondary structure that participates in elongation factor binding. In some embodiments, the 5' UTR is included in any of the constructs described herein. Non-limiting examples of 5' UTRs include those from the following genes: albumin, serum amyloid A, lipoprotein A/B/E, transferrin, alpha-fetoprotein, erythropoietin, and factor VIII , can be used to enhance the performance of nucleic acid molecules such as mRNA.

3'UTR is known to have a stretch of adenosine and uridine (in RNA form) or thymidine (in DNA form) embedded therein. These AU-rich markers are particularly common in genes with high conversion rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be divided into three categories (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 the AUUUA motif in the U-rich region. For example, c-Myc and MyoD mRNAs contain Class I AREs. Class II AREs have two or more overlapping UUAUUUA(U/A) (U/A) 9-mers. GM-CSF and TNF-α mRNAs are examples of containing class II AREs. Class III AREs are less well defined. These U-rich regions do not contain the AUUUA motif, and c-Jun and myogenin mRNAs are two well-studied examples of this class.

Most proteins that bind to AREs are known to destabilize the message, and members of the ELAV family, most notably HuR, have been documented to increase mRNA stability. HuR binds to all three classes of AREs. Engineering a HuR-specific binding site into the 3' UTR of a nucleic acid molecule results in HuR binding and, therefore, message stabilization in vivo.

In some embodiments, the introduction, removal or modification of the 3'UTR ARE can be used to modulate the stability of mRNA encoding an inhibitor of the ISR protein. In other embodiments, the ARE can be removed or mutated to increase intracellular stability and thus increase the translation and production of the inhibitor of the ISR protein.

In some embodiments, non-ARE sequences can be incorporated into the 5' or 3' UTR. In some embodiments, introns or portions of intronic sequences can be incorporated into the flanking regions of polynucleotides in any of the constructs, particles, polynucleotides, compositions, kits, and methods provided herein. Incorporation of intronic sequences can increase protein production and mRNA levels. e. Internal Ribosome Entry Site ( IRES )

In some embodiments, the construct encoding an inhibitor of an ISR protein may include an internal ribosome entry site (IRES). The IRES forms a complex secondary structure that allows translational initiation to occur anywhere in the mRNA immediately downstream of the location of the IRES (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). Several IRES sequences are known to those skilled in the art, including sequences from, for example, foot and mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), human rhinovirus (HRV), cricket paralysis virus, human immunodeficiency virus (HIV), hepatitis A virus (HAV), 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 is incorporated herein by reference for the purposes described herein).

In some embodiments, the IRES sequence incorporated into the construct encoding the inhibitor of the ISR protein is the foot-and-mouth disease virus (FMDV) 2A sequence. The foot-and-mouth disease virus 2A sequence is a small peptide (about 18 amino acids in length) that has been shown to mediate 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 been previously demonstrated in artificial systems including plasmids and gene therapy constructs such as 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).

In some embodiments, IRES can be used in AAV constructs. In some embodiments, a construct encoding an inhibitor of an ISR protein may include a polynucleotide internal ribosome entry site (IRES). In some embodiments, an IRES can be part of a composition containing more than one construct. In some embodiments, an IRES is used to generate more than one polypeptide from a single gene transcript. f.Splicing site

In some embodiments, any of the constructs provided herein may include splice donor and/or splice acceptor sequences that function during RNA processing that occurs during transcription. In some embodiments, the splice sites are involved in trans-splicing. g. Polyadenylation sequences

In some embodiments, constructs provided herein may include a polyadenylation (poly(A)) signal sequence. Most nascent eukaryotic mRNAs have a poly(A) tail at their 3' end that is added during a complex process that includes cleavage of the primary transcript and cleavage of the primary transcript driven by a poly(A) signal sequence. Coupled polyadenylation reactions (see, eg, Proudfoot et al., Cell 108:501-512, 2002, which is incorporated by reference for the purposes described herein). The poly(A) tail confers stability and transferability to mRNA (see, e.g., Molecular Biology of the Cell , 3rd ed. B. Alberts et al., Garland Publishing, 1994, which is incorporated by reference for the purposes described herein. into this article). In some embodiments, the poly(A) signal sequence is located 3' to the coding sequence.

As used herein, "polyadenylation" refers to the covalent linkage of a polyadenosine moiety or a modified variant thereof to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 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) that is added to the pre-mRNA by the action of an enzyme (polyadenylation polymerase). In some embodiments, the poly(A) tail is added to transcripts containing a specific sequence (e.g., a poly(A) signal). The poly(A) tail and associated proteins help protect the mRNA from exonuclease degradation. Polyadenylation also plays a role in transcriptional termination, export of mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but can also occur later in the cytoplasm. After transcriptional termination, the mRNA chain is cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, an adenosine residue is added to the free 3' end of the cleavage site.

As used herein, a "poly(A) signal sequence" or "polyadenylation signal sequence" is a sequence that triggers endonuclease cleavage of an mRNA and adds a string of adenosines to the 3' end of the cleaved mRNA.

There are several poly(A) signal sequences that may be used in some embodiments, including sequences derived from: bovine growth hormone (bGH) (Woychik et al., Proc. Natl. Acad Sci. USA 81(13):3944 -3948, 1984; U.S. Patent No. 5,122,458, each of which is incorporated by reference for the purposes described herein), mouse-beta-globulin, mouse-alpha-globulin (Orkin et al., EMBO J 4(2):453-456, 1985; Thein et al., Blood 71(2):313-319, 1988, each of which is incorporated by reference for the purposes described herein. ), human collagen, polyomavirus (Batt et al., Mol. Cell Biol. 15(9):4783-4790, 1995, which is incorporated by reference for the purposes described herein), simple Herpes virus thymidine kinase gene (HSV TK), IgG heavy chain gene polyadenylation signal (US 2006/0040354, which is incorporated by reference for the purposes described herein), human growth hormone (hGH ) (Szymanski et al., Mol Therapy 15(7):1340-1347, 2007, which is incorporated by reference for the purposes described herein), and/or by proteins such as 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). middle).

In some embodiments, the poly (A) signal sequence may be AATAAA. The AATAAA sequence may be replaced by other hexanucleotide sequences that are homologous to AATAAA and capable of emitting a polyadenylation signal, including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATTAAC, AATTAGA, AATTAAA or AATAAG (see, e.g., WO 06/12414, which is incorporated herein by reference for the purposes described herein). In some embodiments, the poly(A) signal sequence can be a synthetic polyadenylation site (see, e.g., Promega's pCl-neo expression construct 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 h. Other sequences

In some embodiments, constructs of the invention may comprise 2A elements or sequences. In some embodiments, constructs of the invention may include one or more colonization sites. In some such embodiments, the colonization site may not be completely removed prior to manufacture for administration to an individual. In some embodiments, the selection site may have a functional role, including as a linker sequence or as part of a Kozak site. Those skilled in the art will appreciate that the site of selection can vary significantly in the primary sequence while maintaining its desired function.

In some embodiments, the 2A element is a T2A, P2A, E2A and/or F2A element. In some embodiments, the 2A sequence may include 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 SEQ ID NO: 30 - Exemplary P2A amino acid sequence SEQ ID NO: 31 - Exemplary E2A amino acid sequence SEQ ID NO: 32 - Exemplary F2A Amino Acid Sequence 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 i. Destabilize the domain

In some embodiments, any of the constructs provided herein may optionally include a sequence encoding a destabilizing domain ("destabilizing sequence") for temporal and/or spatial control of protein expression. Non-limiting examples of destabilizing sequences include sequences encoding FK506 sequences, dihydrofolate reductase (DHFR) sequences, or other exemplary destabilizing sequences.

In the absence of a stabilizing ligand, a protein sequence operably 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 operably linked to a destabilizing sequence to be actively expressed. As a positive control for stabilization of protein expression, protein expression can be detected by known methods including: enzyme assays, radiographic assays, colorimetric assays, fluorescent or other spectroscopic assays, fluorescent activating cell sorting (FACS) assays, and/or immunoassays (e.g., enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and immunohistochemistry).

Other examples of destabilizing sequences are known in the art. In some embodiments, the destabilizing sequences are FK506 binding protein (FKBP12) and rapamycin binding protein sequences, and the stabilizing ligand is Shield-I (Shld1) (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, the destabilizing sequence is a DHFR sequence and the stabilizing ligand is trimethoprim (TMP) (see, eg, Iwamoto et al., (2010) Chem Biol 17:981-988, found herein (which is incorporated herein by reference for the purposes described in ). j. Reporter sequence or element

In some embodiments, constructs provided herein optionally include sequences encoding reporter polypeptides and/or proteins ("reporter sequences"). Non-limiting examples of reporter sequences include DNA sequences encoding: beta-lactamase, beta-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), red fluorescent protein Photoprotein, mCherry fluorescent protein, yellow fluorescent protein, chloramphenicol acetyltransferase (CAT) and luciferase. Other examples of reporter sequences are known in the art. When the reporter sequence is associated with a control element that drives its expression, the reporter sequence can provide a signal that is detected by conventional methods including: enzymatic assays, radiographic assays, colorimetric assays, fluorescent assays method or other spectroscopic analysis methods, fluorescence-activated cell sorting (FACS) analysis methods and/or immunoassay methods (such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry).

In some embodiments, the reporter sequence is a Lacz gene, and the presence of a construct carrying the Lacz gene in a cell is detected by an assay for β-galactosidase activity. In some embodiments, the reporter sequence is a fluorescent protein (e.g., green fluorescent protein (GFP)) or luciferase. In embodiments in which the reporter sequence is a fluorescent protein or luciferase, the presence of a construct carrying a fluorescent protein or luciferase in a cell can be measured by light generation in a fluorescent imaging technique (e.g., fluorescent microscopy or FACS) or a luminometer (e.g., a spectrophotometer or IVIS imaging instrument). In some embodiments, the reporter sequence can be used to verify the tissue-specific targeting ability and/or tissue-specific promoter regulation and/or control activity of any of the constructs described herein. An exemplary GFP tag sequence is provided as SEQ ID NO: 37.

In some embodiments, the reporter sequence is a FLAG tag (e.g., 3xFLAG tag) and is detected by protein binding or detection assays (e.g., Western blotting, immunohistochemistry, radioimmunoassay (RIA), mass spectrometry) to detect the presence of FLAG-tagged constructs in cells. An exemplary 3xFLAG tag sequence is provided as SEQ ID NO: 36. SEQ ID NO: 36 - Exemplary 3xFLAG tag polynucleotide sequence SEQ ID NO: 37 - Exemplary GFP polynucleotide sequence IV. Administration of constructs and / or compositions

In some embodiments, the constructs, particles, polypeptides, polynucleotides and/or compositions described herein may be included in formulations with one or more additional therapeutic agents.

In some embodiments, the constructs, particles, polypeptides, polynucleotides and/or compositions described herein may be included in a formulation, wherein the formulation comprises a pharmaceutically acceptable excipient.

In some embodiments, a construct, particle, polypeptide, polynucleotide, and/or composition described herein is administered to a subject in need thereof. In some embodiments, the subject may have, may be diagnosed with, or may be susceptible to developing Down syndrome (DS), Charcot-Marie-Duchesne disease, major depressive disorder (MDD), schizophrenia, Alzheimer's disease, Huntington's 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 some embodiments, the individual is a mammal. In some embodiments, the individual is a livestock animal. In some embodiments, the individual is a farm animal. In some embodiments, the individual is a zoo animal. In some embodiments, the individual is a dog or cat. In some embodiments, the individual is a cow, horse, sheep, or goat. In some embodiments, the individual may be, but is not limited to, a dog, cat, ferret, rabbit, cow, duck, pig, goat, chicken, horse, llama, camel, ostrich, deer, turkey, pigeon, Sheep, geese, bulls and/or reindeer. In some embodiments, the individual is a human. In some embodiments, the individual is equal to, less than, or older than 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 old.

In some embodiments, the constructs, particles, polypeptides, polynucleotides and/or compositions described herein according to the present invention can be administered by intraspinal and/or intracerebral administration. In some embodiments, intraspinal administration comprises or consists of intrathecal and epidural administration.

In some embodiments, constructs, particles, polypeptides, polynucleotides, and/or compositions described herein are administered intracerebrately. In some embodiments, intracerebral administration is performed at a site selected from the group consisting of or consisting of: the hippocampus and/or hippocampus (such as, for example, cornu ammonis (e.g., CA1, CA2, and/or CA3), subiculum complex, entorhinal cortex and/or dentate gyrus), striatum (such as, for example, putamen, caudate nucleus, nucleus accumbens, Olfactory tubercle, external globus pallidus and/or internal globus pallidus), thalamus, hypothalamus, epithalamus, subthalamic base, parenchyma, cerebrum, medulla, deep cerebellar nuclei (such as, for example, substantia nigra ), dentate nucleus, embolus nucleus, globular nucleus and/or fastigii nucleus), cerebrospinal fluid (CSF), meninges, dura mater, arachnoid mater, pia mater, subarachnoid cisterns ( Such as, for example, cisterna magna, pontine cistern, interpeduncular cistern, chiasmatic cistern, cistern of lateral cerebral fossa, superior cistern and /or cistern of lamina terminalis), subarachnoid space, cortex, septum, pons and cerebellum.

In some embodiments, the constructs, particles, polypeptides, polynucleotides, and/or compositions described herein pass intrahippocampally (e.g., in the hippocampus, e.g., CA1, CA2, and/or CA3), intrastriatum (e.g., CA1, CA2, and/or CA3), For example, in the striatum, such as in the putamen, caudate nucleus, nucleus basalis, olfactory tubercle, external globus pallidus and/or internal globus pallidus), within the thalamus (e.g. in the thalamus) and/or within the cisterns (e.g. in the subarachnoid cistern) , such as cistern magna, pontine cistern, interpeduncular cistern, interdigital cistern, lateral cerebral fossa cistern, superior cistern and/or thalamic terminal plate cistern).

In some embodiments, the constructs, particles, polypeptides, polynucleotides and/or compositions described herein are administered intraventricularly, intrahippocampally, intraparenchymally, intrastriatically, intrathalamically, intracisternalally and/or intrathecally.

In some embodiments, a treatment regimen comprising a construct, particle, polypeptide, polynucleotide, and/or composition described herein may comprise administering 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 therapies may be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the first and second therapies are administered in separate compositions. In some embodiments, the first and second therapies are in the same composition.

In some embodiments, a treatment regimen comprising a construct, particle, polypeptide, polynucleotide and/or composition described herein comprises administering more than one composition, such as 2 compositions, 3 compositions, 4 compositions, or more than 4 compositions. Various combinations of agents may be used. In some embodiments, the therapeutic agents of the invention may be administered by the same route of administration or by different routes of administration. In some embodiments, a construct, particle, polypeptide, polynucleotide and/or composition described herein and/or an additional therapeutic agent is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the 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, the appropriate dosage can be determined based on the type of disease being treated, the severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to treatment, and the judgment of the attending physician.

In some embodiments, treatment may include various "unit doses." A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amounts administered, as well as the specific routes and preparations administered, are within the judgment of a person skilled in the clinical skill. The unit dose need not be administered as a single injection, but may consist of a continuous infusion over a set period of time. In some embodiments, unit dosages comprise doses that can be administered in a single sitting.

In some embodiments, the amount administered is determined by the desired therapeutic effect, both in terms of treatment number and unit dose. It is understood that an effective dose refers to the amount required to achieve a specific effect. In the practice of certain embodiments, it is contemplated that doses in the range of 0.10 mg/kg to 200 mg/kg may achieve the protective capabilities of these agents. In certain embodiments, it is contemplated that a dosage may comprise a composition containing AAV particles at a concentration of from about ¹⁰⁸ to about ¹⁰¹⁴ viral genomes per milliliter. Furthermore, such doses may be administered multiple times throughout the day, and/or over days, weeks, or months.

In certain embodiments, the exact amount of the therapeutic composition is also determined by the judgment of the practitioner and is unique to each individual. Factors affecting dosage include the patient's physiological and clinical state, route of administration, desired therapeutic goal (relief of symptoms versus cure), and the efficacy, stability, and toxicity of the specific therapeutic substance or other therapies the individual may be undergoing.

It is also understood that uptake is species and organ/tissue dependent. Applicable conversion factors and the physiological assumptions made regarding uptake and concentration measurements are well known and will permit one skilled in the art to convert one concentration measurement to another and make decisions regarding dosage, Reasonable comparison of efficacy and results and drawing conclusions.

In some cases, it will be necessary to administer the composition multiple times, such as 2, 3, 4, 5, 6 or more. Administrations may be administered at intervals of 1, 2, 3, 4, 5, 6, 7, 8 to 5, 6, 7, 8, 9, 10, 11, 12 weeks, or more than 12 weeks, including all ranges therebetween.

The phrase "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce harmful, allergic or other adverse reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antimicrobial and antifungal 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. Unless any conventional media or agent is incompatible with the active ingredient, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infectious agents and vaccines, can also be incorporated into the compositions.

The active compounds may be formulated for parenteral administration, for example, for injection via the intravenous, intramuscular, subcutaneous or intraperitoneal route. Generally, such compositions may be prepared in the form of liquid solutions or suspensions; solid forms suitable for preparation of solutions or suspensions after the addition of liquid prior to injection may be prepared; and the formulations may also be emulsified.

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 can be easily syred. It should also be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.

In some embodiments, when the composition is a protein, the protein composition can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed from free amine groups of the protein) and are formed from inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid and the like). Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or iron hydroxide; and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like.

In some embodiments, the pharmaceutical composition may include a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. For example, proper fluidity may be maintained by the use of a coating such as lecithin, by the maintenance of the desired particle size in the case of dispersions, and by the use of a surfactant. Prevention of microbial activity may be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like). In many cases, it will be preferred to include an isotonic agent, such as a sugar or sodium chloride. Prolonged absorption of the injectable composition may be achieved by the use in the composition of an agent that delays absorption, such as aluminum monostearate and gelatin.

In some embodiments, sterile injectable solutions are prepared by incorporating the active compound in the required amount in an appropriate solvent, along with various other ingredients enumerated above, as appropriate, followed by filtered sterilization or equivalent procedures. Generally, dispersions are prepared by incorporating the various sterilizing 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 other desired ingredients from its previously sterile-filtered solution.

In some embodiments, after formulation, the compositions described herein can be administered in a manner compatible with the dosage form and in such amounts as to be effective therapeutically or prophylactically. In some embodiments, the formulations are administered in various dosage forms, such as the injectable solutions described above. A. Pharmaceutical Compositions

In certain aspects, the constructs, particles, polypeptides, polynucleotides and/or compositions or agents used in methods such as delaying the onset of, treating, slowing the progression of, reducing the risk of, and/or preventing a cognitive disorder in a subject (e.g., a human subject) are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible, and selected so as not to adversely affect the biological activity of the agent. In some aspects of the invention, the medicament can be formulated for local delivery (i.e., to a specific location in the body, such as brain tissue or other tissues) or systemic delivery in solid, semisolid, gel, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, depots, inhalants, and injections that allow oral, parenteral or surgical administration. Certain aspects of the invention also encompass local administration of the composition by coating medical devices and the like.

In some embodiments, carriers suitable for parenteral and topical delivery by injection, infusion or irrigation include distilled water, phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propylene glycol. In addition, sterile, nonvolatile oils can be used as solvents or suspension media. For this purpose, any biocompatible oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injectables. The carrier and the agent can be compounded into a liquid, a suspension, a polymerizable or non-polymerizable gel, a paste, or an ointment.

In certain embodiments, a carrier may also include a delivery vehicle that maintains (i.e., prolongs, delays, or modulates) delivery of the agent, or enhances the delivery, uptake, stability, or pharmacokinetics of the therapeutic agent. As non-limiting examples, such delivery vehicles may include microparticles, microspheres, nanospheres, or nanoparticles comprising proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymerized hydrogels and aggregated microcells.

In certain aspects, the actual dosage of the composition administered to a patient or individual can be determined by physical and physiological factors such as body weight, severity of condition, type of disease being treated, previous or concurrent therapeutic interventions, patient idiopathic conditions, and route of administration. The practitioner responsible for administration will in any case determine the concentration of the active ingredient in the composition and the dosage appropriate for an individual individual.

In some embodiments, solutions of pharmaceutical compositions may be prepared in water, suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and in oils. Under ordinary storage and use conditions, these preparations contain a preservative to prevent the growth of microorganisms.

In some aspects, the pharmaceutical compositions are preferably administered as injectable compositions as liquid solutions or suspensions; suitable solid forms or solutions or suspensions in liquids may also be prepared prior to injection. These preparations can also be emulsified. Typical compositions for such purposes include pharmaceutically acceptable carriers. For example, 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.

In some embodiments, non-limiting examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, such as ethyl oleate. 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 fluids and nutritional supplements. Preservatives include antimicrobials, antifungals, antioxidants, chelating agents and inert gases. The pH and exact concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

In certain embodiments, formulations and/or co-administered formulations comprising the constructs described herein may be suitable for oral administration. In some embodiments, oral formulations include typical excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. The composition is in the form of a solution, suspension, tablet, pill, capsule, sustained release formulation, or powder.

The effective amount of the pharmaceutical composition is determined based on the intended goal. The term "unit dose" or "dose" refers to a physically discrete unit suitable for use in an individual, each unit containing a predetermined amount of the pharmaceutical composition calculated to produce the desired response discussed above in connection with its administration (i.e., appropriate route and treatment regimen). The amount administered depends on the protection or effect desired, based on both the number of treatments and the unit dose.

The exact amount of the pharmaceutical composition also depends on the judgment of the practitioner and is peculiar to each individual. Factors affecting dosage include the patient's physical and clinical condition, the route of administration, the desired therapeutic goal (e.g., relief of symptoms versus cure), and the potency, stability, and toxicity of the particular therapeutic substance.

The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventor to function well in the practice of the invention, and therefore may be considered to constitute preferred modes for its practice. However, in light of the present invention, those skilled in the art will appreciate that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention. Materials and methods -

Unless otherwise indicated, the experiments and procedures described herein were performed as follows. Generation of Ppp1r15b ^R658C mice

Microinjection with specific gRNA (e.g., AGTGGTGATGAGGATCGCAA AGG (SEQ ID NO: 38); PAM sequence is underlined and not required to be included in the gRNA sequence, (Synthego)), Cas9 mRNA (Sigma), and donor DNA containing the R658C mutation Mouse iPSCs. Donor DNA was 500 bp in length and, in addition to point mutations of interest, silent mutations were introduced to prevent the mutants from editing back to WT. The donor also contains approximately 250 bp of flanking mutation regions to facilitate homologous recombination. Edited cells were implanted into replacement females and pups were genotyped by sequencing of the loci. These steps are performed at core facilities at Baylor College of Medicine (BCM). Puppies carrying the desired mutation are bred and phenotypically tested. cell-based assays

For stress experiments, stable cells resulting from lentiviral transduction were generated and selected with puromycin using standard transduction procedures. Next, cells were treated with 1 μM oligomycin, 100 nM thapsigargin, or transfected with 1 μg of poly(IC) using Lipofectamine 2000 for three hours, and then harvested. Lysates were prepared and standardized prior to analysis of designated proteins by SDS-PAGE and western blotting. Immunoprecipitation

Cells were transfected with equivalent amounts of DNA plasmids expressing the indicated GFP-tagged genes and DP71L/ΔCREP chimeras using standard procedures. 16 hours after transfection, 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 a magnetic GFP capture resin (Proteintech Group) by rolling overnight at 4°C. Complexes were separated from the crude lysate using a magnet and washed three times with ice-cold lysis buffer. Complexes were eluted from the resin with 1X Laemmli buffer and analyzed by SDS-PAGE and Western blotting. Adeno-associated virus ( AAV )

Adeno-associated viruses were produced by colonizing the indicated sequences into the AAV2 plasmid backbone. The DNA was then purified and delivered to a core facility to produce AAV serotyped with the neurotropic DJ/8. AAV was purified using a commercially available kit (Cell Biolabs) and quantified by a qPCR-based assay. Typically, approximately 10 ¹² viral genomes per ml were injected into each hippocampus in both the CA1 and CA3 regions (hippocampal subfields 1 and 3) of mice. AAV was allowed to express the transgene for at least 14 days before behavioral experiments began. Subsequently, mouse brains were excised and samples were prepared for SDS-PAGE and Western blotting. Behavioral tests

Specific procedures were performed as previously disclosed by us (see, e.g., Zhu et al., 2019). Briefly, mice were handled for 5 min for three consecutive days to acclimate them to the experimenter. Subsequently, mice were placed in a conditioning chamber to acclimate for 20 min every two days. Mice were then trained with aversive foot shock. Two training shocks were administered at 0.7 mA, each separated by 90 s, with freezing recorded between and after the training shocks. Finally, 24 h after training, mice were placed in the same chamber for 5 min and the percentage of time spent in freezing was recorded using Freezeview software. Object discrimination was performed as previously described by us (see, e.g., Zhu et al., 2019) with only slight modifications. Briefly, mice were treated for 5 days (5 to 10 minutes per day) and then acclimated to a black plastic glass rectangular chamber (31×24 cm, height 27 cm) under dim ambient light for 10 minutes for 5 consecutive days. Two identical objects were presented to the mice for exploration for 5 minutes, after which the mice were returned to the cage. Twenty-four hours later, one object was replaced with a novel object and the mice were placed in the chamber again for 5 minutes. The novel objects had the same height and volume, but had different shapes and appearances. Exploration of the object was defined as sniffing the object within 2 cm of the radius of the object (nose touching or head pointing to the object). Sitting or standing on the object was not scored as exploration. Behavior was recorded from a camera placed above the training chamber. The discrimination index (DI) was calculated as DI = (novel object exploration time - familiar object exploration time / total exploration time) × 100. To control for odor cues, the open space arena and objects were thoroughly cleaned with ethanol, dried, and ventilated between mice. Electrophysiology

Electrophysiological recordings were performed as previously described (see e.g. Zhu et al., 2019). Field recordings were made from CA1 level hippocampal sections (320 μm thick) prepared from artificial cerebrospinal fluid solution (ACSF) at 4°C using a vibratome (Leica VT 1000S, Leica Microsystems, Buffalo Grove, IL); The brains of adult mice (3 to 6 months old) were cut in 95% O2 and 5% CO2) (2 to 3 ml/min). The artificial cerebrospinal fluid solution contained in mM: 124 NaCl, 2.0 KCl, 1.3 MgSO4, 2.5 CaCl2, 1.2 KH2PO4, 25 NaHCO3 and 10 glucose. Prior to recording, sections were incubated in the interface chamber for at least 60 minutes and continuously perfused with artificial cerebrospinal fluid (ACSF) at a flow rate of 2 to 3 ml/min at 28 to 29°C. The recording electrode is placed in the stratum radiatum. Field excitatory postsynaptic potentials (fEPSP) were recorded using ACSF-filled micropipette, and these potentials were induced by bipolar stimulating electrodes placed in the actinographic layer of CA1 to excite Schaffer collateral fibers and commissure fibers. . Adjust the intensity of the 0.1-ms pulse to elicit 30% to 35% of the maximum response. Establish a stable response baseline at 0.033 Hz for at least 30 minutes. Tetanic LTP was induced by applying four high-frequency stimulation sessions (100 Hz, 1 s) separated by 5 min. Whole-cell recordings were performed using a MultiClamp 700B amplifier (Molecular Devices, Union City, CA) using conventional patch-clamp technique in an immersion chamber (2 to 3 ml/min) at 31 to 32°C. CA1 neurons were visually identified by infrared differential interference contrast video microscopy on the stage of an upright microscope (Axioskope FS2, Carl Zeiss, Oberkochen, Germany). Diaphragm pipettes (resistance 2 to 5 MW) filled with (in mM): 140 CsCl, 10 HEPES, 10 Na2-creatine phosphate, 0.2 BAPTA, 2 Mg3-ATP, 0.2 Na3-GTP; use Wescor 5500 vapor A pressure osmometer (Wescor, Logan, UT) adjusted the pH to 7.2 and the osmolality to 295 to 300 mOsm. In the presence of NBQX (5 μM, 2,3-dihydroxy-6-nitro-7-amidosulfonyl-benzo[f]quinolin disodium salt), AP5 (25 μM, DL-2-amino In the presence of -5-phosphinopentanoic acid) and TTX (1 μM, tetrodotoxin), microinhibitory postsynaptic currents at -60 mV were recorded. To record excitatory currents, CsCl was replaced with 130 mM K-gluconate and 10 mM KCl. In the presence of 100 μM picrotoxin, the excitatory postsynaptic current (EPSC) was recorded at -70 mV. All drugs were obtained from Tocris (Ellisville, MO). Polysome analysis and RNA isolation

Polysome analysis followed by RNA sequencing was performed as previously described (see, e.g., Hinnebusch et al., 2016). Briefly, fresh 12 ml of a 10% to 50% sucrose density gradient [10 mM HEPESKOH (pH 7.6), 5 mM MgCl , 150 mM KCl, 200 U/ml RNasin RNase inhibitor (Promega, Madison, WI)]. Keep the gradient at 4°C for at least 2 hours before use. Mouse brain tissue was cultured in cutting solution [1X HBSS, 2.5 mM HEPES-KOH (pH 7.6), 35 mM glucose, 4 mM NaHCO3, 100 μg/ml cycloheximide (Sigma-Aldrich, St. Louis, MO) ] and washed by centrifugation at 3000 rpm for 10 min at 4°C in ice-cold PBS containing 100 μg/ml cycloheximide. Next, the tissue was lysed in polysome lysis buffer [10 mM HEPES-KOH (pH 7.4), 5 mM MgCl2, 150 mM KCl, 0.5 mM DTT, 100 U/ml RNasin RNase inhibitor (Promega, Madison, WI ), 100 μg/ml cycloheximide, and EDTA-free protease inhibitor (Roche Indianapolis, IN)] and centrifuged at 2000 × g for 10 min at 4°C. The supernatant was then transferred to pre-cooled quench tubes supplemented with 0.5% NP-40 and kept on ice for 10 minutes. Centrifuge the sample at 14,000 rpm for 10 minutes at 4°C. The supernatant was layered onto a sucrose gradient or retained for total RNA isolation. Centrifuge the gradient at 35,000 rpm for 2 hours at 4°C in a SW-40Ti rotor and then pass 70% sucrose through the bottom of the tube by piercing the tube with a Brandel tube piercer and using an ISCO UA-6 UV detector Analyze by monitoring the absorbance of material eluted from the tube. Fractions were collected throughout the process and RNA was extracted with TRIzol according to the manufacturer's instructions (Life Technologies, Carlsbad, CA). Experiments were performed in three biological replicates of each group. Translated Surface Sensing ( SUnSET )

Protein synthesis was measured using SUnSET, a non-radioactive labeling method for monitoring protein synthesis, as previously described (see, e.g., Zhu et al., 2019). Briefly, hippocampal sections (300 μm) were cut with a McIlwain tissue microtome (Mickle, UK) and incubated in oxygen (95% O2, Incubation in 5% CO2) ACSF for 1 hour was followed by incubation in oxygenated (95% O2, 5% CO2) ACSF for 1 hour at 32°C before treatment. Puromycin (10 μg/μl, dissolved in oxygenated ACSF) was applied to the sections as a bath for 20 minutes, followed by washing with untreated oxygenated ACSF. Sections were then snap frozen on dry ice and stored at -80°C until use. Dissolve frozen sections in homogenization buffer (in mM: 40 Tris HCl, pH 8.0, 150 NaCl, 25 b-glycerophosphate, 50 NaF, 2 Na3VO3, 1X protease inhibitor cocktail, 10% glycerol, 1% Triton X-100). Puromycin was detected by Western blotting using the 12D10 antibody against puromycin (cat. no. MABE343, 1:5000, EMD Millipore Corp, Darmstadt, Germany) as previously described (see e.g. Zhu et al., 2019). Elements are incorporated. The density of the resulting bands was quantified using ImageJ and statistical significance assessed by Student's t-test. Example 1 - Generation and characterization of Ppp1r15b ^R658C mice . Ppp1r15b ^R658C mice exhibit abnormal translational control and an active ISR .

Generation of a new mouse model ( Ppp1r15b ^R658C mice) carrying a selective mutation (R658C) in PPP1R15B that 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 et al., 2016). Briefly, CRISPR was used to generate mice using guide RNA targeting the PPP1R15B region, which is very close to the base position encoding the R658C mutation. Briefly, donor DNA containing the R658C mutation, Cas9 mRNA, and guide RNA were co-injected into embryos, which were subsequently implanted into replacement mice. Four pups were identified with homotypic combination targeting of the desired loci ( Fig. 2A ). Mice were bred and mutants and littermate controls were obtained. Consistent with the microcephaly observed in individuals carrying isotypic combination mutations (see, e.g., Abdulkarim et al., 2015; and Kernohan et al., 2015), Ppp1r15bR658C mice were also found to exhibit slightly smaller brains ( Figure 2B ). In humans, the R658C mutation inhibits the activity of the CReP·PP1 phosphatase complex, causing an increase in eIF2-P content (see, e.g., Abdulkarim et al., 2015; and Kernohan et al., 2015). Consistent with human data, CReP content was found to be unchanged ( Figure 2C ), but eIF2-P content was found to be increased in the brain ( Figure 2D ) and primary fibroblasts ( Figure 2E ) of Ppp1r15b ^R658C mice. Therefore, the ISR is selectively disrupted in Ppp1r15b ^R658C mice.

Since activation of the ISR inhibits general translation (see e.g. Costa-Mattioli et al., 2009), we next investigated whether polyribosome deposition in sucrose gradients reduces the overall protein synthesis rate in Ppp1r15b ^R658C mice, as recently described (see e.g. Zhu et al., 2019). In this technique, the position of a given mRNA in a sucrose gradient is determined by the number of associated ribosomes. Under-translated or untranslated mRNA accumulated near the top, while translationally active mRNA was associated with multiple ribosomes (polysomes) and sedimented to the bottom of the gradient ( Figure 2F ). The results showed that general translation was inhibited in the brains of Ppp1r15b ^R658C mice, as indicated by a decrease in polyribosomes and a corresponding increase in monoribosomes ( Figure 2G to Figure 2H ), which was consistent with increased eIF2-P content ( See, for example, Costa-Mattioli et al., 2007; and Harding et al., 2000). Furthermore, when the inventors measured protein synthesis in vivo by assessing puromycin incorporation into the nascent polypeptide chain (see, e.g., Zhu et al., 2019; and Di Prisco et al., 2014), it was found that incorporation of puromycin from Ppp1r15b Puromycin incorporation was reduced in fibroblasts from ^R658C mice ( Figure 2I to Figure 2J ). Therefore, when the ISR is activated in the brain of Ppp1r15b ^R658C mice, the rate of protein synthesis is reduced. Long-term memory is impaired in Ppp1r15bR658C mice.

The inventors and others have demonstrated 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 Ppp1r15b ^R658C mice selectively activate the ISR, the inventors next determined whether these mice had long-term memory impairment. To this end, hippocampal-dependent contextual fear memory was examined, in which the context (conditioned stimulus; CS) was paired with a foot shock (unconditioned stimulus; US). Twenty-four hours after training, mice were exposed to the CS and the fear response ("freezing behavior") was measured as an index of the strength of their long-term memory ( Figure 3A ). The data showed that before training, freezing ^was similar in Ppp1r15bR658C mice and untreated control littermates, but 24 hours after training, Ppp1r15bR658C ^mice displayed a significant reduction in freezing, indicating impairment of long-term memory ( Figure 3B ).

In addition, long-term object recognition memory was assessed, which is also dependent on the hippocampus (see, e.g., Vann et al., 2011). In the long-term object recognition memory task, animals need to distinguish between familiar and novel objects. Wild-type (WT) mice are known to preferentially explore novel objects. During training, two identical objects were placed in a box and mice were allowed to explore the objects ( Figure 3C ). The data showed that Ppp1r15bR658C and WT ^littermates spent an average of equal time investigating the objects during training ( Figure 3D ). However, when the novel objects were replaced 24 hours later, Ppp1r15bR658C ^mice showed reduced object discrimination, indicating a deficit in their long-term object recognition memory ( Figure 3E ). Therefore, Ppp1r15b ^R658C mice have impaired long-term memory. Ppp1r15b ^R658C mice have impaired long-term potentiation.

The inventors measured long-term potentiation (LTP) in Ppp1r15b ^R658C mice, a cellular model of memory formation (see, eg, Neves et al., 2008). In hippocampal slices, four training sessions with 100 Hz stimulation induce long-lasting L-LTP that is dependent on new protein synthesis (see, e.g., Costa-Mattioli et al., 2009; Kandel et al., 2001; and Kelleher et al., 2004). Whereas the present inventors and others have demonstrated that the ISR regulates L-LTP bidirectionally: that is, activation of the ISR impairs L-LTP, whereas inhibition of the ISR promotes L-LTP (see, e.g., Costa-Mattioli et al., 2007; Zhu et al., 2011; Jiang et al., 2010; and Huang et al., 2016), and immediate results showed that the ISR was selectively activated in Ppp1r15b ^R658C mice, the inventors wanted to know whether L-LTP was impaired in these mice. Indeed, consistent with the field, four training sessions at 100 Hz induced sustained L-LTP in WT slices ( Fig. 3F ). However, the same stimulation protocol failed to do so in Ppp1r15b ^R658C sections ( Fig. 3F ). Notably, the basal synapse, as measured by input-output plots of field excitatory potentials as a function of presynaptic fiber volley and paired pulse facilitation, Contact conduction was similar in WT and Ppp1r15b ^R658C sections (data not shown). Therefore, long-term potentiation is impaired in Ppp1r15b ^R658C mice. Example 2 - Inhibition of the Integrated Stress Response ( ISR ) Viral strategies to inhibit the ISR .

Since modulation of eIF2-P phosphatase may prove to be an effective mechanism for modulating ISR activity, the inventors exploited a strategy used by viruses to inhibit ISR. Given that increased eIF2-P (i.e., ISR activation) impairs both cellular and viral protein synthesis, many viruses have evolved mechanisms to inactivate the ISR (see, eg, Garcia et al., 2007). In fact, for example, the mammalian homologs GADD34 and CReP, γ34.5 protein from herpes simplex virus, DP71L protein from African swine fever virus, CNPV231 protein from canarypox virus (CNPV), kangaroo herpesvirus (MaHV) ), and the AmEPV193 protein from Entomopoxvirus "L" (AmEPV), all of which are believed to recruit PP1 to dephosphorylate eIF2 (see, e.g., Rojas et al., 2015) ( Figure 4A ). In yeast, these proteins inhibit ISR by reducing eIF2-P (see, e.g., Rojas et al., 2015). However, A) the inventors know that yeast genomes do not naturally encode eIF2-phosphatase cofactor orthologs that inhibit ISR (e.g., GADD34 or CreP-like proteins), and B) PP1 is recruited in yeast to The mechanism of eIF2 recruitment is different from that in human cells (see, e.g., Rojas et al., Protein phosphatase PP1/GLC7 interaction domain in yeast eIF2γ bypasses targeting subunit requirement for eIF2α dephosphorylation. PNAS , 2014, 111(14) E1344-E1353; out of are incorporated herein by reference for the purposes described herein). Until now, it was unclear whether these proteins would function in a similar manner in human cells. Furthermore, to date, it is unclear whether viral proteins (eg, DP71L) can inhibit the ISR, thereby reducing, treating, preventing, and/or reversing cognitive dysfunction and/or other diseases and conditions associated with ISR activation.

Here, the inventors first focused on DP71L because it is the smallest protein (62 amino acids) of the family containing both PP1 and eIF2 binding domains ( Figure 4A ). The inventors first examined whether DP71L could inhibit ISR activation in human cells induced by oligomycin, which is known to activate the ISR kinase HRI. As expected, oligomycin induced ISR in GFP-transfected HEK293 cells as measured by increases in eIF2-P. In contrast, expression of DP71L-GFP completely prevented oligomycin-induced ISR activation ( Fig. 4B ). Similarly, DP71L-GFP inhibits ISR activation induced by poly(I:C) and thapsigargin (thap), two well-known activators of ISR kinases PKR and PERK, respectively. Thus, DP71L is a potent pan-inhibitor of ISR in human cells ( Figure 4C - 4D ) and can be used to inhibit ISR activation in human cells regardless of the activated ISR branch. Molecular characterization of DP71L function.

To elucidate the underlying mechanism of DP71L's efficacy in ISR inhibition, the inventors first compared the effect of DP71L with that of a truncated CReP, which is the same length as DP71L and contains PP1 and eIF2 binding motifs (ΔCREP, Figure 5A ; for example SEQ ID NO: 6). After thapsigargin induced ISR, the inventors found that DP71L-GFP was a more potent inhibitor of ISR when compared to ΔCREP-GFP ( Figure 5B ). DP71L is a scaffold protein that bridges the phosphatase PP1 with its target eIF2, thus making the phosphatase specific for its target. Based on this, the inventors hypothesized that the enhanced ISR inhibitory properties of DP71L could be attributed to increased binding to PP1, eIF2, or both proteins. To test this hypothesis, the inventors compared the binding of ΔCREP-GFP or DP71L-GFP to endogenous PP1 and eIF2 one by one. Although both ΔCREP-GFP and DP71L-GFP bind eIF2 similarly, DP71L-GFP was shown to bind to PP1 more strongly than ΔCREP-GFP ( Figure 5C ), indicating that DP71L can bind to other PP1- and eIF2-binding proteins of similar size. Demonstrates increased PP1 binding.

To identify the protein region that distinguishes DP71L-GFP from ΔCREP-GFP, the inventors generated different chimeric proteins containing various portions of CREP and DP71L ( Figure 6A ). First, the mid-terminal and C-terminal parts of DP71L were replaced with ΔCREP-GFP [C8 only contains the N-terminal (PP1 binding) motif of ΔCREP]. As noted above, full-length DP71L-GFP was observed to bind to PP1 more strongly than ΔCREP-GFP ( Fig. 6B ). Interestingly, C8 showed strong binding to PP1, similar to full-length DP71L. Subsequently, the N-terminus of ΔCREP-GFP containing both the PP1 binding domain and the linker region (eg, the region between the PP1 binding domain and the eIF2 binding domain) was fused to the C-terminus of DP71L (eg, the eIF2 binding domain). This construct, C20, exhibited reduced PP1 binding compared to DP71L ( Fig. 6B ). Finally, the N-terminus of DP71L, including the PP1 binding motif and linker region, was added to ΔCREP-GFP to generate D20, and this clone exhibited improved binding to PP1 when compared to ΔCREP-GFP ( Fig. 6B ). Therefore, the data indicate that the linker region between the PP1 binding domain and the eIF2 binding domain can quantitatively contribute to the binding efficiency of DP71L or ΔCREP-GFP to PP1. To further test this observation, the inventors only exchanged the linker regions of DP71L and ΔCREP-GFP ( Figure 6C ). Based on earlier observations, ΔCREP-GFP containing a linker for DP71 (eg, "DP71-linker") binds to PP1 more strongly when compared to ΔCREP-GFP. In contrast, adding a linker of ΔCREP-GFP (eg, "ΔCREP-linker") to DP71L resulted in reduced binding of the chimeric protein to PP1 when compared to WT DP71L ( Figure 6D ). These results are surprising and unexpected because it was not previously known that the linker region might contribute to binding to PP1.

The linker of DP71L contains 12 amino acids, while the linker of ΔCREP contains 13 amino acids. The α-fold prediction of the two protein structures together with PP1 and the N-terminal domain (NTD) of eIF2α ( Figure 7A to Figure 7B ) shows that DP71L contains glutamine (tyrosine in ΔCREP) that is predicted to form a new hydrogen bond with PP1. . Given that hydrogen bonding is known to stabilize protein-protein interactions/structure and molecular recognition, the inventors hypothesized that mutation of the glutamic acid on DP71L would disrupt PP1 binding. Consistent with this hypothesis, mutating glutamate (E12) and replacing glutamate with tyrosine significantly reduced DP71L binding to PP1 ( Fig. 7C ). Finally, computational analysis revealed that there are >440 proteins of varying sizes containing both PP1 and eIF2 binding domains in the animal kingdom (e.g. including viruses directed against these animals) (e.g. a subset of which are presented in Table 1 herein). Notably, DP71L appears to be the shortest protein identified and is the only protein on this list that contains glutamic acid in this position. Example 3 - Reversal of cognitive deficits associated with ISR DP71L : A novel gene therapy approach to reverse cognitive deficits in mouse models of Down syndrome and Alzheimer's disease.

Tuning down the ISR is emerging as a promising approach to reversing cognitive dysfunction in various memory disorders. Development of small molecule inhibitors of some ISR kinases (see, e.g., Halliday et al., 2015; Nakamura et al., 2018; Axten et al., 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) provide valuable tools for understanding the cellular functions of these kinases and their related targets. Regrettably, however, the therapeutic promise of these small molecule inhibitors remains largely unrealized due to their relative lack of specificity (see e.g. Rojas-Rivera et al., 2017) and significant toxic effects, such as This is hampered by pancreatic toxicity associated with PERK inhibitors (see, e.g., Yu et al., 2015). To date, ISRIB is the best characterized ISR inhibitor. Biochemical, genetic, and structural results show that ISRIB binds directly 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 only moderately active ISRs (see, e.g., Rabouw et al., 2019). That is, ISRIB is unable to inhibit the ISR when it is strongly activated (see, e.g., Halliday et al., 2015; and Rabouw et al., 2019). In addition, ISRIB has very poor solubility, which significantly hinders its ability to penetrate into the brain across the blood-brain barrier and thus hinders its development as a drug. Therefore, there is a great need for new, more specific and/or more potent ISR inhibitors. In view of this need, the present inventors have developed new and highly effective therapy-based methods that silence the ISR and/or inhibit the ISR. Adeno-associated virus (AAV) is the main platform for gene delivery for targeted treatment of various diseases. Recent developments have made substantial contributions to the preclinical and clinical success of AAV-mediated gene therapy by optimizing the AAV capsid. However, a major limitation of AAV-based therapies is the associated relatively small encapsulation size, which is limited to approximately 4.7 kb, for example.

Due to the small size of DP71L and its potent ISR inhibitory ability, the inventors determined that the DP71L protein and/or constructs comprising the polynucleotide sequence encoding it would be a promising method for improving cognition and/or alleviating and/or preventing activated ISR. Ideal gene therapy for symptoms of other diseases. To this end, the inventors cloned DP71L-GFP into AAV-based constructs to form AAV particles, and delivered AAV to the hippocampus (a brain structure required for long-term memory formation in mice) ( Figure 8A ) . Stereotactic injection of AAV expressing DP71L-GFP resulted in detectable protein expression in the mouse hippocampus ( Fig. 8A ). The inventors and others have previously demonstrated that inhibition of the ISR rescues the association between 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) related memory deficits. Therefore, the inventors tested whether injection of DP71L AAV rescues long-term memory in this model of cognitive dysfunction.

DS remains the most common genetic form of intellectual disability. Ts65Dn mice are a well-characterized mouse model of DS that reproduces the memory deficits characteristic of DS patients and exhibits long-term memory deficits (see, e.g., Zhu et al., 2019). Surprisingly, injection of DP71L-GFP (but not GFP alone) into the hippocampus of Ts65Dn mice was sufficient to completely reverse the long-term memory deficits in this model ( Figure 8B ). Next, the inventors examined whether DP71L also restored the deficits in synaptic connection strength in the hippocampus of Ts65Dn mice. For this purpose, the inventors measured hippocampal L-LTP, a cellular model for memory formation. In hippocampal sections, four training sessions at 100 Hz induced persistent L-LTP. It has been previously shown that L-LTP is 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 DP71L-GFP AAV injection rescued the L-LTP deficit found in DS mice when compared to GFP-injected control animals ( Figure 8C ). These data suggest that DP71L AAV therapy reverses cognitive decline and potentially persistent deficits in synaptic function associated with Down syndrome.

Alzheimer's disease is the most common form of dementia. While in 2020, 5.8 million Americans were living with Alzheimer's disease, this number is expected to triple to 14 million by 2060. Currently, effective treatments that can alleviate and/or cure the memory decline associated with Alzheimer's disease (AD) do not exist. APP/PS1 is a double transgenic mouse that expresses chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and mutant human presenilin 1 (PS1-dE9), both of which target CNS neurons. Both mutations are associated with early-onset Alzheimer's disease. This "humanized" APP/PS1 model has been used to study pathologies associated with AD. As previously reported, the inventors found that APP/PS1 mice exhibited considerable long-term memory and L-LTP deficits ( Fig. 8D ). Significantly, DP71L AAV therapy reversed both the long-term memory and L-LTP deficits found in APP/PS1 mice ( Fig. 8E ).

In conclusion, the present inventors have identified a new and potent gene therapy (e.g., comprising an ISR inhibitor, e.g., comprising DP71L) that improves long-term memory formation in a broad range of cognitive disorders that activate the ISR. * * *

All methods disclosed and claimed herein can be made and performed in accordance with the invention without undue experimentation. Although the compositions and methods of the invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods described herein without departing from the concept, spirit and scope of the invention. and in the steps or sequence of steps of a method. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted by agents described herein while achieving the same or similar results. 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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The following drawings form part of this specification and are included to further illustrate certain aspects of the invention. The present invention may be better understood by reference to one or more of these drawings, in conjunction with the detailed description of specific embodiments presented herein.

Figure 1 , (prior art) molecular wiring of ISR. ISR kinases phosphorylate eIF2 in response to different cellular stresses. Phosphorylation of eIF2 causes the inhibition of eIF2B activity in cells, thereby reducing the content of the ternary complex (eIF2·GTP·methiodinyl-initiating tRNA), which is controlled by the GDP/GTP exchange of eIF2B on eIF2 . The concentration of the ternary complex determines the translational status of general protein synthesis (green) and specific mRNA (such as ATF4) translation (red). Two phosphatase complexes antagonize the ISR in response to ISR activation, PP1·CReP in a constitutive mechanism and PP1·GADD34 in a feedback mechanism.

FIG. 2A - 2J , ISR activation and reduced transcription in Ppp1r15b ^R658C mice. ( 2A ) Sanger sequencing of the indicated mutations in Ppp1r15b ^R658C mice. ( 2B ) Brain size in WT and Ppp1r15b ^R658C mice. ( 2C - 2D ) Representative Western blots and quantification of CReP levels ( 2C ) and eIF2-P levels ( 2D ) in hippocampal extracts from WT and Ppp1r15b ^R658C mice. ( 2E ) Representative Western blots and quantification of eIF2-P levels in primary fibroblasts from WT and Ppp1r15b ^R658C . ( 2F ) Schematic representation of polysome profiling sedimentation. After ultracentrifugation, 40S, 60S, and 80S and polysomes were separated based on size. ( 2G to 2H ) Polysome profile ( 2G ) and quantification ( 2H ) in the hippocampus of WT and Ppp1r15b ^R658C mice. ( 2I to 2J ) Puromycin was incorporated into nascent peptides using an anti-puromycin antibody. Data are mean ± sem. *P < 0.05, **P < 0.01.

Figure 3A - 3F , Impairment of long-term fear and object discrimination memory in Ppp1r15b ^R658C mice. (3A) Schematic diagram of fear conditioning. (3B) Freezing behavior was assessed during a 2-min period before conditioning (untrained) and then during a 5-min period 24 hours after training in WT and Ppp1r15b ^R658C mice. (3C) Schematic diagram of the object discrimination task. (3D) Object exploration time during training. (3E) The novel object discrimination index (DI) was calculated as DI = (novel object exploration time - familiar object exploration time / total exploration time) × 100 (see, e.g., Zhu et al., 2019). (3F) Late long-term potentiation (L-LTP) was induced by 4 training sessions at 100 Hz in WT and Ppp1r15b ^R658C mice (p < 0.01 at 220 min). Data are mean ± sem. *P < 0.05, **P < 0.01.

Figure 4A to Figure 4D , DP71L is a potent pan-inhibitor of ISR. (4A) PP1 binding motif and eIF2 binding motif conserved in CReP and viral phosphatase. (4B to 4D) DP71L-GFP prevents the induction of ISR by different stresses (oligomycin, poly(I:C), and thapsigargin, respectively (n=4 per group)).

Figure 5A to Figure 5C , DP71L strongly binds to PP1 to weaken the activation of ISR. (5A) Schematic diagram of DP71L and ΔCREP. The protein domain is involved in the interaction of eIF2α phosphatase cofactor with PP1 and eIF2. (5B) Western blot reveals activation of ISR in cells transfected with GRP, GFP-DP71L or GFP-ΔCREP. (5C) In vivo interaction between GFP-tagged DP71L or CREP and endogenous PP1 or eIF2α. Contents of PP1γ, PP1α and eIF2 in immunoprecipitates from GFP-DP71L or GFP-ΔCREP.

Figures 6A - 6D illustrate that the interconnecting (also known as "linker" or "peptide linker") region between the PP1 binding domain and the eIF2α binding domain is necessary and sufficient for DP71L to bind to PP1. (6A) Schematic representation of different chimeric proteins and protein domains involved in PP1 and eIF2 binding (eg, DP71L, ΔCREP, and linker mutant proteins (C8, C20, and D20, respectively)). (6B) Human HEK293T cells were transfected with different GFP-constructs and subsequently immunoprecipitated with anti-GFP antibodies. The levels of PP1γ and PP1α in immunoprecipitates from different GFP-tagged chimeric proteins are shown. (6C) Schematic diagrams of DP71L, ΔCREP and linker chimeric proteins “DP71L-linker” and “ΔCREP-linker” respectively. (6D) HEK293T cells were transfected with different GFP-constructs and proteins of interest were subsequently immunoprecipitated with anti-GFP antibodies. Results showing endogenous PP1γ in GFP-tagged chimeric proteins and immunoprecipitates from GFP-tagged DP71L, ΔCREP, and linker-swapped chimeric proteins (DP71L-linker and ΔCREP-linker, respectively) and the level of in vivo interaction between PP1α.

FIG7A - 7C , Mutations of hydrogen-bonded glutamines in the linker of DP71L impair PP1 binding. (7A) Alpha-fold prediction of DP71L in complex with eIF2 and PP1. (7B) Zoomed-in alpha-fold prediction showing PP1 (cyan) and similar regions of DP71L (magenta) and CreP (yellow). Glutamates involved in hydrogen bonding are highlighted next to the analogous threonine in ΔCREP. (7C) IP experiments performed with glutamine mutants (E12T) and control tryptophan mutants (W14Y) of DP71L.

FIG8A - 8E , Injection of DP71L rescues long-term memory deficits in mouse models of Down's and Alzheimer's disease. (8A) Representative images showing the expression of DP71L-GFP in mouse brain. (8B) Freezing behavior in Ts65Dn mice (Down's model) injected with DP71L-GFP or GFP. Freezing behavior was assessed during 2-min cycles before conditioning (untrained) and then during 5-min cycles 24 hours after training. (8C) Long-term potentiation (LTP) in Ts65Dn mice injected with DP71L-GFP or GFP. LTP was induced by 4 training sessions at 100 Hz (p < 0.01 at 220 min; n = 8 to 10 per group). (8D) Freezing behavior in APP/PS1 mice (a "humanized" model of Alzheimer's disease) injected with DP71L-GFP or GFP. Freezing behavior was assessed during 2-min cycles before conditioning (untrained) and then during 5-min cycles 24 h after training. (8E) Long-term potentiation (LTP) in APP/PS1 mice injected with DP71L-GFP or GFP. LTP was induced by 4 training sessions at 100 Hz (p < 0.001 at 220 min; n = 10 per group). Data are mean ± sem. *P < 0.05, ***P < 0.001.

TW202408545A_112125405_SEQL.xml

Claims (44)

A non-natural polynucleotide construct comprising a polynucleotide sequence encoding one or more inhibitors of an integrated stress response (ISR), wherein the one or more inhibitors comprise protein phosphatase-1 (PP1) binding domain and eukaryotic initiation factor 2 (eIF2) binding domain, wherein the non-natural polynucleotide construct includes: A) a heterologous promoter operably linked to one or more repressors encoding the ISR the polynucleotide sequence of an agent, wherein the heterologous promoter is not a galactose-inducible promoter; and/or B) a recombinant viral vector comprising a heterologous promoter operably linked to a gene encoding the Polynucleotide sequences for one or more inhibitors of ISR, wherein the heterologous promoter is not a galactose-inducible promoter. The construct of claim 1, further comprising a peptide linker sequence connecting the PP1 binding domain and the eIF2 binding domain. The construct of claim 1 or 2, wherein the peptide linker sequence is at least or exactly 73%, 82%, 91% or 100% identical to SEQ ID NO: 21. The construct of any one of claims 1 to 3, wherein the linker sequence comprises glutamine at position E12 of the DP71L protein according to SEQ ID NO: 18. The construct of any one of claims 1 to 4, wherein the inhibitor contains at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of SEQ ID NO: 4 %identical amino acid sequence. The construct of any one of claims 1 to 5, wherein the inhibitor comprises an amino acid sequence according to SEQ ID NO: 4. The construct of any one of claims 1 to 4, wherein the inhibitor comprises an amino acid sequence that is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 18. The construct of any one of claims 1 to 5, wherein the inhibitor comprises an amino acid sequence according to SEQ ID NO: 18. The construct of any one of claims 1 to 8, wherein the heterologous promoter is a CAG promoter. The construct of any one of claims 1 to 9, wherein the non-natural construct is contained in a retroviral capsid. An AAV particle, comprising the construct of any one of claims 1 to 10, wherein the construct is contained in an adeno associated virus (AAV) capsid. The AAV particle of claim 11, wherein the AAV particle is any one of the AAV-DJ/8, AAV9, AAV Php.B and/or AAV Php.eB serotypes and/or pseudotypes. The AAV particle of claim 11 or 12, wherein the AAV particle is capable of retrograde infection. An AAV particle comprising a nucleotide construct comprising a polynucleotide sequence that is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 22. The AAV particle of claim 14, comprising the polynucleotide sequence according to SEQ ID NO: 22. The AAV particle of claim 14 or 15, wherein the AAV capsid is AAV-DJ/8, AAV9, AAV Php.B and/or AAV Php.eB capsid. A polypeptide comprising an amino acid sequence that is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4. A polypeptide as claimed in claim 17, comprising an amino acid sequence according to SEQ ID NO: 4. A pharmacologically acceptable composition comprising the non-natural construct, polypeptide or AAV particle of any one of claims 1 to 18. A human cell comprising the non-natural construct, polypeptide, AAV particle or composition of any one of claims 1 to 19. A method for 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 non-natural construct, polypeptide, AAV particle, or composition of any one of claims 1 to 20. The method of claim 21, wherein the administration is to the central nervous system. The method of claim 21, wherein the administration is to the peripheral nervous system. The method of claim 21, wherein the administration is into cerebrospinal fluid (CSF). Such as the method of claim 21 or 22, wherein the hippocampus is administered. The method of claim 25, wherein the CA1, CA2 and/or CA3 regions of the hippocampus are administered. The method of any one of claims 21 to 26, wherein the human individual has been diagnosed with a disease and/or condition associated with cognitive impairment or dysfunction. The method of any one of claims 21 to 27, wherein the individual suffers from a neurodevelopmental disorder. The method of any one of claims 21 to 27, wherein the subject suffers from a neurodegenerative disorder. Such as requesting the method of any one of items 21 to 29, wherein the individual suffers from and/or is expected to develop Down Syndrome (Down Syndrome), Alzheimer's disease (Alzheimer's disease), traumatic brain injury, white matter loss (vanishing white matter; VWM) disease, frontotemporal dementia, and/or aging-related cognitive decline. A method for delaying the onset of, treating the disease, slowing down the progression of the disease, reducing the risk of the disease and/or preventing the disease in a human subject, wherein Comprised of administering to the human subject the non-natural construct, polypeptide, AAV particle or composition of any one of claims 1 to 20. The method of claim 31, wherein the disease is a cognitive disorder, neurodegeneration, cancer, diabetes and/or a metabolic disorder. A method of delaying the onset of cognitive dysfunction, treating cognitive dysfunction, slowing the progression of cognitive dysfunction, reducing the risk of cognitive dysfunction, and/or preventing cognitive dysfunction in a human subject, comprising administering to the human subject: A) A polynucleotide encoding an inhibitor of ISR, the inhibitor comprising the amino acid sequence according to SEQ ID NO: 18; B) a polynucleotide encoding an inhibitor of the ISR, the inhibitor comprising the amino acid sequence according to SEQ ID NO: 4; C) A polynucleotide encoding an inhibitor of the ISR, the inhibitor comprising at least or exactly 85%, 90%, 95%, 97%, 98%, 99 of the amino acid sequence according to SEQ ID NO: 18 % or 100% identical amino acid sequence, wherein the inhibitor includes a protein phosphatase-1 (PP1) binding domain and a eukaryotic initiation factor 2 (eIF2) binding domain; or D) A polynucleotide encoding an inhibitor of the ISR, the inhibitor comprising at least or exactly 85%, 90%, 95%, 97%, 98%, 99 of the amino acid sequence according to SEQ ID NO: 4 % or 100% identical amino acid sequence, wherein the inhibitor includes a protein phosphatase-1 (PP1) binding domain and a eukaryotic initiation factor 2 (eIF2) binding domain. The method of claim 33, wherein the polynucleotide comprises a sequence encoding an inhibitor amino acid sequence that is at least or exactly 85%, 90%, 95%, or 96 identical to SEQ ID NO: 18 %, 97%, 98%, 99% or 100% agreement. The method of claim 33, wherein the polynucleotide comprises a sequence encoding an inhibitory amino acid sequence that is at least or exactly 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4. The method of any one of claims 33 to 35, wherein the polynucleotide comprises a sequence encoding a peptide linker sequence that is at least or exactly 73%, 82%, 91% or 100% identical to SEQ ID NO: 21. The method of any one of claims 33 to 36, wherein the polynucleotide is operably linked to a heterologous promoter. The method of claim 37, wherein the heterologous promoter is not a galactose-inducible promoter. The method of any one of claims 33 to 38, wherein the disease is associated with activation, overactivation and/or abnormal activation of the ISR in the human subject. A transgenic mouse comprising a mutation in the Ppp1r15b gene. For example, the transgenic mouse of claim 40, wherein the mutation is an R658C mutation. The transgenic mouse of claim 40 or 41, wherein the mutation is generated using an endonuclease. For example, the transgenic mouse of claim 42, wherein the endonuclease is Cas9. The transgenic mouse of claim 43, wherein the generating comprises contacting the Ppp1r15b gene with a guide RNA comprising SEQ ID NO: 38.