WO2023092152A1 - Therapeutic applications of coronavirus nsp1 protein - Google Patents

Abstract

Systems, formulations, and methods utilizing NSP1 or an NSP1 peptide fragment within a medicament are described. Further, systems, formulations, and methods utilizing an antagonist NSP1 or an antagonist of protein synthesis within a medicament are described. In some instances, NSP1 or an NSP1 peptide fragment is utilized for the treatment of a neurodegenerative disorder, a neoplasm, or a cancer. In some instances, an antagonist NSP1 or an antagonist of protein synthesis is utilized for the treatment of coronavirus.

Description

THERAPEUTIC APPLICATIONS OF CORONAVIRUS NSP1 PROTEIN

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Ser. No. 63/264,436, entitled “Therapeutic Application of Coronavirus NSP1 Protein,” filed November 22, 2021 , which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under contracts AR074875 and NS084412 awarded by the National Institutes of Health. The Government has certain rights in the invention.

TECHNICAL FIELD

[0003] The disclosure is generally directed to systems and methods of utilizing coronavirus NSP1 protein for therapeutic uses. The disclosure also is generally directed to systems and methods of treating coronavirus by targeting NSP1.

INCORPORATION OF SEQUENCE LISTING

[0004] This application hereby incorporates by reference the material of the electronic Sequence Listing filed concurrently herewith. The material in the electronic Sequence Listing is submitted as an XML file entitled “07592PCT_Seq_List.xml” created on November 22, 2022, which has a file size of approximately 25 KB, and is herein incorporated by reference in its entirety.

BACKGROUND

[0005] The coronavirus family includes SARS-CoV, SARS-CoV-2, Middle-East Respiratory Syndrome Coronavirus (MERS-CoV), and several others that are known to infect humans or other animals. These viruses cause severe diseases, suggesting that the proteins they encode are highly virulent. SARS-CoV-2 encodes the spike (S), envelope (E), membrane (M), nucleocapsid (N), non-structural (NSP1 -16), and accessory (0RF3a, 3b, 6, 7a, 7b, 8, 9b, 9c, 10, and 14) proteins. NSP1 is one of the first SARS-CoV proteins synthesized upon cell entry and a major virulence factor. Various studies have implicated NSP1 in inhibiting the translation initiation of host genes through blocking the mRNA entry channel of the 40S ribosome, and by promoting mRNA degradation. NSP1 has also been implicated in viral evasion of host innate immune response, although the underlying mechanism is unclear. Furthermore, neuromuscular complications are commonly associated with COVID-19, however, little is known about the underlying molecular mechanisms.

SUMMARY

[0006] Several embodiments are directed towards the use and manufacture of the coronavirus protein NSP1 (or a peptide fragment thereof) as a therapeutic. In many embodiments, the NSP1 protein (or a peptide fragment thereof) is manufactured and/or utilized as a therapeutic for a neurodegenerative disease. In several embodiments, the NSP1 protein (or a peptide fragment thereof) is manufactured and/or utilized as a therapeutic for a neoplasm or cancer. In many embodiments, the NSP1 protein (or a peptide fragment thereof) is manufactured and/or utilized for systemic delivery.

[0007] Several embodiments are directed towards the use and manufacture of a nucleic acid compound capable of expressing the coronavirus protein NSP1 (or a peptide fragment thereof) as a therapeutic. In many embodiments, the nucleic acid compound capable of expressing the NSP1 protein (or a peptide fragment thereof) is manufactured and/or utilized as a therapeutic for a neurodegenerative disease. In several embodiments, the nucleic acid compound capable of expressing the NSP1 protein (or a peptide fragment thereof) is manufactured and/or utilized as a therapeutic for a neoplasm or cancer. In many embodiments, the nucleic acid compound capable of expressing the NSP1 protein (or a peptide fragment thereof) is manufactured and/or utilized for systemic delivery.

[0008] Several embodiments are directed towards inhibiting or mitigating NSP1 as a treatment for coronavirus, especially SARS-COV2. In many embodiments, a compound capable of inhibiting NSP1 function is utilized and/or manufactured as a treatment for coronavirus, especially SARS-COV2. In several embodiments, an oligomeric compound capable of decreasing NSP1 RNA expression and/or function is utilized and/or manufactured as a treatment for coronavirus, especially SARS-COV2.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The description and claims will be more fully understood with reference to the following figures and data graphs, which are presented as exemplary embodiments and should not be construed as a complete recitation of the scope of the disclosure.

[0010] Fig. 1 provides a schematic of a coronavirus and its genome.

[0011] Fig. 2 provides images showing effect of muscle expression of SARS-CoV-2- encoded viral proteins on wing posture in wild type condition. Bar graph shows quantification of wing posture defect (n=8 per group) and quantification of effect of viral protein expression on locomotion (n=8 per group).

[0012] Fig. 3 provides images, showing abnormal wing postures in Mhc>APP.C99 flies, quantification of wing posture defect in Mhc>APP.C99 flies co-expressing various SARS-CoV-2 encoded viral proteins aged at 35 days (n=6 per group), immunostainings and quantification (n=6) showing Nsp1 protein expression in the muscle in Mhc>Nsp1 flies, and immunoblots and quantification (n=3) showing expression of Nsp1 in Mhc>Nsp1 flies.

[0013] Fig. 4 provides images showing effect of muscle expression of SARS-CoV-2 - encoded viral proteins on wing posture in Mhc>APP.C99 flies, and quantification showing effect of Nsp1 on wing posture in Mhc>APP.C99 flies at different ages (n=12 per group). [0014] Fig. 5 provides immunofluorescent images showing effect of Nsp1 on protein aggregation (lib, p62, and 6E10 staining), lysosomes (Lamp1-GFP), and early endosomes (Rab5) in Mhc>APP.C99 fly muscle (n=6 per group).

[0015] Fig. 6 provides quantification showing effect of Nsp1 on protein aggregation (lib, p62, and 6E10 staining), lysosomes (Lamp1-GFP), and early endosomes (Rab5) in Mhc>APP.C99 fly muscle (n=6 per group), and effect of Nsp1 on locomotion (n=6) and lifespan of Mhc>APP.C99 flies.

[0016] Fig. 7 provides immunostainings and quantification (n=6) showing effect of panneuronal (driven by elav-Gal4) or mushroom body neuron-specific (driven by R13F02- Gal4) expression of Nsp1 on APP.C99-induced proteostasis failure, immunoblots and quantification (n=3) showing effect of Nsp1 on APP.C99 protein level in elav>APP.C99 fly brain, quantification (n=6) showing effect of Nsp1 on climbing activity in elav>APP.C99 flies, and effect of Nsp1 co-expression on the aversive taste memory deficit in elav>APP.C99 flies (n=15).

[0017] Fig. 8 provides effect of Nsp1 on wing posture of Mhc>FL-APP flies (n=6 per group), effect of Nsp1 on learning and memory in the aversive taste memory assay in elav>FL-APP/BACE flies (n=15 per group), and effect of Nsp1 on proteostasis and DA neuron number in the PPL1 cluster in elav>FL-APP/BACE flies (n=6 per group).

[0018] Fig. 9 provides immunostaining and quantification (n=6) showing effect of Nsp1 on proteostasis in Mhc>FL-APP/BACE fly muscle, and quantification (n=6) of wing posture defect in Mhc>FL-APP/BACE flies co-expressing Nsp1.

[0019] Fig. 10 provides immunoblots and quantification (n=3) showing effect of Nsp1 , and Nsp2 on FL-APP.C99 (upper band) and internally stalled APP.C99 (lower band) levels, immunostaining and quantification (n=6) showing effect of Nsp1 on APP.C99 expression in Mhc>APP.C99 fly muscle, immunoblots and quantification (n=3) showing effect of Nsp1 on APP.C99 protein level in Mhc>APP.C99 flies at different ages, and immunoblots and quantification (n=3) showing effect of chloroquine treatment on APP.C99 level in Mhc>APP.C99 flies co-expressing Nsp1.

[0020] Fig. 11 provides immunoblots showing effect of Nsp1 on FL-APP and APP.C99 levels in Mhc>FL-APP/BACE fly muscle. Values below the blots show relative levels of the indicated protein band in this and other figures, immunoblots showing detection of FL- APP. C99 and internally stalled APP.C99 with 6E10 and C.1/6.1 antibodies, immunoblots showing effect of Nsp1 on FL-APP and APP.C99 levels in Mhc>FL-APP/BACE or Mhc>FL-APP \y muscle (30 days old flies were used), immunostaining showing effect of one copy or two copies of Nsp1 on APP.C99 level in GMR-Gal4>APP.C99 fly brain extracts, and immunoblots showing effect of Nsp1 expression on ABCE1 level in Mhc>APP.C99 flies.

[0021] Fig. 12 provides immunoblots and quantification (n=3) showing effect of Nsp1 on FL-APP level in Mhc>APP flies, and immunoblots and quantification (n=3) showing effect of Nsp1 on FL-APP and APP.C99 levels in brain tissues of elav>FL-APP/BACE flies. Cat-C99: CAT-tailed C99. [0022] Fig. 13 provides images and quantification (n=6) comparing body sizes of control and tubulin-Gal4>Nsp1 flies at the larvae, pupae, and adult stages, and immunoblots and quantification (n=3) showing Pum labeling of newly synthesized peptides in Mhc>APP.C99 flies with or without Nsp1 co-expression.

[0023] Fig. 14 provides quantification of mRNA levels by qRT-PCR in Mhc>APP.C99 flies with or without Nsp1 co-expression (n=3).

[0024] Fig. 15 provides immunoblots and quantification (n=3) showing effect of Nsp1 and Nsp1 -KH mutant on APP.C99 expression in HeLa cells (eRF1 serves as loading control), immunostaining and quantification (n=3) showing effect of Nsp1 on APP.C99 protein expression in HeLa cell, and immunoblots and quantification (n=3) showing effect of Nsp1 and Nsp1-KH on FL-APP expression. The weak signal in cells not transfected with APP cDNA likely represents endogenous APP.

[0025] Fig. 16 provides immunoblots and quantification (n=3) showing expression levels of Nsp1 and Nsp1 -KH in Mhc-Gal4 driven transgenic flies, immunoblots and quantification (n=3) showing effect of Nsp1 and Nsp1 -KH on APP.C99 expression in Mhc>APP.C99 flies, immunostaining and quantification (n=6) showing effect of Nsp1 -KH on APP.C99-induced proteostasis failure in Mhc>APP.C99 flies, quantification of effect of Nsp1 -KH on APP.C99-induced wing posture defect in Mhc>APP.C99 flies (n=6), and immunostainingand quantification (n=3) of HeLa cells co-transfected with APP.C99 and Nsp1 or Nsp1 -KH with the amyloid conformation-specific mOC78 (M78) antibody and anti-Pum, which labeled stalled NPCs after active ribosomes were let run off by HHT treatment.

[0026] Fig. 17 provides immunoblots and data quantification (n=3) showing effect of Nsp1 -WT and Nsp1 -KH mutant on RFP and GFP expression from the stall reporter GFP- P2A-K20-P2A-RFP and control reporter GFP-P2A-K0-RFP.

[0027] Fig. 18 provides immunoblots showing effect of Nsp1 on GFP, Flag-K20, and mKate2 expression from the GFP-P2A-Flag-K20-P2A-mKate2 reporter. Actin serves as loading control, immunoblots showing effect of ASCC3 and ZNF598 silencing on GFP, Flag-K20, and mKate2 expression from the GFP-P2A-Flag-K20-P2A-mKate2 reporter in HEK293 cells with or without Nsp1 co-transfection, and immunoblots showing knockdown efficiency in ZNF598 KO cells and ASCC3 shRNA transfected cells. [0028] Fig. 19 provides immunoblots and quantification (n=3) showing effect of Nsp1 and Nsp1-KH on ribosome collision-induced Rps3 ubiquitination in APP.C99 transfected HeLa cell with or without Ans treatment. Arrow marks ubiquitinated Rps3.

[0029] Fig. 20 provides sucrose gradient analysis of ribosomes showing the effect of Nsp1 on collided ribosomes in HeLa cells, immunostainings showing the effect of Nsp1 on cGAS cytoplasmic vs. nuclear localization in anisomycin-treated LI2OS cells with or without silencing of ASCC3 or ZNF598, and representative line scanning of cGAS immunosignal intensity in the cytoplasmic vs. nuclear compartments in cells of the indicated genotypes. For sucrose gradient analysis, lysates of control and Nsp1 transfected HeLa cells with or without anisomycin treatment were separated by analytical 10-50% sucrose gradient and proteins in the fractions from top to bottom (1-11 ) were analyzed by immunoblots for Rps3, ASCC3, and EDF1. Fraction 11 might be contaminated by cell debris and was not included as polysome fraction for analysis. Bar graph shows quantification of relative polysome fraction signal intensity of the indicated proteins in cells with or without Nsp1 expression (n=3).

[0030] Fig. 21 provides immunoblots showing effect of Nsp1 on relative nuclear fraction cGAS level in control and ZNF598 KO or ASCC3 shRNA transfected cells.

[0031] Fig. 22 provides immunoblots showing effect of Nsp1 on innate immune signaling (p-JNK, p-Jun) and interferon stimulated gene (RSAD2) expression promoted by Ans-induced ribosome collision (data represents two independent experiments), and images and data quantification (n=6) showing effect of STING RNAi on the wing posture and locomotion in Mhc>APP.C99 flies.

[0032] Fig. 23 provides immunoblots and quantification (n=3) showing effects of ABCE1 -shRNA and Rackl -shRNA knockdown efficiency, immunoblots showing effect of eRF1 RNAi on the removal of stalled APP.C99 species by Nsp1 , immunoblot and quantification (n=3) showing effect of eRF1 -shRNA knockdown efficiency, immunoblots showing effect of elF3E-shRNA on the removal of stalled APP.C99 species by Nsp1 (Rackl RNAi serves as a positive control), and quantification showing age-dependent effect of ABCE1 RNAi on the wing posture defect rescued by Nsp1 in Mhc>APP.C99 flies (n=6). [0033] Fig. 24 provides immunoblots and quantification (n=3) showing the effect of ABCE1 RNAi and Rackl RNAi on Nsp1 inhibition of APP.C99 protein expression in HeLa cells, and images and data quantification (n=6) showing effect of ABCE1 RNAi on the rescue of wing posture and locomotion defects by Nsp1 in Mhc>APP.C99 flies.

[0034] Fig. 25 provides immunoblots and quantification (n=3) showing effect of ABCE RNAi on the inhibition of stalled APP.C99 protein levels by Nsp1 , immunostainings and quantification (n=6) showing the effect of ABCE1 RNAi on the rescue of proteostasis failure and endolysosomal defects in Mhc>APP.C99 flies by Nsp1 , and effect of ABCE1 RNAi on the rescue of aversive taste memory deficit in elav>FL-APP/BACE flies by Nsp1 (n=15).

[0035] Fig. 26 provides RT-PCR analysis of age-dependent ABCE1 -RNAi efficiency, immunoblots and quantification (n=3) showing age-dependent effect of ABCE1 -RNAi efficiency on protein level, immunoblots showing effect of eRF1 RNAi on the removal of stalled APP.C99 species by Nsp1 in Mhc>APP. C99 fly muscle. Graph on the right shows RT-PCR analysis of eRF1 RNAi efficacy, quantification showing effect of eRF1 RNAi on the rescue by Nsp1 of the aversive taste memory deficit in elav>APP/BACE flies (n=15), and immunoblots showing effect of eRF1 RNAi on the level of FL-APP and APP.C99 removed by Nsp1 in elav>FL-APP/BACE fly brain.

[0036] Fig. 27 provides immunoblots and quantification (n=3) showing effect of ABCE1 RNAi on the level of FL-APP and APP.C99 removed by Nsp1 in elav>FL-APP/BACE fly brains, and immunostainings and quantification (n=6) showing effect of ABCE1 RNAi on the level of lib and p62 positive aggregates in elav>FL-APP/BACE fly brains.

[0037] Fig. 28 provides immunoblot showing effect of ATG1 RNAi and AKT RNAi on the level of stalled APP.C99 species removed by Nsp1 in Mhc>APP.C99 fly muscle. Bar graph on the right show RT-PCR analysis of ATG1 RNAi and ABCE1 RNAi knockdown efficacy (n=3), immunoblots showing effect of Atg1 -OE or Atg1-RNAi, Atg8-RNAi on FL- APP. C99 and stalled APP.C99 levels in Mhc>APP.C99 flies, quantification of effect of Atg1 -RNAi or Atg8-RNAi on wing posture defect caused by APP.C99 (n=6), immunoblots showing effect of combined ATG1 RNAi and ABCE1 RNAi on the level of stalled APP.C99 species removed by Nsp1 in Mhc>APP.C99 fly muscle. FL-APP. C99, CAT-tailed APP.C99, and internally stalled APP.C99 are indicated, image showing abnormal wing posture phenotype recovered by combined ATG1 RNAi and ABCE1 RNAi in Mhc>APP.C99 flies co-expressing Nsp1 , and immunoblots showing effect of anisomycin treatment on the level of stalled APP.C99 species removed by Nsp1 in the various genetic backgrounds.

[0038] Fig. 29 provides images showing rescue of the abnormal wing posture phenotype of PINK1^B9 mutant flies and Mhc>GR80 flies by Nsp1 , and the impact of ABCE1 RNAi on the Nsp1 effect, and quantification (n=6) of the wing posture phenotype in PINK1^B9 mutant flies and Mhc>GR80 flies after various genetic manipulations.

[0039] Fig. 30 provides quantification (n=6) of locomotion phenotypes in PINK1^B9 mutant flies and Mhc>GR80 flies after various genetic manipulations, immunostainings and quantification (n=6) showing effect of Nsp1 on mitochondrial morphology and proteostasis in PINK1^B9 mutant flies and Mhc>GR80 fly muscle. Mitochondria were stained with anti-ATP5A, protein aggregates with anti-p62 and anti-Ub, and muscle fiber with Phalloidin for F-actin, and quantification showing effect of TH-Gal4 driven Nsp1 expression on DA neuron number in PINK1^B9 mutant flies (n=6).

[0040] Fig. 31 provides quantification of mitochondrial morphology of the images shown in Fig. 31 (n=6), immunostainings showing the effect of Nsp1 on DA neuron mitochondrial morphology in PINK1^B9 mutant fly brains. Mitochondrial morphology was monitored with a mito-GFP reporter driven by TH-Gal4. DA neurons in the PPL1 cluster were shown, and quantification of data shown. The relative abundance of tubular vs. swollen mitochondria was quantified (n=6).

[0041] Fig. 32 provides immunoblots and quantification (n=3) showing effect of Nsp1 on stalled translation product of C-I30 in PINK1^B9 mutant flies, with or without ABCE1 or eRF1 RNAi, and immunoblots and quantification (n=3) showing effect of Nsp1 on stalled GR80 products in Mhc>GR80 fly muscle, with or without ABCE1 or eRF1 RNAi. * marks a non-specific band.

[0042] Fig. 33 provides images and quantification (n=4) showing effect of Nsp1 coexpression on photoreceptor neuron degeneration caused by dFoxo, polyQ, or tau, and immunoblots showing effect of Nsp1 on tau protein expression in GMR>tauV337M or sev>tau-WT flies. [0043] Fig. 34 provides immunostaining and data quantification showing reduction of mOC78-positive amyloid aggregates in Nsp1 -transfected FAD iPSC neurons, immunostaining and data quantification showing reduction of Rab5-positive early endosomes in Nsp1 -transfected FAD iPSC neurons, and immunostaining and data quantification showing reduction of p-Tau signals in Nsp1 -transfected FAD iPSC neurons. [0044] Fig. 35 provides immunostaining and data quantification showing reduction of mOC78-positive amyloid aggregates in Nsp1 -C treated FAD iPSC neurons, immunostaining and data quantification showing reduction of Rab5-positive early endosomes in Nsp1 -C treated FAD iPSC neurons, and immunostaining and data quantification showing reduction of p-Tau signals in Nsp1 -C treated FAD iPSC neurons. [0045] Fig. 36 provides MTT assay showing inhibition of HeLa cell proliferation by Nsp1 , MTT assay showing lack of significant effect on non-cancer HEK293 cell proliferation, colony formation assay showing inhibition of HeLa cell colony formation by Nsp1 , and MTT assay showing inhibition of glioblastoma cell (GBM387) proliferation by Nsp1.

[0046] Fig. 37 provides an immunoblot showing reduction of c-Myc, ERK2, elF4E, p- 4EBP levels by Nsp1 in HeLa cells, and an immunoblot showing reduction of c-Myc, p- mTOR, and ZNF598 levels by Nsp1 in HeLa cells. Actin serves as loading control.

[0047] Fig. 38 provides immunoblot and qRT-PCR analyses showing repression of Myc protein level without mRNA level change by Nsp1 in transgenic flies expressing Nsp1 in the eye using GMR-Gal4 driver.

[0048] Fig. 39 provides immunoblot analysis showing effect of Nsp1 on c-Myc and c- Myc-mut protein level in HeLa cells. HeLa cells co-transfected with Nsp1 and c-Myc or Nsp1 and c-Myc-mut were used for western blot analysis by probing for the level of c- Myc. Actin serves as loading control.

[0049] Fig. 40 provides immunostaining images showing relationship between Nsp1 and Myc in brain tumor model. Images and quantification of NB number showing inhibition of Notch-induced brain tumor phenotype by Nsp1 and the blockage of Nsp1 effect by Myc. [0050] Fig. 41 provides eye images showing rescue by Nsp1 of the overgrowth phenotypes induced by AKT-OE, Wts-RNAi, or Ras-CA-OE, and the blockage of Nsp1 effect by Myc overexpression in the AKT-OE tumor model.

[0051] Fig. 42 provides results of neurosphere formation assay showing inhibition of GBM387 tumor sphere formation by the treatment of Nsp1-C peptide.

[0052] Fig. 43 provides immunoblot analysis showing reduction of cancer-related signal molecule expression by the treatment of Nsp1-C peptide. GBM387 cells treated with different concentration of Nsp1-C peptide for 60 hours were used to prepare cell lysates for western blot analysis. Immunoblots probed with the indicated antibodies are shown. Actin serves as a loading control.

DETAILED DESCRIPTION

[0053] Turning now to the drawings and data, systems, formulations, and methods to manufacture and utilize medicaments for treatment of various disorders, in accordance with various embodiments, are described. Various embodiments described within this disclosure are based on the discovery that NSP1 unexpectedly rescues experimental models of neurodegeneration, including models of Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis. Accordingly, in some embodiments, a medicament comprises NSP1 protein (or a peptide fraction thereof) for manufacture and/or use as a treatment for a neurodegenerative disorder. The therapeutic effect of NSP1 is mediated through resolving ribosome collisions, aborting stalled translation, and removing faulty translation product. These complications are not only common in neurodegenerative diseases, but also within neoplasms and cancers. Accordingly, in some embodiments, a medicament comprises NSP1 protein (or a peptide fraction thereof) for manufacture and/or use as a treatment for a neoplasm or a cancer. Furthermore, in some embodiments, a medicament comprises a nucleic acid compound for expression of NSP1 protein (or a peptide fraction thereof) for manufacture and/or use as a treatment for a neurodegenerative disease, a neoplasm, or a cancer.

[0054] In addition, the role of NSP1 during coronavirus infection has been elucidated (see Exemplary Embodiments). It is now understood NSP1 increases protein production of infected cells and thus targeting NSP1 or other targets within protein synthesis pathways can be utilized to treat a coronavirus infection. Accordingly, in some embodiments, a medicament comprises a compound that inhibits or mitigates the function of NSP1 and/or other targets within protein synthesis pathways for manufacture and/or use as a treatment for coronavirus infection.

NSP1 for use within medicaments

[0055] Various embodiments are directed to utilizing NSP1 for the treatment of a neurodegenerative disorder, a neoplasm, or a cancer. In many embodiments, an individual is administered NSP1 (or a peptide fragment thereof) to mitigate and/or prevent onset of a neurodegenerative disorder, a neoplasm, or a cancer. As described in the Exemplary Embodiments, it is now known that NSP1 unexpectedly rescues several neurodegenerative phenotypes. In particular, it was found that NSP1 improves protein synthesis and reduces aberrant protein production. These discoveries provide a treatment approach for medical disorders related to complications of aberrant translation production, ribosome collisions, and stalled translation, such as a neurodegenerative disorder, a neoplasm, or a cancer.

[0056] Neurodegenerative disorders that can be treated, in accordance with various embodiments, include (but are not limited to) Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic Lateral Sclerosis (ALS), ataxia, Huntington’s disease (HD), motor neuron disease, and multiple system atrophy. Neoplasms and cancers that can be treated include (but are not limited to) anal cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, brain cancer (including glioblastoma), breast cancer, breast adenocarcinoma (BRCA), cervical cancer, chronic myeloproliferative neoplasms, colorectal cancer, endometrial cancer, ependymoma, esophageal cancer, diffuse large B-cell lymphoma (DLBCL), esthesioneuroblastoma, Ewing sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, hepatocellular cancer, hypopharyngeal cancer, Kaposi sarcoma, Kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, liver cancer, lung cancer, melanoma, Merkel cell cancer, mesothelioma, mouth cancer, neuroblastoma, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors, pharyngeal cancer, pituitary tumor, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, skin cancer, small cell lung cancer, small intestine cancer, squamous neck cancer, T-cell acute lymphoblastic leukemia (T-ALL), testicular cancer, thymoma, thyroid cancer, uterine cancer, vaginal cancer, and vascular tumors.

[0057] In some embodiments, NSP1 is utilized within a medicament to treat an individual having a neurodegenerative disorder, a neoplasm, or a cancer. In some embodiments, a subject is administered a medicament comprising NSP1 to treat an individual having a neurodegenerative disorder, a neoplasm, or a cancer. It is to be understood that the various proteins and peptides utilized for treatment and/or administration can be truncated, modified, chimerized, and/or conjugated, as would be understood in the art. In some embodiments, a specific region of a protein or a peptide is truncated, modified, chimerized, and/or conjugated.

[0058] In some embodiments, a nucleic acid compound for expressing NSP1 (or a peptide fragment thereof) is utilized within a medicament to treat an individual having a neurodegenerative disorder, a neoplasm, or a cancer. In some embodiments, a subject is administered a medicament comprising a nucleic acid compound for expressing NSP1 (or a peptide fragment thereof) to treat an individual having a neurodegenerative disorder, a neoplasm, or a cancer. It is to be understood that any nucleic acid compound that can express NSP1 (or a peptide fragment thereof) can be utilized for treatment and/or administration, such as (for example) a DNA expression construct, an RNA expression transcript, and/or a viral vector, as would be understood in the art.

[0059] In various embodiments a compound that mimics NSP1 (or a peptide fragment thereof) capable of improving protein synthesis is utilized for treatment. In various embodiments, a compound that induces higher levels of endogenous protein synthesis in an individual is utilized as a treatment. In some embodiments, a compound that targets and modulates activity of one or more endogenous proteins involved with translation is utilized for treatments. Endogenous proteins that can be targeted include (but is not limited to) ABCE1 , Rackl , ZNF598, ASCC3, elF5A, ZAK-alpha, p38, JNK, GCN2, and elF2alpha.

[0060] In some embodiments, proteins, peptides and compounds described herein are utilized in a therapeutically effective amount as part of a course of treatment. As used in this context, to "treat" means to ameliorate or prophylactically prevent at least one symptom of the disorder to be treated or to provide a beneficial physiological effect. For example, one such amelioration of a symptom could be motor movement and/or cognitive ability. Assessment of improvement can be performed in many ways, as understood in the art.

NSP1 proteins and peptides

[0061] Several embodiments are directed to the use of NSP1 or a peptide fragment of NSP1 as a medicinal biologic for the treatment of neurodegeneration and cancer. This treatment strategy is based on the fact that NSP1 improves proper protein synthesis within cells, yielding less aberrant protein production. Further, as detailed in the Exemplary Embodiments, NSP1 and peptide fragments of NSP1 provide a therapeutic benefit to neurodegenerative disorders and cancer. Based on these findings, NSP1 protein and peptides of NSP1 can be manufactured into a medicament for treatment.

[0062] Provided in Fig. 1 is schematic depicting a SARS-CoV-2 virus and its genome (image obtained from Q. Zhang, et. al., Sig Transduct Target Ther G, 233 (2021 ), the disclosure of which is herein incorporated by reference) . As can be seen, NSP1 exists within the ORF1 a region and is the first translated protein in the genome. The use of NSP1 and peptides as described herein can be based on the coronavirus genome. Accordingly, various embodiments can utilize ORF1A, the N-terminal portion of ORF1A, NSP1 , and peptides of NSP1 .

[0063] SARS-CoV-2 NSP1 is 180 amino acids long and is generally broken further down into two regions: an N-terminal region (AAs 13-127) and a C-terminal region (AAs 128-180). Various studies have found that both the N-terminal region and the C-terminal region interact with host ribosomes and thus either regions alone or in combination could be utilized within a medicament. It has also been found that shorter peptides are sufficient to provide a therapeutic benefit. As detailed in the Exemplary Embodiments, AAs 148- 180 are sufficient to yield therapeutic benefit. Provided in Table 1 is an exemplary list sequences of NSP1 proteins and peptides that can be utilized in a medicament.

[0064] In various embodiments, a peptide or protein of NSP1 for use in a medicament comprises an amino acid sequence having a length of at least ten amino acids and up to the full length NSP1 (e.g., 180 AAs in SARS-CoV-2 NSP1 ). In various embodiments, a peptide of NSP1 for use in a medicament comprises an amino acid sequence having a length of about 10 AAs, about 20 AAs, about 30 AAs, about 40 AAs, about 50 AAs, about 60 AAs, about 70 AAs, about 80 AAs, about 90 AAs, about 100 AAs, about 110 AAs, about 120 AAs, about 130 AAs, about 140 AAs, about 150 AAs, about 160 AAs, about 170 AAs, or 180 AAs. It should be understood that an NSP1 peptide can comprise any of the contiguous AAs of NSP1 having a length as described. In some particular embodiments, an NSP1 peptide for use in a medicament comprises N-terminal region AAs 13-127 or C-terminal region AAs 128-180. In some particular embodiments, an NSP1 peptide for use in a medicament comprises AAs 148-180.

[0065] Medicaments of NSP1 protein or peptides of NSP1 can be derived from various coronaviruses. In many embodiments, NSP1 protein or a peptide of NSP1 for use within a medicament is derived from a human coronavirus, such as (for example) SARS-CoV, SARS-CoV-2, MERS-CoV, 229E, NL63, OC43, and HKU1. In some embodiments, NSP1 protein or peptides of NSP1 for use within a medicament is derived from a nonhuman coronavirus. In some embodiments, NSP1 protein or a peptide of NSP1 for use within a medicament is derived from a zoonotic coronavirus. In some embodiments, NSP1 protein or a peptide of NSP1 for use within a medicament is derived from an emergent coronavirus. In some embodiments, NSP1 protein or peptides of NSP1 for use within a medicament is a chimera derived from two or more coronaviruses.

[0066] As is well understood in the field of virology, virus genomic sequences mutate and evolve quickly, which can yield an NSP1 amino acid sequence that is deviated from its parent NSP1 sequence. Furthermore, as is well understood in the field molecular biology, site-directed mutagenesis can be performed to yield an NSP1 amino acid sequence that is deviated from its parent NSP1 sequence. Accordingly, several embodiments are directed to NSP1 protein or a peptide of NSP1 for use within a medicament having a deviated sequence from a parent sequence, whether the deviation is naturally or unnaturally derived. In various embodiments, NSP1 protein or a peptide of NSP1 for use within a medicament has a change in one or more amino acids, an addition of one or more amino acids, a removal of one or more amino acids, or any combination thereof, as determined by a parent sequence. In various embodiments, NSP1 protein or a peptide of NSP1 for use within a medicament comprises a sequence that is greater than 99% homologous to its parent sequence, greater than 98% homologous to its parent sequence, greater than 97% homologous to its parent sequence, greater than 96% homologous to its parent sequence, greater than 95% homologous to its parent sequence, greater than 90% homologous to its parent sequence, greater than 80% homologous to its parent sequence, or greater than 70% homologous to its parent sequence.

[0067] NSP1 protein or a peptide of NSP1 can be generated via chemical synthesis or a biological expression system. Solid-phase peptide synthesis (SPPS) is utilized to generate NSP1 protein or a peptide of NSP1 via chemical synthesis. Any appropriate SPPS protocol can be utilized. The solid support can be any appropriate solid support, such as (for example) the Merrifield resin, the PAM resin, the Wang resin, or 2-chlorotrity I resin. Any appropriate protecting groups can be utilized, such as (for example) Fmoc or Boc. Peptides are generally synthesized in reverse order as compared to natural synthesis via ribosomes. In other words, synthetic peptides are generally synthesized from the C-terminus to the N-terminus.

[0068] To generate NSP1 protein or a peptide of NSP1 via a biological system, an expression system is utilized comprising a nucleic acid polymer-based expression vector and a host cell system. Nucleic acid molecules may be used to express large quantities of NSP1 protein or a peptide of NSP1 .

[0069] An expression vector can be utilized to express NSP1 protein or a peptide of NSP1. Nucleic acids encoding the protein or the peptide are inserted into expression vectors such that the gene product sequence is operatively linked to transcriptional and translational regulatory sequences. The term “regulatory sequence” refers to nucleic acid sequences that are necessary to affect the expression of transgene sequences to which they are operably linked. Such regulatory sequences may include a promoter, a splice junction, translation initiation codon, restriction enzyme sites for introducing an insert into the vector. The term “operably linked’ refers to a juxtaposition of a regulatory sequence with a transgene permitting them to function in their intended manner. A regulatory sequence “operably linked to a transgene sequence is ligated in such a way that expression of the transgene is achieved under conditions compatible with the control sequences. Examples of regulatory sequences permitting expression in eukaryotic host cells include (but are not limited to) the yeast regulator sequences A0X1 or GAL1 and the human regulatory sequences CMV- promoter, SV40- promoter, RSV-promoter, CMV- enhancer, SV40-enhancer and a globin intron. Regulatory elements may also include transcription termination signals, such as (for example) the SV40 poly-A site or the tk- poly-A site, typically operably linked downstream of the transgene.

[0070] Typically, expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as “flanking sequences” typically include one or more of the following operatively linked regulatory sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.

[0071] Numerous expression systems exist that include at least a part or all of the expression vectors discussed above. Prokaryote- and/or eukaryote-based systems can be employed for use with an embodiment to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Commercially and widely available systems include in but are not limited to bacterial, mammalian, yeast, and insect cell systems. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. Those skilled in the art are able to express a vector to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide using an appropriate expression system.

[0072] Several embodiments are also directed to the medicinal use of nucleic acid molecules for expression of NSP1 protein or a peptide of NSP1 within the recipient to be treated. In some instances, RNA molecules comprising a nucleic acid sequence encoding NSP1 protein or a peptide of NSP1 are utilized as a treatment. In some instances, expression vectors comprising a nucleic acid sequence encoding NSP1 protein or a peptide of NSP1 are utilized as a treatment. In either instance, nucleic acid molecules can be prepared via biological systems as described herein. The nucleic acid molecules can also be prepared in a way to promote translation within the host. For instance, nucleic acid molecules can be inserted into lipid vesicles, liposomes, or other delivery carriers that are capable of delivery the nucleic acid into a host cell for protein or peptide translation. In some instances, nucleic acid molecules are modified or have their sequence altered to promote survivability within the recipient. It should be understood, however, any mechanism for expression of NSP1 protein or a peptide of NSP1 within the individual to be treated can be utilized.

Antagonists of NSP1 or protein synthesis for use within medicaments

[0073] Various embodiments are directed to antagonizing NSP1 or protein synthesis for the treatment of a coronavirus infection. In many embodiments, an individual is administered an antagonist of NSP1 or protein synthesis to mitigate and/or prevent onset of medical disorders related to coronavirus infection (e.g. COVID). As described in the attached manuscript, it is now known that the mechanism of NSP1 is to dramatically increase protein synthesis for viral production. These discoveries provide a treatment approach for medical disorders related to coronavirus infection.

[0074] In some embodiments, a compound that targets and modulates activity of one or more endogenous proteins involved with translation is utilized for a treatment of coronavirus infection. Endogenous proteins that can be targeted include (but is not limited to) ABCE1 , Rackl , ZNF598, ASCC3, elF5A, ZAK-alpha, p38, JNK, GCN2, and elF2alpha.

[0075] In some embodiments, an antagonist of NSP1 or protein synthesis is utilized within a medicament to treat an individual for coronavirus infection. In some embodiments, a subject is administered a medicament comprising an antagonist of NSP1 or protein synthesis to treat an individual for coronavirus infection. In some embodiments, a medicament is utilized as prophylaxis to prevent coronavirus infection. In some embodiments, a medicament is administered during active coronavirus infection to reduce viral load.

[0076] In some embodiments, proteins, peptides and compounds described herein are utilized in a therapeutically effective amount as part of a course of treatment. As used in this context, to "treat" means to ameliorate or prophylactically prevent at least one symptom of the disorder to be treated or to provide a beneficial physiological effect. For example, one such amelioration of a symptom could be reduction of coronavirus load or improvement of symptoms related to coronavirus infection. Assessment of improvement can be performed in many ways, as understood in the art.

Formulations

[0077] Provided herein are various embodiments of pharmaceutical compositions which incorporate one or more of certain compounds disclosed herein, or one or more pharmaceutically acceptable salts, prodrugs, or solvates thereof, optionally together with one or more pharmaceutically acceptable carriers thereof and optionally one or more other therapeutic ingredients. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art. The pharmaceutical compositions disclosed herein may be manufactured in any manner known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes. Pharmaceutical compositions may also be formulated as a modified release dosage form, including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmed-release, and gastric retention dosage forms. These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art (see, Remington: The Science and Practice of Pharmacy, 21 st Edition; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Modified-Release Drug Delivery Technology, Rathbone et al, Eds., Drugs and the Pharmaceutical Science, Marcel Dekker, Inc., New York, NY, 2002; Vol. 126).

[0078] The terms "active ingredient," "active compound," and "active substance" refer to a compound, which is administered, alone, in combination with other active compounds, or in combination with one or more pharmaceutically acceptable excipients or carriers, to a subject for treating, preventing, or ameliorating one or more symptoms of a disorder. [0079] The compounds disclosed herein can exist as therapeutically acceptable salts. The term "therapeutically acceptable salt," as used herein, represents salts or zwitterionic forms of the compounds disclosed herein which are therapeutically acceptable as defined herein. The salts can be prepared during the final isolation and purification of the compounds or separately by reacting the appropriate compound with a suitable acid or base. Therapeutically acceptable salts include acid and basic addition salts. For a more complete discussion of the preparation and selection of salts, refer to "Handbook of Pharmaceutical Salts, Properties, and Use," Stah and Wermuth, Ed., (Wiley-VCH and VHCA, Zurich, 2002) and Berge et al, J. Pharm. Sci. 1977, 66, 1 -19.

[0080] Several embodiments incorporate at least one active ingredient for the treatment of a subject for a neurodegenerative disorder, a neoplasm, a cancer, or a coronavirus infection. In some embodiments, an active ingredient is NSP1 (or a peptide fragment thereof). In some embodiments, an active ingredient is a nucleic acid compound for expressing NSP1 (or a peptide fragment thereof). In some embodiments, an active ingredient is an antagonist of NSP1 .

[0081] Numerous coating agents can be used in accordance with various embodiments of the invention. In some embodiments, the coating agent is one which acts as a coating agent in conventional delayed release oral formulations, including polymers for enteric coating. Examples include hypromellose phthalate (hydroxy propyl methyl cellulose phthalate; HPMCP); hydroxypropylcellulose (HPC; such as KLUCEL®); ethylcellulose (such as ETHOCEL®); and methacrylic acid and methyl methacrylate (MAA/MMA; such as EUDRAGIT®).

[0082] Various embodiments of formulations also include at least one disintegrating agent, as well as diluent. In some embodiments, a disintegrating agent is a super disintegrant agent. One example of a diluent is a bulking agent such as a polyalcohol. In many embodiments, bulking agents and disintegrants are combined, such as, for example, PEARLITOL FLASH®, which is a ready to use mixture of mannitol and maize starch (mannitol/maize starch). In accordance with a number of embodiments, any polyalcohol bulking agent can be used when coupled with a disintegrant or a super disintegrant agent. Additional disintegrating agents include, but are not limited to, agar, calcium carbonate, maize starch, potato starch, tapioca starch, alginic acid, alginates, certain silicates, and sodium carbonate. Suitable super disintegrating agents include, but are not limited to crospovidone, croscarmellose sodium, AMBERLITE (Rohm and Haas, Philadelphia, Pa.), and sodium starch glycolate.

[0083] In certain embodiments, diluents are selected from the group consisting of mannitol powder, spray dried mannitol, microcrystalline cellulose, lactose, dicalcium phosphate, tricalcium phosphate, starch, pregelatinized starch, compressible sugars, silicified microcrystalline cellulose, and calcium carbonate.

[0084] Several embodiments of a formulation further utilize other components and excipients. For example, sweeteners, flavors, buffering agents, and flavor enhancers to make the dosage form more palatable. Sweeteners include, but are not limited to, fructose, sucrose, glucose, maltose, mannose, galactose, lactose, sucralose, saccharin, aspartame, acesulfame K, and neotame. Common flavoring agents and flavor enhancers that may be included in the formulation of the present invention include, but are not limited to, maltol, vanillin, ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol and tartaric acid.

[0085] Multiple embodiments of a formulation also include a surfactant. In certain embodiments, surfactants are selected from the group consisting of Tween 80, sodium lauryl sulfate, and docusate sodium.

[0086] Many embodiments of a formulation further utilize a binder. In certain embodiments, binders are selected from the group consisting of povidone (PVP) K29/32, hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC), ethylcellulose (EC), com starch, pregelatinized starch, gelatin, and sugar.

[0087] Various embodiments of a formulation also include a lubricant. In certain embodiments, lubricants are selected from the group consisting of magnesium stearate, stearic acid, sodium stearyl fumarate, calcium stearate, hydrogenated vegetable oil, mineral oil, polyethylene glycol, polyethylene glycol 4000-6000, talc, and glyceryl behenate.

[0088] Modes of administration, in accordance with multiple embodiments, include, but are not limited to, oral, transdermal, transmucosal (e.g., sublingual, nasal, vaginal or rectal), or parenteral (e.g., subcutaneous, intramuscular, intravenous, bolus or continuous infusion). The actual amount of drug needed will depend on factors such as the size, age and severity of disease in the afflicted subject. The actual amount of drug needed will also depend on the effective concentration ranges of the various active ingredients.

[0089] A number of embodiments of formulations include those suitable for oral administration. Formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Typically, these methods include the step of bringing into association a compound of the subject invention or a pharmaceutically salt, prodrug, or solvate thereof ("active ingredient") with the carrier which constitutes one or more accessory ingredients.

[0090] Embodiments of formulations disclosed herein suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a nonaqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. Multiple embodiments also compartmentalize various components within a capsule, cachets, or tablets, or any other appropriate distribution technique.

[0091] Several embodiments of pharmaceutical preparations include tablets, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Tablets, in a number of embodiments, may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with binders, inert diluents, or lubricating, surface active or dispersing agents. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein. All formulations for oral administration should be in dosages suitable for such administration. Push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0092] In some embodiments, formulations described herein are administered in a therapeutically effective amount as part of a course of treatment of a subject for a neurodegenerative disorder, a neoplasm, a cancer, or a coronavirus infection. As used in this context, to "treat" means to ameliorate at least one symptom of a disorder to be treated or to provide a beneficial physiological effect. For example, one such amelioration of a symptom could be improvement of motor function or cognitive ability.

[0093] A therapeutically effective amount can be an amount sufficient to prevent reduce, ameliorate or eliminate the symptoms of diseases or pathological conditions susceptible to such treatment.

[0094] Dosage, toxicity and therapeutic efficacy of the compounds for clinical applications can be determined, e.g., by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to non-neoplastic cells and, thereby, reduce side effects.

[0095] Data obtained from cell culture assays or animal studies can be used in formulating a range of dosage for use in humans. If the medicament is provided systemically, the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration or within the local environment to be treated in a range that includes the IC50 (i.e. , the concentration of the test compound that achieves a half-maximal inhibition of neoplastic growth) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by liquid chromatography coupled to mass spectrometry.

[0096] An "effective amount" is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a composition depends on the composition selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compositions described herein can include a single treatment or a series of treatments. For example, several divided doses may be administered daily, one dose, or cyclic administration of the compounds to achieve the desired therapeutic result.

[0097] In various embodiments, a compound is administered at a concentration of about 100 nM to 100 mM. In various embodiments, a compound is administered at about a unit of: 100 nM, 1000 nM, 10 pM, 100 pM, 1000 pM, 10 mM, or 100 mM. In various embodiments, a compound is administered at a concentration of about 10 ng/kg to 100 mg/kg. In various embodiments, a compound is administered at about a unit of: 10 ng/kg. 100 ng/kg, 1000 ng/kg, 10 pg/kg, 100 pg/kg, 1000 pg/kg, 10 mg/kg or 100 mg/kg.

[0098] Preservatives and other additives, like antimicrobial, antioxidant, chelating agents, and inert gases, can also be present. (See generally, Remington's Pharmaceutical Sciences, cited supra.) Genetic Modulation

[0099] Genetic modulation can be performed on subjects by varying methodologies. In several embodiments, to increase expression, an expression cassette of the gene to be increased can be provided. Expression cassettes can be provided by transfecting into cells a plasmid, utilizing a viral vector, or integrating an expression cassette into the host genome. Integration of expression cassettes can be performed by recombination methods or the CRISPR/Cas9 system.

[0100] In many embodiments, to decrease expression, expression can be disrupted by various genetic techniques, including (but not limited to) introducing to a cell antisense oligos, introducing to a cell short-hairpin RNA, introducing into a cell a repressor, or ablating expression by disrupting the endogenous gene. Disrupting the endogenous gene can be done by recombination methods or the CRISPR/Cas9 system.

EXEMPLARY EMBODIMENTS

[0101] Biological data support the systems, formulations, and methods of treatments described herein. In the attached manuscript and figures, exemplary treatments for neurodegeneration are provided.

EXAMPLE 1 : Prevention of ribosome collision-induced neuromuscular degeneration by SARS CoV-2-encoded Nsp1

[0102] Here, the in vivo effects of SARS-CoV-2 encoded viral proteins in Drosophila neuromuscular tissues under normal and disease conditions was investigated. This led to an unexpected finding of amelioration of neuromuscular degeneration by Nsp1 in multiple neurodegenerative disease models and new insights into the biochemical function of Nsp1 in manipulating the host translation machinery. Results

The AD-related proteostasis failure and neuromuscular degeneration in amyloid precursor protein C-terminal fragment (APP.C99) transgenic flies are rescued by Nsp1

[0103] Although SARS-CoV-2 causes severe respiratory disease, it also affects other organ systems, including the musculoskeletal system. To test potential effect of individual viral proteins on muscle function, viral proteins that are likely to engage in intracellular virus-host interactions were tissue-specifically expressed using the Mhc-Gal4 driver and the UAS-Gal4 system. Twelve SARS-CoV-2 proteins, Nsp1 , Nsp2, Nsp3, Nsp6, Orf3a, Orf3b, Orf6, Orf7a, Orf7b, Orf8, Orf9b and Orf10, were individually expressed. Using two assays, wing posture (Fig. 2), a reflection of indirect flight muscle integrity, and locomotor activity (Fig. 2), a measure of muscle function. No obvious effect of the individual viral proteins upon muscle function was found.

[0104] It was next tested whether the expression of SARS-CoV-2 viral proteins might exacerbate already compromised neuromuscular function in disease conditions. APP.C99 corresponds to the C-terminal fragment of APP resulting from b-secretase cleavage. APP.C99 and its aberrant translation products are emerging as key players in AD pathogenesis. Muscle can be a useful system for studying APP-induced toxicity, as APP-derived amyloid pathology in muscle similar as that seen in AD brain is associated with inclusion body myositis (IBM), a muscle disease, and APP.C99 transgenic mice have been used to model IBM. Expression of APP.C99 in the fly muscle resulted in indirect flight muscle degeneration manifested as abnormal held-up or droopy wing postures (Fig. 3). Surprisingly, whereas co-expression of most of the SARS-CoV-2 viral proteins tested had no effect on APP.C99-induced wing posture defect, the co-expression of Nsp1 completely suppressed such defect at different ages (Figs. 3 and 4). Expression of Nsp1 protein was confirmed by western blot and immunostaining (Fig. 3). The wing posture defect is known to correlate with APP.C99-induced proteostasis failure manifested as the formation of protein aggregates immunopositive for APP.C99 (detected with 6E10 antibody), ubiquitin (Ubi), and the autophagy receptor p62, and endolysosomal defects such as enlargement of Rab5-positive early endosome and accumulation of enlarged lysosomes detected with Lamp1 -GFP that are likely caused by altered autophagy flux. Similar APP.C99-inducd endolysosomal defects are observed in mammalian AD models. APP.C99-induced proteostasis failure (Figs. 5 and 6) and endolysosomal defects (Figs. 5 and 6 were effectively suppressed by Nsp1. Muscle expression of APP.C99 also resulted in locomotor defects (Fig. 6) and significantly shortened lifespan (Fig. 6), which were also effectively suppressed by Nsp1. Neuronal expression of APP.C99 using the pan-neuronal elav-Gal4 driver or the mushroom body-specific R13F02-Gal4 driver caused proteostasis failure, locomotor and learning and memory deficits, which were also effectively rescued by Nsp1 coexpression (Fig. 7). These results were totally unexpected, as it was initially expected to see exacerbation of the pre-existing neuromuscular defects in the APP.C99 model by SARS-CoV-2 viral proteins, considering that post-infection patients with the long-COVID syndrome presented neuromuscular deficits that became persistent with time and caused disabilities, and AD patients were reported to be more susceptible to COVID-19, albeit with undefined mechanism.

The proteostasis failure, neuromuscular degeneration, and cognitive deficits in full-length APP-based models are rescued by Nsp1

[0105] The effect of Nsp1 on AD-related phenotypes was tested in full-length APP (FL- APP) contexts. Overexpression of FL-APP in the muscle caused wing posture defect, although the penetrance was not as high as in the APP.C99 model, presumably due to the lower level of APP.C99 produced from FL-APP. This phenotype was completely rescued by Nsp1 (Fig. 8).

[0106] The effect of Nsp1 on APP/APP.C99-induced defects in learning and memory was also tested. Transgenic flies co-expressing FL-APP and b-secretase (BACE1 ) in neurons under elav-Gal4 control were used for the behavioral assay. BACE1 was included to facilitate APP.C99 production from FL-APP. Aversive taste memory was measured as controlled by a neural circuit involving dopaminergic input to neurons in mushroom body - the memory center of the flies, and mushroom body output neurons directing taste response. Applying sucrose solution (appetitive tastant) to the tarsi (feet) of a starved fly induced robust feeding behavior as measured by the proboscis extension reflex (PER). After rounds of paired application of sucrose to the tarsi and quinine (aversive tastant) to the proboscis, normal flies learned from experience and showed attenuated PER response to subsequent application of sucrose alone. Compared to control flies, elav-Gal4>FL-APP/BACE flies presented impaired aversive taste memory as indicated by the higher PER response (Fig. 8). Importantly, Nsp1 co-expression effectively attenuated the PER response in elav>FL-APP/BACE flies, whereas UAS-RFP control had no effect (Fig. 8). Nsp1 co-expression also rescued the proteostasis defect in elav>FL-APP/BACE fly brain, as shown by the removal of ubiquitin- and p62-positive protein aggregates (Fig. 8). The elav>FL-APP/BACE flies present neurodegeneration phenotypes as shown by the loss of dopaminergic neurons (DN) in the PPL1 cluster. This phenotype was also rescued by Nsp1 (Fig. 8). Muscle expression of FL-APP/BACE in Mhc>FL-APP/BACE flies also caused proteostasis failure manifested as the formation of protein aggregates immunopositive for ubiquitin and p62 and abnormal wing posture, which were both suppressed by Nsp1 (Fig. 9). These data demonstrate that Nsp1 potently rescues AD-related key pathological features induced by FL-APP and FL-APP-derived APP.C99.

Nsp1 promotes removal of aberrant APP.C99 species resulting from inadequate RQC of ribosome stalling

[0107] The molecular mechanism by which Nsp1 rescues APP.C99-induced pathologies was investigated. It was recently shown that during the co-translational translocation of APP and APP.C99, ribosomes stall on the ER translocon at two locations, one at the stop codon site and another at an internal site ~ 30-40 AA upstream of the stop codon site. Stalling at the stop codon site was associated with decreased level of the ribosome splitting and recycling factor ABCE1 , whereas the internal stalling might be caused by ER-targeting, translocon-gating, and co-translational protein folding and membrane insertion, events intrinsic to APP.C99 biogenesis and membrane topogenesis that are likely to slow down translation and cause ribosome collision. In Mhc>APP.C99 flies co-expressing Nsp1 , but not Nsp2, internally stalled APP.C99 was virtually undetectable by western blot in newly eclosed flies (Fig. 10). Only upon longer exposure was a faint signal detected (Fig. 11 ). That the lower band corresponded to internally stalled APP.C99 was based on its differential reactions to antibodies against the very N- terminus (6E10) or the very C-terminus (C.1/6.1 ) of APP.C99. Whereas stop codon stalled FL-APP.C99 reacted with both 6E10 and C.1/6.1 , internally stalled APP.C99 only reacted with 6E10 (Fig. 11 ). FL-APP.C99 level was also significantly reduced in Nsp1 coexpressed flies compared to flies without Nsp1 co-expression, consistent with immunostaining for APP.C99 (Fig. 10). The remaining FL-APP.C99 protein in Nsp1 coexpressing flies was interpreted as those APP.C99 proteins that have successfully passed the stall, completed the co-translational translocation, and entered the ER lumen. As flies aged, the level of FL-APP.C99 species also gradually diminished (Fig. 10), suggesting that Nsp1 might act through yet another mechanism to downregulate APP.C99 protein level, for example by inhibition of new round of translation or promoting protein turnover. Treatment with chloroquine, which blocks the binding of autophagosomes to lysosomes, partially blocked the Nsp1 effect on APP.C99 protein abundance (Fig. 10), supporting the involvement of autophagy/lysosomes.

[0108] The effect of Nsp1 on FL-APP was next examined. The level of FL-APP was significantly reduced in Mhc>FL-APP (Fig. 12) and Mhc>FL-APP/BACE muscle tissues expressing Nsp1 (Fig. 11 ). Moreover, in Mhc>FL-APP/BACE (Fig. 11 ) and Mhc>FL-APP (Fig. 11 ) flies both the internally stalled and stop-codon stalled FL-APP. C99 species were effectively removed by Nsp1. In the brain tissues of elav>FL-APP/BACE flies coexpressing Nsp1 , internally stalled APP.C99 was dramatically reduced, whereas FL- APP. C99 was less affected (Fig. 12), suggesting that FL-APP. C99 level might be differentially regulated by Nsp1 in different cell types. FL-APP level was also significantly reduced by Nsp1 in elav>FL-APP/BACE fly brain (Fig. 12). Moreover, a dose-dependent effect of Nsp1 on APP.C99 level was observed when one copy or two copies of the UAS- Nsp1 transgene were co-expressed with APP.C99 in photoreceptor neurons using the GMR-Gal4 driver (Fig. 11 ). Nsp1 thus rescues APP.C99-associated toxicity by removing translationally stalled APP/APP.C99 species and downregulating overall APP/APP.C99 levels.

Nsp1 does not cause global shutdown of protein synthesis or turnover of APP.C99 mRNA in Drosophila

[0109] Nsp1 is a virulence factor proposed to restrict cellular gene expression by inhibiting translation through blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting global mRNA degradation. Whether Nsp1 affects host mRNA translation or degradation at an organismal level has not been examined. Muscle, neuronal, or body-wide expression of Nsp1 alone had no obvious detrimental effect in flies. Ubiquitous Nsp1 also had no effect on body size (Fig. 13), a sensitive readout of global translation activity in flies as shown by the “minute” phenotype caused by inhibition of translation. It was suspected that Nsp1 might not act as a strong repressor of global translation, at least in Drosophila tissues. This was supported by the relatively unchanged expression levels of actin and other proteins we checked. If anything, at least in the case of ABCE1 its level might be modestly increased by Nsp1 (Fig. 11 ). To further test if Nsp1 affects global translation in Drosophila tissues, a new approach was used to measure protein synthesis in Drosophila tissues, which is based on the incorporation of puromycin (Pum) into NPCs. Pum-labelled newly synthesized peptides were then detected using an anti-Pum antibody. With this method, a significant difference was not found in new protein synthesis in the muscle tissues of animals with or without Nsp1 co-expression (Fig. 13). qRT-PCR was performed to analyze mRNA level and evidence of degradation of APP.C99 mRNA was not found (Fig. 14). The mRNA levels of several other host genes we tested were either not affected or increased (Fig. 14). Although further proteomics and transcriptom ics studies are needed to assess Nsp1 effect on host protein and mRNA expression, this data indicate that the effect of Nsp1 on APP/APP.C99 expression is unlikely through mRNA degradation or inhibition of global translation.

Nsp1 aborts stalled translation

[0110] Translation stalling of APP.C99 also occurs in mammalian cells. As in fly tissues, Nsp1 dramatically lowered stalled translation products of APP.C99 (Fig. 15) and overall APP.C99 expression as detected by immunostaining (Fig. 15) in HeLa cells. In contrast, an Nsp1 mutant containing the K164A/H165A (Nsp1 -KH) mutations, which abolish the ribosome-binding of Nsp1 , was less effective in reducing stalled translation products of APP.C99, although itself was expressed at a higher level than wild type Nsp1 (Fig. 15). Nsp1 , but not Nsp1 -KH, also significantly reduced FL-APP (Fig. 15). To test the in vivo effect of Nsp1 -KH, UAS-Nsp1-KH transgenic flies we generated. When expressed at comparable level as wild type Nsp1 (Fig. 16), Nsp1 -KH had no obvious effect on stalled APP.C99 translation (Fig. 16), nor APP.C99-induced proteostasis failure (Fig. 16) and wing posture defect (Fig. 16). Mammalian cells were used to investigate the mechanism of Nsp1 regulation of stalled translation. First, Pum labeling was performed on stalled NPCs. This involved pre-treatment of cells with homoharringtonine (HHT), which allows elongating/active ribosomes to run off but prevents new rounds of translation. This was followed by a combined emetine and Pum treatment to incorporate tRNA-like Pum to the C-termini of ribosome-stalled NPCs. This way, Nsp1 but not Nsp1-KH was found to significantly reduce the level of stalled NPCs in APP.C99 expressing cells (Fig. 16).

[0111] Next, the GFP-P2A-K20-P2A-RFP reporter was used to assess the effect of Nsp1 on ribosome stalling. In this reporter, the GFP, K20, and RFP reporters are used to monitor overall mRNA translation, translational stalling by 20 consecutive K residues, and readthrough of the stall site, respectively, with the self-cleaving P2A allowing each reporter to be independent marker of translation. As a control, the GFP-P2A-K0-RFP reporter without a stall signal was used. In contrast to situations in which the early RQC pathway was disabled, as in ZNF598 knockdown condition, that resulted in readthrough of the K20 stall and increased RFP/GFP ratio, Nsp1 co-transfection significantly reduced the ratio of RFP/GFP expressed from the GFP-P2A-K20-P2A-RFP reporter compared to without Nsp1 (Fig. 17), suggesting that readthrough of the K20 stall was further blocked. The Nsp1 -KH mutant was less effective in this assay (Fig. 17). No effect on the ratio of RFP/GFP expressed from the non-stalling GFP-P2A-K0-RFP reporter was observed for Nsp1 or Nsp1 -KH (Fig. 17), although Nsp1 apparently inhibited overall translation of both reporters in mammalian cells. Using another translation stall reporter, GFP-P2A-Flag- K20-P2A-mKate2, in which the arrested translation product can be detected with the Flag antibody, it was found that Nsp1 reduced the mKate2/GFP ratio, consistent with the GFP- P2A-K20-P2A-RFP reporter data, and levels of both FL-Flag-K20 and arrested Flag-K20 products, suggesting that it aborted stalled translation (Fig. 18). Importantly, the effect of Nsp1 in aborting stalled translation was dependent on early RQC factors - the ribosome collision sensor ZNF598 and the ribosome disassembly factor ASCC3, as FL-Flag-K20 level and mKate2/GFP ratio were both increased when ZNF598 or ASCC3 was inhibited, irrespective of Nsp1 presence (Fig. 18). Together with the results of Pum labeling of stalled NPCs, these data support the hypothesis that a normal activity of Nsp1 is to abort stalled translation. In SARS CoV-2 infection condition, this presumably serves to promote the disassembly of stalled ribosomes on viral RNAs to prevent the accumulation of aberrant viral proteins, or to recycle ribosomes stalled on host mRNAs to make them available for viral translation. The reduction of total FL-APP and APP.C99 levels by Nsp1 observed earlier is also consistent with this abortive termination mode of NSP1 action on stalled ribosomes.

Nsp1 promotes resolution of collided ribosomes and inhibits cGAS/STING signaling

[0112] To further test the mechanism of Nsp1 action in handling stalled translation, the effect of Nsp1 on the ubiquitination of 40S subunit Rps3 was assessed, which is an indicator of ribosome collision. Consistent with APP.C99 translation causing ribosome collision, Rps3 ubiquitination was elevated in APP.C99 transfected HeLa cells. Low concentration of anisomycin (Ans) was used to induce ribosome collision and Rps3 ubiquitination. Nsp1 significantly reduced collision-induced Rps3 ubiquitination in APP.C99-expressing and Ans treatment conditions, whereas Nsp1-KH was less able to do so (Fig. 19), consistent with Nsp1 resolving collided ribosomes. To further test this hypothesis, sucrose gradient analysis of ribosomes was performed and the distribution of ASCC3 and EDF1 was examined, which are factors preferentially associating with collided ribosomes. Nsp1 did not change the distribution of 40S protein Rps3 across the sucrose gradient but reduced the abundance of ASCC3 and EDF1 in the polysome fractions in HeLa cells (Fig. 20). This was also observed for EDF1 in Ans treated HeLa cells (Fig. 20). Together, these results support the notion that Nsp1 promotes resolution of collided ribosomes.

[0113] The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway senses cytosolic DNA and induces interferon signaling to activate the innate immune system. Translation stress and collided ribosomes can serve as coactivators of cGAS, with ribosome collision leading to cytosolic localization of cGAS, which preferentially interacts with collided ribosomes, and the ribosome association stimulates cGAS activity. Using cytosolic cGAS localization as a proxy of its activation, it was found that Nsp1 significantly attenuated ribosome collision-induced cGAS cytoplasmic distribution and thus activation (Fig. 20). Western blot analysis of subcellular fractionation confirmed the relative nuclear enrichment of cGAS in Nsp1 expressing cells (Fig. 21 ). These results were correlated with attenuated innate immune signaling as monitored with p-JUN and p-JNK, and expression of interferon-stimulated gene RSAD2 (Fig. 22). Moreover, the effect of Nsp1 in suppressing the cytosolic distribution of cGAS was at least partially dependent on the early acting RQC factors ZNF598 and ASCC3 that disassemble collided ribosomes (Figs. 20 and 21 ). These data further corroborate the novel role of Nsp1 in resolving collided ribosomes and offer a new mechanism of innate immune evasion by SARS-CoV-2. It was also tested if ribosome collision-induced cGAS- STING signaling is relevant to the neuromuscular toxicity of APP.C99. The abnormal wing posture and locomotor defects in Mhc>APP.C99 flies was effectively rescued by RNAi of the fly homolog of STING (Fig. 22), supporting that cGAS-STING signaling contributes to ribosome collision-related neuromuscular toxicity.

The ribosome splitting and recycling factor ABCE1 mediates Nsp1 effect on stalled APP.C99 translation

[0114] To understand the mechanism of action of Nsp1 in regulating ribosome stalling, published reports on the Nsp1 -interactome were searched for potential interaction between Nsp1 and RQC-related factors. One study reported the presence of ABCE1 , together with translation initiation and elongation factors, in the immunocomplex captured with a 3xFlag-Nsp1 construct , although the functional significance of these interactions was unknown. Given the critical role of ABCE1 in regulating ribosome splitting and recycling during RQC, it was tested if ABCE1 is required for Nsp1 to abort stalled APP.C99 translation. ABCE1 knockdown, with the knockdown efficiency confirmed by western blot (Fig. 23), resulted in recovery of stalled APP.C99 species removed by Nsp1 in HeLa cells (Fig. 24). Other RQC factors were also tested for possible role in mediating the effect of Nsp1 on stalled APP.C99 translation. Knockdown of Rackl , a 40S ribosomal protein previously implicated in regulating 40S subunit ubiquitination and resolving stalled ribosomes, also resulted in partial recovery of stalled APP.C99 species removed by Nsp1 (Figs. 23 and 24). Intriguingly, although ABCE1 and eRF1 work in a complex to terminate normal translation at stop codons, unlike ABCE1 , the knockdown of eRFI had no obvious effect on the removal of stalled APP.C99 by Nsp1 (Fig. 23). Furthermore, although the elF3 complex involved in initiation was shown to associate with Nsp1 directly or indirectly, knocking down a key elF3 component elF3E had no obvious effect on NspTs ability to abort stalled APP.C99 translation (Fig. 23). These data support a specific role of ABCE1 in mediating the effect of Nsp1 in regulating stalled APP.C99 translation.

Nsp1 employs a multipronged strategy to manipulate stalled APP.C99 translation in Drosophila AD models

[0115] The role of ABCE1 in mediating the effect of Nsp1 in suppressing APP.C99- induced neuromuscular pathology in vivo was further examined. ABCE1 RNAi partially blocked the effect of Nsp1 on APP.C99-induced wing posture (Fig. 24) and locomotion (Fig. 24) defects in an age-dependent manner (Fig. 23), concomitant with ABCE1 RNAi age-dependently recovering the stalled APP.C99 species removed by Nsp1 (Fig. 25). The age-dependent effect was likely due to incomplete knockdown of ABCE1 by the RNAi transgene and age-related decline of ABCE1 level, such that the knockdown of ABCE1 was more complete in older flies. This was confirmed by RT-PCR and western blot analyses of ABCE1 RNA and protein levels (Fig. 26). As in mammalian cells, eRF1 RNAi was ineffective in blocking the Nsp1 effect (Fig. 26). This lack of effect by eRF1 RNAi ruled out any possible explanation of the ABCE1 effect as caused by the RNAi process or titration of Gal4 by an added copy of UAS transgene, therefore supporting the specificity of the ABCE1 -Nsp1 interaction. ABCE1 RNAi also blocked the effect of Nsp1 in rescuing the proteostasis failure and endolysosomal defects caused by APP.C99 (Fig. 25). In the aversive taste memory assay, ABCE1 RNAi effectively blocked the Nsp1 effect in rescuing the memory deficit in elav>FL-APP/BACE flies (Fig. 25), whereas eRF1 RNAi had no significant effect (Fig. 26). Moreover, the effect of Nsp1 in reducing FL-APP and APP.C99 expression in elav>FL-APP/BACE flies was also partially blocked by ABCE1 RNAi (Fig. 27), but not eRF1 RNAi (Fig. 26). Correspondingly, ABCE1 RNAi attenuated the rescuing effect of Nsp1 on proteostasis failure in brain tissues of elav>FL-APP/BACE flies (Fig. 26).

[0116] Previous studies implicated mechanistic target of rapamycin complex 2 (mTORC2)-AKT signaling in regulating the RQC process. It was found that knockdown of fly AKT partially recovered FL-APP.C99 species removed by Nsp1 , suggesting that AKT signaling also mediates the effect of Nsp1 in the handling of stalled APP.C99 (Fig. 28), although the internally stalled APP.C99 species was not as efficiently recovered as in ABCE1 RNAi condition. Furthermore, knockdown of ATG1 , a key regulator of the autophagy process that was previously implicated in regulating APP.C99 toxicity, also partially recovered FL-APP.C99 but not the internally stalled lower APP.C99 species removed by Nsp1 (Fig. 28). Consistent with autophagy playing a key role in regulating aberrant translation products, ATG1 OE efficiently removed internally stalled APP.C99 species (Fig. 28), whereas knockdown of ATG1 and another autophagy gene ATG8 resulted in increased levels of internally stalled APP.C99 species (Fig. 28), and enhanced wing posture defect caused by APP.C99 (Fig. 28).

[0117] Intriguingly, although ABCE1 -RNAi and ATG1 RNAi individually each resulted in recovery of primarily the FL-APP.C99 protein in young flies, their combined RNAi recovered both FL-APP.C99 and internally stalled APP.C99 species (Fig. 28), concomitant with the reappearance of the wing posture defect (Fig. 28). Moreover, induction of ribosome collision with Ans facilitated the recovery of internally stalled APP.C99 species removed by Nsp1 in ABCE1 -RNAi and ATG1 RNAi conditions but had less effect in ABCE1 RNAi/ATG1 RNAi conditions (Fig. 28). Although the biochemical mechanisms by which Nsp1 impinges on ABCE1 , AKT, and ATG1 to regulate the abundance of stalled APP.C99 species remain to be elucidated, these results support the notion that Nsp1 deploys a multi-pronged strategy to handle stalled APP.C99 translation.

Nsp1 specifically and robustly rescues the neuromuscular degeneration phenotypes in Drosophila PD and ALS models that also feature translation stalling [0118] Encouraged by the robust effect of Nsp1 in rescuing the disease phenotypes in AD models, its effect was further tested in other disease models. Inefficient resolution of stalled translation also contributes to neuromuscular degeneration in the PINK1 model of PD, and the poly(GR) model of C9ORF72-ALS/FTD. Overexpression of Nsp1 completely rescued the muscle degeneration-induced wing posture defect in PINK1 mutant flies and Mhc>GR80 flies expressing 80 GR dipeptide repeats in the fly muscle (Fig. 29). This was correlated with rescue of the locomotor deficits in these flies (Fig. 30). At the cellular level, Nsp1 restored proteostasis in the flight muscle as indicated by the significant removal of p62- and Ub-positive protein aggregates (Fig. 30), and it improved mitochondrial morphology (Figs. 30 and 31 ). In the PINK1 model, Nsp1 overexpression also rescued neuron loss (Fig. 30) and restored mitochondrial morphology (Fig. 31 ) in the PPL1 cluster DNs. Importantly, the ability of Nsp1 to rescue the proteostasis failure and mitochondrial morphology defect in both the PINK1 and Mhc>GR80 flies was significantly blocked by the knockdown of ABCE1 (Figs. 30 and 31 ). The ability of Nsp1 to rescue the wing posture defect caused by muscle degeneration in both the PINK1 and Mhc>GR80 flies was also significantly blocked by the knockdown of ABCE1 , but not eRF1 (Figs. 29 and 30).

[0119] In PINK1 mutant flies, inadequate RQC of stalled translation of complex-l 30 kD subunit (C-130) mRNA at the stop codon site resulted in reduced normal C-130 protein level and the formation of a CAT-tailed C-130 species (C-130-u). Nsp1 efficiently removed C-I30-U and restored normal C-I30 level (Fig. 32), indicating that Nsp1 resolved C-I30 translation stalling. This effect of Nsp1 was significantly attenuated by the knockdown of ABCE1 , but not eRFI , demonstrating that ABCE1 specifically mediates the effect of Nsp1 in resolving stalled C-I30 translation (Fig. 32). Similarly, aberrant poly(GR) translation products resulting from inadequate RQC of stalled GR80 translation were also robustly removed by Nsp1 , and ABCE1 -RNAi, but not eRF1 RNAi, partially blocked this Nsp1 effect (Fig. 32).

[0120] The specificity of Nsp1 action in rescuing neurodegeneration phenotypes was assessed. Photoreceptor neuron degeneration caused by dFoxo overexpression or polyglutamine (polyQ) expansion was not affected by Nsp1 (Fig. 33). Photoreceptor neuron degeneration caused by overexpression of AD-related tau was also not affected by Nsp1 (Fig. 33). At the molecular level, Nsp1 did not affect tau protein expression (Fig. 33). This lack of effect of Nsp1 on tau protein expression or toxicity also argues against its rescue of the neuromuscular degeneration phenotypes in the AD, PD, and ALS models being attributable to a general translational inhibition of disease-associated proteins. Since there is no evidence that the translation of tau is stalled or subjected to RQC, these data support that Nsp1 exhibits specificity in its modulation of neuromuscular pathology, preferentially affecting those caused by inadequate RQC of stalled translation. Methods

Drosophila genetics

[0121] Fly culture and crosses were performed according to standard procedures. Adult flies were generally raised at 25°C and with 12/12 hr dark/light cycles. Fly food was prepared with a standard receipt (Water, 17 L; Agar, 93 g; Cornmeal, 1 ,716 g; Brewer’s yeast extract, 310 g; Sucrose, 517 g; Dextrose, 1033 g).

[0122] The UAS-SARS-CoV-2 viral protein transgenic fly lines UAS-Nsp1, Nsp2, Nsp3, Nsp6, Orf 3a, Orf 3b, Orf6, Orf7a, Orf7b, Orf8, Orf9b and Orf 10 were utilized. The following flies were obtained from the Bloomington Drosophila Stock Center: elav-GAL4 (8765), UA S-APP.C99 (33783), UA S-APP (6700), UAS-APP;UAS-BACE (33798), UAS- LAMP1-GFP (42714), UAS-ABCE1-RNAi (57740), UAS-ZNF598-RNAi (61288), UAS- eRF1-RNAi (67900), UAS-ATG1-RNAi (16133);, UAS-STING-RNAi (31565). Obtained were UAS-CIbn-RNAi (v103351 ) and UAS-AKT-RNAi (v103703) from Vienna Drosophila Stock Center. Obtained were UAS-ABCE1 (F001097), UAS-ZNF598 (F001909), UAS- Pelo (F003036) from FLYORF. Drs. T. Littleton provided MHC-GAL4, S. Birman provided TH-GAL4, Eric Baehrecke provided UAS-ATG1, Fen-biao Gao provided UAS-GR80, Mel Feany provided UAS-tau, Jongkyeong Chung provided PINK1-B9, E. Hafen provided UAS-dFoxo. UAS-Q82-YFP was generated as follows: The Q82-YFP cDNA (a gift from Dr. Richard Morimoto) was cloned into pUAST vector, and the resulting pUAST-87Q-YFP construct was injected into w- embryos to generate transgenic lines. The indicated UAS RNAi and OE fly lines were crossed to Mhc-Gal4 or elav-Gal4 driver lines for muscle or pan-neuronal expression, respectively. To make the UAS-Nsp1-KH transgenic flies, Nsp1-KH cDNA with a C-terminal Flag tag amplified from the pcDNA- Nsp1 -KH plasmid (Addgene 164522) by PCR was cloned into the pUAST vector. Sequence-confirmed construct DNA was injected into embryos by Bestgene to make transgenic flies.

Pum labeling of fly tissue

[0123] Pum labeling of newly synthesized proteins in Drosophila larvae was performed to similar protocols in prior studies. Briefly, 5-10 third instar larvae were transferred into Schneider’s media containing 10 pg/ml Pum and incubated in a nutator for 40 min at room temperature. Subsequently, the larvae were snap frozen in dry ice. The muscle tissues were dissected in ice cold PBS and used to prepare protein lysates for western blot analyses.

Lifespan analysis

[0124] Flies were reared in vials containing standard cornmeal food. Flies were anesthetized using CO2 and collected at a density of 20 male flies/vial. All flies were kept at humidified, 12h on/off light cycle at 25°C. Flies were flipped into fresh vial every 3 days and the number of dead animals was recorded.

Cell lines

[0125] HeLa, LI2OS, and HEK293T cells were purchased from ATCC. Cells were cultured under standard tissue culture conditions (1x DMEM medium - GIBCO, 10% FBS, 5% CO2, 37°C).

Drug treatments

[0126] HeLa cells were treated with the following drug concentration and times as indicated in the main text. Cycloheximide: 50 pg/ml for 4 hrs; HHT: 5 pM for 10 mins; emetine: 100 pM for 15 mins; Pum: 100 pM for 15 mins; Ans: 0.19 pM for 30 mins. For drug treatment in Drosophila, adult flies were raised in standard fly food supplemented with 0.25 pg/ml of emetine, or 3 mM Ans for 7 days. To provide chloroquine to flies, 7- day old flies were firstly starved for 6-8 hrs. Then a 2 mM chloroquine solution made in 100 mM sucrose was provided to the starved flies via soaked Kimwipe paper for 3-4 days. 10-11 days old flies were dissected to evaluate the level of APP.C99 protein expression in thoracic muscle by western blot.

Translation stalling reporter assays

[0127] Analysis of translation readthrough at K20 stall sequences using stalled vs. non-stalled reporter constructs (GFP-P2A-K20-P2A-RFP vs. GFP-P2A-K0-RFP or GFP- P2A-FLAG-K20-P2A-mKate2 vs. GFP-P2A-K0-mKate2) was performed essentially as previously described (12). The pmGFP-P2A-K0-P2A-RFP (#105686) and pmGFP-P2A- K(AAG)20-P2A-RFP (#105689) plasmids were purchased from Addgene. HeLa cells were transfected with KO and K20 reporters for 24 hrs, and thereafter NSP1 -WT and NSP1-KH were co-transfected for another 36 hrs. Cells were lysed and processed for immune blot assay. Translation readthrough of stall sequence was analysed by calculation of RFP and GFP ratio. For analysis of GFP-P2A-FLAG-K20-P2A-mKate2 vs. GFP-P2A-K0-mKate2 reporters, HEK293T cells were transfected with reporter constructs using polyethylenimine (1 mg/ml) for two days, followed by transfection with Nsp1 plasmid with or without ZNF598 or ASCC3 knockdown. Cellular GFP, FLAG and mKate2 proteins were measured by Western blot analysis.

Climbing activity and wing posture assays

[0128] Around 10-20 male flies were transferred to a clean plastic vial. The flies were allowed to get accustomed to the new environment for 3-4 min and subsequently measured for bang-induced vertical climbing distance. The performance was scored as percentage of flies crossing the 8 cm mark within 12 seconds. Each experiment was performed > 4 times. To assay wing posture, cohorts of flies raised at 25 or 29 degrees at the indicated ages were visually for straight, held-up, or droopy wing postures. The number of flies with normal (straight) or abnormal (held-up or droopy) wing postures were counted and quantified as the percentage of the total number of flies.

Aversive taste memory

[0129] Drosophila taste memory assay was performed similar to prior studies. Briefly, one-week old flies were starved for 12-18h in an empty vial on wet Kimwipe paper before test. Flies were anesthetized on ice and fixed on a glass slide by applying nail polish to their wings. 10-15 flies were used for each set of experiment. Flies were then incubated in a humid chamber for 2 h to allow recovery from the procedure. In the pretest phase, flies were presented with 500 mM sucrose stimuli (attractive tastant) to their legs using Kimwipe wick. Flies that showed positive proboscis extension to the stimulus were used for the next phases. In the second training phase, flies were presented with 500 mM sucrose stimuli at their legs while being simultaneously punished by 10 mM quinine (aversive tastant) applied to their extended proboscis. Training was repeated 15 times for each fly. The last phase is the test phase where the flies were given 500 mM sucrose at their legs at different time intervals (0, 5, 15, 30, 45, and 60 min), and proboscis extension was recorded. Each experiment was carried out > 4 times.

Extraction of fly proteins for western blot analysis

[0130] Around 5 fly thoraces or 10 fly heads were homogenized in 80 pl of either regular lysis buffer (50 mM Tris-HCI, 150 mM NaCI, 1 % Triton X100, protease inhibitors) or Urea lysis buffer (6 M Urea, 50 mM Tris-HCI, 150 mM NaCI, 0.1 % Triton X100, protease inhibitors) on ice. Samples were homogenized using a hand-held mechanical homogenizer for 30 secs. The homogenized samples were incubated on ice for 30 mins before centrifuging at 15000 rpm for 20 mins at 4°C. 30 pl of supernatant was mixed with 10 pl of 4x Lammaelli buffer (BioRad #161 -0747) and boiled for 5 mins at 100°C. The protein lysate was cooled, centrifuged and loaded onto 4-12% Bis-Tris gel (Invitrogen #NP0321 ) or 16% Tricine gel (Invitrogen #EC66955) with 1x MES (Invitrogen #NP0002) as running buffer. For analysis of C-I30-U in PINK1 mutant flies, a home-made gel system was used as described previously (2).

Analysis of gene expression by RT-qPCR

[0131] TRIzol (Invitrogen) was used to extract mRNA from fly thorax and iscript cDNA synthesis kit (Bio-Rad) to synthesize cDNA. Real time quantitative PCR (RT-qPCR) was performed using SYBR Green.

Protein extraction from cultured cells and western blotting

[0132] HeLa or LI2OS cells were transfected with the respective plasmids. Cells were washed with 1X PBS 30h post transfection and lysed in lysis buffer (50 mM Tris-HCI, 150 mM NaCI, 1 % Triton X100, protease inhibitors), followed by centrifugation at 13000 rpm for 20 mins at 4°C. Protein concentration was measured using the Bradford method. The supernatant was then mixed with 4x protein loading buffer and loaded onto either 4-12% bis-tris gels using MES as running buffer or on 16% Tricine gel and immunoblotted onto PVDF membranes. The membranes were blocked with blocking buffer (5% BSA in TBST) and incubated with following primary antibodies (Anti-Flag, Sigma-Aldrich F1804, 1 :2000; Anti-GFP, ProteinTech 66002, 1 :1000; anti-RFP, anti-mKate2, Invitrogen TagRFP Polyclonal Antibody, R10367; Anti-Actin, Sigma-Aldrich A2228, 1 :500; 6E10, Bio Legend 803001 , 1 : 1000; Anti-Myc, ProteinTech 16286, 1 :1000; Anti-Nsp1 , Cell Signaling #57896, 1 :200; Anti-ABCE1 , abeam (ab32270), 1 :1000; Anti-ZNF598, GeneTex GTX119245, 1 :250; Anti-Rack1 , Santa Cruz sc-17754, 1 : 1000; Anti-RSAD2, Proteintech 28089-1 -AP; anti-JUN, Proteintech 24909-1 -AP; anti-p-JUN, Proteintech 28891 -1 -AP; anti-JNK, Proteintech 24164-1 -AP; anti-p-JNK, Cell Signaling #9251 ). Goat anti-Rabbit IgG HRP, Santa Cruz sc2004 or Goat anti-Mouse IgG-HRP, Santa Cruz sc2005 antibodies were used for detection at 1 :10000 dilution. Special steps were taken during SDS PAGE to better resolve the different APP.C99 species as described before. For quantification of western blot data, signal intensity was measured and calculated using NIH Image J.

Immunohistochemistry

[0133] Immunostaining of adult fly muscle was performed similar to prior studies. Briefly, fly thoraxes were dissected and fixed with 4% paraformaldehyde (Electron Microscopy Sciences, cat. no. 15710) in phosphate buffered saline and 0.3% Triton X- 100 (PBS-T). Tissues were washed three times with PBS-T and then incubated for 30 min at room temperature in blocking buffer: 0.5% goat serum in PBS-T. The indicated primary antibodies (anti-Ubiquitin, Abeam ab140601 , 1 :1000; anti-Rab5, Abeam ab31261 , 1 : 1000; anti-P62, Abeam ab178440, 1 : 1000; 6E10, Bio legend 803001 , 1 :1000; anti-LAMP1 , DSHB 1 D4B, 1 :100) were added and samples were incubated overnight at 4 °C. The samples were washed three times with PBS-T and subsequently incubated with the indicated secondary antibodies (Alexa Flour 488 (A32723), Alexa flour 594 (A11036), Invitrogen, 1 :200) for 4h at 4 °C. After washing three times with PBS-T, samples were mounted in slow fade gold buffer (Invitrogen).

[0134] Immunostaining of adult brains was performed similar to prior studies. Briefly, adult fly brains were dissected and fixed on ice for 30-45 min in fixing buffer (940 pl of 1 % PBS-T and 60 pl of 37% formaldehyde). Tissues were washed three times in 0.1 % PBS- T and blocked overnight at 4°C in blocking buffer (1 ml 1x PBS, 0.1 % Triton-X, 5 mg/ml BSA). Following incubation at 4°C for 16 h with the indicated primary antibodies (anti-TH, Pel-Freez P40101 -150, 1 :1000; anti-Ubiquitin, Abeam ab140601 , 1 :1000; 6E10, Bio legend 803001 , 1 :1000; anti-Rab5, Abeam ab31261 , 1 :1000; anti-P62, Abeam ab178440, 1 :1000; Nsp1 , Cell Signaling #57896, 1 :200; ATP5A1 monoclonal antibody (15H4C4) from Thermo Fisher Catalog # 43-9800, 1 :1500), tissues were washed three times with 0.1 % PBS-T and subsequently incubated with the appropriate secondary antibodies [Alexa Flour 488 (A32723), Alexa flour 594 (A11036), Invitrogen, 1 :200], or F-actin attaining with Alexa Fluor™ 647 Phalloidin from Thermo Fisher (Catalog number: A22287; 1 :1000) for 4h at 4°C. Samples were washed three times with 0.1 % PBS-T and finally mounted in slow fade gold buffer (Invitrogen) and viewed using a Leica SP8 confocal microscope.

Immunostaining of cultured cells

[0135] For immunostaining of cultured cells, HeLa or LI2OS cells on coverslips were washed with PBS twice and fixed in 4% paraformaldehyde/PBS solution for 15 mins at RT. Cells were washed repeatedly with 1x PBS prior to incubating with PBS containing 0.1 % Triton X-100 for 20 mins. Cells were then incubated in blocking buffer (1x PBS, 0.1 % Triton X100, 2% BSA) for 30 mins. After blocking, desired primary antibodies (Anti- cGAS, Proteintech 26416-1 -AP; 6E10, BioLegend 803001 ; Anti-Puromycin, Millipore MABE343; mOC78, Gift from Dr. Glabe) were added to the blocking buffer at 1 :1000 concentration and cells were incubated with the antibody solution overnight. The following day, after washing with 1x PBS the following day, cells were incubated in appropriate secondary antibodies for 1 hr. Cells were washed again and the coverslips were mounted on slides using DAPI-containing mounting medium.

[0136] For analysis of cGAS distribution in Nsp1 transfected cell with RQC manipulation, ZNF598 KO U2OS cells and ASCC3 KD U2OS cells were transfected with NSP1 and RFP at the ratio of 3 to 1 for two days before harvesting. All cells were harvested and fixed with 3% paraformaldehyde in PBS for 10 minutes at room temperature. This was followed by permeabilization with 0.5% Triton X-100 for 15 minutes. Cells were washed with 1 ml PBS twice and blocked with PBS containing 5% goat serum for 30 minutes at room temperature. Primary antibodies (cGAS) were diluted in blocking buffer (PBS containing 5% goat serum) and incubated in cold room overnight. Cells were washed with 1 ml PBS and incubated with secondary antibody in blocking buffer for 1 h at room temperature. After the final wash cells were mounted on glass slides with antifade mounting medium (Vector Laboratories, H-1700). Images were captured with Leica Confocal Microscope.

Plasmid transfections and siRNA knockdown

[0137] Cell transfections were performed by using Lipofectamine 3000 (cat#: L3000015, Invitrogen), and siRNA knockdown experiments were performed using Lipofectamine RNAiMAX reagent (cat#: 13778150, Invitrogen), according to manufacturer’s instructions. For plasmid transfection, cells were plated at 70% confluency the day before in DMEM without antibiotics. On the day of transfection, plasmid DNA and lipofectamine reagent were individually mixed in OptiMEM such that the plasm id/reagent = 1 :3. After incubation of 10 mins at room temperature, the plasmid-reagent mixture was added dropwise to the cells. Medium was changed the following day and cells were analyzed 36-48h post transfection. For siRNA treatments, a similar protocol was followed with the final concentration of siRNA at 450 pmol for a 10cm dish (for a 10cm dish: 10 pg of DNA and 20 pl of Lipofectamine 3000, for 6cm dish: 3-5 pg of DNA and 5pl of Lipofectamine 3000 was added). For siRNA and plasmid co-transfection, cells were first treated with siRNA. 24h post transfection with siRNA, medium change and plasmid DNA transfection were carried out. For analysis of the effect of Nsp1 on APP and APP.C99 expression, NSP1 and APP/APP.C99 plasmids mixed at 2:1 ratio were co-transfected in HeLa cells. After 48 hrs, cell lysates were prepared and processed for western blot analysis. For analysis of effect on Nsp1 by RQC factor RNAi, HeLa cells were first transfected with ABCE1 (Stealth RNAi™ siRNA, Invitrogen HSS109285), eRF1 (Stealth RNAi™ siRNA, Invitrogen HSS103392), elF3E (Stealth RNAi™ siRNA, Invitrogen HSS1 79956), or RACK1 (Stealth RNAi™ siRNA, Invitrogen HSS115921 , Invitrogen) siRNA for 24 hrs. Thereafter, C99 and NSP1 plasmids mixed at 1 :2 ratio were cotransfected for 36 hrs. Cell lysates were prepared and western blot analysis was performed. Nsp1 -WT and Nsp1 -KH mutant plasmids were obtained from Addgene (141255 and 164522). Puromycin labeling of ribosome stalled newly synthesized proteins

[0138] Puromycin labeling of stalled NPCs was performed similar to prior studies. Briefly, Hela cells were seeded on coverslips in a 6-well plate and transfected with pCAX- C99 (Addgene #30146). After 48 hr transfection, cells were treated with HHT for 5 minutes, thereafter Puromycin (50pg) and Emetine (100pg) were added and cells were incubated for 5-7 minutes. After this, cells were permeabilized by 0.02% digitonin in Permeabilization buffer (50 mM Tris-HCI, pH7.5, 5 mM MgCI2, 25 mM KCI, 355 mM cyclohexamide, 10 units RNAseOut and 0.02% digitonin) for 2 minutes. Permeabilized cells were washed twice with washing buffer (permeabilization buffer without digitonin) and fixed in 4% paraformaldehyde for 30 minutes. The Permeabilization and washing steps were performed in ice-old buffers. Cells were immunostained with the amyloid conformation-specific antibody mOC78 that recognizes aggregation-prone APP.C99 (4) and Puromycin antibody and observed under the confocal microscope.

Sucrose gradient analysis of ribosomes

[0139] For each experimental condition, one 10 cm plate of cells at around 80% confluency were first washed with ice-cold PBS and harvested by scraping. After sedimentation at 4°C at 5000 rpm for 3 min, cell pellets were resuspended in 200 pl of 1xRNC buffer containing 0.01 % digitonin, 1x protease inhibitor cocktail (EDTA-free complete from Roche) and 1 mM DTT. After 15 min incubation on ice, cells were disrupted using a pre-chilled 26G needle appended to 1 mL syringe. Lysates were clarified by 15 min centrifugation at 15,000 g at 4°C. Lysate containing 150 pg of total RNA was loaded onto a 10%-50% analytical sucrose gradients (2 ml) and spun for 30 min at 55,000 rpm in TLS-55 rotor at 4°C using slowest acceleration and deceleration settings. Eleven fractions of 200 pl were collected manually from the top of the gradient. Protein was precipitated from the solution using a final concentration of 20% Trichloroacetic acid (TCA). The resulting pellets were washed with 10% TCA, followed by 100% acetone, and dried. Samples were resuspended in Laemmli sample buffer containing [3- mercaptoethanol, boiled at 95°C for 5 min, and subjected to western blot analysis. EXAMPLE 2: Rescue of Alzheimer’s disease-related phenotypes in human induced pluripotent stem cell (iPSC) model of AD by Nsp1 and Nsp1-C peptide

[0140] Using human cells to test the conservation of disease-relevant genes and pathways initially identified in animal models can help winnow the candidates down to those directly relevant to human disease. Patient iPSC-based neuronal models offer an excellent system to study disease mechanism and for drug screening/testing, and they have been successfully implemented for AD studies. Using a method that facilitates the differentiation of iPSCs into cortical neurons through the introduction of the transcription factor Ngn2, human neurons were obtained from iPSCs carrying a familial AD (FAD) mutation involving APP duplication, or from iPSCs derived from Down syndrome (DS) patients who develop AD due to an extra copy of APP on Chr. 21 . Compared to control neurons, the FAD and DS iPSC-derived neurons exhibited higher ROS level and lower NAD⁺/NADH ratio, suggesting mitochondrial dysfunction, consistent with the mitochondrial hypothesis of AD. The FAD iPSC-derived neurons also exhibit other AD- related phenotypes, such as mOC78-positive amyloid aggregates, enlarged early endosomes, and tau hyperphosphorylation. To test the effect of Nsp1 in this iPSC model, lentivirus was used to introduce the expression of Nsp1 (Fig. 34) or treated the neurons with the Nsp1 -C peptide corresponding the C-terminal 33 amino acids (Fig. 35). Both treatments attenuated the AD-related phenotypes, as illustrated by the reduction of mOC78-positive amyloid aggregates (Figs. 34 and 35), the number and size of Rab5- positive early endosomes (Figs. 34 and 35), and the level of phosphorylated tau (Figs. 34 and 35). These data support the further development of Nsp1 and Nsp1 -C peptide into therapeutics for the treatment of AD.

EXAMPLE 3: Anti-cancer activity of Nsp1 and Nsp1-C peptide

[0141] Translational control at the initiation, elongation, and termination steps exerts immediate effects on the rate as well as the spatiotemporal dynamics of new protein synthesis, shaping the composition of the proteome. Translational control is particularly important for cells under stress as during viral infection or in disease conditions such as cancer and neurodegenerative diseases. Much has been learned about the control mechanisms acting at the translational initiation step under normal or pathological conditions. However, problems during the elongation or termination steps of translation can lead to ribosome stalling and ribosome collision, which will trigger ribosome- associated quality control (RQC) mechanism. The rapid and continuous proliferation of cancer cells require increased protein synthesis and ribosome content. Deregulated translation initiation in cancer has been well studied. For example, upregulation of the initiation complex elF4F is observed in cancer, and overexpression of elF4E is sufficient to cause transformation of fibroblasts. Inactivation of 4EBP, an inhibitor of elF4E, by mTORCI -mediated phosphorylation is also a common event in cancer. Moreover, phosphorylation of elF2a, an event that negatively regulates tertiary complex formation, was deregulated in cancer, although the exact function of elF2a phosphorylation in cancer biology may be context-dependent. Translational regulation at the elongation and termination steps is also altered in cancer cells. For example, negative regulation of translation elongation at the eEF2 step by eEF2 kinase (eEF2K) is countered by mTORCI -mediated phosphorylation of eEF2K in cancer cells. Cancer cells also overexpress elF5A, a protein initially identified as an initiation factor but later shown to be important for ribosomes to readthrough difficult-to-translate regions enriched in Pro, Gly, and basic residues, suggesting that cancer cells may upregulate elF5A to resolve stalled translation. This is supported by studies in yeast showing that deletion of elF5A leads to accumulation of stalled ribosomes.

[0142] Having implicated Nsp1 in the translational regulation of problematic mRNAs in AD, PD, and ALS settings, it was tested if it is also effective in cancer settings. First, the sensitivity of cancer cells and non-cancer cells to Nsp1 was tested. It was found that Nsp1 preferentially inhibited the viability of cancer cells (Fig. 36). The effect of Nsp1 on the expression of signaling molecules involved in cancer was tested. It was found that the expression of many signaling molecules involved in cancer, including p-mTOR, p-4EBP, c-Myc, and c-Myc target elF4E, were significantly reduced by Nsp1 in cancer cells (Fig. 37). Nsp1 also reduced the expression of Drosophila Myc in transgenic flies, and this occurred without any significant change of Myc mRNA expression (Fig. 38). c-Myc is a master regulator of cell growth and a pervasive oncogenic driver that is deregulated in more than 70% of human cancers and thus presenting an attractive drug target, but so far it has been deemed “undruggable” due to the lack of clear ligand-binding site or enzymatic activity. Given the newly discovered role of Nsp1 in regulating stalled translation, the possibility that ribosome stalling may occur during cMyc translation elongation was considered, which is targeted for aborted translation by Nsp1 . To test this model, 5 PP motifs were mutated in the N-terminus of cMyc into AA (c-Myc-5PPmut). Such motifs were identified as stall signals by recent disome-seq studies. It was found that while cMyc-WT was downregulated by Nsp1 , c-Myc-5PPmut was not affected (Fig. 39), supporting the notion that stalled translation of cMyc makes it a target of Nsp1 induced quality control and translational repression.

[0143] Myc is a regulator of NSC growth and indispensable in Notch-induced dedifferentiation from intermediate progenitors (IPs) to cancer stem cell (CSC)-like neuroblasts (NBs) in the fly brain. It was tested if, by downregulating Myc, Nsp1 would affect Notch-induced NB overproliferation. Indeed, Nsp1 rescued Notch-induced brain tumor phenotype in Drosophila (Fig. 40). Consistent with Myc being a key target of Nsp1 in this process, overexpression of Myc blocked the Nsp1 effect (Fig. 40). The effect of Nsp1 on cell growth was also tested in other cancer settings that also involve Myc. Activation of the insulin/PI3K signaling pathway, Ras pathway, or Yorki pathway, by AKT overexpression (AKT-OE), constitutively active Ras (Ras-CA) OE, or Warts knockdown (Wts-RNAi), respectively, all lead to overgrowth of the fly eye. These cell growth pathways all depend on dMyc. Nsp1 co-expression restored fly eyes to normal sizes, and this effect can be blocked by the co-expression of dMyc (Fig. 41 ). These data support the notion that Nsp1 acts through the RQC of Myc to restrain Myc-driven cell growth in cancer.

[0144] Human glioblastoma (GBM) cells were used to test the therapeutic effect of Nsp1 in brain tumor settings. It was found that overexpression of full-length Nsp1 (Fig. 36), or treatment with Nsp1 -C peptide, which corresponds to the C-terminal 33 amino acids of Nsp1 (Fig. 42), effectively inhibited GBM tumor sphere formation (Fig. 42). This is correlated with the ability of Nsp1 -C to downregulate c-Myc and p-mTOR expression level (Fig. 43). Together, these data support the further development of Nsp1 and Nsp1 - C peptide into therapeutics for the treatment of brain tumors and possibly other tumors that are driven by c-Myc. DOCTRINE OF EQUIVALENTS

[0145] While the above description contains many specific embodiments of the invention, these should not be construed as limitations on the scope of the invention, but rather as an example of one embodiment thereof. Accordingly, the scope of the invention should be determined not by the embodiments illustrated, but by the appended claims and their equivalents.

Table 1. Sequence Listing

Claims

WHAT IS CLAIMED IS:

1. Use of coronavirus NSP1 or a peptide fragment of coronavirus NSP1 in manufacture of a medicament for treatment of a neurodegenerative disorder.

2. The use of claim 1 , wherein the neurodegenerative disorder is one of: Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic Lateral Sclerosis (ALS), ataxia, Huntington’s disease (HD), motor neuron disease, or multiple system atrophy.

3. The use of claim 1 , wherein the coronavirus is a human coronavirus.

4. The use of claim 3, wherein the human coronavirus is SARS-CoV, SARS-CoV-2,

MERS-CoV, 229E, NL63, OC43, or HKUI .

5. The use of claim 1 , wherein the coronavirus is a nonhuman coronavirus.

6. The use of claim 1 , wherein the peptide fragment of coronavirus NSP1 is derived from the N-terminal region.

7. The use of claim 1 , wherein the peptide fragment of coronavirus NSP1 is derived from the C-terminal region.

8. The use of claim 7, wherein the peptide fragment of coronavirus NSP1 is derived from the C-terminal region.

9. The use of claim 1 , wherein the coronavirus NSP1 or the peptide fragment of coronavirus NSP1 is truncated, modified, chimerized, or conjugated.

10. Use of nucleic acid encoding coronavirus NSP1 or a peptide fragment of coronavirus NSP1 in manufacture of a medicament for treatment of a neurodegenerative disorder.

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11. Use of coronavirus NSP1 or a peptide fragment of coronavirus NSP1 in manufacture of a medicament for treatment of a neoplasm or cancer.

12. The use of claim 11 , wherein the neoplasm or cancer is one of: anal cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, brain cancer (including glioblastoma), breast cancer, breast adenocarcinoma (BRCA), cervical cancer, chronic myeloproliferative neoplasms, colorectal cancer, endometrial cancer, ependymoma, esophageal cancer, diffuse large B-cell lymphoma (DLBCL), esthesioneuroblastoma, Ewing sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, hepatocellular cancer, hypopharyngeal cancer, Kaposi sarcoma, Kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, liver cancer, lung cancer, melanoma, Merkel cell cancer, mesothelioma, mouth cancer, neuroblastoma, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors, pharyngeal cancer, pituitary tumor, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, skin cancer, small cell lung cancer, small intestine cancer, squamous neck cancer, T-cell acute lymphoblastic leukemia (T-ALL), testicular cancer, thymoma, thyroid cancer, uterine cancer, vaginal cancer, or vascular tumors.

13. The use of claim 11 , wherein the coronavirus is a human coronavirus.

14. The use of claim 13, wherein the human coronavirus is SARS-CoV, SARS-CoV-2, MERS-CoV, 229E, NL63, OC43, or HKUI .

15. The use of claim 11 , wherein the coronavirus is a nonhuman coronavirus.

16. The use of claim 11 , wherein the peptide fragment of coronavirus NSP1 is derived from the N-terminal region.

17. The use of claim 11 , wherein the peptide fragment of coronavirus NSP1 is derived from the C-terminal region.

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18. The use of claim 17, wherein the peptide fragment of coronavirus NSP1 is derived from the C-terminal region.

19. The use of claim 11 , wherein the coronavirus NSP1 or the peptide fragment of coronavirus NSP1 is truncated, modified, chimerized, or conjugated.

20. Use of nucleic acid encoding coronavirus NSP1 or a peptide fragment of coronavirus NSP1 in manufacture of a medicament for treatment of a neoplasm or a cancer.

21 . A method of treating a neurodegenerative disorder, comprising: administering to a subject having the neurodegenerative disorder a medicament comprising coronavirus NSP1 or a peptide fragment of coronavirus NSP1 .

22. The method of claim 21 , wherein the neurodegenerative disorder is one of: Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic Lateral Sclerosis (ALS), ataxia, Huntington’s disease (HD), motor neuron disease, or multiple system atrophy.

23. The method of claim 21 , wherein the coronavirus is a human coronavirus.

24. The method of claim 23, wherein the human coronavirus is SARS-CoV, SARS- CoV-2, MERS-CoV, 229E, NL63, OC43, or HKU1.

25. The method of claim 21 , wherein the coronavirus is a nonhuman coronavirus.

26. The method of claim 21 , wherein the peptide fragment of coronavirus NSP1 is derived from the N-terminal region.

27. The method of claim 21 , wherein the peptide fragment of coronavirus NSP1 is derived from the C-terminal region.

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28. The method of claim 27, wherein the peptide fragment of coronavirus NSP1 is derived from the C-terminal region.

29. The method of claim 21 , wherein the coronavirus NSP1 or the peptide fragment of coronavirus NSP1 is truncated, modified, chimerized, or conjugated.

30. A method of treating a neurodegenerative disorder, comprising: administering to a subject a medicament comprising nucleic acid encoding coronavirus NSP1 or a peptide fragment of coronavirus NSP1 .

31 . A method of treating a neoplasm or cancer, comprising: administering to a subject having the neoplasm or the cancer a medicament comprising coronavirus NSP1 or a peptide fragment of coronavirus NSP1 .

32. The method of claim 31 , wherein the neoplasm or cancer is one of: anal cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, brain cancer (including glioblastoma), breast cancer, breast adenocarcinoma (BRCA), cervical cancer, chronic myeloproliferative neoplasms, colorectal cancer, endometrial cancer, ependymoma, esophageal cancer, diffuse large B-cell lymphoma (DLBCL), esthesioneuroblastoma, Ewing sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, hepatocellular cancer, hypopharyngeal cancer, Kaposi sarcoma, Kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, liver cancer, lung cancer, melanoma, Merkel cell cancer, mesothelioma, mouth cancer, neuroblastoma, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors, pharyngeal cancer, pituitary tumor, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, skin cancer, small cell lung cancer, small intestine cancer, squamous neck cancer, T-cell acute lymphoblastic leukemia (T-ALL), testicular cancer, thymoma, thyroid cancer, uterine cancer, vaginal cancer, or vascular tumors.

33. The method of claim 31 , wherein the coronavirus is a human coronavirus.

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34. The method of claim 33, wherein the human coronavirus is SARS-CoV, SARS- CoV-2, MERS-CoV, 229E, NL63, OC43, or HKU1.

35. The method of claim 31 , wherein the coronavirus is a nonhuman coronavirus.

36. The method of claim 31 , wherein the peptide fragment of coronavirus NSP1 is derived from the N-terminal region.

37. The method of claim 31 , wherein the peptide fragment of coronavirus NSP1 is derived from the C-terminal region.

38. The method of claim 37, wherein the peptide fragment of coronavirus NSP1 is derived from the C-terminal region.

39. The method of claim 31 , wherein the coronavirus NSP1 or the peptide fragment of coronavirus NSP1 is truncated, modified, chimerized, or conjugated.

40. A method of treating a neurodegenerative disorder, comprising: administering to a subject a medicament comprising nucleic acid encoding coronavirus NSP1 or a peptide fragment of coronavirus NSP1 .

41. Use of a modulator of ABCE1 , Rackl , ZNF598, elF5A, ZAK-alpha, p38, JNK, GCN2, elF2alpha or ASCC3 in manufacture of a medicament for treatment of a neurodegenerative disorder.

42. Use of a modulator of ABCE1 , Rackl , ZNF598, elF5A, ZAK-alpha, p38, JNK, GCN2, elF2alpha or ASCC3 in manufacture of a medicament for treatment of a neoplasm or cancer.

43. A method of treating a neurodegenerative disorder, comprising: administering to a subject a medicament comprising a modulator of ABCE1 , Rackl , ZNF598, elF5A, ZAK-alpha, p38, JNK, GCN2, elF2alpha, or ASCC3.

44. A method of treating a neoplasm or a cancer, comprising: administering to a subject a medicament comprising a modulator of ABCE1 , Rackl , ZNF598, elF5A, ZAK-alpha, p38, JNK, GCN2, elF2alpha, or ASCC3.

45. Use of an antagonist of NSP1 or an antagonist of protein synthesis in manufacture of a medicament for treatment of a coronavirus infection.

46. A method of treating a coronavirus infection, comprising: administering to a subject a medicament comprising an antagonist of NSP1 or an antagonist of protein synthesis.

47. Use of a modulator of ABCE1 , Rackl , ZNF598, elF5A, ZAK-alpha, p38, JNK, GCN2, elF2alpha, or ASCC3 in manufacture of a medicament for treatment of a coronavirus infection.

48. A method of treating a coronavirus infection, comprising: administering to a subject a medicament comprising a modulator of ABCE1 , Rackl , ZNF598, elF5A, ZAK-alpha, p38, JNK, GCN2, elF2alpha or ASCC3.