Patent Publication Number: US-2023151369-A1

Title: Compositions and methods for inhibiting tdp-43 and fus aggregation

Description:
RELATED APPLICATION 
     This is a Patent Cooperation Treaty Application which claims the benefit of 35 U.S.C. § 119 based on the priority of U.S. Provisional Patent Application No. 63/011,786, filed Apr. 17, 2020 which is herein incorporated in its entirety by reference. 
    
    
     INCORPORATION OF SEQUENCE LISTING 
     A computer readable form of the Sequence Listing “P61012PC00 Sequence Listing_ST25” (89,532 bytes), submitted via EFS-WEB and created on Apr. 16, 2021, is herein incorporated by reference. 
     FIELD 
     The present disclosure relates to oligomeric antisense compounds for use in gene modulation of RACK1 and methods for reducing TDP-43 and FUS aggregation in disease cells. Specifically, the disclosure pertains to oligomeric antisense compounds and their use for treating TDP-43-opathy and FUS-opathy neurodegenerative diseases. 
     BACKGROUND 
     RACK1 (Receptor for Activated C Kinase 1) is a highly conserved scaffold protein that has many normal functions, including PKC transduction, miRNA regulation, and protein translation by binding to the eukaryotic small (40S) ribosomal subunit (1). Cellular RACK1 has been reported to aggregate in cells displaying TDP43 or tau pathology (2,3). 
     RACK1 is a tryptophan, aspartic acid repeat (WD-repeat) protein that adopts a seven-bladed p-propeller structure. RACK1 is a core ribosomal protein of the eukaryotic 40S ribosomal subunit; a scaffold protein interacting with &gt;100 proteins, thereby regulating a variety of signaling pathways critical for cell proliferation, transcription, protein synthesis, and neuronal functions; involved in translational regulation and ribosome quality control; and expressed in the cytosol, endoplasmic reticulum (ER), and nuclei. RACK1 is highly conserved through evolution. The amino acid sequence identity of  Homo sapiens  RACK1 to  Mus musculus  is 100%, to  Rattus norvegicus  is 100%, to  Drosophila melanogaster  is 76%, to  Arabidopsis thaliana  is 64%, and to  Saccharomyces cerevisiae  is 53% (4). 
     It has been reported that RACK1 interacts with wild-type and mutant huntingtin (HTT), a gene associated with Huntington&#39;s disease (10). 
     TAR DNA-binding protein 43 (TDP-43) is a well-known RNA/DNA binding protein involved in the pathogenesis of ALS and Frontotemporal Lobar Dementia (FTLD) (5). TDP-43 mainly localizes in the nucleus, where it participates in the expression and splicing of RNAs, whereas, when in the cytoplasm, its functions range from transport to translation of specific mRNAs (6). Binding of TDP-43 to the translational machinery is mediated by an interaction with RACK1 and that an increase in cytoplasmic TDP-43 represses global protein synthesis, an effect that is rescued by overexpression of wild-type RACK1 (2). TDP-43 represents a repressor for overall translation and its binding to polyribosomes through RACK1 may promote the formation of cytoplasmic inclusions (2). In the presence of a ribosomal binding deficient mutant (DE-RACK1) protein, nuclear localization signal-deficient (dNLS) TDP-43 protein aggregation is reduced, less associated with the translational machinery, and global translational suppression by dNLS TDP-43 is relieved (2). 
     Fused in Sarcoma/Translocated in Sarcoma (FUS/TLS) FUS is an RNA/DNA binding protein mainly localized in the nucleus of most cell types (6). Cytoplasmic aggregation of FUS has been reported in brain and spinal cord neurons of ALS patients with FUS mutations (6), and in ˜10% of FTLD without mutations (i.e., wild-type protein) (11). 
     Molecules that increase or decrease RACK1 expression have been described. 
     PCT/GB2007/003447 describes dopamine receptor interacting proteins as markers of disease and describes determining the presence or absence of a variant form of one or more nucleic acid sequences including in the GNB2L1 (RACK1) gene, wherein the presence of the variant is indicative of disease or susceptibility to disease. 
     U.S. Pat. No. 8,916,530 patent describes methods for individualized cancer therapy and mentions specific antisense/shRNA/siRNA sequences for use in knocking down upregulated RACK1 gene expression for treatment of cancer. 
     U.S. Ser. No. 15/844,601 describes a method for increasing the expression levels of genes including GNB2L1, by administering an agent as a cancer treatment. 
     PCT/EP2019/065116 describes affinity-based isolation and purification of drug-loaded extracellular vesicles, such as exosomes, wherein the exosomes are engineered to enable affinity purification. 
     CN101985037 describes the use of specific siRNA or antisense oligonucleotides to inhibit the RACK1 gene for treatment of tumors. 
     Additional treatments for TDP-43-opathies or FUS-opathies are desirable. 
     SUMMARY 
     Disclosed herein in a first aspect is an oligomeric compound comprising a portion that is complementary to at least part of a nucleic acid target selected from any one of SEQ ID NOs: 1-16, 49-51 or 289-499. 
     In an embodiment, the oligomeric compound is 14 to 40 nucleotides in length. 
     In an embodiment, the nucleic acid target sequence is selected from any one of SEQ ID NOs: 2-6, 8, 10-16, 49-51, 292-294 and 296-499. In an embodiment, the nucleic acid target sequence is selected from any one of SEQ ID NOs: 2-6, 8, 10-16, 49-51, 292-294 and 296-298. In an embodiment, the nucleic acid target is sequence selected from any one of SEQ ID NOs: 2, 3, 292, 297 and 298. 
     The target sequences are in RACK1 mRNA or pre-mRNA. The sequence of human RACK1 mRNA is provided in for example NCBI Reference Sequence Accession code NM_006098.5 and having SEQ ID NO: 500. The sequence of human RACK1 pre-mRNA is provided in for example Accession code NC_000005.10 sequence index 181236897 to 181248096. 
     In an embodiment, the portion is complementary to the nucleic acid target sequence and the nucleic acid target sequence is or comprises a sequence selected from any one of SEQ ID NOs: 1-16, 49-51 and 289-499. In an embodiment, the portion is complementary to the nucleic acid target sequence and the nucleic acid target sequence is or comprises a sequence selected from any one of SEQ ID NOs: 2-6, 8, 10-16, 49-51, 292-294 and 296-499. In an embodiment, the portion is complementary to the nucleic acid target sequence and the nucleic acid target sequence is or comprises a sequence selected from any one of SEQ ID NOs: SEQ ID NOs: 2-6, 8, 10-16, 49-51, 292-294 and 296-298. In an embodiment, the portion is complementary to the nucleic acid target sequence and the nucleic acid target sequence is or comprises any one of SEQ ID NOs: 2, 3, 292, 297 and 298. 
     The oligomeric compounds can be comprised of naturally occurring or modified monomers or combinations thereof. The oligomeric compounds can be single or double stranded and can be RNA, DNA or DNA/RNA hybrids (e.g. single stranded or double stranded). 
     The oligomeric compound can be an antisense oligonucleotide, for example comprising the sequence of any one of SEQ ID NOs: 78-288, preferably any one of SEQ ID NOs: 81-83 and 85-87, and more preferably any one of SEQ ID NOs: 81, 86 and 87. 
     The oligomeric compound can be an siRNA compound that targets one of the nucleic acid targets and comprising a native or non-native overhang sequence. 
     In an embodiment, the siRNA comprises a guide strand that comprises a sequence of any one of SEQ ID NOs: 17-32 and 52-54. 
     Double stranded oligomeric compounds such as siRNA sequences can have identical 3′-overhang sequences or non-identical 3′ overhang sequences. One may be native and one may be non-native. 
     The oligomeric compound may be an shRNA. In an embodiment, the oligomeric compound comprises one or more cell penetrating moieties. 
     In a further aspect, there is disclosed a vector comprising the oligomeric compound herein disclosed. 
     In a further aspect, a composition comprising said oligomeric compound or vector and a diluent is disclosed. 
     An aspect disclosed herein relates to a method of treating a TDP43-opathy or a FUS-opathy neurodegenerative disease optionally selected from amyotrophic lateral sclerosis (ALS), Alzheimer&#39;s disease (AD), frontotemporal lobar dementia (FTLD), Huntington&#39;s disease (HD), neuronal intermediate filament inclusion disease (NIFID), basophilic inclusion body disease (BIBD) or limbic-predominant age-related TDP-43 encephalopathy (LATE), the method comprising knocking down RACK1 RNA, optionally RACK1 mRNA and/or RACK1 pre-mRNA in cells of the central nervous system, in particular in neurons and/or astrocyte cells of a subject in need thereof. 
     Another aspect disclosed herein is a method of treating a TDP43-opathy or a FUS-opathy neurodegenerative disease optionally selected from amyotrophic lateral sclerosis (ALS), Alzheimer&#39;s disease (AD), frontotemporal lobar dementia (FTLD), Huntington&#39;s disease (HD), neuronal intermediate filament inclusion disease (NIFID), basophilic inclusion body disease (BIBD) or limbic-predominant age-related TDP-43 encephalopathy (LATE), the method comprising administering to a subject in need thereof one or more antisense molecule(s), optionally one or more of said oligomeric compounds disclosed herein. 
     Another aspect is a method of reducing or inhibiting TDP-43 and/or FUS aggregation in a cell, the method comprising introducing into the cell one or more antisense molecule(s) optionally one or more of said oligomeric compounds targeting RACK1, compositions and/or vectors disclosed herein in a sufficient amount and for a sufficient time to decrease RACK1 levels in the cell. 
     A further aspect is the use of one or more antisense molecule(s), compositions, vectors and/or a methods described herein, for treating a TDP-43opathy optionally selected from amyotrophic lateral sclerosis (ALS), Alzheimer&#39;s disease (AD), frontotemporal lobar dementia (FTLD) e.g. TDP-43 type FTLD or FUS-type FTLD, Huntington&#39;s disease (HD), neuronal intermediate filament inclusion disease (NIFID), basophilic inclusion body disease (BIBD) or limbic-predominant age-related TDP-43 encephalopathy (LATE) in a subject in need thereof, or for reducing or inhibiting TDP-43 and/or FUS aggregation in a cell. 
     Also provided in an aspect is one or more antisense molecule(s), compositions, vectors and/or a methods described herein for use in the treatment of a TDP-43opathy optionally selected from amyotrophic lateral sclerosis (ALS), Alzheimer&#39;s disease (AD), frontotemporal lobar dementia (FTLD), Huntington&#39;s disease (HD), neuronal intermediate filament inclusion disease (NIFID), basophilic inclusion body disease (BIBD) or limbic-predominant age-related TDP-43 encephalopathy (LATE) in a subject in need thereof. 
     Further, an aspect comprises use of one or more antisense molecule(s), compositions, vectors and/or a methods described herein for the preparation of a medicament for the treatment of a TDP-43opathy optionally selected from amyotrophic lateral sclerosis (ALS), Alzheimer&#39;s disease (AD), frontotemporal lobar dementia (FTLD), Huntington&#39;s disease (HD), neuronal intermediate filament inclusion disease (NIFID), basophilic inclusion body disease (BIBD) or limbic-predominant age-related TDP-43 encephalopathy (LATE). 
     In an embodiment, the antisense molecule, optionally the oligomeric compound is an antisense oligonucleotide, an siRNA or an shRNA. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       An embodiment of the present disclosure will now be described in relation to the drawings in which: 
         FIG.  1    is a series of images of cells stained for wild type and dNLS TDP-43 (HA) and RACK1. 
         FIG.  2    is a series of images of cells stained for wild type and different mutants of FUS(HA) and RACK1. 
         FIG.  3    is a series of images of cells stained for different mutants of SOD1 (SOD100) and RACK1. 
         FIG.  4    is a series of images of cells stained for DE-RACK1, R495x-FUS, and RACK1. 
         FIG.  5    is a series of images of cells stained for DE-RACK1, P525L-FUS, and RACK1. 
         FIG.  6 A  depicts the gel electrophoresis Western blotting results for surface sensing of translation which uses puromycin to tag newly synthesized protein (SUnSET).  FIG.  6 B  depicts a graph that illustrates global translational levels normalized to a-tubulin.  FIG.  6 C  depicts a graph that illustrates the ratio of global translational levels+/− RACK1 siRNA. 
         FIG.  7    is a series of images of cells stained for R495x-FUS, Puromycin (PMY), and nucleus (DAPI). 
         FIG.  8    is a series of images of cells stained for dNLS TDP-43, Puromycin (PMY), and nucleus (DAPI). 
         FIG.  9    is a series of images of cells stained for RACK1 and dNLS TDP-43+/−siRNA. 
         FIG.  10    is a series of images of cells stained for RACK1 and Pan TDP-43+/−siRNA. 
         FIG.  11    is a series of images of cells stained for RACK1 and R495x-FUS+/−siRNA. 
         FIG.  12    is a series of images of cells stained for RACK1 and P525L-FUS+/−siRNA. 
         FIG.  13    is a series of images of cells stained for RACK1 and R495x-FUS+siRNA. 
         FIG.  14    is a series of images of cells stained for RACK1 and Pan FUS+/−siRNA. 
         FIG.  15    is a series of images of cells stained for RACK1, DAPI, 40S ribosomal subunit (Rps6), and dNLS TDP-43. 
         FIG.  16    is a series of images of cells stained for RACK1, DAPI, 40S ribosomal subunit (Rps6), and R495x-FUS. 
         FIG.  17    is a series of images of cells stained for RACK1, DAPI, 40S ribosomal subunit, and P525L-FUS. 
         FIG.  18    is a series of images of cells stained for RACK1, DAPI, 60S ribosomal subunit (RPL14), and dNLS TDP-43. 
         FIG.  19    is a series of images of cells stained for RACK1, DAPI, 60S ribosomal subunit (RPL14), and R495x-FUS. 
         FIG.  20    is a series of images of cells stained for RACK1, DAPI, 60S ribosomal subunit (RPL14), and P525L-FUS. 
         FIG.  21 A  is a series of images of cells stained for RACK1, 40S ribosomal subunit (Rps6), and dNLS TDP-43+ RACK1 siRNA.  FIG.  21 B  is a series of images of cells stained for RACK1, 60S ribosomal subunit (RPL14), and dNLS TDP-43+ RACK1 siRNA. 
         FIG.  22 A  is a series of images of cells stained for RACK1, 40S ribosomal subunit (Rps6), and P525L-FUS+ RACK1 siRNA.  FIG.  22 B  is a series of images of cells stained for RACK1, 60S ribosomal subunit (RPL14), and P525L-FUS+ RACK1 siRNA. 
         FIG.  23    is a model of the rescue of global translation by RACK1 knockdown. 
         FIG.  24    is a plot of the hotspot score of siRNA prediction on RACK1 mRNA. Circle markers are the peaks of the hotspot score, and correspond to regions that has potential to be targeted by siRNA. Plus markers show the position of existing effective siRNA from literature (Table 1). Star markers correspond to the siRNA that has been made and tested herein (Table 2). Triangle markers are the negative control of the prediction (Table 5). 
         FIG.  25    is a series of plots depicting the hotspot score (HS) of siRNA prediction on RACK1 pre-mRNA. Here 8 exon regions are extracted, showing the intron/exon boundaries. Location 4406, 5750 and 10382 are potential splice-blocking siRNA designs (Table 4). 
         FIG.  26    is an image of a Western Blot testing siRNAs of Table 2 for efficacy in knocking down RACK1. Santa Cruz Biotechnology is a positive control and has the same sequences as [7]. 
         FIG.  27    is a schematic of the UAS-Gal4 expression system used for producing flies expressing either wild-type or mutant hTDP43 or not, with or without RACK1-RNAi. 
         FIGS.  28 A to  28 L  are representative photographs of fly eyes of various genotypes. GMR drives expression of transgenes, shown at A1 ( FIG.  28 A- 28 D ) or at A6 ( FIGS.  28 E- 28 H,  28 K,  28 L ). Undriven controls are shown at A6 ( FIGS.  28 I,  28 J ). 
         FIG.  29    is a graph showing the percentage of flies in which degeneration score remains at 1. 
         FIG.  30    shows Western Blotting results for the detection of RACK1 in HeLa cells treated with different ASOs. Lane loading control: tubulin 
         FIG.  31    is a bar graph showing RACK1 protein expression in ASO treated HeLa cells relative to untreated (UT) cells, set to 1 and represented by upper dotted line. 
     
    
    
     DETAILED DESCRIPTION 
     Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. For example, the term “a cell” includes a single cell as well as a plurality or population of cells. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art (see, e.g. Green and Sambrook, 2012). 
     As used herein, the term “administration” means to provide or give a subject a compound or molecule, such as a composition comprising an antisense molecule, optionally an oligomeric compound disclosed herein or a vector comprising an antisense molecule, e.g. an shRNA by any effective route such as an intrathecal, intraventricular, intraparenchymal or intranasal administration route. 
     As used herein, the term “effective amount” refers to an amount of a compound or molecule, such as an antisense molecule, for example an antisense oligonucleotide or an anti-RACK1 siRNA that is sufficient to generate a desired response, such as to reduce or eliminate RACK1 protein, TDP-43 aggregation and/or FUS aggregation or to treat a TDP43-opathy or a FUS-opathy neurodegenerative disease such as amyotrophic lateral sclerosis (ALS), Alzheimer&#39;s disease (AD), frontotemporal lobar dementia (FTLD), Huntington&#39;s disease (HD), neuronal intermediate filament inclusion disease (NIFID), basophilic inclusion body disease (BIBD) or limbic-predominant age-related TDP-43 encephalopathy (LATE). 
     The term “treating” or “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. “Treating” and “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. For example, a subject with early stage ALS or FTLD can be treated with an antisense molecule(s) such as an oligomeric compound described herein to prevent progression of disease e.g. to prevent worsening of neurodegeneration. 
     As used herein, the term “diluent” refers to a pharmaceutically acceptable carrier which does not inhibit a physiological activity or property of an active compound to be administered and does not irritate the subject and does not abrogate the biological activity and properties of the administered compound. Diluents include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives, salts, preservatives, gels, binders, excipients, disintegration agents, lubricants, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington&#39;s Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated. 
     As used herein, the term “complementarity” or “complementary” means the ability of an antisense molecule such as an oligomeric compound disclosed herein, or a portion thereof, to hybridize to the target sequence of RACK1 RNA e.g. RACK1 mRNA and/or RACK1 pre-mRNA thereby “knocking down” RACK1 (e.g. reducing RACK1 mRNA and/or pre-mRNA by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% or greater). Complementarity between the antisense molecule and the target RNA may be perfect (100% complementary) but some mismatches are tolerated. For example, the antisense molecule can be 70%, 80%, 85%, 90% or 95% complementary to the target RNA or comprise up to 1, 2 or 3 mismatches in any 10 monomer stretch. 
     As used herein, the term “reverse complement” means the complementary strand of a nucleic acid sequence in the direction of its 5′ to 3′ end. For example, where a sequence in the 5′ to 3′ direction is TCCAGAGACAATCTGCCGGT (SEQ ID NO: 81), its reverse complement is ACCGGCAGATTGTCTCTGGA (SEQ ID NO: 292). 
     As used herein, “complementary to at least part” refers to an antisense molecule such as an oligomeric compound disclosed herein having sufficient complementarity to RACK1 RNA such as RACK1 mRNA or RACK1 pre-mRNA to decrease RACK1 levels, as measured for example an in vitro assay. “Complementary to at least part” includes for example complementary to at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous nucleotides of RACK1 RNA. 
     As used herein, the terms “antisense molecule” including for example any one of the oligomeric compounds disclosed herein comprises a compound at least a portion of which is a nucleic acid and includes for example antisense oligonucleotides, molecules comprising antisense oligonucleotides, siRNAs and molecules comprising siRNAs. The term antisense molecule includes for example antisense oligonucleotides that are typically single stranded as well as siRNA compounds which are typically double stranded as well as shRNA molecules. The antisense molecules are anti-RACK1 antisense molecules that are complementary to at least a portion of the RACK1 mRNA or pre-mRNA transcript. 
     As used herein, the term “oligomeric compound” relates to a compound herein disclosed that comprises an oligonucleotide, at least a portion of which is complementary to RACK1 RNA such as RACK1 mRNA or RACK1 pre-mRNA, or a part thereof. The oligomeric compound can comprise DNA, RNA, or a hybrid of DNA/RNA, and can comprise one or more modified (i.e. non-naturally occurring) monomers. “Oligomeric compound” includes antisense oligonucleotides, siRNAs and shRNA constructs. The oligomeric compound can consist of the portion that is complementary to RACK1 RNA but can also comprise additional one or more additional molecule, group or moiety (e.g. cell penetrating moiety). 
     As used herein, the term “antisense oligonucleotide” or “ASO” is a nucleic acid, e.g. a single stranded nucleic acid, that comprises a nucleotide sequence, which is complementary to at least a part of RACK1 RNA such as RACK1 mRNA or RACK1 pre-mRNA, and includes without limitation mixmers, gapmers, tailmers, headmers and blockmers, morpholinos, peptide nucleic acids (PNAs), 2′-O-substituted antisense oligonucleotides (e.g. 2′-O-methyl phosphorothioates, 2′-O-methoxyethyl phosphorothioates), locked nucleic acids (LNAs) and the like. Accordingly, an antisense oligonucleotide can hydrogen bond to a sense nucleic acid. For example, the antisense oligonucleotide can comprise DNA, RNA and/or a chemical analog (i.e. modified base) that binds to the target RNA. 
     As used herein, the term “siRNA” refers to an siRNA comprising a guide strand that is complementary to at least a part of the RACK1 mRNA or pre-mRNA transcript. 
     As used herein, the term “guide strand” refers to the portion or strand of an antisense molecule such as a double stranded siRNA that is complementary to the RNA sequence to which it is targeting to bind. It can comprise naturally occurring and/or modified bases. “Guide strand” can be used when referring to siRNAs and “portion” can be used when referring to antisense oligonucleotides and/or other antisense molecules. 
     As used herein, the term “shRNA construct” refers to a construct comprising a vector and a shDNA insert that when expressed can knock down expression of RACK1, the vector including viral vectors such as lentiviral and non-viral vectors, wherein the shDNA can be expressed to produce a short hairpin RNA comprising a guide strand that is complementary to at least a portion of the RACK1 mRNA or pre-mRNA transcript. As used herein, the term “guide strand” refers to the strand of an expressed double stranded shRNA that is complementary to the RNA sequence to which it is targeting to bind. 
     As used herein, the term “locked nucleic acid” or “LNA” refers to a bicyclic RNA analogue in which the ribose is locked in a C3′-endo conformation by introduction of a 2′-O,4′-C methylene bridge. Desirable LNA monomers and their method of synthesis also are disclosed in U.S. Pat. Nos. 6,043,060, 6,268,490, PCT Publications WO 01/07455, WO 01/00641, WO 98/39352, WO 00/56746, WO 00/56748 and WO 00/66604 as well as in the following papers: Morita et al., Bioorg. Med. Chem. Lett. 12(1):73-76, 2002; Hakansson et al., Bioorg. Med. Chem. Lett. 11(7):935-938, 2001; Koshkin et al., J. Org. Chem. 66(25):8504-8512, 2001; Kvaerno et al., J. Org. Chem. 66(16):5498-5503, 2001; Halkansson et al., J. Org. Chem. 65(17):5161-5166, 2000; Kvaerno et al., J. Org. Chem. 65(17):5167-5176, 2000; Pfundheller et al., Nucleosides Nucleotides 18(9):2017-2030, 1999; and Kumar et al, Bioorg. Med. Chem. Lett. 8(16):2219-2222, 1998, all of which are herein incorporated by reference in their entirety. 
     The term “mixmer” refers to an antisense oligonucleotide that comprises both naturally and non-naturally occurring nucleotides. However, unlike gapmers, tailmers, headmers and blockmers, there is no contiguous sequence of more than 5 naturally occurring nucleotides. 
     The term “gapmer” as used herein refers to for example an antisense oligonucleotide in which an internal DNA-based region (e.g. “gap”) having a plurality of nucleosides that support RNase H cleavage is flanked by one or more RNA-based nucleosides (e.g. 5′ and 3′ “wings”) that promote target binding. The gap nucleosides are distinct from the wing nucleosides. In a non-limiting example, the gapmer comprises DNA residues flanked by 2-MOE modified RNA residues, as described in Table 8. The 5′ and 3′ wings may have the same chemical modifications however different modifications between the 5′ and 3′ wings are contemplated as well as differences in nucleotide length. 
     The term “morpholino oligonucleotides” as used herein refers to a non-natural oligonucleotide comprising morpholino monomers such as methylenemorpholine rings replacing the ribose or deoxyribose sugar moieties and non-ionic phosphorodiamidate linkages replacing the anionic phosphates of DNA and RNA. Antisense morpholino oligonucleotides, for example that are targeted to intronic elements can modulate RNA splicing (12). Morpholino oligonucleotides can be short chains of about 25 morpholino monomers. Each morpholino oligonucleotide would block small (˜25 base) regions of the base-pairing surfaces of ribonucleic acid (RNA). The term “morpholino monomer” refers to a subunit comprising a nucleic acid base, a 6 membered morpholine ring and a non-ionic phosphorodiamidate intersubunit linkage. 
     As used herein, the term “cell penetrating moiety” refers to a compound or a functional group which mediates transfer of a compound, such as an oligomeric compound herein disclosed, from an extracellular space to within a cell. 
     As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus for example, a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. 
     As used in this application and claim(s), the word “consisting” and its derivatives, are intended to be close ended terms that specify the presence of stated features, elements, components, groups, integers, and/or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and/or steps. 
     The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about”. 
     The terms “about”, “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% or at least ±10% of the modified term if this deviation would not negate the meaning of the word it modifies. 
     The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. 
     As is demonstrated herein, RACK1 co-aggregates with mutant FUS and SOD1, which with mutant TDP43 could constitute a common pathway for the toxicity of these mutation-validated inclusions in for example ALS. 
     It is also demonstrated herein that knockdown of RACK1 in cultured cells can diminish or inhibit formation of FUS or TDP43 inclusions, accompanied by partial nuclear repatriation of mutant proteins which lack a nuclear localization sequence, perhaps due to diffusion of the de-aggregated protein into the nucleus [Pinarbasi et al., 2018]. Without wishing to be bound by theory the recruitment of polyribosomes to RACK1 co-aggregates may contribute to a toxic gain-of-function in misfolding and propagation of ALS/FTLD-implicated proteins, by virtue of recruitment of the 60s ribosomal subunit possessing the PFAR. The data described herein shows that co-aggregation of RACK1 with mutant TDP-43 or FUS suppresses global translation by sequestration of ribosomal subunits, and that siRNA knockdown of RACK1 can rescue global translation as well as the possible pathological chaperone activity of the 60s ribosome PFAR. 
     Neurotoxicity of protein aggregate-recruited RACK1 may be due to many factors, including loss-of-function for normal RACK1 activities. However, toxic gain-of-function of aggregated RACK1 could be one cause of the protein translational defects observed in ALS and other TDP-43 proteinopathies (i.e. TDP-43opathies). 
     It is also demonstrated herein that cell-specific in vivo knockdown of RACK1 ameliorates the neurodegeneration caused by transgenic overexpression of wildtype or mutant human TDP-43. 
     Accordingly, in an aspect is provided an oligomeric compound comprising a portion that is complementary to at least part of a nucleic acid target sequence selected from any one of SEQ ID NOs: 1-16, 49-51 and 289-499. 
     The portion of the oligomeric compound that is complementary to at least part of the nucleic acid target sequence can be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In an embodiment, the oligomeric compound is 14 to 60 nucleotides in length. In an embodiment, the oligomeric compound is 14 to 50 nucleotides in length. In an embodiment, the oligomeric compound is 14 to 40 nucleotides in length. In an embodiment, the oligomeric compound corresponds to the portion complementary to at least part of the target sequence and comprises 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In another embodiment, the oligomeric compound includes one or more additional nucleotides in the 5′ and/or 3′ direction of the portion complementary to the target sequence. For example, the oligomeric compound can comprise up to 15 or up to 20 nucleotides upstream and downstream of the portion. In an embodiment, the oligomeric compound is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. 
     The nucleic target sequence can be a sequence in Tables 2, 3, 4 or 8 or a part of any of the sequences therein. In an embodiment, the nucleic target sequence does not have the same sequence as a nucleic target sequence from Table 1. In an embodiment, the nucleic target sequence does not have the same sequence as a nucleic target sequence from Table 5. The oligomeric compound can be or comprise the reverse complement of a sequence in any of Tables 2, 3, 4 or 8, or a part thereof. 
     In an embodiment, the nucleic acid target sequence is selected from any one of SEQ ID NOs: SEQ ID NOs: 2-6, 8, 10-16, 49-51, 292-294 and 296-499. 
     In an embodiment, the nucleic acid target sequence is selected from any one of SEQ ID NOs: 2-6, 8, 10-16, 49-51, 292-294 and 296-298. 
     In an embodiment, the nucleic acid target sequence selected from any one of SEQ ID NOs: 2, 3, 292, 297 and 298. 
     In an embodiment, wherein the portion is complementary to the nucleic acid target sequence and the nucleic acid target sequence is or comprises a sequence selected from any one of SEQ ID NOs: 2-6, 8, 10-16, 49-51, 292-294 and 296-499. 
     In an embodiment, the portion is complementary to the nucleic acid target sequence and the nucleic acid target sequence is or comprises a sequence selected from any one of SEQ ID NOs: 2-6, 8, 10-16, 49-51, 292-294 and 296-298. 
     In an embodiment, the portion is complementary to the nucleic acid target sequence and the nucleic acid target sequence is or comprises any one of SEQ ID NOs: 2, 3, 292, 297 and 298. 
     In an embodiment, the portion is complementary to 
                            (SEQ ID NO: 2)           GAACTGAAGCAAGAAGTTATC           or                       (SEQ ID NO: 3)           CTCTGGATCTCGAGATAAA            
In a preferred embodiment, the portion is complementary to GAACTGAAGCAAGAAGTTATC (SEQ ID NO: 2). In a preferred embodiment, the portion is complementary to CTCTGGATCTCGAGATAAA (SEQ ID NO: 3). In a preferred embodiment, the portion is complementary to SEQ ID NO: 81. In a preferred embodiment, the portion is complementary to SEQ ID NO: 86. In a preferred embodiment, the portion is complementary to SEQ ID NO: 87.
 
     The oligomeric compound can be RNA or DNA or a hybrid thereof optionally comprising one or more modified residues. The target is RNA. Although, the targets may be represented as DNA herein, a person skilled in the art would recognize that thymidine (T) is replaced by uracil (U) in the sequences. Similarly, although an oligomeric compound may be represented as RNA herein, a person skilled in the art would recognize that the DNA compound comprises thymidine (T) instead of uracil (U). 
     Antisense molecules may be chemically synthesized using naturally occurring nucleotides and/or variously modified (non-naturally occurring) nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with the target RNA or DNA. Derivatives such as phosphorothioate derivatives and acridine substituted nucleotides can be used. Other examples of modified nucleotides can include those with N3′-P5′ phosphoramidates, 2′-deoxy-2′-fluoro-p-D-arabino nucleic acid analogue (FANA), morpholino monomers as well as those found in cyclohexene nucleic acids (CeNAs) (i.e. furanose moiety of DNA replaced by a cyclohexene ring) and tricyclo-DNA (tcDNA) (i.e. nucleotide comprising additional ethylene bridge between the centers C (3′) and C (5′) of the nucleosides, to which a cyclopropane unit is fused), peptide nucleic acid (PNA) (i.e. N-(2-aminoethyl)-glycine units), and/or be locked nucleic acid (LNA). The antisense molecule can be complementary to a target strand, or only to a portion thereof. 
     Antisense molecules can comprise at least one non-naturally occurring monomer which can function similarly to non-modified oligonucleotides. The chemical modification can for example be one found in locked nucleic acid (LNA) or can be 2′-fluoro (2′-F), 2′-O-methoxyethyl(2′-MOE) or 2′-O-methyl (2′-O-Me), which are modifications at the 2′ position of the ribose moiety or morpholino monomer where a six-membered morpholine ring replaces the sugar moiety or phosphorothioate (PS) linkage where sulfur replaces one of the non-bridging oxygen atoms in the phosphate group. Phosphorothioate and phosphoramidate linkages can be incorporated into any of the above-mentioned antisense molecules. Other internucleoside linkages include for example phosphorodithioate, methylphosphonate, alkylphosphonate, alkylphosphonothioate, phosphotriester, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone internucleoside linkages. Such modified or substituted nucleic acids may be preferred over naturally occurring forms because of properties such as increased stability in the presence of nucleases. The term also includes chimeric nucleic acids that contain two or more chemically distinct regions. For example, chimeric nucleic acids may contain at least one region of modified nucleotides that confer beneficial properties (e.g., increased nuclease resistance, increased uptake into cells), or two or more nucleic acids of the disclosure may be joined to form a chimeric nucleic acid. 
     Antisense molecules can be produced using a variety of methods, for example as described in Agrawal S. &amp; Gait M. J. (2019). History and Development of Nucleotide Analogues in Nucleic Acid Drugs.  Advances in Nucleic Acid Therapeutics , (pp 1-21). Royal Society of Chemistry, incorporated herein by reference. The antisense molecules or the nucleic acid component thereof can be produced biologically using for example an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high-efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced. Additionally, antisense molecules, for example siRNA, can be purchased from manufacturers, for example Santa Cruz Biotechnology (Dallas, Tex., USA). 
     In another embodiment, the oligomeric compound comprises non-modified RNA, DNA or a mixture of DNA/RNA. 
     In an embodiment, the oligomeric compound comprises modified RNA, DNA or a mixture of DNA/RNA. 
     In a further embodiment, the oligomeric compound comprises one or more nucleotide monomers which is chemically modified. In a further embodiment, the chemical modification comprises modification at a 2′ position. In another embodiment, the chemical modification is selected from 2′Omethyl (2′)-O-Me), 2′-O-methoxyethyl(2′O-MOE), 2′fluoro (2′F) and 2′-0,4′-C methylene bridge i.e. locked nucleic acid monomer (LNAM). 
     The oligomeric compound can comprise a modified backbone. In an embodiment, the oligomeric compound comprises at least one modified occuring internucleoside linkage. In an embodiment, at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage. In an embodiment, at least one internucleoside linkage is a phosphoramidate linkage. For example, all of the internucleoside linkages are phoshorothioate modified, as described for example in Example 4. Phosphorothioate linkages may be mixed Rp and Sp enantiomers, or they may be made stereoregular or substantially stereoregular in either Rp or Sp form. 
     In a further embodiment, the oligomeric compound comprises a modification of a plurality of nucleotide monomers. In another embodiment, all of the nucleotide monomers are modified. For example, referring to Table 8, the antisense oligonucleotides have phosphorothioate bonds between all bases and the RNA bases flanking the central DNA bases are 2′-MOE modified. 
     As described herein, antisense oligonucleotides of the present disclosure were found to reduce RACK1 levels in vivo. 
     In an embodiment, the oligomeric compound is an antisense oligonucleotide. 
     The antisense oligonucleotide can be DNA, RNA or a DNA/RNA hybrid thereof e.g. a mixture of DNA and RNA and can comprise one or more modified nucleotide. 
     In a further embodiment, the antisense oligonucleotide comprises a plurality of locked nucleic acid monomers (LNAM). 
     In a further embodiment, the antisense oligonucleotideis a locked nucleic acid (LNA), a LNA/DNA mixmer or a LNA/RNA mixmer. 
     In another embodiment, the antisense oligonucleotide is a gapmer, for example comprising a plurality of DNA nucleotides, e.g. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 DNA nucleotides, flanked by a plurality of RNA nucleotides e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 RNA nucleotides, for example a gapmer described in Example 4 (Table 8). 
     In an embodiment, the antisense oligonucleotide comprises or is the sequence of any one of SEQ ID NOs: 78-288. In an embodiment, the antisense oligonucleotide comprises or is the sequence of any one of SEQ ID NOs: 81-83 or 85-288. In an embodiment, the antisense oligonucleotide comprises or is the sequence of any one of SEQ ID NOs: 81-83 or 85-87. In an embodiment, the antisense oligonucleotide comprises or is the sequence of SEQ ID NO: 81. In an embodiment, the antisense oligonucleotide comprises or is the sequence of SEQ ID NO: 82. In an embodiment, the antisense oligonucleotide comprises or is the sequence of SEQ ID NO: 83. In an embodiment, the antisense oligonucleotide comprises or is the sequence of SEQ ID NO: 85. In an embodiment, the antisense oligonucleotide comprises or is the sequence of SEQ ID NO: 86. In an embodiment, the antisense oligonucleotide comprises or is the sequence of SEQ ID NO: 87. 
     In an embodiment, the antisense oligonucleotide is a morpholino oligonucleotide. 
     As demonstrated herein, siRNA sequences successfully knocked down RACK1. 
     In an embodiment, the oligomeric compound is a small interfering RNA (siRNA). 
     The siRNA can comprise a guide strand that comprises the reverse complement of a sequence in any of Tables 2, 3, 4, or 8, a portion thereof or a longer sequence extending 5′ or 3′ in the RACK1 mRNA. For example, with reference to Tables 2, 3 or 4, the guide strand can comprise the reverse complement of nucleotides shown in brackets. Double-stranded antisense molecules such as siRNA can include a single stranded overhang, for example corresponding to native sequence such as the nucleotides shown in brackets in Tables 2, 3 and 4 or non-native overhangs residues. Accordingly, the siRNA can include or not include the sequence shown in brackets or it can be replaced with non-native nucleotides such as tt, or in the RNA context uu. 
     The target can include additional nucleotides upstream or downstream of the RACK1 target sequence. For example, the target sequence can include 2 nucleotides 5′ to the recited RACK1 target sequences, for example TTTAGAGGGAAAGATCATT (SEQ ID NO: 1) with a 5′ GA overhang, GAACTGAAGCAAGAAGTTATC (SEQ ID NO: 2) with a 5′ AT overhang, CTCTGGATCTCGAGATAAA (SEQ ID NO: 3) with a 5′ GT overhang, GCTAACTGCAAGCTGAAGA (SEQ ID NO: 4) with a 5′ TG overhang, GACAAGCTGGTCAAGGTAT (SEQ ID NO: 5) with a 5′ GG overhang, GGATGGCCAGGCCATGTTA (SEQ ID NO: 6) with a 5′ AA overhang, ACACCTTTACACGCTAGAT (SEQ ID NO: 7) with a 5′ AA overhang, CTATCTGAACACGGTGACT (SEQ ID NO: 8) with a 5′ GG overhang, CAGGGATGAGACCAACTAT (SEQ ID NO: 9) with a 5′ AC overhang, CCAACAGCAGCAACCCTAT (SEQ ID NO: 10) with a 5′ GC overhang, CTTTGTTAGTGATGTGGTT (SEQ ID NO: 11) with a 5′ CA overhang, CCCTGGGTGTGTGCAAATA (SEQ ID NO: 12) with a 5′ TA overhang, GCTGATGGCCAGACTCTGT (SEQ ID NO: 13) with a 5′ CT overhang, GATTTGTGGGCCATACCAA (SEQ ID NO: 14) with a 5′ GC overhang, GTAACCCAGATCGCTACTA (SEQ ID NO: 15) with a 5′ GG overhang, CGCAGTTCCCGGACATGAT (SEQ ID NO: 16) with a 5′ CG overhang, GTACGGACTAAGGTAGATT (SEQ ID NO: 49) with a 5′ AG overhang, TTTTACCTCCTTTAGATAA (SEQ ID NO: 50) with a 5′ TG overhang and TGTTCCCCAGGATTTAGAG (SEQ ID NO: 51) with a 5′ CC overhang, respectively. In oligomeric compounds that comprise an overhang the overhang may correspond to the reverse compliment of the residues in brackets or can be non-target residues such as tt, where undercase denotes a sequence is non-native. 
     In another embodiment, the guide strand is complementary to GAACTGAAGCAAGAAGTTATC (SEQ ID NO: 2) with a 5′ AT overhang, or CTCTGGATCTCGAGATAAA (SEQ ID NO: 3) with a 5′ GT overhang. In a preferred embodiment, the guide strand is complementary to GAACTGAAGCAAGAAGTTATC (SEQ ID NO: 2) with a 5′ AT overhang. In a preferred embodiment, the guide strand is complementary to CTCTGGATCTCGAGATAAA (SEQ ID NO: 3) with a 5′ GT overhang. 
     The overhang can for example be any 2 nucleotide combination from A, U, C, G, dA, dT, dC, dG as well as modified bases. 
     In an embodiment, the siRNA is or comprises a guide strand comprising a sequence 5′-3′ GAUAACUUCUUGCUUCAGUUC (SEQ ID NO: 18). In another embodiment, the sequence is 5′-3′ UUUAUCUCGAGAUCCAGAG (SEQ ID NO: 19). 
     In an embodiment, the siRNA is or comprises a guide strand comprising a sequence 5′ to 3′ GAUAACUUCUUGCUUCAGUUC (SEQ ID NO: 18) with an 3′ (AU) overhang (i.e. additional AU nucleotides at the 3′ end). In another embodiment, the sequence is 5′-3′ UUUAUCUCGAGAUCCAGAG (SEQ ID NO: 19) with a 3′ (AC) overhang. 
     In another embodiment, the siRNA is or comprises a guide strand comprising a sequence of 5′-3′ GAUAACUUCUUGCUUCAGUUC (SEQ ID NO: 18) with a 3′ (AU) overhang and/or UUUAUCUCGAGAUCCAGAG (SEQ ID NO: 19) with a 3′ (gu) overhang. In a further embodiment, the sequence is 5′-3′ GAUAACUUCUUGCUUCAGUUC (SEQ ID NO: 18) with an 3′ (AU) overhang. In another embodiment, the sequence is 5′-3′ UUUAUCUCGAGAUCCAGAG (SEQ ID NO: 19) with a 3′ (gu) overhang. 
     In an embodiment, the guide strand comprises GAUAACUUCUUGCUUCAGUUC (SEQ ID NO: 18) with an 3′ (AU) overhang, or UUUAUCUCGAGAUCCAGAG (SEQ ID NO: 19) with a 3′ (gu) overhang. 
     The siRNA can for example be single stranded or double stranded. The oligomeric compound can be double stranded for example having: 
     ss5′-3′ GAACUGAAGCAAGAAGUUAUC (SEQ ID NO: 34) with a 3′ (au) overhang, and
 
as5′-3′ GAUAACUUCUUGCUUCAGUUC (SEQ ID NO: 18) with a 3′ (AU) overhang, wherein “ss” refers here to sense strand or passenger strand and “as” refers to antisense strand which can be the guide strand. The guide strand can also be a portion thereof or include additional residues.
 
     The siRNA can be double stranded for example having: 
     ss5′-3′ CUCUGGAUCUCGAGAUAAA (SEQ ID NO: 35) with a 3′ (gu) overhang; and
 
as5′-3′ UUUAUCUCGAGAUCCAGAG (SEQ ID NO: 19) with a 3′ (gu) overhang, wherein “ss” refers here to sense strand and “as” refers to antisense strand.
 
     In one embodiment, the siRNA is about 21-25 residues and optionally double stranded. In one embodiment, the siRNA is 21 residues in length. In one embodiment, the siRNA is 22 residues in length. In one embodiment, the siRNA is 23 residues in length. In one embodiment, the siRNA is 24 residues in length. In one embodiment, the siRNA is 25 residues in length. 
     In one embodiment, the oligomeric compound is a short hairpin RNA (shRNA). Using the non-limiting example of siR-2 and siR-3, the shRNA can comprise for example; 
     
       
         
           
               
               
            
               
                   
                 siR-2 5′-3′: 
               
               
                   
                 (SEQ ID NO: 34) 
               
               
                   
                 GAACUGAAGCAAGAAGUUAUC 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 18) 
               
               
                   
                 (loop)GAUAACUUCUUGCUUCAGUUC 
               
               
                   
                   
               
               
                   
                 siR-3 5′-3′: 
               
               
                   
                 (SEQ ID NO: 35) 
               
               
                   
                 CUCUGGAUCUCGAGAUAAA 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 35) 
               
               
                   
                 (loop)UUUAUCUCGAGAUCCAGAG.  
               
            
           
         
       
     
     In an embodiment, the antisense molecule is comprised in a vector, for example a plasmid, or viral vector such as a lentiviral vector an adenoviral vector or an adeno associated viral (AAV) vector. 
     In the context of the shRNA, the loop region could be any combination of nucleotide that could form a stable loop, and normally composed of 5-10nt. The termini of the shRNA can be chemically modified and/or comprise additional overhang nucleotides. 
     In some embodiments, the target is a part of the sequence specified herein. For example, the target can be 19-30 nucleotides in length. In some embodiments, the portion of the oligomeric compound that is complementary to at least part of the target sequence comprises one or more alternate nucleotides. For example, the portion may comprise one or more alternate nucleotides in the 3′ half of the compound, particularly the 3′ overhang. It has been found for example that the sequence between the 5′ end and the middle of the antisense siRNA is responsible for recognizing mRNA and the middle residues (nt 10-11) are typically the cleavage site recognition. 
     The oligomeric compound can comprise a cell penetrating moiety, be comprised in a transport reagent, or a vector for example a recombinant plasmid or viral vector that expresses the oligomeric compound or compounds. 
     In an embodiment, the oligomeric compound comprises one or more cell penetrating moieties. Non limiting examples of cell penetrating moieties (or cell attaching moieties) that promote intracellular uptake include peptides e.g. Penetrin, Pip&#39;s (PMO/PNA internalization peptide), sugars e.g. N-acetylgalactosamine (GaINAc), antibodies, e.g. a Fab fragment, carbohydrates, lipids e.g. cholesterol, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. The cell penetrating moiety can be operably linked or conjugated to the 5′ end, the 3′ end and/or to internal nucleotides of the portion of the oligomeric compound that is complementary to the target sequence. In an embodiment, the cell penetrating moiety is conjugated to the 5′ end and/or the 3′ end. In the context of a double stranded siRNA, the cell penetrating moiety is preferably attached to the passenger strand, for example at the 3′ terminus. The oligomeric compound can be coupled to the cell penetrating moiety using a variety of methods. For example, the oligomeric compound can be covalently linked to the moiety, as described for example in International patent application publication no. WO2008/063113 to Langel et al. and United States patent application publication no. US2005/0260756 to Troy et al. The moiety can also be linked to the oligomeric compound via chemical linkers, as described for example in WO2008/033285 to Troy et al and WO2007/069068 to Alluis et al. 
     Another aspect is a vector comprising the oligomeric compound or the portion thereof that is complementary to at least part of the target sequence. For example, the oligomeric compound is comprised in a viral vector such as an adeno-associated virus (AAV), an adenovirus, a lentivirus, or a γ-retroviral vector. The vector can be an integrating vector optionally for providing constitutive expression or can be an extranuclear vector optionally for transient expression. 
     Another aspect is a composition comprising an oligomeric compound, optionally an anti-RACK1 siRNA, anti-RACK1 shRNA construct, or an antisense oligonucleotide (e.g. anti-RACK1 gapmer or morpholino oligonucleotide) and a diluent. The diluent can for example be RNase free water or saline, optionally sterile. 
     The composition can comprise lipid particles such as liposomes, nanoparticles, exosomes, or nanosomes for delivering the antisense molecules. 
     As mentioned above the antisense molecules can be comprised in a vector. The vector can for example be a plasmid, bacterial or viral vector such as lentiviral particles or AAV. The composition can comprise multiple oligomeric compounds and/or other antisense molecules, for example for targeting RACK1. 
     The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions that can be administered to subjects, optionally as a vaccine, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. 
     Pharmaceutical compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially compatible with the tissues or the blood of an intended recipient. Other components that may be present in such compositions include water, surfactants (such as Tween), alcohols, polyols, glycerin and vegetable oils, for example. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions. The composition may be supplied, for example but not by way of limitation, as a lyophilized powder which is reconstituted with sterile water or saline prior to administration to the subject. 
     The composition may be in the form of a pharmaceutically acceptable salt which includes, without limitation, those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylarnino ethanol. 
     The compositions, oligomeric compounds and vectors described herein can be formulated for example for intrathecal, intraventricular, intracranial, intraspinal, intraorbital, ophthalmic, intracisternal, intraparenchymal, intraperitoneal, intranasal, aerosol or oral administration. In a preferred embodiment, compositions, oligomeric compounds and vectors are formulated for intrathecal administration. 
     Also provided in another aspect is a method of treating a TDP43-opathy or a FUS-opathy neurodegenerative disease optionally selected from amyotrophic lateral sclerosis (ALS), Alzheimer&#39;s disease (AD), frontotemporal lobar dementia (FTLD), Huntington&#39;s disease (HD), neuronal intermediate filament inclusion disease (NIFID), basophilic inclusion body disease (BIBD) or limbic-predominant age-related TDP-43 encephalopathy (LATE), the method comprising knocking down RACK1 in cells of the central nervous system such as neurons and/or astrocyte cells of a subject in need thereof. 
     The “knocking down” can be achieved using an antisense molecule, such as an oligomeric compound described herein, targeting RACK1 mRNA and/or pre-mRNA. 
     Also provided in another aspect is a method of treating a TDP43-opathy or a FUS-opathy neurodegenerative disease optionally selected from amyotrophic lateral sclerosis (ALS), Alzheimer&#39;s disease (AD), frontotemporal lobar dementia (FTLD), Huntington&#39;s disease (HD), neuronal intermediate filament inclusion disease (NIFID), basophilic inclusion body disease (BIBD) or limbic-predominant age-related TDP-43 encephalopathy (LATE), the method comprising administering to a subject in need thereof one or more antisense molecule(s), for example one or more oligomeric compound disclosed herein. 
     Also provided in another aspect is a method of reducing or inhibiting TDP-43 and/or FUS aggregation in a cell such as a disease cell comprising TDP-43 and/or FUS aggregation, the method comprising administering to the cell or introducing into the cell one or more antisense molecule(s) targeting RACK1 in a sufficient amount and for a sufficient time to decrease RACK1 levels in the cell. In one embodiment, the amount and/or time is sufficient to reduce TDP-43 aggregation and/or partially restore nuclear TDP-43. In one embodiment, the amount and/or time is sufficient to reduce FUS aggregation and/or partially restore nuclear FUS. 
     The antisense molecules, for example the oligomeric compounds of the present disclosure, may be administered alone, as naked antisense molecules. As used herein “naked” means that the antisense molecule is not administered using a delivery vehicle (e.g. viral vector) or delivery agent (e.g. liposome) e.g. viral vector, transport reagent. 
     In one embodiment, the antisense molecule(s) is/are administered and/or introduced into the cell via with a transport reagent, as a recombinant plasmid or as a viral vector that expresses the antisense molecule(s). In a further embodiment, the antisense molecules(s) are introduced into the cell via electroporation. 
     In another embodiment, the antisense molecule(s) comprise one or more cell penetrating moieties. In such context, the antisense molecule can be injected alone i.e. naked, for example intrathecally, and other elements of the antisense molecule are relied upon, e.g. chemical modification(s), for facilitating delivery into the cell. In another embodiment, the one or more antisense molecule is an antisense oligonucleotide, an siRNA, or an shRNA construct. In another embodiment, the antisense molecule(s) is one or more of the aforementioned oligomeric compounds. 
     In other embodiments, the one or more antisense molecules further targets a nucleic acid target sequence listed in Table 1. 
     For example, the one or more antisense molecule is an antisense oligonucleotide molecule disclosed herein, for example comprising or consisting of any one of SEQ ID NOs: 81, 86 or 87. 
     For example, the one or more antisense molecules can be an siRNA molecule, for example comprising sense 5′-CCUUUACACGCUAGAUGGU (SEQ ID NO: 501) with a 3′ tt overhang and antisense 5′-ACCAUCUAGCGUGUAMGG (SEQ ID NO: 502) with a 3′ tg targeting CCTTTACACGCTAGATGGT (SEQ ID NO: 75). 
     In another embodiment, the one or more antisense molecule(s) is introduced via the aforementioned composition. 
     In an embodiment, the cell is a diseased cell. In an embodiment, the cell is a cell of the central nervous system such as a neuron or an astrocyte. In an embodiment, the cell is in a subject, with a TDP43-opathy or a FUS-opathy neurodegenerative disease such as amyotrophic lateral sclerosis (ALS), frontotemporal lobar dementia (FTLD) proteinopathies or a protein folding disease where the disease protein interacts with RACK1. For example, the TDP43-opathy is amyotrophic lateral sclerosis (ALS), Alzheimer&#39;s Disease (AD), frontotemporal lobar dementia (FTLD), Huntington&#39;s Disease (HD) or limbic-predominant age-related TDP-43 encephalopathy (LATE). In another embodiment, the FUS-opathy neurodegenerative disease is neuronal intermediate filament inclusion disease (NIFID) or basophilic inclusion body disease (BIBD). 
     In another embodiment, the one or more antisense molecule(s) is the aforementioned oligomeric compound and/or is comprised in the aforementioned composition. In an embodiment, the antisense molecule and/or composition is administered or introduced into a cell together with a transport reagent, or as a recombinant plasmid or viral vector that expresses the antisense molecule. The transport reagent can be lipid particles such as liposomes, nanoparticles, or nanosomes. In an embodiment, the transport reagent is a liposome. 
     In another embodiment, the antisense molecule and/or composition is administered in a suitable parenteral or enteral route of administration, including intranasal, mucosal, oral, sublingual, transdermal, topical, inhalation, aerosol, intraocular, intratracheal, intrarectal, vaginal, by gene gun, dermal patch, eye drop or mouthwash form or intravascular administration; in particular intrathecal, intraventricular, intraparenchymal or intracerebroventricular administration; e.g., a catheter or other placement device for example using an implanted reservoir that is connected to the ventricles within the brain or spinal cord via an outlet catheter. 
     In other embodiments, the pharmaceutical composition is administered directly to the brain or other portion of the CNS. For example, such methods include the use of an implantable catheter and a pump, which would serve to discharge a pre-determined dose through the catheter to the infusion site. A person skilled in the art would further recognize that the catheter may be implanted by surgical techniques that permit visualization of the catheter so as to position the catheter adjacent to the desired site of administration or infusion in the brain. Such techniques are described in Elsberry et al. U.S. Pat. No. 5,814,014 “Techniques of Treating Neurodegenerative Disorders by Brain Infusion”, which is herein incorporated by reference. Also contemplated are methods such as those described in US patent application 20060129126 (Kaplitt and During “Infusion device and method for infusing material into the brain of a patient”. Devices for delivering drugs to the brain and other parts of the CNS are commercially available (eg. SynchroMed® EL Infusion System, Medtronic, Minneapolis, Minn.). 
     In another embodiment, the pharmaceutical composition is administered to the brain using methods such as modifying the compounds to be administered to allow receptor-mediated transport across the blood brain barrier. 
     Other embodiments contemplate the co-administration of the antisense molecules with biologically active molecules known to facilitate the transport across the blood brain barrier. 
     Also contemplated in certain embodiments, are methods for administering antisense molecules described herein across the blood brain barrier such as those directed at transiently increasing the permeability of the blood brain barrier as described in U.S. Pat. No. 7,012,061 “Method for increasing the permeability of the blood brain barrier”, herein incorporated by reference. 
     When the route of administration is oral, the pharmaceutical composition can be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% antisense molecule and preferably from about 25 to 90% antisense molecule. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of the antisense molecule or from about 1 to 50% antisense molecule. 
     Where the administration is parenteral, mucosal delivery, oral, sublingual, transdermal, topical, inhalation, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, by gene gun, dermal patch or in eye drop or mouthwash form, the antisense molecule can be in the form of a pyrogen-free, parenterally acceptable aqueous solution, and may, in addition to the antisense molecule(s), contain an isotonic vehicle such as Sodium Chloride Injection, Ringer&#39;s Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer&#39;s Injection or other vehicle as known in the art. The pharmaceutical composition may also contain stabilizers, preservatives, buffers, antioxidants or other additives known to those of skill in the art. 
     The amount of antisense molecule in the pharmaceutical composition will depend upon the nature and severity of the condition being treated, and on the nature of prior and concurrent treatments which the subject has undergone or is undergoing. It is contemplated that the various pharmaceutical compositions used to practice the presently disclosed method may comprise about 1 micrograms to about 50 mg of antisense molecule per kg body per day. The duration of the treatment with the pharmaceutical composition herein disclosed will vary, depending on the disease, severity of the disease and the condition and potential idiosyncratic response of each individual subject. 
     In another embodiment, the TDP43-opathy neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Alzheimer&#39;s Disease (AD) or frontotemporal lobar dementia (FTLD), or limbic-predominant age-related TDP-43 encephalopathy (LATE). In another embodiment, the FUS-opathy neurodegenerative disease is neuronal intermediate filament inclusion disease (NIFID) or basophilic inclusion body disease (BIBD). 
     In another embodiment, the subject is a human. 
     Another aspect is the use of one or more antisense molecules, for example the aforementioned oligomeric compounds such as antisense oligonucleotide(s) or siRNA molecule(s), and/or the methods, to treat amyotrophic lateral sclerosis (ALS), Alzheimer&#39;s disease (AD), frontotemporal lobar dementia (FTLD) or Huntington&#39;s disease (HD) in a subject in need thereof, or to reduce and/or disaggregate TDP-43 and/or FUS in a cell such as a diseased cell. 
     Another aspect is one or more antisense molecules, for example oligomeric compounds herein disclosed for use in the treatment of a TDP43-opathy or a FUS-opathy neurodegenerative disease optionally selected from amyotrophic lateral sclerosis (ALS), Alzheimer&#39;s disease (AD), frontotemporal lobar dementia (FTLD), Huntington&#39;s disease (HD), neuronal intermediate filament inclusion disease (NIFID), basophilic inclusion body disease (BIBD) or limbic-predominant age-related TDP-43 encephalopathy (LATE). 
     In an embodiment is use of the aforementioned anti-RACK1 antisense molecules including the oligomeric compounds, such as antisense oligonucleotides, siRNA molecule(s) and/or composition for use in the manufacture of a medicament. 
     Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. 
     The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation. 
     The following non-limiting examples are illustrative of the present disclosure: 
     EXAMPLES 
     Knockdown of RACK1 in cultured cells can diminish or inhibit aggregation of FUS and TDP43 mutants, which is accompanied by partial nuclear repatriation of mutant proteins lacking a nuclear localization sequence. 
     Herein, is data showing that co-aggregation of RACK1 with TDP43 or FUS suppresses global translation by sequestration of ribosomal subunits, and that siRNA knockdown of RACK1 can rescue global translation and prevent TDP-43 mediated neurodegeneration. 
     Example 1 
     Human embryonic kidney 293T (HEK293T) cell line was purchased from American Type Culture Collection (ATCC, Rockville, Md.), and maintained in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), GlutaMax™-1 (2 mM) and antibiotics (50 U/ml penicillin and 50 mg/ml streptomycin) at 37 C in 5% CO 2 . HEK293T cells were transfected with HA-tagged dNLS TDP-43, R495x-FUS, or P525L-FUS cDNA plasmid using Lipofectamine LTX reagent (ThermoFisher Scientific) following the manufacturer&#39;s instruction, and cells were analyzed 48 hrs post-transfection. 
     RACK1 knockdown was achieved by introducing a pool of 3 19-25 nucleotide siRNAs specifically targeting human RACK1 (Santa Cruz Biotechnology, sc-36354) with Lipofectamine RNAiMAX transfection reagent (ThermoFisher Scientific) and incubated for 72 hrs according to the manufacturer&#39;s instruction, followed by transfection of cDNA plasmids of HA-tagged dNLS TDP-43, R495-FUS, or P525L-FUS as described above. 
     Surface Sensing of Translation (SUnSET) was performed to monitor global translation. 48 hrs post-cDNA transfection, cells were incubated with 5 μg/ml of puromycin (ThermoFisher Scientific) in conditioned media for 10 min at 37 C, immediately followed by immunocytochemical or biochemical procedures. 
     Immunocytochemistry (ICC) was performed to visualize the expressions of HA-tagged dNLS TDP-43, R495x-FUS, P525L-FUS, SOD1 mutants, RACK1, and global protein translation. Cells were washed twice with Phosphate Saline Buffer (PBS) and fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature (RT), followed by wash with 20 mM glycine for 10 min at RT with constant rocking. Cells were then incubated with blocking buffer containing PBS, 1% Bovine Serum Albumin (BSA), 10% normal goat serum, and 0.1% Triton-X-100 for 30 min at RT. The following primary antibodies were incubated for 1 h at RT or overnight at 4 C: rabbit polyclonal anti-HA (Abcam, ab9110, 1:1000), chicken polyclonal anti-HA (Abcam, ab9111, 1:8,000), mouse monoclonal anti-RACK1 (BD Biosciences, 610178, 1:500), and mouse monoclonal anti-puromycin (ThermoFisher Scientific, clone 12D10, 1:1000). Cells were then washed with PBS/0.1% Triton-X-100 3×10 min with constant rocking, followed by incubation with Alexa Fluor® goat anti-rabbit, -mouse, or -chicken secondary antibody (ThermoFisher Scientific, 1:1000) for 30 min at RT in the dark. Cells were then washed with PBS/0.1% Triton-X-100 3×10 min, dipped in 5% PBS, and mounted with ProLong Gold Anti-fading mounting media with DAPI (ThermoFisher Scientific, P36931). Cells were analyzed by confocal microscopy (Leica TCS SP8 MP). 
     To quantify global translational levels, following SUnSET described above, cells were washed twice with cold PBS, and lysed in 2% SDS followed by sonication at 30% power for 15 sec to extract total protein. Protein concentration was determined by BCA assay (ThermoFisher Scientific). 10 pg of protein from each transfection was separated on 4-12% NuPAGE SDS-PAGE (ThermoFisher Scientific), transferred onto a PVDF membrane, and blocked in Tris buffered saline (TBS) containing 5% skim milk and 0.1% Tween-20 for 1 h at RT. The following primary antibodies were incubated overnight at 4 C: rabbit anti-HA (Abcam, ab9110, 1:1000), mouse anti-RACK1 (BD Biosciences, 610178, 1:2000), mouse anti-puromycin (ThermoFisher Scientific, clone 12D10, 1:10,000), mouse anti-a-tubulin (ProteinTech, 66031-1-Ig, 1:20,000). Membranes were washed with TBS/0.1% Tween (TBST) 3×10 min at RT with constant rocking, followed by horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibody (GE, 1:5000) incubation for 30 min at RT. Membranes were then washed with TBST 3×10 min, and developed with SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific). 
     Results 
     Using the methods described herein, it is demonstrated that cytoplasmic aggregates of dNLS TDP-43 induce RACK1 aggregation and co-aggregation ( FIG.  1   ) and dNLS TDP-43 aggregates suppress global translation in transfected cells ( FIG.  8   ). It is further demonstrated that cytoplasmic aggregates of mutant SOD1 induce RACK1 aggregation and co-aggregation ( FIG.  3   ). 
     It is demonstrated that cytoplasmic aggregates of dNLS FUS, R495x-FUS and P525L-FUS, induce RACK1 aggregation and co-aggregation ( FIG.  2   ), and dNLS-FUS transfected individual cells demonstrate global translational suppression ( FIG.  7   ). It is also demonstrated that ribosomal binding deficient mutant (DE-RACK1) disrupts mutant FUS, R495x-FUS, and RACK1 co-aggregation ( FIG.  4   ) and partially disrupts P525L-FUS and RACK1 co-aggregation ( FIG.  5   ). It is further demonstrated that mutant FUS suppresses global translation, which can be rescued by RACK1 knockdown ( FIGS.  6 A,  6 B, and  6 C ). 
     siRNA targeted to RACK1 (RACK 1 siRNA) knocks down RACK1 and attenuates dNLS TDP-43 aggregation in the cytoplasm and partially restores nuclear expression ( FIG.  9   ), while it does not affect endogenous nuclear TDP43 expression in empty vector transfected cells ( FIG.  10   ). 
     RACK1 siRNA attenuates mutant FUS, R495x-FUS( FIG.  11   ) and P525L-FUS( FIG.  12   ), aggregation in the cytoplasm, and partially restores the nuclear expression of mutant FUS, R495x-FUS( FIG.  13   ). RACK1 siRNA does not affect endogenous nuclear FUS expression in empty vector transfected cells ( FIG.  14   ). 
     dNLS TDP-43, RACK1, and 40S (small ribosomal subunit, Rps6 as marker) co-aggregate ( FIG.  15   ), dNLS R495x-FUS, RACK1, and 40S co-aggregate ( FIG.  16   ), and dNLS P525L-FUS, RACK1, and 40S co-aggregate ( FIG.  17   ). Additionally, dNLS TDP-43, RACK1, and 60S (large ribosomal subunit, RPL14 as marker) co-aggregate ( FIG.  18   ), dNLS R495x-FUS, RACK1, and 60S co-aggregate ( FIG.  19   ), and dNLS P525L-FUS, RACK1, and 60S co-aggregate ( FIG.  20   ). 
     Upon RACK 1 knockdown, “rescued” nuclear dNLS TDP-43 ( FIGS.  21 A and  21 B ) or dNLS FUS, P25L-FUS( FIGS.  22 A and  22 B ), does not associate with either ribosomal subunit. Where dNLS TDP-43 ( FIGS.  21 A and  21 B ) or dNLS FUS, P525L-FUS( FIGS.  22 A and  22 B ), does remain in the cytoplasm, it often displays a more diffused pattern, as opposed to the typical large aggregates, and remains interacting with the ribosome. 
     dNLS FUS or TDP 43 and RACK1 co-aggregates sequester polyribosome 40S and 60S subunits, resulting in global translational suppression ( FIGS.  15 - 20   ). RACK1 knockdown disperses dNLS FUS or TDP-43 aggregates in the cytoplasm, or even restores their nuclear expressions in a proportion of cells, which as a result releases polyribosomes from the aggregates and rescues global translation ( FIGS.  21 - 23   ). SUnSET ICC shows that, unlike dNLS TDP-43 aggregates, filamentary/diffuse dNLS TDP-43 expressing cells display normal global translation ( FIG.  8   ). This data suggests that knocking down RACK1 presents a great potential to normalize pathological TDP 43/FUS aggregates and translational machinery function without affecting endogenous nuclear TDP-43, which makes RACK1 an extremely attractive therapeutic target for ALS and FTLD. 
     Example 2 
     siRNAs were designed targeting RACK1 mRNA using the following method. 
     Step 1. The siRNA meta-prediction result was collected from five servers (listed below). For the starting position of the candidate siRNA, a server-based prediction score is recorded for the 5 servers. The score definitions for each of the servers are different, and defined as follows. 
     BLOCK-It™ RNAi Designer tool by Thermo Fisher: Gives the quality of prediction as zero to five stars (0-5) with an interval of half star (Link: https://rnaidesigner.thermofisher.com/rnaiexpress/setOption.do?designOption=sirna). Score was normalized to a max score of unity with an interval 0.1. 
     The RNAi design tool of siDirect: This server gives a binary yes/no prediction, which is given a score of one or zero (1 or 0) for each start position in the sequence. (Link: http://sidirect2.rnai.jp/design.cgi) 
     OligoWalk siRNA design tool of Mathews Lab at University of Rochester Medical Center: This server gives a continuous probability between 0 and 1 for a given sequence to be an efficient siRNA (Link: http://rna.urmc.rochester.edu/cgi-bin/server_exe/oligowalk/oligowalk_form.cgi). This probability is directly converted to a score. 
     siRNA wizard design tool of Invivogen: This server categorizes their prediction into either effective siRNA, moderate siRNA, or ineffective siRNA when no prediction is made (Link: https://www.invivogen.com/sirnawizard/design advanced.php). These categories are converted to scores of 1, 0.5, or 0 respectively. 
     siRNA target finder of Genescript: This server gives an unnormalized score for each prediction (Link: https://www.genscript.com/tools/sirna-target-finder). The score values were subsequently normalized to unity by dividing by the maximum prediction score. 
     Step 2. After normalization, the scores from the five servers were summed, resulting in a sum S (x). S (x) is highly variable site to site, i.e. rugged, because each base pair is either being assigned a score or may be zero. In order to smooth the rugged distribution of S (x), a Gaussian filter with sigma=8 bp is applied, which gives a smoothed hotspot score HS (x). ( FIG.  24    shows HS (x) for RACK1 post-splicing exonic mRNA, and  FIG.  25    shows HS (x) for the 8 intron regions of pre-spliced RACK1 mRNA). 
     Step 3. The peaks of HS (x) indicate zones of the RNA sequence which are predicted to give effective siRNA prediction. 
     Known siRNA/shRNA are provided in Table 1 and their starting positions are labeled as plus sign in  FIG.  24   . The Santa Cruz siRNA is a mixture of three sequences that bind mRNA starting at position starting at 246, 631 and 892. The sequence shown in brackets in lower case “(aa)” is not a target sequence but an overhang sequence that can be incorporated when the antisense molecule is a siRNA. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Known siRNA/shRNA 
               
            
           
           
               
               
               
            
               
                 mRNA 
                   
                 SEQ 
               
               
                 Starting 
                   
                 ID 
               
               
                 Position 
                 Target Sequence 
                 NO 
               
               
                   
               
               
                 242 
                 ACCAGGGATGAGACCAACT [9]   
                 70 
               
               
                   
               
               
                 246 
                 (aa)GGGATGAGACCAACTATGG  [7][8]   
                 71 
               
               
                   
               
               
                 247 
                 GGATGAGACCAACTATGGAAT  [9]   
                 72 
               
               
                 (shRNA) 
                   
                   
               
               
                   
               
               
                 631 
                 (AA)GGTATGGAACCTGGCTAACG [8]   
                 73 
               
               
                   
               
               
                 892 
                 (aa)GGGAAAGATCATTGTAGAT [7][8]   
                 74 
               
               
                   
               
               
                 784 
                 (aa)CCTTTACACGCTAGATGGT [3]   
                 75 
               
               
                   
               
            
           
         
       
     
     Synthesized siRNA for RACK1 mRNA are provided in Table 2. Their corresponding peaks are labeled as star marker in  FIG.  24   . 
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Synthesized siRNA for RACK1 mRNA 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 SEQ 
               
               
                 Peak 
                 mRNA 
                   
                 ID 
               
               
                 location 
                 sequence 
                 Target Sequence 
                 NO 
               
               
                   
               
               
                 909 
                 909-931 
                 (AT)GAACTGAA 
                 2 
               
               
                   
                 (siR-2) 
                 GCAAGAAGTTATC 
                   
               
               
                   
               
               
                 474 
                 467-487 
                 (GT)CTCTGGAT 
                 3 
               
               
                   
                 (siR-3) 
                 CTCGAGATAAA 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 SEQ  
                   
                 SEQ 
               
               
                 Peak 
                 antisense 
                 ID 
                 Sense 
                 ID 
               
               
                 location 
                 (5′ to 3′) 
                 NO 
                 (5′ to 3′) 
                 NO 
               
               
                   
               
               
                 909 
                 GAUAACUUCUUG 
                 18 
                 GAACUGAAGC 
                 34 
               
               
                   
                 CUUCAGUUC(AU) 
                   
                 AAGAAGUUAUC 
                   
               
               
                   
                   
                   
                 (au) 
                   
               
               
                   
               
               
                 474 
                 UUUAUCUCGAGA 
                 19 
                 CUCUGGAUCU 
                 35 
               
               
                   
                 UCCAGAG(gu) 
                   
                 CGAGAUAAA(gu) 
                   
               
               
                   
               
            
           
         
       
     
     siRNA targeting mRNA: Within the coding region (sequence 108-1059), other significant peaks in  FIG.  24    include positions 887, 909, 474, 212, 646, 618, 748, 779, 685, 242, 584, 295, 508, 988, 405, 160 and 178. Their corresponding targeting sequences are listed in Table 3. The sequences are listed in the order from higher HS (x) to lower HS (x). 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 siRNA design for RACK1 mRNA 
               
            
           
           
               
               
               
               
            
               
                   
                 Target mRNA 
                 Target mRNA 
                 SEQ ID 
               
               
                   
                 sequence 
                 Sequence 
                 NO of 
               
               
                   
                 index 
                 including 
                 target 
               
               
                 Peak 
                 including 
                 overhang 
                 mRNA 
               
               
                 location 
                 overhang 
                 brackets 
                 sequence 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 887 
                 884-904 
                 (GA)TTTAGAGGGAAAGATCATT 
                 1 
               
               
                   
               
               
                 909 
                 909-931 (siR-2) 
                 (AT)GAACTGAAGCAAGAAGTTATC 
                 2 
               
               
                   
               
               
                 474 
                 467-487 (siR-3) 
                 (GT)CTCTGGATCTCGAGATAAA 
                 3 
               
               
                   
               
               
                 646 
                 642-662 
                 (TG)GCTAACTGCAAGCTGAAGA 
                 4 
               
               
                   
               
               
                 618 
                 615-635 
                 (GG)GACAAGCTGGTCAAGGTAT 
                 5 
               
               
                   
               
               
                 748 
                 740-750 
                 (AA)GGATGGCCAGGCCATGTTA 
                 6 
               
               
                   
               
               
                 779 
                 779-799 
                 (AA)ACACCTTTACACGCTAGAT 
                 7 
               
               
                   
               
               
                 685 
                 683-703 
                 (GG)CTATCTGAACACGGTGACT 
                 8 
               
               
                   
               
               
                 242 
                 242-262 
                 (AC)CAGGGATGAGACCAACTAT 
                 9 
               
               
                   
               
               
                 584 
                 577-597 
                 (GC)CCAACAGCAGCAACCCTAT 
                 10 
               
               
                   
               
               
                 295 
                 296-316 
                 (CA)CTTTGTTAGTGATGTGGTT 
                 11 
               
               
                   
               
               
                 508 
                 505-525 
                 (TA)CCCTGGGTGTGTGCAAATA 
                 12 
               
               
                   
               
               
                 988 
                 981-1001 
                 (CT)GCTGATGGCCAGACTCTGT 
                 13 
               
               
                   
               
               
                 405 
                 403-423 
                 (GC)GATTTGTGGGCCATACCAA 
                 14 
               
               
                   
               
               
                 160 
                 156-176 
                 (GG)GTAACCCAGATCGCTACTA 
                 15 
               
               
                   
               
               
                 178 
                 178-198 
                 (CC)CGCAGTTCCCGGACATGAT 
                 16 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Antisense 
                   
                 Sense(5′ to 3′)  
                   
               
               
                   
                 (5′ to 3′) 
                   
                 passenger  
                   
               
               
                   
                 guide strand 
                 SEQ 
                 strand 
                 SEQ 
               
               
                 Peak 
                 including  
                 ID 
                 including 
                 ID 
               
               
                 location 
                 overhang 
                 NO 
                 overhang 
                 NO 
               
               
                   
               
               
                 887 
                 AAUGAUCUUUCCCUCUAAA(UC) 
                 17 
                 UUUAGAGGGAAAGAUCAUU(UC) 
                 33 
               
               
                   
               
               
                 909 
                 GAUAACUUCUUGCUUCAGUUC(AU) 
                 18 
                 GAACUGAAGCAAGAAGUUAUC(au) 
                 34 
               
               
                 (siR-2) 
                   
                   
                   
                   
               
               
                   
               
               
                 474 
                 UUUAUCUCGAGAUCCAGAG(AC) 
                 19 
                 CUCUGGAUCUCGAGAUAAA(ac) 
                 35 
               
               
                 (siR-3) 
                   
                   
                   
                   
               
               
                   
               
               
                 646 
                 UCUUCAGCUUGCAGUUAGC(CA) 
                 20 
                 GCUAACUGCAAGCUGAAGA(ca) 
                 36 
               
               
                   
               
               
                 618 
                 AUACCUUGACCAGCUUGUC(CC) 
                 21 
                 GACAAGCUGGUCAAGGUAU(CC) 
                 37 
               
               
                   
               
               
                 748 
                 UAACAUGGCCUGGCCAUCC(UU) 
                 22 
                 GGAUGGCCAGGCCAUGUUA(UU) 
                 38 
               
               
                   
               
               
                 779 
                 AUCUAGCGUGUAAAGGUGU(UU) 
                 23 
                 ACACCUUUACACGCUAGAU(UU) 
                 39 
               
               
                   
               
               
                 685 
                 AGUCACCGUGUUCAGAUAG(CC) 
                 24 
                 CUAUCUGAACACGGUGACU(CC) 
                 40 
               
               
                   
               
               
                 242 
                 AUAGUUGGUCUCAUCCCUG(GU) 
                 25 
                 CAGGGAUGAGACCAACUAU(gu) 
                 41 
               
               
                   
               
               
                 584 
                 AUAGGGUUGCUGCUGUUGG(GC) 
                 26 
                 CCAACAGCAGCAACCCUAU(gc) 
                 42 
               
               
                   
               
               
                 295 
                 AACCACAUCACUAACAAAG(UG) 
                 27 
                 CUUUGUUAGUGAUGUGGUU(ug) 
                 43 
               
               
                   
               
               
                 508 
                 UAUUUGCACACACCCAGGG(UA) 
                 28 
                 CCCUGGGUGUGUGCAAAUA(ua) 
                 44 
               
               
                   
               
               
                 988 
                 ACAGAGUCUGGCCAUCAGC(AG) 
                 29 
                 GCUGAUGGCCAGACUCUGU(ag) 
                 45 
               
               
                   
               
               
                 405 
                 UUGGUAUGGCCCACAAAUC(GC) 
                 30 
                 GAUUUGUGGGCCAUACCAA(gc) 
                 46 
               
               
                   
               
               
                 160 
                 UAGUAGCGAUCUGGGUUAC(CC) 
                 31 
                 GUAACCCAGAUCGCUACUA(CC) 
                 47 
               
               
                   
               
               
                 178 
                 AUCAUGUCCGGGAACUGCG(GG) 
                 32 
                 CGCAGUUCCCGGACAUGAU(gg) 
                 48 
               
               
                   
               
            
           
         
       
     
     siRNA targeting pre-mRNA (Splice-blocking siRNA): Splice-blocking siRNA is designed to bind the boundary of intron and Extron region of RACK1 pre-mRNA. The hotspot score, HS (x), is constructed the same way as mRNA. The hotspot score of Extron-intron boundaries are extracted and shown in  FIG.  25   . The proposed target sequences are in Table 4. The sequence are listed from 5′ to 3′, or from N-terminal to C-terminal of the protein translation. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 siRNA targeting RACK1 pre-mRNA 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Pre-mRNA 
                 Target 
                   
               
               
                   
                   
                 sequence 
                 Sequence 
                 SEQ  
               
               
                   
                 Peak 
                 including 
                 including 
                 ID 
               
               
                   
                 location 
                 overhang 
                 overhang 
                 NO 
               
               
                   
                   
               
               
                   
                  4406 
                 4404-4424 
                 (AG)GTACGGACTA 
                 49 
               
               
                   
                   
                   
                 AGGTAGATT 
                   
               
               
                   
                   
               
               
                   
                 −5750 
                 5735-5755 
                 (TG)TTTTACCTCC 
                 50 
               
               
                   
                   
                   
                 TTTAGATAA 
                   
               
               
                   
                   
               
               
                   
                 10382 
                 10366-10386 
                 (CC)TGTTCCCCAG 
                 51 
               
               
                   
                   
                   
                 GATTTAGAG 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 Antisense 
                   
                 Sense 
                   
               
               
                   
                   
                 (5′ to 3′) 
                   
                 (5′ to 3′) 
                   
               
               
                   
                   
                 guide 
                   
                 passenger 
                   
               
               
                   
                   
                 strand 
                 SEQ 
                 strand 
                 SEQ  
               
               
                   
                 Peak 
                 including 
                 ID 
                 including 
                 ID 
               
               
                   
                 location 
                 overhang 
                 NO 
                 overhang 
                 NO 
               
               
                   
                   
               
               
                   
                  4406 
                 AAUCUACCUUAG 
                 52 
                 GUACGGACUAA 
                 55 
               
               
                   
                   
                 UCCGUAC(CU) 
                   
                 GGUAGAUU(CU) 
                   
               
               
                   
                   
               
               
                   
                 −5750 
                 UUAUCUAAAGGA 
                 53 
                 UUUUACCUCCU 
                 56 
               
               
                   
                   
                 GGUAAAA(CA) 
                   
                 UUAGAUAA(ca) 
                   
               
               
                   
                   
               
               
                   
                 10382 
                 CUCUAAAUCCUG 
                 54 
                 UGUUCCCCAGG 
                 57 
               
               
                   
                   
                 GGGAACA(GG) 
                   
                 AUUUAGAG(cg) 
               
               
                   
                   
               
            
           
         
       
     
     Negative control siRNA: To test the effectiveness of the prediction method, the low HS (x) score region was used as a negative control. The middle of each zero-score-region in  FIG.  24    are listed in Table 5, in the order from wider to narrower zero-score-region in  FIG.  24   . 
     
       
         
           
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 Negative control of siRNA design 
               
               
                 for RACK1 mRNA 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Target Sequence  
                 SEQ 
               
               
                 mRNA 
                 including 
                 ID 
               
               
                 sequence 
                 overhang 
                 NO 
               
               
                   
               
               
                 100-120 
                 (CG)CCGCCATGACTGAGCAGAT 
                 58 
               
               
                   
               
               
                 362-382 
                 (AC)CCTGCGCCTCTGGGATCTC 
                 59 
               
               
                   
               
               
                 546-566 
                 (AC)TCAGAGTGGGTGTCTTGTG 
                 60 
               
               
                   
               
               
                 830-850 
                 (AG)CCCTAACCGCTACTGGCTG 
                 61 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Antisense 
                   
                 Sense 
                   
               
               
                   
                 (5′ to 3′) 
                 SEQ 
                 (5′ to 3′) 
                 SEQ 
               
               
                 mRNA 
                 including  
                 ID 
                 including 
                 ID 
               
               
                 sequence 
                 overhang 
                 NO 
                 overhang 
                 NO 
               
               
                   
               
               
                 100-120 
                 AUCUGCUCAG 
                 62 
                 CCGCCAUGA 
                 66 
               
               
                   
                 UCAUGGCGG 
                   
                 CUGAGCAGA 
                   
               
               
                   
                 (CG) 
                   
                 U(cg) 
                   
               
               
                   
               
               
                 362-382 
                 GAGAUCCCAG 
                 63 
                 CCUGCGCCU 
                 67 
               
               
                   
                 AGGCGCAGG 
                   
                 CUGGGAUCU 
                   
               
               
                   
                 (GU) 
                   
                 C(gu) 
                   
               
               
                   
               
               
                 546-566 
                 CACAAGACAC 
                 64 
                 UCAGAGUGG 
                 68 
               
               
                   
                 CCACUCUGA 
                   
                 GUGUCUUGU 
                   
               
               
                   
                 (GU) 
                   
                 G(gu) 
                   
               
               
                   
               
               
                 830-850 
                 CAGCCAGUAG 
                 65 
                 CCCUAACCG 
                 69 
               
               
                   
                 CGGUUAGGG 
                   
                 CUACUGGCU 
                   
               
               
                   
                 (CU) 
                   
                 G(cu) 
               
               
                   
               
            
           
         
       
     
     Results 
     siR-2 and siR-3 siRNA sequences successfully knocked down RACK1 ( FIG.  26   ). HEK293T cells were seeded onto a 6-well plate (ThermoFisher Scientific) at a density of 250,000 cells per well the day prior to siRNA transfection. 10 μM stock of negative control or RACK1 siRNAs were introduced into the cell using Lipofectamine RNAiMAX transfection reagent (ThermoFisher Scientific) according to the manufacturer&#39;s instruction to achieve a final concentration of 25 pmol per well (or 1 pmol per 10,000 cells). 72 hrs post-transfection, cells were lysed in 2% SDS, followed by sonication at 30% power for 15 sec to extract total protein. Protein concentration was determined by BCA assay (ThermoFisher Scientific). 10 pg of protein from each sample was separated on 4-12% NuPAGE SDS-PAGE (ThermoFisher Scientific), transferred onto a PVDF membrane, and Western blotted for RACK1 and loading control α-tubulin as described above. Western blot band intensity was quantified using ImageJ. RACK1 intensity was normalized to corresponding α-tubulin intensity in each lane. Normalized RACK1 intensity of each transfection was then compared with un-transfected (UT) cells. 
     Example 3: Knockdown of Rack1 Prevents hTDP-43-Induced Neurodegeneration In Vivo 
     As shown in Example 1 RACK1 knockdown in cultured cells ameliorates the phenotype caused by hTDP-43 expression in a number of ways, including by: reducing aggregation; restoring nuclear localization; and relieving TDP-43-induced suppression of protein synthesis. To extend these findings, it was further demonstrated herein that reduction of hTDP43-induced toxicity by RACK1 knockdown also takes place in vivo, in neurons functioning in a living network. 
     A  Drosophila melanogaster  expression system which allows modular, targeted expression was used. Using the UAS-Gal4 expression system (Rodriguez et al., 2012; explained in  FIGS.  27 A and  27 B ), expression of the alleles of interest was driven by the GMR promoter thus largely limiting expression to retinal neurons, a cell population widely used for its read-out of neuronal degeneration. Human TDP43 alleles wild-type (WT) and an ALS-associated point mutation (Q331K) (Elden et al. 2010) were used. Flies expressing hTDP43 either WT or Q331K, with or without RACK1-RNAi, in retinal neurons were generated ( FIG.  27 B ). 
     With reference to  FIG.  27 A , in general, one line of flies harbors a transgene consisting of a promotor specific for the chosen cell population driving expression of the protein Gal4. A separate stable line of flies harbors a transgene with an upstream activating sequence (UAS) to drive expression of the sequence of interest, which may be protein-coding or RNAi. The UAS is not active, and these flies express no transgene. However, when these two lines of flies are crossed, producing offspring with one copy of each transgene, the F1 flies produce Gal4 protein only in the cells of interest, which then binds to and activates the UAS and turns on production of the gene/target of interest (Rodriguez et al., 2012). With reference to  FIG.  27 B , the GMR-Gal4 driver line (obtained from Bloomington  Drosophila  Stock Centre (BDSC) line #9146) which expresses Gal4 in retinal neurons, was used and crossed with one of five UAS lines: 
     1) UAS-hTDP43 WT  (Elden et al., 2010; obtained from BDSC #79587) 
     2) UAS-hTDP43 Q331K  (Elden et al., 2010; obtained from BDSC #79590) 
     3) UAS-RACK1-RNAi (Perkins et al., 2015; obtained from BDSC #60399) 
     4) 1 recombined onto the same chromosome with 3 
     5) 2 recombined onto the same chromosome with 3 
     These crosses produce flies expressing either wild-type or mutant hTDP43 or not, with or without RACK1-RNAi, in retinal neurons. Short hairpin RNA used to prepare the RNAi has Hairpin ID #SH047-D12; forward oligo is CAAGACCATCAAGCTGTGGAA (SEQ ID NO: 76), and reverse oligo is TTCCACAGCTTGATGGTCTTG (SEQ ID NO: 77). Since the parental lines are heterozygous for each transgene, having also a balancer chromosome with marker, siblings of the experimental flies are also produced which harbor only the driver or only the undriven UAS transgene. These flies are used as controls. 
     Cohorts of flies of each genotype were monitored for the first six days of adulthood (A1 to A6), and scored each day for retinal neuron degeneration. Control flies of a variety of genotypes provide a baseline for normal eye morphology. Representative photographs are provided in  FIGS.  28 A to  28 L , and detailed numbers with statistical analysis are given in  FIG.  29    and Tables 6 and 7 below. In eyes displaying mild degeneration, ommatidia are often missing from the ventral margin (arrows), in contrast to eyes without degeneration in which this margin is clearly intact. Additionally, darker dots of dying ommatidia can be observed. 
     As shown in  FIGS.  28 A,  28 E,  28 I  (left column), hTDP43 WT  causes mild neurodegeneration at A1 ( FIG.  28 A ) which persists to A6 ( FIG.  28 E ) and is absent in control ( FIG.  28 I ). As shown in  FIGS.  28 B,  28 F,  28 J  (second column), flies co-expressing of RACK1-RNAi with hTDP43 WT  have no degeneration at A1 ( FIG.  28 B ) or A6 ( FIG.  28 F ), indistinguishable from control ( FIG.  28 J ). As shown in  FIGS.  28 C,  28 G,  28 K  (third column), hTDP43 Q331K  causes degeneration which is mild at A1 ( FIG.  28 C ), but worsens over time leading to some mild ( FIG.  28 G ) and some moderate ( FIG.  28 K ) cases at A6. When RACK1-RNAi is co-expressed with hTDP43 Q331K  degeneration remains mild from A1 ( FIG.  28 D ) to A6 ( FIG.  28 H ).  FIG.  28 L  is an additional control showing that GMR expression of RACK1-RNAi alone causes no phenotype. Flies were scored according to the system published by Li et al., 2010: 0=normal; 1=&lt;25% ommatidia loss; 2=25-50% ommatidia loss; 3=50-75% ommatidia loss with small regions of necrosis (black patches); 4=&gt;75% ommatidia loss with massive regions of necrosis. In each panel, the number at top right indicates the score which that eye received. 
     Quantification of retinal degeneration is shown in  FIG.  29   . Table 6 shows results of neurodegeneration scores for fly eyes. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Neurodegeneration scores for fly eyes 
               
               
                 PERCENT with each score 
               
            
           
           
               
               
            
               
                   
                 age: 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 genotype: 
                 score: 
                 A1 
                 A2 
                 A3 
                 A4 
                 A5 
                 A6 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 GMR &gt; hTDP43 WT   
                 0 
                   
                   
                   
                   
                   
                   
               
               
                   
                 1 
                 100 
                 100 
                 100 
                 100 
                 100 
                 100 
               
               
                   
                 2 
               
               
                 GMR &gt; hTDP43 WT   
                 0 
                 100 
                 100 
                 100 
                 100 
                 100 
                 100 
               
               
                 RACK1-RNAi 
                 1 
               
               
                   
                 2 
               
               
                 GMR &gt; hTDP43 Q331K   
                 0 
               
               
                   
                 1 
                 100 
                 100 
                 92 
                 84 
                 75 
                 72 
               
               
                   
                 2 
                   
                   
                 8 
                 16 
                 25 
                 28 
               
               
                 GMR &gt; hTDP43 Q331K   
                 0 
               
               
                 RACK1-RNAi 
                 1 
                 100 
                 100 
                 100 
                 100 
                 100 
                 100 
               
               
                   
                 2 
               
               
                 GMR &gt; RACK1-RNAi 
                 0 
                 100 
                 100 
                 100 
                 100 
                 100 
                 100 
               
               
                 GMR alone 
                 0 
                 100 
                 100 
                 100 
                 100 
                 100 
                 100 
               
               
                 hTDP43 WT  (undriven) 
                 0 
                 100 
                 100 
                 100 
                 100 
                 100 
                 100 
               
               
                 hTDP43 WT  RACK1-RNAi 
                 0 
                 100 
                 100 
                 100 
                 100 
                 100 
                 100 
               
               
                 (undriven) 
               
               
                 hTDP43 Q331K  (undriven) 
                 0 
                 100 
                 100 
                 100 
                 100 
                 100 
                 100 
               
               
                   
               
            
           
         
       
     
     Approximately 50 flies per genotype were scored each day. For the experimental flies, 3 rows indicate the percentage of flies which received a score of 0, 1 or 2 on each of days A1 to A6. 100% of GMR &gt;hTDP43 WT  scored 1 every day, while 100% of GMR &gt;hTDP43 WT  RACK1-RNAi scored 0 every day. GMR &gt;hTDP43 Q331K  flies all scored 1 on A1 and A2, but an increasing proportion worsened to score 2 on subsequent days. GMR &gt;hTDP43 Q331K  RACK1-RNAi all scored 1 at A1-A6. The various controls scored 0 at all ages. As shown in Table 7, Chi-squared tests were carried out as pair-wise comparisons, and extremely low p values show that all the indicated pairs of cohorts were significantly different from each other: hTDP43 W T is different from control (line 1); mutant TDP43 is different from WT (line 3); and the addition of RACK1-RNAi makes a significant difference to both hTDP43 WT  (line 2) and hTDP43 Q331K  (line 4). In  FIG.  29   , a Kaplan-Meier curve for GMR &gt;hTDP43 Q331K  (the only genotype which worsens with age) shows the percentage of flies whose score remains at 1 (rather than declining to 2) on any given day. This is shown in comparison to GMR &gt;hTDP43 Q331K  RACK1-RNAi, from which it is highly significantly different (Log-rank test: p=0.002. Error bars are 95% confidence intervals). 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Chi-squared tests 
               
            
           
           
               
               
               
            
               
                 genotype 1 
                 genotype 2 
                 p (Chi 2  test) 
               
               
                   
               
               
                 hTDP43 WT  (undriven) 
                 GMR &gt; hTDP43 WT   
                 0.84 × 10 −28   
               
               
                 GMR &gt; hTDP43 WT   
                 GMR &gt; hTDP43 WT   
                 0.19 × 10 −21   
               
               
                   
                 RACK1-RNAi 
               
               
                 GMR &gt; hTDP43 WT   
                 GMR &gt; hTDP43 Q331K  (A6) 
                 0.30 × 10 −4    
               
               
                 GMR &gt; hTDP43 Q331K  (A6) 
                 GMR &gt; hTDP43 Q331K   
                 0.0059 
               
               
                   
                 RACK1-RNAi 
               
               
                   
               
            
           
         
       
     
     It was found that all flies expressing hTDP-43 WT  in retinal neurons displayed mild neurodegeneration, replicating published findings (Elden et al., 2010). This was evident at A1 ( FIG.  28 A ) and did not change over the next five days ( FIG.  28 E , and Tables 6 and 7, top rows). In striking contrast, 100% of flies co-expressing RACK1-RNAi with hTDP-43 WT  displayed normal eye morphology with no degeneration, at all ages ( FIG.  28 B,  28 F , Tables 6 and 7, second rows). Thus, RACK1 knockdown completely rescues hTDP-43 WT -induced degeneration. Expression of mutant hTDP-43 Q331K  also caused retinal neuron degeneration in 100% of flies ( FIG.  28 C,  28 G,  28 K ). This was more severe than that caused by hTDP-43 WT  expression, and also significantly worsened over time (Tables 6 and 7, third rows,  FIG.  29   ), thus modeling two features of disease. In contrast, flies co-expressing RACK1-RNAi with hTDP-43 Q331K  displayed mild degeneration that remained mild from A1 to A6 ( FIGS.  2 D,  2 H , Tables 6 and 7, fourth rows,  FIG.  29   ). Thus, RACK1 knockdown completely prevents the worsening of neurodegeneration overtime caused by hTDP-43 Q331K    
     Example 4: Antisense Oligonucleotides 
     Antisense oligonucleotides (ASOs) binding human RACK1 mRNA were generated and are detailed in Table 8. Modifications to the bases are as follows. The ASOs have phosphorothioate bonds between all bases. The 2′-O-methoxyethyl(2′-MOE) modification is used for the 5 RNA bases on each end, with 10 DNA bases in the middle to form a ‘gapmer’ structure. The mRNA start position at which the ASO sequences bind RACK1 are indicated. Although the ASO sequences may be represented as DNA, RNA where thymidine (T) is uracil (U) also contemplated. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 ASO sequences 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SEQ 
                 Target 
                 Target 
                 Corresponding mRNA 
                 SEQ 
               
               
                   
                 ID 
                 mRNA 
                 mRNA 
                 (reverse complement 
                 ID 
               
               
                 ASO sequence 
                 NO 
                 start 
                 end 
                 of ASO) 
                 NO 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 TGGTCTCATCCCTGGTCAGT 
                 78 
                 238 
                 257 
                 ACTGACCAGGGATGAGACCA 
                 289 
               
               
                 [ASO #1] 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 CACGAAGGGTCATCTGCTCA 
                 79 
                 112 
                 131 
                 TGAGCAGATGACCCTTCGTG 
                 290 
               
               
                 [ASO #2] 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 CAGGTTCCATACCTTGACCA 
                 80 
                 624 
                 643 
                 TGGTCAAGGTATGGAACCTG 
                 291 
               
               
                 [ASO #3] 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 TCCAGAGACAATCTGCCGGT 
                 81 
                 456 
                 475 
                 ACCGGCAGATTGTCTCTGGA 
                 292 
               
               
                 [ASO #4] 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 ACCGTGTTCAGATAGCCTGT 
                 82 
                 680 
                 699 
                 ACAGGCTATCTGAACACGGT 
                 293 
               
               
                 [ASO #5] 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 ATCATGTCCGGGAACTGCGG 
                 83 
                 179 
                 198 
                 CCGCAGTTCCCGGACATGAT 
                 294 
               
               
                 [ASO #6] 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 CGTTGTGAGATCCCAGAGGC 
                 84 
                 369 
                 388 
                 GCCTCTGGGATCTCACAACG 
                 295 
               
               
                 [ASO #7] 
                   
                   
                   
                   
                   
               
               
                 GCCGGTTGTCAGAGGAGAAG 
                 85 
                 442 
                 461 
                 CTTCTCCTCTGACAACCGGC 
                 296 
               
               
                 [ASO #8] 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 CCAGAGACAATCTGCCGGTT 
                 86 
                 455 
                 474 
                 AACCGGCAGATTGTCTCTGG 
                 297 
               
               
                 [ASO #9] 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 ACGATGATAGGGTTGCTGCT 
                 87 
                 584 
                 603 
                 AGCAGCAACCCTATCATCGT 
                 298 
               
               
                 [ASO #10] 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 AAGGGTCATCTGCTCAGTCA 
                 88 
                 108 
                 127 
                 TGACTGAGCAGATGACCCTT 
                 299 
               
               
                   
               
               
                 GAAGGGTCATCTGCTCAGTC 
                 89 
                 109 
                 128 
                 GACTGAGCAGATGACCCTTC 
                 300 
               
               
                   
               
               
                 CGAAGGGTCATCTGCTCAGT 
                 90 
                 110 
                 129 
                 ACTGAGCAGATGACCCTTCG 
                 301 
               
               
                   
               
               
                 ACGAAGGGTCATCTGCTCAG 
                 91 
                 111 
                 130 
                 CTGAGCAGATGACCCTTCGT 
                 302 
               
               
                   
               
               
                 CCACGAAGGGTCATCTGCTC 
                 92 
                 113 
                 132 
                 GAGCAGATGACCCTTCGTGG 
                 303 
               
               
                   
               
               
                 GCCACGAAGGGTCATCTGCT 
                 93 
                 114 
                 133 
                 AGCAGATGACCCTTCGTGGC 
                 304 
               
               
                   
               
               
                 TGCCACGAAGGGTCATCTGC 
                 94 
                 115 
                 134 
                 GCAGATGACCCTTCGTGGCA 
                 305 
               
               
                   
               
               
                 GTGCCACGAAGGGTCATCTG 
                 95 
                 116 
                 135 
                 CAGATGACCCTTCGTGGCAC 
                 306 
               
               
                   
               
               
                 GGTGCCACGAAGGGTCATCT 
                 96 
                 117 
                 136 
                 AGATGACCCTTCGTGGCACC 
                 307 
               
               
                   
               
               
                 GGGTGCCACGAAGGGTCATC 
                 97 
                 118 
                 137 
                 GATGACCCTTCGTGGCACCC 
                 308 
               
               
                   
               
               
                 AGGGTGCCACGAAGGGTCAT 
                 98 
                 119 
                 138 
                 ATGACCCTTCGTGGCACCCT 
                 309 
               
               
                   
               
               
                 GAGGGTGCCACGAAGGGTCA 
                 99 
                 120 
                 139 
                 TGACCCTTCGTGGCACCCTC 
                 310 
               
               
                   
               
               
                 TGAGGGTGCCACGAAGGGTC 
                 100 
                 121 
                 140 
                 GACCCTTCGTGGCACCCTCA 
                 311 
               
               
                   
               
               
                 TTGAGGGTGCCACGAAGGGT 
                 101 
                 122 
                 141 
                 ACCCTTCGTGGCACCCTCAA 
                 312 
               
               
                   
               
               
                 CTTGAGGGTGCCACGAAGGG 
                 102 
                 123 
                 142 
                 CCCTTCGTGGCACCCTCAAG 
                 313 
               
               
                   
               
               
                 CCTTGAGGGTGCCACGAAGG 
                 103 
                 124 
                 143 
                 CCTTCGTGGCACCCTCAAGG 
                 314 
               
               
                   
               
               
                 CCCTTGAGGGTGCCACGAAG 
                 104 
                 125 
                 144 
                 CTTCGTGGCACCCTCAAGGG 
                 315 
               
               
                   
               
               
                 GCCCTTGAGGGTGCCACGAA 
                 105 
                 126 
                 145 
                 TTCGTGGCACCCTCAAGGGC 
                 316 
               
               
                   
               
               
                 GGCCCTTGAGGGTGCCACGA 
                 106 
                 127 
                 146 
                 TCGTGGCACCCTCAAGGGCC 
                 317 
               
               
                   
               
               
                 TGCGGGGTAGTAGCGATCTG 
                 107 
                 164 
                 183 
                 CAGATCGCTACTACCCCGCA 
                 318 
               
               
                   
               
               
                 CTGCGGGGTAGTAGCGATCT 
                 108 
                 165 
                 184 
                 AGATCGCTACTACCCCGCAG 
                 319 
               
               
                   
               
               
                 ACTGCGGGGTAGTAGCGATC 
                 109 
                 166 
                 185 
                 GATCGCTACTACCCCGCAGT 
                 320 
               
               
                   
               
               
                 AACTGCGGGGTAGTAGCGAT 
                 110 
                 167 
                 186 
                 ATCGCTACTACCCCGCAGTT 
                 321 
               
               
                   
               
               
                 GAACTGCGGGGTAGTAGCGA 
                 111 
                 168 
                 187 
                 TCGCTACTACCCCGCAGTTC 
                 322 
               
               
                   
               
               
                 GGAACTGCGGGGTAGTAGCG 
                 112 
                 169 
                 188 
                 CGCTACTACCCCGCAGTTCC 
                 323 
               
               
                   
               
               
                 GGGAACTGCGGGGTAGTAGC 
                 113 
                 170 
                 189 
                 GCTACTACCCCGCAGTTCCC 
                 324 
               
               
                   
               
               
                 CGGGAACTGCGGGGTAGTAG 
                 114 
                 171 
                 190 
                 CTACTACCCCGCAGTTCCCG 
                 325 
               
               
                   
               
               
                 CCGGGAACTGCGGGGTAGTA 
                 115 
                 172 
                 191 
                 TACTACCCCGCAGTTCCCGG 
                 326 
               
               
                   
               
               
                 TCCGGGAACTGCGGGGTAGT 
                 116 
                 173 
                 192 
                 ACTACCCCGCAGTTCCCGGA 
                 327 
               
               
                   
               
               
                 GTCCGGGAACTGCGGGGTAG 
                 117 
                 174 
                 193 
                 CTACCCCGCAGTTCCCGGAC 
                 328 
               
               
                   
               
               
                 TGTCCGGGAACTGCGGGGTA 
                 118 
                 175 
                 194 
                 TACCCCGCAGTTCCCGGACA 
                 329 
               
               
                   
               
               
                 ATGTCCGGGAACTGCGGGGT 
                 119 
                 176 
                 195 
                 ACCCCGCAGTTCCCGGACAT 
                 330 
               
               
                   
               
               
                 CATGTCCGGGAACTGCGGGG 
                 120 
                 177 
                 196 
                 CCCCGCAGTTCCCGGACATG 
                 331 
               
               
                   
               
               
                 TCATGTCCGGGAACTGCGGG 
                 121 
                 178 
                 197 
                 CCCGCAGTTCCCGGACATGA 
                 332 
               
               
                   
               
               
                 GATCATGTCCGGGAACTGCG 
                 122 
                 180 
                 199 
                 CGCAGTTCCCGGACATGATC 
                 333 
               
               
                   
               
               
                 GGATCATGTCCGGGAACTGC 
                 123 
                 181 
                 200 
                 GCAGTTCCCGGACATGATCC 
                 334 
               
               
                   
               
               
                 AGGATCATGTCCGGGAACTG 
                 124 
                 182 
                 201 
                 CAGTTCCCGGACATGATCCT 
                 335 
               
               
                   
               
               
                 GAGGATCATGTCCGGGAACT 
                 125 
                 183 
                 202 
                 AGTTCCCGGACATGATCCTC 
                 336 
               
               
                   
               
               
                 AGAGGATCATGTCCGGGAAC 
                 126 
                 184 
                 203 
                 GTTCCCGGACATGATCCTCT 
                 337 
               
               
                   
               
               
                 GAGAGGATCATGTCCGGGAA 
                 127 
                 185 
                 204 
                 TTCCCGGACATGATCCTCTC 
                 338 
               
               
                   
               
               
                 GGAGAGGATCATGTCCGGGA 
                 128 
                 186 
                 205 
                 TCCCGGACATGATCCTCTCC 
                 339 
               
               
                   
               
               
                 CGGAGAGGATCATGTCCGGG 
                 129 
                 187 
                 206 
                 CCCGGACATGATCCTCTCCG 
                 340 
               
               
                   
               
               
                 GCGGAGAGGATCATGTCCGG 
                 130 
                 188 
                 207 
                 CCGGACATGATCCTCTCCGC 
                 341 
               
               
                   
               
               
                 GGCGGAGAGGATCATGTCCG 
                 131 
                 189 
                 208 
                 CGGACATGATCCTCTCCGCC 
                 342 
               
               
                   
               
               
                 AGGCGGAGAGGATCATGTCC 
                 132 
                 190 
                 209 
                 GGACATGATCCTCTCCGCCT 
                 343 
               
               
                   
               
               
                 GAGGCGGAGAGGATCATGTC 
                 133 
                 191 
                 210 
                 GACATGATCCTCTCCGCCTC 
                 344 
               
               
                   
               
               
                 AGAGGCGGAGAGGATCATGT 
                 134 
                 192 
                 211 
                 ACATGATCCTCTCCGCCTCT 
                 345 
               
               
                   
               
               
                 GAGAGGCGGAGAGGATCATG 
                 135 
                 193 
                 212 
                 CATGATCCTCTCCGCCTCTC 
                 346 
               
               
                   
               
               
                 CGAGAGGCGGAGAGGATCAT 
                 136 
                 194 
                 213 
                 ATGATCCTCTCCGCCTCTCG 
                 347 
               
               
                   
               
               
                 TCAGTTTCCACATGATGATG 
                 137 
                 223 
                 242 
                 CATCATCATGTGGAAACTGA 
                 348 
               
               
                   
               
               
                 GTCAGTTTCCACATGATGAT 
                 138 
                 224 
                 243 
                 ATCATCATGTGGAAACTGAC 
                 349 
               
               
                   
               
               
                 GGTCAGTTTCCACATGATGA 
                 139 
                 225 
                 244 
                 TCATCATGTGGAAACTGACC 
                 350 
               
               
                   
               
               
                 TGGTCAGTTTCCACATGATG 
                 140 
                 226 
                 245 
                 CATCATGTGGAAACTGACCA 
                 351 
               
               
                   
               
               
                 CTGGTCAGTTTCCACATGAT 
                 141 
                 227 
                 246 
                 ATCATGTGGAAACTGACCAG 
                 352 
               
               
                   
               
               
                 CCTGGTCAGTTTCCACATGA 
                 142 
                 228 
                 247 
                 TCATGTGGAAACTGACCAGG 
                 353 
               
               
                   
               
               
                 CCCTGGTCAGTTTCCACATG 
                 143 
                 229 
                 248 
                 CATGTGGAAACTGACCAGGG 
                 354 
               
               
                   
               
               
                 TCCCTGGTCAGTTTCCACAT 
                 144 
                 230 
                 249 
                 ATGTGGAAACTGACCAGGGA 
                 355 
               
               
                   
               
               
                 ATCCCTGGTCAGTTTCCACA 
                 145 
                 231 
                 250 
                 TGTGGAAACTGACCAGGGAT 
                 356 
               
               
                   
               
               
                 CATCCCTGGTCAGTTTCCAC 
                 146 
                 232 
                 251 
                 GTGGAAACTGACCAGGGATG 
                 357 
               
               
                   
               
               
                 TCATCCCTGGTCAGTTTCCA 
                 147 
                 233 
                 252 
                 TGGAAACTGACCAGGGATGA 
                 358 
               
               
                   
               
               
                 CTCATCCCTGGTCAGTTTCC 
                 148 
                 234 
                 253 
                 GGAAACTGACCAGGGATGAG 
                 359 
               
               
                   
               
               
                 TCTCATCCCTGGTCAGTTTC 
                 149 
                 235 
                 254 
                 GAAACTGACCAGGGATGAGA 
                 360 
               
               
                   
               
               
                 GAGGCGCAGGGTTCCATCCC 
                 150 
                 354 
                 373 
                 GGGATGGAACCCTGCGCCTC 
                 361 
               
               
                   
               
               
                 AGAGGCGCAGGGTTCCATCC 
                 151 
                 355 
                 374 
                 GGATGGAACCCTGCGCCTCT 
                 362 
               
               
                   
               
               
                 CAGAGGCGCAGGGTTCCATC 
                 152 
                 356 
                 375 
                 GATGGAACCCTGCGCCTCTG 
                 363 
               
               
                   
               
               
                 CCAGAGGCGCAGGGTTCCAT 
                 153 
                 357 
                 376 
                 ATGGAACCCTGCGCCTCTGG 
                 364 
               
               
                   
               
               
                 CCGTTGTGAGATCCCAGAGG 
                 154 
                 370 
                 389 
                 CCTCTGGGATCTCACAACGG 
                 365 
               
               
                   
               
               
                 CCCGTTGTGAGATCCCAGAG 
                 155 
                 371 
                 390 
                 CTCTGGGATCTCACAACGGG 
                 366 
               
               
                   
               
               
                 GCCCGTTGTGAGATCCCAGA 
                 156 
                 372 
                 391 
                 TCTGGGATCTCACAACGGGC 
                 367 
               
               
                   
               
               
                 TGCCCGTTGTGAGATCCCAG 
                 157 
                 373 
                 392 
                 CTGGGATCTCACAACGGGCA 
                 368 
               
               
                   
               
               
                 GTGCCCGTTGTGAGATCCCA 
                 158 
                 374 
                 393 
                 TGGGATCTCACAACGGGCAC 
                 369 
               
               
                   
               
               
                 GGTGCCCGTTGTGAGATCCC 
                 159 
                 375 
                 394 
                 GGGATCTCACAACGGGCACC 
                 370 
               
               
                   
               
               
                 TGGTGCCCGTTGTGAGATCC 
                 160 
                 376 
                 395 
                 GGATCTCACAACGGGCACCA 
                 371 
               
               
                   
               
               
                 GTGGTGCCCGTTGTGAGATC 
                 161 
                 377 
                 396 
                 GATCTCACAACGGGCACCAC 
                 372 
               
               
                   
               
               
                 GGTGGTGCCCGTTGTGAGAT 
                 162 
                 378 
                 397 
                 ATCTCACAACGGGCACCACC 
                 373 
               
               
                   
               
               
                 TGGTGGTGCCCGTTGTGAGA 
                 163 
                 379 
                 398 
                 TCTCACAACGGGCACCACCA 
                 374 
               
               
                   
               
               
                 GTGGTGGTGCCCGTTGTGAG 
                 164 
                 380 
                 399 
                 CTCACAACGGGCACCACCAC 
                 375 
               
               
                   
               
               
                 CGTGGTGGTGCCCGTTGTGA 
                 165 
                 381 
                 400 
                 TCACAACGGGCACCACCACG 
                 376 
               
               
                   
               
               
                 TCGTGGTGGTGCCCGTTGTG 
                 166 
                 382 
                 401 
                 CACAACGGGCACCACCACGA 
                 377 
               
               
                   
               
               
                 CTCGTGGTGGTGCCCGTTGT 
                 167 
                 383 
                 402 
                 ACAACGGGCACCACCACGAG 
                 378 
               
               
                   
               
               
                 CCTCGTGGTGGTGCCCGTTG 
                 168 
                 384 
                 403 
                 CAACGGGCACCACCACGAGG 
                 379 
               
               
                   
               
               
                 AGAAGGCCACACTCAGCACA 
                 169 
                 427 
                 446 
                 TGTGCTGAGTGTGGCCTTCT 
                 380 
               
               
                   
               
               
                 GAGAAGGCCACACTCAGCAC 
                 170 
                 428 
                 447 
                 GTGCTGAGTGTGGCCTTCTC 
                 381 
               
               
                   
               
               
                 GGAGAAGGCCACACTCAGCA 
                 171 
                 429 
                 448 
                 TGCTGAGTGTGGCCTTCTCC 
                 382 
               
               
                   
               
               
                 AGGAGAAGGCCACACTCAGC 
                 172 
                 430 
                 449 
                 GCTGAGTGTGGCCTTCTCCT 
                 383 
               
               
                   
               
               
                 GAGGAGAAGGCCACACTCAG 
                 173 
                 431 
                 450 
                 CTGAGTGTGGCCTTCTCCTC 
                 384 
               
               
                   
               
               
                 AGAGGAGAAGGCCACACTCA 
                 174 
                 432 
                 451 
                 TGAGTGTGGCCTTCTCCTCT 
                 385 
               
               
                   
               
               
                 CAGAGGAGAAGGCCACACTC 
                 175 
                 433 
                 452 
                 GAGTGTGGCCTTCTCCTCTG 
                 386 
               
               
                   
               
               
                 TCAGAGGAGAAGGCCACACT 
                 176 
                 434 
                 453 
                 AGTGTGGCCTTCTCCTCTGA 
                 387 
               
               
                   
               
               
                 GTCAGAGGAGAAGGCCACAC 
                 177 
                 435 
                 454 
                 GTGTGGCCTTCTCCTCTGAC 
                 388 
               
               
                   
               
               
                 TGTCAGAGGAGAAGGCCACA 
                 178 
                 436 
                 455 
                 TGTGGCCTTCTCCTCTGACA 
                 389 
               
               
                   
               
               
                 TTGTCAGAGGAGAAGGCCAC 
                 179 
                 437 
                 456 
                 GTGGCCTTCTCCTCTGACAA 
                 390 
               
               
                   
               
               
                 GTTGTCAGAGGAGAAGGCCA 
                 180 
                 438 
                 457 
                 TGGCCTTCTCCTCTGACAAC 
                 391 
               
               
                   
               
               
                 GGTTGTCAGAGGAGAAGGCC 
                 181 
                 439 
                 458 
                 GGCCTTCTCCTCTGACAACC 
                 392 
               
               
                   
               
               
                 CGGTTGTCAGAGGAGAAGGC 
                 182 
                 440 
                 459 
                 GCCTTCTCCTCTGACAACCG 
                 393 
               
               
                   
               
               
                 CCGGTTGTCAGAGGAGAAGG 
                 183 
                 441 
                 460 
                 CCTTCTCCTCTGACAACCGG 
                 394 
               
               
                   
               
               
                 TGCCGGTTGTCAGAGGAGAA 
                 184 
                 443 
                 462 
                 TTCTCCTCTGACAACCGGCA 
                 395 
               
               
                   
               
               
                 CTGCCGGTTGTCAGAGGAGA 
                 185 
                 444 
                 463 
                 TCTCCTCTGACAACCGGCAG 
                 396 
               
               
                   
               
               
                 TCTGCCGGTTGTCAGAGGAG 
                 186 
                 445 
                 464 
                 CTCCTCTGACAACCGGCAGA 
                 397 
               
               
                   
               
               
                 ATCTGCCGGTTGTCAGAGGA 
                 187 
                 446 
                 465 
                 TCCTCTGACAACCGGCAGAT 
                 398 
               
               
                   
               
               
                 AATCTGCCGGTTGTCAGAGG 
                 188 
                 447 
                 466 
                 CCTCTGACAACCGGCAGATT 
                 399 
               
               
                   
               
               
                 CAATCTGCCGGTTGTCAGAG 
                 189 
                 448 
                 467 
                 CTCTGACAACCGGCAGATTG 
                 400 
               
               
                   
               
               
                 ACAATCTGCCGGTTGTCAGA 
                 190 
                 449 
                 468 
                 TCTGACAACCGGCAGATTGT 
                 401 
               
               
                   
               
               
                 GACAATCTGCCGGTTGTCAG 
                 191 
                 450 
                 469 
                 CTGACAACCGGCAGATTGTC 
                 402 
               
               
                   
               
               
                 AGACAATCTGCCGGTTGTCA 
                 192 
                 451 
                 470 
                 TGACAACCGGCAGATTGTCT 
                 403 
               
               
                   
               
               
                 GAGACAATCTGCCGGTTGTC 
                 193 
                 452 
                 471 
                 GACAACCGGCAGATTGTCTC 
                 404 
               
               
                   
               
               
                 AGAGACAATCTGCCGGTTGT 
                 194 
                 453 
                 472 
                 ACAACCGGCAGATTGTCTCT 
                 405 
               
               
                   
               
               
                 CAGAGACAATCTGCCGGTTG 
                 195 
                 454 
                 473 
                 CAACCGGCAGATTGTCTCTG 
                 406 
               
               
                   
               
               
                 ATCCAGAGACAATCTGCCGG 
                 196 
                 457 
                 476 
                 CCGGCAGATTGTCTCTGGAT 
                 407 
               
               
                   
               
               
                 GATCCAGAGACAATCTGCCG 
                 197 
                 458 
                 477 
                 CGGCAGATTGTCTCTGGATC 
                 408 
               
               
                   
               
               
                 AGATCCAGAGACAATCTGCC 
                 198 
                 459 
                 478 
                 GGCAGATTGTCTCTGGATCT 
                 409 
               
               
                   
               
               
                 GAGATCCAGAGACAATCTGC 
                 199 
                 460 
                 479 
                 GCAGATTGTCTCTGGATCTC 
                 410 
               
               
                   
               
               
                 CGAGATCCAGAGACAATCTG 
                 200 
                 461 
                 480 
                 CAGATTGTCTCTGGATCTCG 
                 411 
               
               
                   
               
               
                 TCGAGATCCAGAGACAATCT 
                 201 
                 462 
                 481 
                 AGATTGTCTCTGGATCTCGA 
                 412 
               
               
                   
               
               
                 CTCGAGATCCAGAGACAATC 
                 202 
                 463 
                 482 
                 GATTGTCTCTGGATCTCGAG 
                 413 
               
               
                   
               
               
                 TCTCGAGATCCAGAGACAAT 
                 203 
                 464 
                 483 
                 ATTGTCTCTGGATCTCGAGA 
                 414 
               
               
                   
               
               
                 ATCTCGAGATCCAGAGACAA 
                 204 
                 465 
                 484 
                 TTGTCTCTGGATCTCGAGAT 
                 415 
               
               
                   
               
               
                 TATCTCGAGATCCAGAGACA 
                 205 
                 466 
                 485 
                 TGTCTCTGGATCTCGAGATA 
                 416 
               
               
                   
               
               
                 TTATCTCGAGATCCAGAGAC 
                 206 
                 467 
                 486 
                 GTCTCTGGATCTCGAGATAA 
                 417 
               
               
                   
               
               
                 TTTATCTCGAGATCCAGAGA 
                 207 
                 468 
                 487 
                 TCTCTGGATCTCGAGATAAA 
                 418 
               
               
                   
               
               
                 TTTTATCTCGAGATCCAGAG 
                 208 
                 469 
                 488 
                 CTCTGGATCTCGAGATAAAA 
                 419 
               
               
                   
               
               
                 GTTTTATCTCGAGATCCAGA 
                 209 
                 470 
                 489 
                 TCTGGATCTCGAGATAAAAC 
                 420 
               
               
                   
               
               
                 GGTTTTATCTCGAGATCCAG 
                 210 
                 471 
                 490 
                 CTGGATCTCGAGATAAAACC 
                 421 
               
               
                   
               
               
                 CTGCTGTTGGGCGAGAAGCG 
                 211 
                 569 
                 588 
                 CGCTTCTCGCCCAACAGCAG 
                 422 
               
               
                   
               
               
                 GCTGCTGTTGGGCGAGAAGC 
                 212 
                 570 
                 589 
                 GCTTCTCGCCCAACAGCAGC 
                 423 
               
               
                   
               
               
                 TGCTGCTGTTGGGCGAGAAG 
                 213 
                 571 
                 590 
                 CTTCTCGCCCAACAGCAGCA 
                 424 
               
               
                   
               
               
                 TTGCTGCTGTTGGGCGAGAA 
                 214 
                 572 
                 591 
                 TTCTCGCCCAACAGCAGCAA 
                 425 
               
               
                   
               
               
                 GTTGCTGCTGTTGGGCGAGA 
                 215 
                 573 
                 592 
                 TCTCGCCCAACAGCAGCAAC 
                 426 
               
               
                   
               
               
                 GGTTGCTGCTGTTGGGCGAG 
                 216 
                 574 
                 593 
                 CTCGCCCAACAGCAGCAACC 
                 427 
               
               
                   
               
               
                 GGGTTGCTGCTGTTGGGCGA 
                 217 
                 575 
                 594 
                 TCGCCCAACAGCAGCAACCC 
                 428 
               
               
                   
               
               
                 AGGGTTGCTGCTGTTGGGCG 
                 218 
                 576 
                 595 
                 CGCCCAACAGCAGCAACCCT 
                 429 
               
               
                   
               
               
                 TAGGGTTGCTGCTGTTGGGC 
                 219 
                 577 
                 596 
                 GCCCAACAGCAGCAACCCTA 
                 430 
               
               
                   
               
               
                 ATAGGGTTGCTGCTGTTGGG 
                 220 
                 578 
                 597 
                 CCCAACAGCAGCAACCCTAT 
                 431 
               
               
                   
               
               
                 GATAGGGTTGCTGCTGTTGG 
                 221 
                 579 
                 598 
                 CCAACAGCAGCAACCCTATC 
                 432 
               
               
                   
               
               
                 TGATAGGGTTGCTGCTGTTG 
                 222 
                 580 
                 599 
                 CAACAGCAGCAACCCTATCA 
                 433 
               
               
                   
               
               
                 ATGATAGGGTTGCTGCTGTT 
                 223 
                 581 
                 600 
                 AACAGCAGCAACCCTATCAT 
                 434 
               
               
                   
               
               
                 GATGATAGGGTTGCTGCTGT 
                 224 
                 582 
                 601 
                 ACAGCAGCAACCCTATCATC 
                 435 
               
               
                   
               
               
                 CGATGATAGGGTTGCTGCTG 
                 225 
                 583 
                 602 
                 CAGCAGCAACCCTATCATCG 
                 436 
               
               
                   
               
               
                 GACGATGATAGGGTTGCTGC 
                 226 
                 585 
                 604 
                 GCAGCAACCCTATCATCGTC 
                 437 
               
               
                   
               
               
                 AGACGATGATAGGGTTGCTG 
                 227 
                 586 
                 605 
                 CAGCAACCCTATCATCGTCT 
                 438 
               
               
                   
               
               
                 GAGACGATGATAGGGTTGCT 
                 228 
                 587 
                 606 
                 AGCAACCCTATCATCGTCTC 
                 439 
               
               
                   
               
               
                 GGAGACGATGATAGGGTTGC 
                 229 
                 588 
                 607 
                 GCAACCCTATCATCGTCTCC 
                 440 
               
               
                   
               
               
                 AGGAGACGATGATAGGGTTG 
                 230 
                 589 
                 608 
                 CAACCCTATCATCGTCTCCT 
                 441 
               
               
                   
               
               
                 CAGGAGACGATGATAGGGTT 
                 231 
                 590 
                 609 
                 AACCCTATCATCGTCTCCTG 
                 442 
               
               
                   
               
               
                 ACAGGAGACGATGATAGGGT 
                 232 
                 591 
                 610 
                 ACCCTATCATCGTCTCCTGT 
                 443 
               
               
                   
               
               
                 CACAGGAGACGATGATAGGG 
                 233 
                 592 
                 611 
                 CCCTATCATCGTCTCCTGTG 
                 444 
               
               
                   
               
               
                 CCACAGGAGACGATGATAGG 
                 234 
                 593 
                 612 
                 CCTATCATCGTCTCCTGTGG 
                 445 
               
               
                   
               
               
                 GCCACAGGAGACGATGATAG 
                 235 
                 594 
                 613 
                 CTATCATCGTCTCCTGTGGC 
                 446 
               
               
                   
               
               
                 AGCCACAGGAGACGATGATA 
                 236 
                 595 
                 614 
                 TATCATCGTCTCCTGTGGCT 
                 447 
               
               
                   
               
               
                 CAGCCACAGGAGACGATGAT 
                 237 
                 596 
                 615 
                 ATCATCGTCTCCTGTGGCTG 
                 448 
               
               
                   
               
               
                 CCAGCCACAGGAGACGATGA 
                 238 
                 597 
                 616 
                 TCATCGTCTCCTGTGGCTGG 
                 449 
               
               
                   
               
               
                 CCCAGCCACAGGAGACGATG 
                 239 
                 598 
                 617 
                 CATCGTCTCCTGTGGCTGGG 
                 450 
               
               
                   
               
               
                 TCCCAGCCACAGGAGACGAT 
                 240 
                 599 
                 618 
                 ATCGTCTCCTGTGGCTGGGA 
                 451 
               
               
                   
               
               
                 GACCAGCTTGTCCCAGCCAC 
                 241 
                 609 
                 628 
                 GTGGCTGGGACAAGCTGGTC 
                 452 
               
               
                   
               
               
                 TGACCAGCTTGTCCCAGCCA 
                 242 
                 610 
                 629 
                 TGGCTGGGACAAGCTGGTCA 
                 453 
               
               
                   
               
               
                 TTGACCAGCTTGTCCCAGCC 
                 243 
                 611 
                 630 
                 GGCTGGGACAAGCTGGTCAA 
                 454 
               
               
                   
               
               
                 CTTGACCAGCTTGTCCCAGC 
                 244 
                 612 
                 631 
                 GCTGGGACAAGCTGGTCAAG 
                 455 
               
               
                   
               
               
                 CCTTGACCAGCTTGTCCCAG 
                 245 
                 613 
                 632 
                 CTGGGACAAGCTGGTCAAGG 
                 456 
               
               
                   
               
               
                 ACCTTGACCAGCTTGTCCCA 
                 246 
                 614 
                 633 
                 TGGGACAAGCTGGTCAAGGT 
                 457 
               
               
                   
               
               
                 TACCTTGACCAGCTTGTCCC 
                 247 
                 615 
                 634 
                 GGGACAAGCTGGTCAAGGTA 
                 458 
               
               
                   
               
               
                 ATACCTTGACCAGCTTGTCC 
                 248 
                 616 
                 635 
                 GGACAAGCTGGTCAAGGTAT 
                 459 
               
               
                   
               
               
                 CATACCTTGACCAGCTTGTC 
                 249 
                 617 
                 636 
                 GACAAGCTGGTCAAGGTATG 
                 460 
               
               
                   
               
               
                 CCATACCTTGACCAGCTTGT 
                 250 
                 618 
                 637 
                 ACAAGCTGGTCAAGGTATGG 
                 461 
               
               
                   
               
               
                 TCCATACCTTGACCAGCTTG 
                 251 
                 619 
                 638 
                 CAAGCTGGTCAAGGTATGGA 
                 462 
               
               
                   
               
               
                 TTCCATACCTTGACCAGCTT 
                 252 
                 620 
                 639 
                 AAGCTGGTCAAGGTATGGAA 
                 463 
               
               
                   
               
               
                 GTTCCATACCTTGACCAGCT 
                 253 
                 621 
                 640 
                 AGCTGGTCAAGGTATGGAAC 
                 464 
               
               
                   
               
               
                 GGTTCCATACCTTGACCAGC 
                 254 
                 622 
                 641 
                 GCTGGTCAAGGTATGGAACC 
                 465 
               
               
                   
               
               
                 AGGTTCCATACCTTGACCAG 
                 255 
                 623 
                 642 
                 CTGGTCAAGGTATGGAACCT 
                 466 
               
               
                   
               
               
                 GCTTGCAGTTAGCCAGGTTC 
                 256 
                 637 
                 656 
                 GAACCTGGCTAACTGCAAGC 
                 467 
               
               
                   
               
               
                 AGCTTGCAGTTAGCCAGGTT 
                 257 
                 638 
                 657 
                 AACCTGGCTAACTGCAAGCT 
                 468 
               
               
                   
               
               
                 CAGCTTGCAGTTAGCCAGGT 
                 258 
                 639 
                 658 
                 ACCTGGCTAACTGCAAGCTG 
                 469 
               
               
                   
               
               
                 CCTGTGTGGCCAATGTGGTT 
                 259 
                 665 
                 684 
                 AACCACATTGGCCACACAGG 
                 470 
               
               
                   
               
               
                 GCCTGTGTGGCCAATGTGGT 
                 260 
                 666 
                 685 
                 ACCACATTGGCCACACAGGC 
                 471 
               
               
                   
               
               
                 AGCCTGTGTGGCCAATGTGG 
                 261 
                 667 
                 686 
                 CCACATTGGCCACACAGGCT 
                 472 
               
               
                   
               
               
                 TAGCCTGTGTGGCCAATGTG 
                 262 
                 668 
                 687 
                 CACATTGGCCACACAGGCTA 
                 473 
               
               
                   
               
               
                 ATAGCCTGTGTGGCCAATGT 
                 263 
                 669 
                 688 
                 ACATTGGCCACACAGGCTAT 
                 474 
               
               
                   
               
               
                 GATAGCCTGTGTGGCCAATG 
                 264 
                 670 
                 689 
                 CATTGGCCACACAGGCTATC 
                 475 
               
               
                   
               
               
                 AGATAGCCTGTGTGGCCAAT 
                 265 
                 671 
                 690 
                 ATTGGCCACACAGGCTATCT 
                 476 
               
               
                   
               
               
                 CAGATAGCCTGTGTGGCCAA 
                 266 
                 672 
                 691 
                 TTGGCCACACAGGCTATCTG 
                 477 
               
               
                   
               
               
                 TCAGATAGCCTGTGTGGCCA 
                 267 
                 673 
                 692 
                 TGGCCACACAGGCTATCTGA 
                 478 
               
               
                   
               
               
                 TTCAGATAGCCTGTGTGGCC 
                 268 
                 674 
                 693 
                 GGCCACACAGGCTATCTGAA 
                 479 
               
               
                   
               
               
                 GTTCAGATAGCCTGTGTGGC 
                 269 
                 675 
                 694 
                 GCCACACAGGCTATCTGAAC 
                 480 
               
               
                   
               
               
                 TGTTCAGATAGCCTGTGTGG 
                 270 
                 676 
                 695 
                 CCACACAGGCTATCTGAACA 
                 481 
               
               
                   
               
               
                 GTGTTCAGATAGCCTGTGTG 
                 271 
                 677 
                 696 
                 CACACAGGCTATCTGAACAC 
                 482 
               
               
                   
               
               
                 CGTGTTCAGATAGCCTGTGT 
                 272 
                 678 
                 697 
                 ACACAGGCTATCTGAACACG 
                 483 
               
               
                   
               
               
                 CCGTGTTCAGATAGCCTGTG 
                 273 
                 679 
                 698 
                 CACAGGCTATCTGAACACGG 
                 484 
               
               
                   
               
               
                 CACCGTGTTCAGATAGCCTG 
                 274 
                 681 
                 700 
                 CAGGCTATCTGAACACGGTG 
                 485 
               
               
                   
               
               
                 TCACCGTGTTCAGATAGCCT 
                 275 
                 682 
                 701 
                 AGGCTATCTGAACACGGTGA 
                 486 
               
               
                   
               
               
                 GTCACCGTGTTCAGATAGCC 
                 276 
                 683 
                 702 
                 GGCTATCTGAACACGGTGAC 
                 487 
               
               
                   
               
               
                 AGTCACCGTGTTCAGATAGC 
                 277 
                 684 
                 703 
                 GCTATCTGAACACGGTGACT 
                 488 
               
               
                   
               
               
                 CAGTCACCGTGTTCAGATAG 
                 278 
                 685 
                 704 
                 CTATCTGAACACGGTGACTG 
                 489 
               
               
                   
               
               
                 ACAGTCACCGTGTTCAGATA 
                 279 
                 686 
                 705 
                 TATCTGAACACGGTGACTGT 
                 490 
               
               
                   
               
               
                 GACAGTCACCGTGTTCAGAT 
                 280 
                 687 
                 706 
                 ATCTGAACACGGTGACTGTC 
                 491 
               
               
                   
               
               
                 AGACAGTCACCGTGTTCAGA 
                 281 
                 688 
                 707 
                 TCTGAACACGGTGACTGTCT 
                 492 
               
               
                   
               
               
                 GAGACAGTCACCGTGTTCAG 
                 282 
                 689 
                 708 
                 CTGAACACGGTGACTGTCTC 
                 493 
               
               
                   
               
               
                 AGAGACAGTCACCGTGTTCA 
                 283 
                 690 
                 709 
                 TGAACACGGTGACTGTCTCT 
                 494 
               
               
                   
               
               
                 GAGAGACAGTCACCGTGTTC 
                 284 
                 691 
                 710 
                 GAACACGGTGACTGTCTCTC 
                 495 
               
               
                   
               
               
                 GGAGAGACAGTCACCGTGTT 
                 285 
                 692 
                 711 
                 AACACGGTGACTGTCTCTCC 
                 496 
               
               
                   
               
               
                 TGGAGAGACAGTCACCGTGT 
                 286 
                 693 
                 712 
                 ACACGGTGACTGTCTCTCCA 
                 497 
               
               
                   
               
               
                 CTGGAGAGACAGTCACCGTG 
                 287 
                 694 
                 713 
                 CACGGTGACTGTCTCTCCAG 
                 498 
               
               
                   
               
               
                 TCTGGAGAGACAGTCACCGT 
                 288 
                 695 
                 714 
                 ACGGTGACTGTCTCTCCAGA 
                 499 
               
               
                   
               
            
           
         
       
     
     Example 5: In Vitro and In Vivo Study of Antisense Oligonucleotides 
     Cell Culture 
     ASOs #1 to #10 described in Example 4 were tested in human-derived wild-type HeLa cells. Cells were cultured in Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, GlutaMax™-1 (2 mM), penicillin (50 U/ml), and streptomycin (50 mg/ml) at 37° C. in 5% CO 2  One day before ASO treatment, naïve cells were seeded in 12-well plates (Corning™ Costar™ Flat Bottom Cell Culture Plates, ThermoFisher Scientific) at a density of 75,000 cells/well in 1 ml media and grown overnight to reach 20-30% confluency. 
     ASO Treatment 
     250 nM, 500 nM, or 1 μM of ASOs #1 to #10 described in Example 4 were introduced into the cells using Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher Scientific) at a ratio of 5 μl RNAiMAX per 1 μM ASO. Cells were incubated with fresh media containing ASO/RNAiMAX complexes for 72 hr until lysed. 
     Protein Extraction and Immunoblotting 
     Cells were washed twice with ice-cold PBS, lysed in 2% SDS, and sonicated at 25% amplitude for 10 sec. Lysates were clarified by centrifugation at 14,000 RPM for 10 min and protein concentrations were determined by BCA (ThermoFisher Scientific). 3-5 ug* of each sample was separated by on 4-12% NuPAGE Bis-Tris SDS-PAGE (ThermoFisher Scientific), transferred onto a PVDF membrane, followed by Western Blotting following standard procedure. The following primary antibodies were used for Western blotting: RACK1 (BD Biosciences, 1:1,000) and loading control α-tubulin (Protein Tech, 1:20,000). Band intensities were quantified using ImageJ. Results are shown in  FIG.  30    and  FIG.  31   . As can be seen, a decrease in RACK1 expression is seen in ASO-treated cells compared to untreated cells, in particular in cells treated with ASO #4, #9 or #10. ASO #4 was effective in decreasing RACK1 expression at low dose (0.25 μM), thus was not investigated at higher doses (0.5 μM or 1 μM). 
     In Vivo Study 
     ASOs #9 and #10 were selected for study. 200 uM of ASO in a 1.0 uL volume was unilaterally injected directly into the right striatum of 6 mice, 2 for each ASO, namely ASO #9 or ASO #10, or negative control ASO, with the left striatum of each mouse brain serving as an uninjected control. 7 days post-injection, the  striata  were micro-dissected and homogenized using a stand-up homogenizer in 200 ul of Radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris pH7.5; 150 mM NaCl; 1% Triton-X-100; 1% deoxycholic acid; 0.1% SDS; 1 mM EDTA) supplemented with a protease and phosphatase inhibitor cocktail (Thermo). Samples were centrifuged at 4 C for 5 min at 14,000 rpm, and the protein concentrations of the supernatant were estimated by BCA. 25 ug of each sample was separated on 4-12% NuPage SDS-PAGE. For Western Blotting analyses, the following antibodies were used: RACK1 (BD Biosciences, 1:1,000), a-Tubulin (loading control, ProteinTech, 1:50,000). ImageJ was used to quantify band intensity. 
     Injection of ASO #9 or ASO #10 in the right striatum resulted in less RACK1 compared to injection of control ASO as measured by western blot and normalized to tubulin expression. 
     While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. 
     All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Specifically, the sequences associated with each accession numbers provided herein including for example accession numbers and/or biomarker sequences (e.g. protein and/or nucleic acid) provided in the Tables or elsewhere, are incorporated by reference in its entirely. 
     The scope of the claims should not be limited by the preferred embodiments and examples, but should be given the broadest interpretation consistent with the description as a whole. 
     REFERENCES 
     
         
         1. UniProtKB—P63244 (RACK1_HUMAN) 
         2. Russo A et al. (2017), “Increased cytoplasmic TDP-43 reduces global protein synthesis by interacting with RACK1 on polyribosomes”. Hum Mol Genet. 26(8):1407-1418 
         3. U.S. Pat. No. 8,916,530 
         4. Adams D R, Ron D, and Kiely PA (2011) RACK1, A multifaceted scaffolding protein: Structure and function. Cell Commun Signal. 9: 22. Review 
         5. Mackenzie IRA and Rademakers, R., (2008) The role of TDP-43 in amyotrophic lateral sclerosis and frontotemporal dementia Curr Opin Neurol. 21:693-700. 
         6. Lagier-Tourenne C., Cleveland D. W. (2009) Rethinking A LS: the FUS about TDP-43. Cell, 136, 1001-1004. 
         7. Zhou, Zhuan, et al. “Human rhomboid family-1 suppresses oxygen-independent degradation of hypoxia-inducible factor-1a in breast cancer.” Cancer research 74.10 (2014): 2719-2730. 
         8. Kraus, Sarah, et al. “Receptor for activated C kinase 1 (RACK1) and Src regulate the tyrosine phosphorylation and function of the androgen receptor.” Cancer research 66.22 (2006): 11047-11054. 
         9. Cao, Junxia, et al. “RACK1 Promotes Self-Renewal and Chemoresistance of Cancer Stem Cells in Human Hepatocellular Carcinoma through Stabilizing Nanog.” Theranostics 9.3 (2019): 811. 
         10. Culver B P, Savas J N, Park S K, Choi J H, Zheng S, Zeitlin S O, Yates J R 3rd, Tanese N. Proteomic analysis of wild-type and mutant huntingtin-associated proteins in mouse brains identifies unique interactions and involvement in protein synthesis. J Biol Chem. 2012 June 22,287(26):21599-614. 
         11. Mackenzie I R A et al. (2011) Distinct pathological subtypes of FTLD-FUS  Acta Neurologica  121:207-218. 
         12. Ivone G. Bruno, Wei Jin, Gilbert J. Cote, Correction of aberrant FGFR1 alternative RNA splicing through targeting of intronic regulatory elements,  Human Molecular Genetics , Volume 13, Issue 20, 15 Oct. 2004, Pages 2409-2420. 
         Elden A C, Kim H-J, Hart M P, Chen-Plotkin A S, Johnson B S, Fang X, Armakola M, Geser F, Greene R, Lu M M, Padmanabhan A, Clay D, McCluskey L, Elman L, Juhr D, Gruber P J, Rub U, Auburger G, Trojanowski J Q, Lee V M-Y, Van Deerlin V M, Bonini N M, Gitler A D (2010). Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS.  Nature  466(7310): 1069-1075. doi:10.1038/nature09320. 
         Li Y, Raya P, Raoc E J, Shia C, Guoa W, Chen X, Woodruff E A III, Fushimia K, Wua J Y (2010). A  Drosophila  model for TDP-43 proteinopathy.  PNAS  107(7): 3169-3174 
         Perkins, L. A., Holderbaum, L., Tao, R., Hu, Y., Sopko, R., McCall, K., Yang-Zhou, D., Flockhart, I., Binari, R., Shim, H. S., Miller, A., Housden, A., Foos, M., Randkelv, S., Kelley, C., Namgyal, P., Villalta, C., Liu, L. P., Jiang, X., Huan-Huan, Q., Wang, X., Fujiyama, A., Toyoda, A., Ayers, K., Blum, A., Czech, B., Neumuller, R., Yan, D., Cavallaro, A., Hibbard, K., Hall, D., Cooley, L., Hannon, G. J., Lehmann, R., Parks, A., Mohr, S. E., Ueda, R., Kondo, S., Ni, J. Q., Perrimon, N. (2015). The Transgenic R NAi Project at Harvard Medical School: Resources and Validation. Genetics 201(3): 843-852. 
         Rodriguez A del V, Didiano D, Desplan C (2012). Power tools for gene expression and clonal analysis in  Drosophila. Nat Methods.  9(1):47-55. doi:10.1038/nmeth.1800.Power. 
         Pinarbasi, E. S., Cagatay, T., Fung, H. Y. J. et al. Active nuclear import and passive nuclear export are the primary determinants of TDP-43 localization.  Sci Rep  8, 7083 (2018).