Methods of treating aging-related diseases and disorders associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress

Abstract

Methods and compositions are provided for treating aging-related diseases and disorders associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress by administering zinc transporter protein 7 (ZIP7) or ZIP7 in combination with regulatory particle non-ATPase 11 (Rpn11). Methods of gene therapy are also provided for expressing ZIP7 and Rpn11 in vivo in effective amounts sufficient to treat an aging-related disease or disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress.

Description

METHODS OF TREATING AGING-RELATED DISEASES AND DISORDERS ASSOCIATED WITH PROTEIN MISFOLDING, PROTEIN AGGREGATION, OR ENDOPLASMIC RETICULUM STRESSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit under 35 U.S.C. § 119(e) of provisional application 63 / 656,540, filed June 5, 2024, and provisional application 63 / 608,534, filed December 11, 2023, which applications are hereby incorporated by reference in their entireties.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. R01AG36907 and Grant No. R01GM073164 awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] A Sequence Listing is provided herewith as a Sequence Listing XML file, “UCSB- 553WO” created on November 18, 2024, and having a size of 8,913 bytes. The contents of the Sequence Listing XML file are incorporated by reference herein in their entireties.INTRODUCTION

[0004] Normal development and healthy aging require robust protein quality control. During normal cellular life, up to 30% of protein molecules misfold and are targeted for degradation by the proteasome (Karagbz et al. (2019) Cold Spring Harb. Perspect. Biol. 11. 10.1101 / cshperspect.a033886; Schubert et al. (2000). Nature 404, 770-774. 10.1038 / 35008096). Some proteins are naturally more prone to misfolding than others and some cells experience greater protein folding challenges than others. Mutations can increase the susceptibility of proteins to misfolding, as can small protein aggregates that seed larger ones. Long-lived cells like stem cells and neurons, as well as secretory cells are especially dependent on proteasome activity for survival. Unfortunately, proteasome activity declines with age (Tonoki et al. (2009) Mol. Cell. Biol. 29, 1095-1106; Martinez, Aging Cell (2017) 16, 615-623).

[0005] Healthy cells degrade misfolded proteins. If the load of misfolded proteins increases, inducing endoplasmic reticulum (ER) stress, cells adapt by activating the unfolded protein response (UPR) (Hetz (2012). Nat. Rev. Mol. Cell Biol. 13, 89-102), expanding the ER,enhancing ER-associated degradation (ERAD), reducing the rate of new protein synthesis, and altering transcriptional programs. If the stress resolves, the cell recovers. Tf the stress persists and overwhelms the adaptive response, cells die. There is great interest in identifying mechanisms that enhance ERAD in anticipation that such approaches will prevent or reverse degenerative diseases and promote healthy aging (Njomen et al. (2019) J. Med. Chem. 62, 6469-6481; George et al. (2021) Biomolecules 11. 10.3390 / biomll 121789). Conversely, inhibitors of proteasomal degradation of misfolded proteins are in clinical use to treat cancers, especially malignancies of highly secretory cells, such as B cells.SUMMARY

[0006] Methods and compositions are provided for treating aging-related diseases and disorders associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress by administering zinc transporter protein 7 (ZIP7) or ZIP7 in combination with regulatory particle non-ATPase 11 (Rpnll). Methods of gene therapy are also provided for expressing ZIP7 and Rpnl l in vivo in effective amounts sufficient to treat an aging-related disease or disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress.

[0007] In one aspect, a method of treating an aging-related disease or a disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress of a subject is provided, the method comprising administering a therapeutically effective amount of ZIP7 to the subject.

[0008] In certain embodiments, the treatment increases survival, increases lifespan, or prevents, decreases, delays, or reverses senescence of the subject.

[0009] In certain embodiments, the treatment reduces protein misfolding, protein aggregation, or endoplasmic reticulum stress.

[0010] In certain embodiments, the aging-related disease is a retinopathy, wherein the treatment prevents, delays, decreases, or reverses retinal degeneration.

[0011] In certain embodiments, the method further comprises administering a therapeutically effective amount of Rpnl l in combination with the ZIP7 to the subject.

[0012] In certain embodiments, the ZIP7 and / or the Rpnl 1 are provided by a recombinant nucleic acid. In some embodiments, the recombinant nucleic acid is RNA or DNA. In some embodiments, the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the ZIP7 or Rpnl 1 in the subject.

[0013] In certain embodiments, the ZIP7 and / or the Rpnl l are provided by a vector system comprising one or more viral vectors or plasmids. Exemplary viral vectors include, but are not limited to, adeno-associated viral vectors, adenoviral vectors, lentiviral vectors, anelloviral vectors, and retroviral vectors.

[0014] In certain embodiments, the vector system is administered intravenously.

[0015] In certain embodiments, the vector system is administered locally to a target organ or tissue in need of treatment.

[0016] In certain embodiments, the expression of the ZIP7 and / or the Rpnl 1 is inducible.

[0017] In certain embodiments, the vector system comprises a first vector comprising a first coding sequence encoding ZIP7 and a second vector comprising a second coding sequence encoding Rpnl l, wherein the ZIP7 and the Rpnll are co-expressed in vivo in the subject in effective amounts sufficient to extend lifespan of the subject. In some embodiments, the first vector comprises a first promoter operably linked to the first coding sequence encoding the ZIP7. In some embodiments, the first promoter is a tissue-specific promoter or cell type-specific promoter. In some embodiments, the cell type-specific promoter is specific for a postmitotic cell type such as, but not limited to a neuron, a cardiomyocyte, a skeletal muscle myofiber, a retinal ganglion cell, a cochlear hair cell, an osteocyte, or an adipocyte.

[0018] In certain embodiments, the first coding sequence encoding the ZIP7 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated first coding sequence encoding the ZIP7 at the chromosomal locus.

[0019] In certain embodiments, the second vector comprises a second promoter operably linked to the second coding sequence encoding the Rpnl l. In some embodiments, the second promoter is a tissue-specific promoter or cell type-specific promoter. In some embodiments, the cell type-specific promoter is specific for a postmitotic cell type such as, but not limited to, a neuron, a cardiomyocyte, a skeletal muscle myofiber, a retinal ganglion cell, a cochlear hair cell, an osteocyte, or an adipocyte.

[0020] In certain embodiments, the second coding sequence encoding the Rpnl l is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated second coding sequence encoding the Rpnl l at the chromosomal locus.

[0021] In certain embodiments, the ZIP7 and the Rpnll are provided by a multicistronic vector comprising a first coding sequence encoding ZIP7 and a second coding sequence encoding Rpnll, wherein the ZIP7 and the Rpnl l are co-expressed in vivo in the subject in effective amounts sufficient to extend lifespan of the subject. In some embodiments, the multicistronicvector further comprises a viral T2A peptide or internal ribosome entry site (IRES) sequence operably linked to the first coding sequence encoding the ZIP7 and the second coding sequence encoding the Rpnl 1 to allow co-expression of the ZIP7 and the Rpnl 1.

[0022] In certain embodiments, the ZIP7 is provided by a viral particle comprising a vector comprising a coding sequence encoding the ZIP7.

[0023] In certain embodiments, the Rpnll is provided by a viral particle comprising a vector comprising a coding sequence encoding the Rpnll.

[0024] In certain embodiments, the viral particle is administered intravenously.

[0025] In certain embodiments, the viral particle is administered locally to a target organ or tissue in need of treatment.

[0026] In certain embodiments, the ZIP7 and / or the Rpnll are provided by genetically modifying the genome of the subject to express the ZIP7 and / or the Rpnl l. In some embodiments, the genome of the subject is genetically modified using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

[0027] In certain embodiments, the ZIP7 comprises an amino acid sequence having at least about 80-100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:2, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto.

[0028] In certain embodiments, the Rpnll comprises an amino acid sequence having at least about 80-100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto.

[0029] In certain embodiments, the subject is a mammal.

[0030] In certain embodiments, the subject is a human.

[0031] In another aspect, a composition for use in a method of treating an aging-related disease and extending lifespan of a subject or treating a disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress is provided, the composition comprising ZIP7 or a vector system encoding ZIP7. In certain embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0032] In another aspect, a method of increasing survival or extending lifespan of a cell is provided, organoid, or organism, the method comprising introducing into the cell, organoid, or organism an effective amount of ZIP7.

[0033] In certain embodiments, the method further comprises introducing into the cell, organoid, or organism an effective amount of Rpnl 1 .

[0034] In certain embodiments, the cell is a postmitotic cell such as, but not limited to, a neuron, a cardiomyocyte, a skeletal muscle myofiber, a retinal ganglion cell, a cochlear hair cell, an osteocyte, or an adipocyte.

[0035] In certain embodiments, the ZIP7 and / or the Rpnl 1 are provided by a recombinant nucleic acid. In some embodiments, the recombinant nucleic acid is RNA or DNA. In some embodiments, the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the ZIP7 or Rpnl 1 in the cell, organoid, or organism.

[0036] In certain embodiments, the ZIP7 and / or the Rpnl 1 are provided by a recombinant nucleic acid or vector system comprising one or more viral vectors or plasmids. Exemplary viral vectors include, but are not limited to, adeno-associated viral vectors, adenoviral vectors, lentiviral vectors, anelloviral vectors, and retroviral vectors. In some embodiments, the expression of the ZIP7 and / or the Rpnl 1 is inducible.

[0037] In certain embodiments, the vector system comprises a first vector comprising a first coding sequence encoding ZIP7 and a second vector comprising a second coding sequence encoding Rpnll, wherein the ZIP7 and the Rpnl l are co-expressed in the cell, organoid, or organism in effective amounts sufficient to extend lifespan of the cell, organoid, or organism. In some embodiments, the first vector comprises a first promoter operably linked to the first coding sequence encoding the ZIP7. In some embodiments, the first promoter is a tissue-specific promoter or cell type-specific promoter.

[0038] In certain embodiments, the first coding sequence encoding the ZIP7 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated first coding sequence encoding the ZIP7 at the chromosomal locus.

[0039] In certain embodiments, the second vector comprises a second promoter operably linked to the second coding sequence encoding the Rpnll. In some embodiments, the second promoter is a tissue-specific promoter or cell type-specific promoter.

[0040] In certain embodiments, the second coding sequence encoding the Rpnl l is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated second coding sequence encoding the Rpnl l at the chromosomal locus.

[0041] In certain embodiments, the ZIP7 and the Rpnll are provided by a multicistronic vector comprising a first coding sequence encoding ZIP7 and a second coding sequence encoding Rpnll, wherein the ZIP7 and the Rpnl l are co-expressed in the cell, organoid, or organism ineffective amounts sufficient to extend lifespan of the cell, organoid, or organism. In some embodiments, the multi cistronic vector further comprises a viral T2A peptide or internal ribosome entry site (IRES) sequence operably linked to the first coding sequence encoding the ZIP7 and the second coding sequence encoding the Rpnl 1 to allow co-expression of the ZIP7 and the Rpnll.

[0042] In certain embodiments, the ZIP7 is provided by a viral particle comprising a vector comprising a coding sequence encoding the ZIP7.

[0043] In certain embodiments, the Rpnll is provided by a viral particle comprising a vector comprising a coding sequence encoding the Rpnll.

[0044] In certain embodiments, the viral particle is administered intravenously.

[0045] In certain embodiments, the viral particle is administered locally to a target organ or tissue in need of treatment.

[0046] In certain embodiments, the ZIP7 and / or the Rpnll are provided by genetically modifying the genome of the cell, organoid, or organism to express the ZIP7 and / or the Rpnl l. In some embodiments, the genome of the cell, organoid, or organism is genetically modified using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

[0047] In certain embodiments, the ZIP7 is introduced into the cell, organoid, or organism, by a method comprising: introducing a donor polynucleotide into the cell, organoid, or organism, wherein the donor polynucleotide comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3’ genomic target sequence flanking a nucleotide sequence encoding the ZIP7; and introducing an RNA-guided nuclease into the cell, organoid, or organism; and introducing a guide RNA into the cell, organoid, or organism, wherein the guide RNA forms a complex with the RNA-guided nuclease such that the guide RNA directs the RNA-guided nuclease to a genomic target sequence at a genomic target locus to be modified, wherein the RNA-guided nuclease creates a double-stranded break in the genomic target sequence, wherein the donor polynucleotide is integrated at the genomic target locus recognized by its 5' homology arm and 3' homology arm by homology directed repair (HDR) such that a genetically modified cell, organoid, or organism is produced, wherein the survival or the lifespan of the genetically modified cell, organoid, or organism is increased compared to the unmodified cell, organoid, or organism.

[0048] In certain embodiments, the Rpnl l is introduced into the cell, organoid, or organism, by a method comprising: introducing a donor polynucleotide into the cell, organoid, ororganism, wherein the donor polynucleotide comprises a 5’ homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence encoding the Rpnll; introducing an RNA-guided nuclease into the cell, organoid, or organism; and introducing a guide RNA into the cell, organoid, or organism, wherein the guide RNA forms a complex with the RNA-guided nuclease such that the guide RNA directs the RNA-guided nuclease to a genomic target sequence at a genomic target locus to be modified, wherein the RNA-guided nuclease creates a double- stranded break in the genomic target sequence, wherein the donor polynucleotide is integrated at the genomic target locus recognized by its 5’ homology arm and 3' homology arm by homology directed repair (HDR) such that a genetically modified cell, organoid, or organism is produced, wherein the survival or the lifespan of the genetically modified cell, organoid, or organism is increased compared to the unmodified cell, organoid, or organism.

[0049] In certain embodiments, the donor polynucleotide, the RNA-guided nuclease, and the guide RNA are provided by one or more vectors. In some embodiments, the one or more vectors are viral vectors or plasmids. In some embodiments, the one or more vectors are introduced into the cell, organoid, or organism by transient transfection or stable transfection. In some embodiments, the expression of the RNA-guided nuclease or the guide RNA is inducible.

[0050] In certain embodiments, the RNA-guided nuclease is provided by a mRNA encoding the RNA-guided nuclease, wherein translation of the mRNA results in production of the RNA-guided nuclease in the cell, organoid, or organism.

[0051] In certain embodiments, the RNA-guided nuclease and the guide RNA are provided as a ribonucleoprotein complex of the RNA-guided nuclease with the guide RNA, wherein the ribonucleoprotein complex is introduced into the cell, organoid, or organism by microinjection.

[0052] In certain embodiments, the RNA-guided nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease. In some embodiments, the Cas nuclease is Cas9 or Cas 12a.

[0053] In certain embodiments, the organoid is a retinal organoid.

[0054] In certain embodiments, the retinal organoid is used as a model of retinal degeneration.

[0055] In certain embodiments, the retinal organoid comprises a mutation associated with an inherited retinopathy. In some embodiments, the effective amount of the ZIP7 or thecombination of the effective amount of the ZIP7 and the effective amount of the Rpnl 1 prevents, delays, decreases, or at least partially reverses the retinal degeneration in the retinal organoid.

[0056] In another aspect, a method of preventing, delaying, or reversing senescence of a postmitotic cell is provided, the method comprising introducing into the postmitotic cell an effective amount of ZIP7. In certain embodiments, the method further comprises introducing an effective amount of Rpnl 1 into the postmitotic cell. In some embodiments, the postmitotic cell is a neuron, a cardiomyocyte, a skeletal muscle myofiber, a retinal ganglion cell, a cochlear hair cell, an osteocyte, or an adipocyte.

[0057] In another aspect, a method of treating a retinopathy in a subject is provided, the method comprising administering a therapeutically effective amount of ZIP7 to the subject.

[0058] In certain embodiments, the method further comprises administering a therapeutically effective amount of Rpnl l in combination with the therapeutically effective amount of the ZIP7 to the subject.

[0059] In certain embodiments, the therapeutically effective amount of the ZIP7 or ZIP7 in combination with Rpnl 1 prevents, delays, decreases, or reverses retinal degeneration.

[0060] In certain embodiments, the ZIP7 and / or the Rpnl 1 are administered locally to the retina.

[0061] In another aspect, a method of increasing proteasomal degradation of poly- ubiquitinated proteins in a cell is provided, the method comprising introducing an effective amount of zinc transporter protein 7 (ZIP7) into the cell.

[0062] In certain embodiments, the method further comprises introducing an effective amount of regulatory particle non-ATPase 11 (Rpnll) into the cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0063] FIGS. 1A-1J. dZIP7 knockdown causes border cell migration defects and ER stress. (FIGS. 1A-1B) Developing Drosophila egg chambers expressing dZIP7:GFP. Border cells migrate during stage 9 (FIG. IB) and complete migration by stage 10 (FIG. 1A). (FIGS. 1C-1D) Stage 10 egg chambers with fruitlessGal4 driving expression of UAS-dZiP7RNAi and (C) UAS- GFPnls or (D) UAS-dZIP7::V5 in outer, migratory border cells. (FIGS. 1E-1F’) dZIP7 :GFP expression in control border cells (FIGS. IE, IE’) or c306Gal4>dZIP7RNAi (FIGS. IF, IF’). (FIG. 1G) Quantification of stage 10 migration defects in c306Gal4 and fruitlessGal4 driving the indicated transgenes. Each dot represents the average of >24 egg chambers (n=3 independent experiments). Error bars=SEM. (FIGS. 1H-1H”) A mosaic border cell cluster composed of somecontrol cells (RFP+, which can be dZIP7+ / +or dZIP7‘ / +, FIG. 1H' ) and some homozygous dZIP7 mutant cells (RFP-, outlined). Polar cells (p) express higher levels of RFP compared to outer border cells. Xbpl::EGFP (FIG. 1H”) is a marker for ER stress. ( FIGS. 11-11") Anti-POl antibody staining reveals that cells expressing dZIP7RNAi and GFPnls exhibit ER expansion. (FIGS. 1J-1J") Mosaic clone expressing dZIP7RNAi and dZIP7::V5 and RFP. Scale bars=20 pm.

[0064] FIGS. 2A-2S. dZIP7 enhances ERAD and promotes Rpn 11 -mediated deubiquitination of proteins required for proteasome entry. (FIG. 2A) Stage 10 egg chamber expressing RhlG69Dand the ER stress sensor Xbpl::EGFP in border cells using c306Gal4. RhlG69Daccumulation induced ER stress and blocked migration. (FIGS. 2B-2D) Co-expressing dZip7 rescued migration (FIGS. 2B-2C) and reduced Xbpl EGFP (FIGS. 2B-2D) and RhlG69D(BE). (FIG. 2C) Each dot represents one experiment (n>17 clusters). Error bars=SEM. (FIG. 2D) Each dot represents an individual border cell. Error bars=95% confidence intervals. (FIG. 2E) Each dot represents one experiment (n >17 clusters). Error bars=95% confidence intervals. (FIGS. 2F-2H) Stage 10 egg chambers expressing misfolded RhlG69Din border cells (insets) treated with 10 pM of the MG132 proteasome inhibitor for 5 hours and stained with an antibody against Rhl. dZip7 co-expression with RhlG69Ddid not reduce Rhl protein levels in the presence of MG132 showing that the dZIP7-mediated degradation of RhlG69Dis mediated by the proteasome and that dZIP7 functions upstream of the 20S core enzyme blocked by MG132. (FIG. 21) Schematic of proteasomal processing of misfolded proteins. (FIGS. 2J-2L') Representative images of stage 8 egg chambers stained with an antibody against ubiquitinated proteins (PUB, FIGS. 2B-2D, FIRE LUT in 8'-D'). (FIG. 2M) Quantification of fluorescence intensity of PUB staining in control (lacZ), dZIP7 overexpression and dZIP7RNAi expressing border cells. (FIGS. 2N-2O') dZip7 overexpression reduced ubiquitinated protein levels in egg chambers treated with MG132 (10 pM, 5 hours). Relative fluorescence intensity is quantified in (FIG. 2P). (FIGS. 2Q-2R') dZip7 overexpression does not prevent ubiquitinated protein buildup in egg chambers treated with Rpnl 1 inhibitor Capzimin (Czm, 20 pM, 5 hours); this effect is quantified in (FIG. 2S). Dots represent individual border cell clusters. Error bars=95% confidence intervals. *P < 0.05, **P < 0.01, *** P < 0.0001 Scale bars=20 pm.

[0065] FIGS. 3A-3R. Cytosolic Zn2+is limiting for ERAD. (FIG. 3A) Schematic of transmembrane domains and topology of dZIP7. Point mutations H183A and H187 A res within the second transmembrane domain while H315A and H344A are within the highly conserved HELP domain and CHEXPHEXGD motif on the fourth and fifth transmembrane domainsrequired for Zn2+transport. (FIGS. 3B-3E) Colocalization of V5-tagged, RNAi-resistant dZIP7 mutants with the ER marker PDI in border cells. (FIG. 3F) Quantification of incomplete migration at stage 10 in egg chambers expressing dZIP7RNAi with the indicated mutant forms. N=3 independent experiments. Error bars=SEM. (FIGS. 3G-3N’) Mosaic expression of dZIP7RNAi together with the indicated mutant forms of dZIP7 marked by RFPnls and stained for Notch or EGFR . Scale bars=20 pm. Quantification of the fold change of Notch (FIG. 30) and EGFR (FIG. 3P) expression in dZip7 mutants. (FIG. 3Q) Representative western blot on Hela cell protein extract probed for polyubiquitinated protein and GAPDH. Cells were treated with / without proteasome inhibitor MG132 and zinc pyrithione (ZnPyr), a zinc ionophore. Treatments from left to right: DMSO, 500 nM MG132, 500 nM MG132 + 1 pM ZnPyr, 500 nM ZnPyr. (FIG. 3R) Adding ZnPyr to MG132-treated cells reduces polyubiquitinated protein levels. Error bars=95% confidence intervals. **P < 0. 01, ****P < 0.0001.

[0066] FIGS. 4A-4H. dZip7 knockdown results in Notch & EGFR accumulation and reduced Notch transcriptional activity. (FIGS. 4A-4B') Intracellular Notch (FIGS. 4A, 4A') and EGFR (FIGS. 4B, 4B') accumulated in epithelial follicle cell clones expressing dZIP7RNAi (GFP+, green) relative to neighboring wild type cells. (FIGS. 4C-4D') Mosaic clones of follicle cells expressing RhlG69Dand GFP. RhlG69Dexpression does not cause accumulation of Notch (FIGS. 4C, 4C) or EGFR (FIGS. 4D, 4D') relative to wild type cells. (FIGS. 4E-4H) Notch transcriptional activity visualized with a Notch responsive element reporter (white). (FIGS. 4E- 4F) Notch in dZIP7RNAiexpressing cells (GFP+) compared to neighboring wild type (GFP-) cells. (FIGS. 4G-4H) Notch in RhlG69D-expressing cells (GFP+) compared to wild type cells (GFP-). **** P < 0.0001. Scale bars=20 pm.

[0067] FIGS. 5A-5N. dZIP7 overexpression prevents RhlG69Dretinal degeneration. (FIGS. 5A, 5C, 5E) Representative light photomicrographs of retinal morphology. (FIGS. 5B, 5D, 5F) DIC images of retinal imprint morphology. (FIG. 5G) Quantification of number of flies with rough eye. dZip7 overexpression reduces the proportion of flies with rough eye morphology. Each dot represents an average of >1 0 flies observed in each experiment. (FIGS. 5H-5K) Representative ERG recordings of one-week-old flies. (FIGS. 5L-5N) Quantification of ERG recordings. dZip7 co-expression with RhlG69Dreturns ERG amplitude to control levels. Error bars represent 95% confidence intervals. *P < 0.05, ** P < 0.01, **** P < 0.0001. We co-express UAS- dZIP7::V5 with UAS- RhlG69Din fly photoreceptor cells using GMR-Gal4. Eye morphology is normal in flies expressing dZIP7 alone (FIGS. 5A, SB and 5G) whereas RhlG69Dcauses severe disruption of eye morphology compared to controls17’23-2527and (FIGS. 5C, 5D, and 5G). Co-expression of RhlG69Dand dZIP7 fully rescue eye morphology in the majority of flies examined (FIGS. 5E, 5F and 5G) To test whether dZIP7 overexpression could restore visual function, we carry out electroretinogram (ERG) recordings, which measure the summed responses of all retinal cells to light. Control flies display a corneal negative receptor potential upon turning on a light stimulus, which quickly decays to baseline upon termination of the light (FIG. 5H). The large maintained component of the ERG results principally from activation of the phototransduction cascade. The on- and off-transient responses, which are nearly coincident with the initiation and cessation of the light stimulus (FIG. 5H), depend on synaptic transmission from the photoreceptor cells to postsynaptic cells in the optic lobes. Expression of RhlG69Din photoreceptor cells greatly diminishes the amplitude of the ERG, and eliminates the on- and off transients (FIGS. 51, 5L- 5N).

[0068] FIGS. 6A-6E. Model for ZIP7 function at molecular, cellular, and organismal scales (FIG. 6A) ZIP7 transports Zn2+from ER lumen to the cytosol where it is limiting for the activity of the Rpnll deubiquitinase in the lid of the proteasome. (FIG. 6B) When ZIP7 is knocked down or inhibited, misfolded proteins accumulate in the ER and cause ER stress and the UPR. (FIG. 6C) In the presence of ZIP7 misfolded proteins are degraded and ER stress is prevented. (FIG. 6D) In a fly model of autosomal dominant retinitis pigmentosa, the RhlG69Dmutant form of rhodopsin causes ER stress, photoreceptor cell death and blindness. (FIG. 6E) Overexpression of ZIP7 prevents ER stress, cell death, and blindness.

[0069] FIGS. 7A-7E. dZIP7 functions in migratory border cells Border cell position cluster Control BC dZip7 mutant BC (FIGS. 7A-7A”) An egg chamber with homozygous dZip7 mutant cells (GFP ). Both polar cells (p) and two border cells (b) are mutant. (FIGS. 7B-7B”) An egg chamber in which all outer border cells are GFP_ / ‘ (homozygous dZip7 mutant). (FIG. 7C) Migration distance expressed as a percentage of the migration path for mosaic border cell clusters as a function of the proportion of homozygous mutant cells in each cluster. (FIG. 7D) High magnification view showing the spatial distribution of dZip7+(GFP+) and dZip7' / _(GFP / _) cells in a migrating cluster. (FIG. 7E) Quantification of the percentage of dZip7+vs dZip7_ / ‘ border cells in the front, side, or back of the border cell cluster showing that dZip7 / _cells are more likely to occupy a rear position, “p” indicated polar cells, “b” indicated border cells, green labels control cells, yellow labels mutant cells. ** P<0.01, *** P<0.001, **** P<0.0001. Scale bars=20 pm.

[0070] FIGS. 8A-8T. dZIP7 localizes to the ER (FIGS. 8A, 8B) dZip7::GFP grayscale single channel corresponding to FIG. 1A. (FIGS. 8C-8G) High magnification of a border cell cluster showing the localization of overexpressed dZip7::V5 (yellow), anti-PDI staining for ER(green), phalloidin, and Hoechst. (FIGS. 8H-8J) 2-dimensional intensity histograms showing localization of dZip7::V5 relative to ER, F-actin, and DNA. The colocalization regression Pearson’s coefficient is displayed in the upper right comer. (FIG. 8K) Comparison of Pearson’s coefficients (average of 4 border cell clusters). ** P value < 0.01. (FIGS. 8L-8P) High magnification of a border cell cluster expressing dZip7::GFP under the control of endogenous regulatory sequences, ER (PDI, F-actin (phalloidin) and DNA (Hoechst). (FIGS. 8Q-8T) 2- dimensional intensity histograms showing colocalization and Pearson’s coefficient for dZip7::GFP relative to ER, F-actin, nuclei, as well as ER relative to F-actin. Scale bars=20 pm.

[0071] FIGS. 9A-9C. dZIP7 knockdown causes ER accumulation of Notch and EGFR but not E-cadherin. (FIGS. 9A-9A’) dZIP7RNAi-expressing clones (GFP+) accumulate intracellular Notch protein in border cells relative to neighboring wild type cells. (FIGS. 9B, 9B’) Accumulation of EGFR in dZIP7RNAi-expressing border cells. (FIGS. 9C, 9C’) c306Gal4>dZip7RNAi reduces dZip7::GFP expression but does not cause E-cadherin (magenta) intracellular accumulation. Scale bars=20 pm.

[0072] FIGS. 10A-10N. (FIGS. 10A-10C’) Representative images of stage 8 egg chambers stained with an antibody against ubiquitinated proteins (PUB). Scale bars, 10 pm. (FIG. 10D) Quantification of fluorescence intensity of ubiquitinated proteins. Expressing RhlG69Dcauses buildup of ubiquitinated proteins compared to the control, but this effect is suppressed by co-expressing dZip7. *P<0.05, ** P<0.01. Error bars represent the 95% confidence intervals. Dots represent individual border cell clusters. (FIGS. 10E-10N) Representative images of stage 9 border cell clusters stained against ubiquitinated proteins. (FIGS. 10E-10G’) Co-expressing dZip7 and RhlG69Dreduced buildup of poly ubiquitinated protein in border cells. (FIGS. 10H- 10 J’) dZip7 overexpression prevented buildup of ubiquitinated protein, while dZip7 knockdown increased ubiquitinated protein load. (FIGS. 10K-10L’) dZip7 overexpression also reduced ubiquitinated proteins in egg chambers treated with MG132. (FIGS. 10M-10N’) However, dZip7 overexpression does not prevent ubiquitinated protein buildup in egg chambers treated with the Rpnl 1 inhibitor Capzimin (Czm). Scale bars=20 pm.

[0073] FIGS. 11A-11G. (FIG. 11A) Histidine to alanine mutations predicted to disrupt Zn2+transport or not indicated on the sequence alignment between plant IRT1 (SEQ ID NO:7), human ZIP7 (SEQ ID NO:2), and Drosophila dZIP7 (Catsup, SEQ ID NO:1). (FIGS. 11B-11G) Images of border cell clusters with C306 Gal4 driving dZIP7 RNAi and Xbpl-GFP. (FIG. 11B) ZIP7 RNAi induces the ER stress sensor Xbpl. (FIG. 11C) Wild type ZIP7 rescues ER stress.(FIGS. 11D, HE) Mutations predicted to disrupt Zn2+transport block rescue. (FIGS. HF, 11G) Mutations predicted not to disrupt Zn2+transport do not block rescue. Scale bar= 20 pM.

[0074] FIG. 12. ZIP7 and Rpnl l extend lifespan. Ubiquitous overexpression of the Zn2+transporter dZIP7 extends lifespan in fruitflies. Furthermore, in combination with a protein called Rpnl 1, dZIP7 extends lifespan even more such that at 40 days (at 30°C) all the wild-type control flies were dead whereas nearly all of the dZIP7- and Rpnl l -overexpressing flies were alive. Moreover, our mechanistic studies of dZIP7 offer a plausible explanation for how this could be effective.

[0075] FIGS. 13A-13B. ZIP7 promotes deubiquitination of proteins destined for proteasomal degradation. (FIG. 13A) HeLa cells expressing dox-inducible ZIP7. MG132, a proteasome inhibitor, causes an increase in the abundance of poly-ubiquitinated (PUB) proteins. (FIG. 13B) Inducing ZIP7 overexpression with DOX reduces those levels (likely by stimulating the Rpnll Zn2+metalloproteinase in the proteasome lid). The effect is specific to the stressed cells.

[0076] FIGS. 14A-14C. Hypothesis: DOX-inducible ZTP7 will enhance degradation of misfolded RHOP23H by the proteasome. (FIG. 14A) Schematic of experimental design: HT-1080 cells constitutively expressing wildtype (WT) or mutated (P23H) Rhodopsin and doxycycline- inducible ZIP7. (FIG. 14B) HT-1080 cells with transgenic expression of Rhodopsin (WT) tagged with GFP. The protein reaches the plasma membrane. WT rhodopsin is produced in the ER, folds properly, and progresses to the plasma membrane in HT-1080 cells. (FIG. 14C) HT-1080 cells with transgenic expression of Rhodopsin (P23H) tagged with GFP. The protein misfolds and is stuck in the ER. Rhodopsin (P23H) misfolds in the ER and fails to reach the plasma membrane in HT-1080 cells.

[0077] FIGS. 15A-15C. ZIP7 overexpression decreased the abundance of misfolded Rhodopsin (P23H). (FIG. ISA) HT1080 expressing WT and P23H Rhodopsin were treated with DOX (1 ftg / ml) for 31 hours. (FIG. 15B) Normalized RHO-GFP to actin in HT-1080 cells expressing misfolded Rhodopsin (P23H) with 31 hours of Doxycycline (DOX-1 Jig / ml) treatment. After 31 hours of Doxycycline induction, there is a reduction of RHOP23H (quantified in (FIG. 15B) each color represents an independent experiment) consistent with our model that ZIP7 promotes proteasomal degradation. (FIG. 15C) HT1080-P23H-RHO-GFP cells were treated with DOX(1 gtg / ml) for 31 hours and 8 hrs of cycloheximide (CHX) treatment (CHX inhibits translation).DETAILED DESCRIPTION

[0078] Methods and compositions are provided for treating aging-related diseases and disorders associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress by administering zinc transporter protein 7 (ZIP7) or ZIP7 in combination with regulatory particle non-ATPase 11 (Rpnll). Methods of gene therapy are also provided for expressing ZIP7 and Rpnl l in vivo in effective amounts sufficient to treat an aging-related disease or disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress.

[0079] Before exemplary embodiments of the present invention are described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0080] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0081] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and exemplary methods and materials may now be described. Any and all publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0082] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of such proteins and reference to "theprotein" includes reference to one or more proteins and equivalents thereof, e.g., polypeptides and peptides, known to those skilled in the art, and so forth.

[0083] It is further noted that the claims may be drafted to exclude any element which may be optional. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only” and the like in connection with the recitation of claim elements, or the use of a “negative” limitation.

[0084] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. To the extent such publications may set out definitions of a term that conflicts with the explicit or implicit definition of the present disclosure, the definition of the present disclosure controls.

[0085] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.DEFINITIONS

[0086] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. The term “treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and / or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and / or symptom(s), i.e., arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and / or symptom(s). Those in need of treatment include those already inflicted as well as those in which prevention is desired.

[0087] A therapeutic treatment is one in which the subject is inflicted prior to administration and a prophylactic treatment is one in which the subject is not inflicted prior to administration. In some embodiments, the subject has an increased likelihood of becoming inflicted or is suspected of being inflicted prior to treatment. In some embodiments, the subject is suspected of having an increased likelihood of becoming inflicted.

[0088] The term “subject” as used herein refers to a patient in need of the treatments disclosed herein. The patient may be a mammal, such as, a rodent, a feline, a canine, a primate, or a human, e.g., a child, an adolescent, an adult, such as a young, middle-aged, or elderly human. The patient may have been diagnosed as having an aging-related disease or a disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress or may be suspected of suffering from an aging-related disease or a disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress.

[0089] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[0090] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0091] The term “aging-related disease” refers to a disease that occurs with increasing frequency in a population with individuals of increasing age. Examples of aging-related diseases include, without limitation, cardiovascular disease (e.g., atherosclerosis, heart attack, heart failure, coronary artery disease, and peripheral arterial disease), cerebrovascular diseases (e.g., ischemic stroke and hemorrhagic stroke), cancer, benign prostatic hyperplasia, hypertension, dementia (e.g., Alzheimer's disease, vascular dementia, and Lewy body dementia), frontotemporal disorders, Huntington’s disease, Parkinson's disease, chronic obstructive pulmonary disease, age- related macular degeneration, cataracts, age related endocrine disorders (e.g., type 2 diabetesmellitus, thyroid disorders, adrenal insufficiency, and parathyroid disorders), osteoporosis, osteoarthritis, abdominal obesity, hyperglycemia, dyslipidemia, and metabolic syndrome.

[0092] The term “disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress” includes any disease or condition associated with pathological accumulation of misfolded proteins or protein aggregates, extracellular or cytoplasmic deposits of a fibrillary protein, or endoplasmic reticulum stress that causes organ damage or interferes with organ function. Disorders associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress include, but are not limited to, amyloidosis disorders, including transthyretin variant amyloidosis (ATTRv), transthyretin wild type amyloidosis (ATTRwt), immunoglobulin light chain amyloidosis (AL), immunoglobulin heavy chain amyloidosis (AH), leukocyte chemotactic factor 2 amyloidosis (ALECT2), fibrinogen alpha amyloidosis (AFib), apolipoprotein amyloidosis (various variants, including Al, All, CII, CIII, AIV), lysozyme amyloidosis (ALys), gelsolin amyloidosis (AGel), beta 2m macroglobulin variant amyloidosis (Beta-2m v), beta 2m macroglobulin wild type amyloidosis (Beta- 2m), and amyloidosis involving viral proteins; amyloid beta aggregation-associated diseases such as Alzheimer’s disease, cerebral amyloid angiopathy, and Down syndrome; tauopathies such as Alzheimer's disease, Pick disease, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, primary age-related tauopathy, frontotemporal dementia and parkinsonism linked to chromosome 17, vacuolar tauopathy, lytico-bodig disease, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, pantothenate kinase-associated neurodegeneration, and lipofuscinosis; and synucleinopathies, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA); polyQ aggregation-associated diseases, including Huntington's disease, dentatorubropallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA) also known as Kennedy's disease, and spinocerebellar ataxia (SCA) including SCA Type 1 (SCA1), SCA Type 2 (SCA2) , SCA Type 3 (SCA3) or Machado-Joseph disease, SCA Type 6 (SCA6), SCA Type 7 (SCA7), and SCA Type 17 (SCA17); prion diseases, including Creutzfeldt- Jakob disease (CJD), variant CJD, Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), and kuru; TAR DNA binding protein 43 aggregation diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, chronic traumatic encephalopathy (CTE), Lewy body disease (LBD), Huntington’s disease, argyrophilic grain disease (AGD), and hippocampal sclerosis; islet amyloid polypeptide aggregation diseases, including type 2 diabetes; diseases associated with repeat expansions in the C9orf72 gene, including ALS andfrontotemporal dementia (FTD); diseases associated with mutations in the genes coding for superoxide dismutase- 1 (SOD-1 ) and fused in sarcoma (FUS), including ALS; as well as diseases and conditions that are exacerbated by protein misfolding and / or protein aggregation and / or endoplasmic reticulum stress such as, but not limited to, diabetes, including type 1 and type 2 diabetes, and liver diseases, including non-alcoholic steatohepatitis (NASH), steatosis, nonalcoholic fatty liver disease, alcoholic liver disease, alpha- 1 antitrypsin (AAT) deficiency, fibrosis, cirrhosis, viral hepatitis, liver ischemia, drug toxicity, liver cancers such as hepatocellular carcinoma, intrahepatic cholangiocarcinoma, angiosarcoma, hemangiosarcoma, hepatoblastoma, and metastases to the liver.

[0093] The term “senescence” refers to biological aging or deterioration of physiological function with increasing age.

[0094] The term “organism senescence” refers to deterioration of physiological function, increased risk of aging-related diseases, and increased death rate with increasing age.

[0095] The term "cellular senescence" as used herein refers to cellular aging. Senescent cells may be identified by expression of one or markers of senescence including, but not limited to, senescence-associated P-galactosidase (SA-P-gal), p!6, p21, and p53, and / or a decrease in phosphorylated retinoblastoma protein (pRB). Cellular senescence may result in reduced ability of cells to proliferate or cell cycle arrest at the G1 phase. DNA damage, somatic mutations, oncogene activation, oxidative stress, oxidative damage to chromosome telomere regions or telomere dysfunction, mitochondrial dysfunction, exposure to chemotherapeutic drugs, or agedependent increases in molecular damage to cells may trigger senescence. Decline of immune system function with aging may result in accumulation of senescent cells. Senescent cells may include mitotic or postmitotic cells.

[0096] The term "survival" as used herein means the time from the start of treatment to the time of death.

[0097] By "therapeutically effective dose or amount" of ZIP7 and Rpnl 1 is intended an amount that, when administered separately or in combination, as described herein, brings about a positive therapeutic response with respect to treatment of an individual for an aging-related disease or a disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress, and / or an amount that increases survival, increases lifespan, and / or prevents, decreases, delays, or reverses senescence. Additionally, a therapeutically effective dose or amount may improve ERAD function, increase degradation of misfolded proteins, reduce aggregation of misfolded proteins, and / or reduce ER stress. In some embodiments, the ZIP7 and Rpnl l areprovided by a vector system, wherein the ZIP7 and the Rpnl l are expressed separately or coexpressed in vivo in a subject in effective amounts sufficient for treatment of an individual for an aging-related disease or a disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress or in effective amounts sufficient to increase survival, increase lifespan, or prevent, decrease, delay, or reverse senescence. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the type of condition and the severity of the condition being treated, the particular drug or drugs employed, mode of administration, and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

[0098] The terms "protein", "peptide", and "polypeptide" refer to any compound comprising naturally occurring or synthetic amino acid polymers or amino acid-like molecules including but not limited to compounds comprising amino and / or imino molecules. No particular size is implied by use of the terms "protein", "peptide", and "polypeptide", and these terms are used interchangeably. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic). Thus, synthetic oligopeptides, dimers, multimers (e.g., tandem repeats, linearly-linked peptides), cyclized, branched molecules and the like, are included within the definition. The terms also include molecules comprising one or more peptoids (e.g., N-substituted glycine residues) and other synthetic amino acids or peptides. (See, e.g., U.S. Patent Nos. 5,831,005; 5,877,278; and 5,977,301; Nguyen et al. (2000) Chem Biol. 7(7):463-473; and Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89(20):9367-9371 for descriptions of peptoids). Non-limiting lengths of peptides suitable for use in the present invention includes peptides of 3 to 5 residues in length, 6 to 10 residues in length (or any integer therebetween), 11 to 20 residues in length (or any integer therebetween), 21 to 75 residues in length (or any integer therebetween), 75 to 100 (or any integer therebetween), or polypeptides of greater than 100 residues in length. Typically, polypeptides useful in this invention can have a maximum length suitable for the intended application. Preferably, the polypeptide is between about 3 and 100 residues in length. Generally, one skilled in art can easily select the maximum length in view of the teachings herein. Further, peptides and polypeptides, as described herein, for example synthetic peptides, may include additional molecules such as labels or other chemical moieties.

[0099] Thus, references to polypeptides or peptides also include derivatives of the amino acid sequences of the invention including one or more non-naturally occurring amino acids. A first polypeptide or peptide is "derived from" a second polypeptide or peptide if it is (i) encoded by a first polynucleotide derived from a second polynucleotide encoding the second polypeptide or peptide, or (ii) displays sequence identity to the second polypeptide or peptide as described herein. Sequence (or percent) identity can be determined as described below. Preferably, derivatives exhibit at least about 50% percent identity, more preferably at least about 80%, and even more preferably between about 85% and 99% (or any value therebetween) to the sequence from which they were derived. Such derivatives can include postexpression modifications of the polypeptide or peptide, for example, glycosylation, acetylation, phosphorylation, and the like.

[0100] Amino acid derivatives can also include modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature), so long as the protein (or fragment thereof) maintains the desired activity (e.g., ability to increase survival, extend lifespan, prevent, decrease, delay, or reverse senescence, or ZIP7 or Rpnl l biological activity such as ability of ZIP7 to provide Zn2+to Rpnl l , Rpnl 1 deubiquitinase activity, ability to improve ERAD function, increase degradation of misfolded proteins, reduce aggregation of misfolded proteins, and / or reduce ER stress). These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the proteins or errors due to PCR amplification. Furthermore, modifications may be made that have one or more of the following effects: increasing ability to increase survival and extend lifespan, prevent, delay, decrease, or reverse senescence, increasing ZIP7 or Rpnll biological activity, or facilitating purification, delivery, or cell processing. Proteins or biologically active fragments thereof can be made recombinantly, synthetically, or in tissue culture.

[0101] The term “zinc transporter protein 7” or “ZIP7” as used herein encompasses all forms of ZIP7 and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain biological activity (e.g., ability to increase survival, extend lifespan, prevent, decrease, delay, or reverse senescence, ability ofZIP7 to provide Zn2+to Rpnl l, ability to improve ERAD function, increase degradation of misfolded proteins, reduce aggregation of misfolded proteins, and / or reduce ER stress).

[0102] A ZIP7 polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule need not be physically derived from an organism, but may be synthetically or recombinantly produced. A number of ZIP7 nucleic acid and protein sequences are known. A representative sequence of a ZIP7 protein fromDrosophila melanogaster is presented in SEQ ID NO: 1, and a representative sequence of a human ZIP7 protein is presented in SEQ ID NO:2. Additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NM_001077516, NM_006979, NM_001288777, NM_130931, XM_015449564,XM_015449563, XM_005553337, XM_005553336, XM_040983171, XM_040983170XM_040983169, XM_011976665, XM_011976664, XM_011976663, XM_017522703XM_017522702, XM_017522700, XM_017522701, XM_021185695, XM_021185696NM_001048100, XM_038682747, XM_038682746, XM_036766199, XM_036766198XM_036766197, AQY77122, AQY77121, NP_001070984, NP_008910, and NP_001275706; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used to produce a ZIP7 protein or recombinant polynucleotide comprising a coding sequence encoding a ZIP7 protein for use in the methods described herein.

[0103] The term “regulatory particle non-ATPase 11”, “26S proteasome non-ATPase regulatory subunit 14”, or “Rpnll” as used herein encompasses all forms of Rpnll and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain biological activity (e.g., ability to increase survival, extend lifespan, prevent, decrease, delay, or reverse senescence, deubiquitinase activity, ability to improve ERAD function, increase degradation of misfolded proteins, reduce aggregation of misfolded proteins, and / or reduce ER stress).

[0104] A Rpnll polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule need not be physically derived from an organism, but may be synthetically or recombinantly produced. A number of Rpnll nucleic acid and protein sequences are known. A representative sequence of a Rpnll protein from Drosophila melanogaster is presented in SEQ ID NO:3, and a representative sequence of a human Rpnll protein (also known as 26S proteasome non-ATPase regulatory subunit 14) is presented in SEQ ID NO:4. Additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NM_135061, NM_OO58O5, XM_038584653, XM_019022073, NM_001025689, NM_021526, XM_029470729, XM_025471067, XM_019022073, XM_054679078, XM_045033869, XM_006935316, XM_051816741, NP_608905, NP_005796, NP_067501, NP_001020860,NP_001025636, NP_001077042, XP_003378024, XP_033766068, and XP_051669958; all of which sequences (as entered hy the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used to produce a Rpnll protein or recombinant polynucleotide comprising a coding sequence encoding a Rpnl 1 protein for use in the methods described herein.

[0105] By "fragment" is intended a molecule consisting of only a part of the intact full- length sequence and structure. The fragment can include a C-terminal deletion an N- terminal deletion, and / or an internal deletion of the polypeptide. Active fragments of a particular protein or polypeptide will generally include at least about 5-14 contiguous amino acid residues of the full length molecule, but may include at least about 15-25 contiguous amino acid residues of the full length molecule, and can include at least about 20-50 or more contiguous amino acid residues of the full length molecule, or any integer between 5 amino acids and the full length sequence, provided that the fragment in question retains biological activity (e.g., ability to increase survival, increase lifespan, or prevent, decrease, or delay senescence, increase ZIP7 or Rpnl 1 biological activity, ability to enhance ERAD, increase degradation of misfolded proteins, suppress protein aggregation, and / or reduce ER stress).

[0106] "Substantially purified" generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, peptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample, a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0107] By " isolated" is meant, when referring to a protein, polypeptide, or peptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro molecules of the same type. The term "isolated" with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.

[0108] The term “derived from” is used herein to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means.

[0109] The terms “variant,” “analog” and “mutein” refer to biologically active derivatives of the reference molecule that retain desired activity, such as ability to increase survival, extend lifespan, prevent, decrease, delay, or reverse senescence, ZIP7 or Rpnl 1 biological activity, the ability to improve ERAD function, increase degradation of a misfolded protein, suppress pathological accumulation of misfolded proteins in a cell and / or protein aggregation, and / or reduce ER stress for use in increasing survival or extending lifespan, as described herein. In general, the terms “variant” and “analog” refer to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and / or deletions, relative to the native molecule, so long as the modifications do not destroy biological activity, and which are “substantially homologous” to the reference molecule as defined below. In general, the amino acid sequences of such analogs will have a high degree of sequence homology to the reference sequence, e.g., amino acid sequence homology of more than 50%, generally more than 60%-70%, even more particularly 80%-85% or more, such as at least 90%-95% or more, when the two sequences are aligned. Often, the analogs will include the same number of amino acids but will include substitutions, as explained herein. The term “mutein” further includes polypeptides having one or more amino acid-like molecules including but not limited to compounds comprising only amino and / or imino molecules, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic), cyclized, branched molecules and the like. The term also includes molecules comprising one or more N- substituted glycine residues (a “peptoid”) and other synthetic amino acids or peptides. (See, e.g., U.S. Patent Nos. 5,831,005; 5,877,278; and 5,977,301; Nguyen et al., Chem Biol. (2000) 7:463- 473; and Simon et al., Proc. Natl. Acad. Sci. USA (1992) 89:9367-9371 for descriptions of peptoids). Preferably, the analog or mutein has at least the same biological activity as the native molecule. Methods for making polypeptide analogs and muteins are known in the art and are described further below.

[0110] As explained above, analogs generally include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: (1) acidic —aspartate and glutamate; (2) basic - lysine, arginine, histidine; (3) non-polar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar - glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. For example, the polypeptide of interest may include up to about 5-10 conservative or nonconservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5-25, so long as the desired function of the molecule remains intact. One of skill in the art may readily determine regions of the molecule of interest that can tolerate change by reference to Hopp / Woods and Kyte-Doolittle plots, well known in the art.

[0111] By ‘ ‘derivative” is intended any suitable modification of the native polypeptide of interest, of a fragment of the native polypeptide, or of their respective analogs, such as glycosylation, phosphorylation, polymer conjugation (such as with polyethylene glycol), or other addition of foreign moieties, as long as the desired biological activity of the native polypeptide is retained. Methods for making polypeptide fragments, analogs, and derivatives are generally available in the art.

[0112] "Homology" refers to the percent identity between two polynucleotide or two polypeptide molecules. Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% 98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence.

[0113] In general, "identity" refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 3:353 358,National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman Advances in Appl. Math. 2:482489, 1981 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.

[0114] Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages, the Smith Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs are readily available.

[0115] Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single stranded specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra', DNA Cloning, supra', Nucleic Acid Hybridization, supra.

[0116] 'Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which itis associated in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.

[0117] The term "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction or f-mating are included. The exogenous polynucleotide may be maintained as a nonintegrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.

[0118] 'Recombinant host cells," "host cells," "cells", "cell lines," "cell cultures," and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.

[0119] A "coding sequence" or a sequence which "encodes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3’ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0120] Typical "control elements," include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences.

[0121] "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0122] Encoded by" refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence.

[0123] "Expression cassette" or "expression construct" refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest. An expression cassette generally includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the expression cassette described herein may be contained within a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include, one or more selectable markers, a signal which allows the plasmid construct to exist as single stranded DNA (e.g., a M13 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).

[0124] "Purified polynucleotide" refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about at least 90%, of the protein with which the polynucleotide is naturally associated. Techniques for purifying polynucleotides of interest are well-known in the art and include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0125] The term "transfection" is used to refer to the uptake of foreign DNA by a cell. A cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13: 197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material, and includes uptake of peptide- or antibody-linked DNAs.

[0126] A "vector" is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non- viral vectors, particulate carriers, and liposomes). Typically, "vector construct," "expression vector," and "gene transfer vector," mean any nucleic acid construct capable ofdirecting the expression of a nucleic acid of interest and which can transfer nucleic acid sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0127] Gene transfer" or "gene delivery" refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells. Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmid vectors, viral vectors, non-viral vectors, adenoviruses, lentiviruses, alphaviruses, pox viruses, and vaccinia viruses.

[0128] A polynucleotide "derived from" a designated sequence refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence. The derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.

[0129] A “CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.

[0130] The term "Cas9" as used herein encompasses type II clustered regularly interspaced short palindromic repeats (CRISPR) system Cas9 endonucleases from any species, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks).

[0131] A Cas9 endonuclease binds to and cleaves DNA at a site comprising a sequence complementary to its bound guide RNA (gRNA). For purposes of Cas9 targeting, a gRNA maycomprise a sequence "complementary" to a target sequence (e.g., in an exon or an intron of a gene), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.

[0132] The Cas 9 protein naturally contains DNA endonuclease activity that depends on association of the protein with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNAs). In some cases, the two molecules are covalently linked to form a single molecule (also called a single guide RNA (“sgRNA”)). Thus, the Cas9 associates with a DNA-targeting RNA (which term encompasses both the two-molecule guide RNA configuration and the single-molecule guide RNA configuration), which activates the Cas9 or Cas9-like protein and guides the protein to a target nucleic acid sequence. If the Cas9 protein retains its natural enzymatic function, it will cleave target DNA to create a double-strand break, which can lead to genome alteration (i.e., editing: deletion, insertion (when a donor polynucleotide is present), replacement, etc.), thereby altering gene expression.

[0133] The term “CRISPR agent” as used herein encompasses any agent (or nucleic acid encoding such an agent), comprising naturally occurring and / or synthetic sequences, that can be used in a Cas9-based system (e.g., a Cas9 or Cas9-like protein; any component of a DNA-targeting RNA, e.g., a crRNA-like RNA, a tracrRNA-like RNA, a single guide RNA, etc.; a donor polynucleotide; and the like).

[0134] A Cas9 polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule need not be physically derived from an organism, but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP_002989955, WP_038434062, WP_011528583); Campylobacter jejuni (WP_022552435, YP_002344900), Campylobacter coli (WP_060786116); Campylobacter fetus (WP_059434633); Corynebacterium ulcerans (NC_015683, NC_017317);Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis(WP_033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychroflexus torquisl (NC_018721); Streptococcus thermophilus (YP_820832), Streptococcus mutans (WP_061046374, WP_024786433); Listeria innocua (NP_472073); Listeria monocytogenes (WP_061665472); Legionella pneumophila (WP_062726656);Staphylococcus aureus (WP_001573634); Francisella lularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP_033919308); Lactobacillus rhamnosus (WP_048482595, WP_032965177); and Neisseria meningitidis (WP_061704949,YP_002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein, wherein the variant retains biological activity, such as Cas9 site- directed endonuclease activity. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol. 198(5):797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385- 397, and Chylinski et al. (2014) Nucleic Acids Res. 42(10):6091-6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

[0135] By "selectively binds" with reference to a guide RNA is meant that the guide RNA binds preferentially to a target sequence of interest or binds with greater affinity to the target sequence than to other genomic sequences. For example, a gRNA will bind to a substantially complementary sequence and not to unrelated sequences. A gRNA that selectively binds to a particular target DNA sequence will selectively direct binding of Cas9 to a substantially complementary sequence at the target site and not to unrelated sequences.

[0136] The term "donor polynucleotide" refers to a polynucleotide that provides a sequence of an intended edit to be integrated into the genome at a target locus by homology directed repair (HDR).

[0137] A "target site" or "target sequence" is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide. The target site may be in an exon or an intron or a specific allele.

[0138] By "homology arm" is meant a portion of a donor polynucleotide that is responsible for targeting the donor polynucleotide to the genomic sequence to be edited in a cell. The donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence comprising the intended edit to the genomic DNA. The homology arms are referred to herein as 5' and 3’ (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide. The 5' and 3' homology arms hybridize to regions within thetarget locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively. The nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR or recombineering at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3’ homology arms.

[0139] As used herein, the terms "complementary" or "complementarity" refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine. However, when a uracil is denoted in the context of the present invention, the ability to substitute a thymine is implied, unless otherwise stated. "Complementarity" may exist between two RNA strands, two DNA strands, or between a RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be "complementary" and able to form a duplex despite having less than perfect or less than 100% complementarity. Two sequences are "perfectly complementary" or "100% complementary" if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region. Two or more sequences are considered "perfectly complementary" or "100% complementary" even if either or both polynucleotides contain additional non-complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other. "Less than perfect" complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is a matter of ordinary skill in the art. For purposes of Cas9 targeting, a gRNA may comprise a sequence "complementary" to a target sequence (e.g., in an intron), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.

[0140] A “zinc-finger nuclease” or “ZFN” is an artificial DNA endonuclease generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be engineered totarget desired DNA sequences and this enables zinc-finger nucleases to cleave unique target sequences. When introduced into a cell, ZFNs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. For more information on the use of ZFNs, see, for example: Asuri et al., Mol Ther. 2012 February; 20(2):329-38; Bibikova et al. Science. 2003 May 2; 300(5620):764; Wood et al. Science. 2011 Jul. 15; 333(6040):307; Ochiai et al. Genes Cells. 2010 August; 15(8):875-85; Takasu et. al., Insect Biochem Mol Biol. 2010 October; 40(10) :759-65 ; Ekker et al, Zebrafish 2008 Summer; 5(2): 121-3; Young et al, Proc Natl Acad Sci USA. 2011 Apr. 26; 108(17):7052-7; Goldberg et al, Cell. 2010 Mar. 5; 140(5):678-91; Geurts et al, Science. 2009 Jul. 24; 325(5939):433; Flisikowska et al, PLoS One. 2011; 6(6):e21045. doi: 10.1371 / journal.pone.0021045. Epub 2011 Jun. 13; Hauschild et al, Proc Natl Acad Sci USA. 2011 Jul. 19; 108(29): 12013-7; and Yu et al, Cell Res. 2011 November; 21(11): 1638-40; all of which are herein incorporated by reference for their teachings related to ZFNs. The term “ZFN agent” encompasses a zinc finger nuclease and / or a polynucleotide comprising a nucleotide sequence encoding a zinc finger nuclease.

[0141] A “transcription activator-like effector nuclease” or “TALEN” is an artificial DNA endonuclease generated by fusing a TAL (Transcription activator-like) effector DNA binding domain to a DNA cleavage domain. TALENS can be engineered to bind practically any desired DNA sequence and when introduced into a cell, TALENs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. For more information on the use of TALENs, see, for example: Hockemeyer et al. Nat Biotechnol. 2011 Jul. 7; 29(8):731-4; Wood et al. Science. 2011 Jul. 15; 333(6040):307; Tesson et al. Nat Biotechnol. 2011 Aug. 5; 29(8):695- 6; and Huang et. al., Nat Biotechnol. 2011 Aug. 5; 29(8):699-700; all of which are herein incorporated by reference for their teachings related to TALENs. The term “TALEN agent” encompasses a TALEN and / or a polynucleotide comprising a nucleotide sequence encoding a TALEN.

[0142] "Administering" a nucleic acid, such as a vector encoding ZIP7 and / or Rpnll, a CRISPR system or vector encoding a CRISPR system, a guide RNA, a donor polynucleotide (e.g., for HDR), a vector encoding a ZFN, or a vector encoding a TALEN to a cell comprises transducing, transfecting, electroporating, translocating, fusing, phagocytosing, shooting or ballistic methods, etc., i.e., any means by which a nucleic acid can be transported across a cell membrane.

[0143] The term "stem cell" refers to a cell that retains the ability to renew itself through mitotic cell division and that can differentiate into a diverse range of specialized cell types.Mammalian stem cells can be divided into three broad categories: embryonic stem cells, which are derived from blastocysts, adult stem cells, which are found in adult tissues, and cord blood stem cells, which are found in the umbilical cord. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body by replenishing specialized cells. Totipotent stem cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. Multipotent stem cells can produce only cells of a closely related family of cells (e.g., hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). Unipotent cells can produce only one cell type, but have the property of self-renewal, which distinguishes them from non-stem cells. Induced pluripotent stem cells are a type of pluripotent stem cell derived from adult cells that have been reprogrammed into an embryonic-like pluripotent state. Induced pluripotent stem cells can be derived, for example, from adult somatic cells such as peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose derived stem cells, leukocytes, hematopoietic stem cells, bone marrow cells, or hepatocytes.

[0144] The term “spheroid” refers to a three-dimensional (3D) cluster of cells.

[0145] The term “organoid” refers to a 3D cluster of organ-specific cells of multiple subtypes that exhibit some organ-appropriate organization and physiology. Organoids are a subclass of spheroids.

[0146] As used herein, “reprogramming factors” refers to one or more, i.e., a cocktail, of biologically active factors that act on a cell to alter transcription, thereby reprogramming a cell to multipotency or to pluripotency. Reprogramming factors may be provided individually or as a single composition, that is, as a premixed composition, of reprogramming factors to the cells, e.g., somatic cells from an individual with a family history or genetic make-up of interest, such as a patient who has a neurological disorder or a neurodegenerative disease. The factors may be provided at the same molar ratio or at different molar ratios. The factors may be provided once or multiple times in the course of culturing the cells of the subject invention. In some embodiments the reprogramming factor is a transcription factor, including without limitation, Oct3 / 4; Sox2; Klf4; c-Myc; Nanog; and Lin-28.

[0147] The somatic cells may include, without limitation, peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose derived stem cells, leukocytes, hematopoietic stem cells, bone marrow cells, or hepatocytes, etc., which are contacted with reprogramming factors, as defined above, in a combination and quantity sufficient to reprogram the cell to pluripotency. Reprogramming factors may be provided to the somatic cells individually or as a single composition, that is, as a premixed composition, of reprogramming factors. In some embodiments the reprogramming factors are provided as a plurality of coding sequences on a vector.

[0148] Differentiation of IPSCs into retina- specific cell types such as bipolar cells, retina amacrine cells, horizontal cells, retinal ganglion cells, Muller glia, rod photoreceptor cells, and cone photoreceptor cells may be promoted by using lineage-determining transcription factors and various growth factors and other differentiation agents. For example, inhibitors of Wnt and bone morphogenetic protein (BMP) signaling in combination with insulin like growth factor 1 (IGF-1) promote differentiation of iPSCs into retinal neuronal cell types. BMP4 treatment promotes formation of neuroretinal epithelia, and IGF-1 promotes formation of three-dimensional (3D)- laminated retinal organoids when added to the media during the first 3 months of differentiation. Inhibition of Notch signaling with the gamma y-secretase inhibitor, DAPT ((2S)-N-[(3,5- Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine 1,1 -dimethylethyl ester), significantly increases the proportion of photoreceptor and retinal pigment epithelium (RPE). Rod- genesis factors such as retinoic acid (RA) and taurine increase the number of cells having photoreceptor markers. Use of 9-cis retinal, instead of all-trans RA improves rod photoreceptor differentiation in organoid cultures resulting in higher rhodopsin expression and more mature mitochondrial morphology. Neural induction media with heparin and chemically defined N2 supplement promote aggregation of iPSCs into embryoid bodies and neural retina differentiation. Fetal bovine serum (FBS) and the hedgehog agonist smoothened agonist (SAG) improve retinal differentiation for human stem cells into laminated retinas that express markers of all retinal cell types, including ganglion, amacrine, bipolar, horizontal, Muller, and photoreceptor cells. Thyroid hormone signaling regulation can be used to control the fate of cone subtypes in retinal organoids. Addition of triiodothyronine (T3) induces L and M opsins in nearly all cones. Non-adherent (3D) culturing protocols have been used to produce stratified neural retinas. Retinal ganglion cells typically appear 40 to 50 days after the start of differentiation. Ganglion cell development can be accelerated by encapsulating embryoid body-like aggregates in a 3D Matrigel drop instead of growing in suspension cultures. See, e.g., Example 3 and differentiation protocols described byAfanasyeva et al. (2021) Cellular and Molecular Life Sciences 78:6505-6532, Lamba et al. (2006) Proc Natl Acad Sci USA 103:12769-12774, Osakada et al. (2008) Nat Biotechnol 26:215-224, Nakano et al. (2012) Cell Stem Cell 10:771-785, Reichman et al. (2014) Proc Natl Acad Sci USA 111:8518-8523, Gonzalez-Cordero et al. (2017) Stem Cell Rep 9:820-837, Lowe et al. (2016) Stem Cell Reports 6:743-756, Haynes et al. (2007) Proc Natl Acad Sci 104:20380-20385, Huang et al. (2015) Dev Biol 402: 119-126, Mellough et al. (2015) Stem Cells 33:2416-2430, Chichagova et al. (2020) Stem Cells 38: 195-201, and Kaya et al. (2019) Mol Vis 25:663-678; herein incorporated by reference in their entireties. However, any suitable method of inducing differentiation of IPSCs into retina- specific cell types to produce organoids may be used.

[0149] The IPSC-derived retina-specific cell types may be harvested at an appropriate stage of development, which may be determined based on the expression of photoreceptor markers and phenotypic characteristics of the desired mature differentiated cell type. Cultures may be empirically tested by staining for the presence of the markers of interest, by morphological determination, etc. for example, photoreceptor markers include, without limitation, RHO, OPN1LW, 0PN1MW, OPN1 SW, PNA, lectin, R0M1 , RCVRN, RIBEYE, NRL, ARR3, PRPH2, SYNTAXIN3, NR2E3, and ARL13B. The mature IPSC-derived retina-specific cell types may be purified prior to assembly into organoids by positive selection for one or more markers expressed on mature retina-specific cell types. The cells are optionally enriched before or after the positive selection step by drug selection, panning, density gradient centrifugation, etc. In addition, a negative selection can be performed, where the selection is based on expression of one or more of the markers found on the somatic cells they are derived from (e.g., PBMCs, fibroblasts, epithelial cells, endothelial progenitor cells, leukocytes, hematopoietic stem cells, mesenchymal stem cells, bone marrow cells, hepatocytes), or retinal progenitor cells, and the like. Selection may utilize panning methods, magnetic particle selection, particle sorter selection, and the like.

[0150] The somatic cells, the IPSCs derived therefrom, or the mature IPSC-derived retinaspecific cell types may be genetically modified for a variety of purposes, e.g., to introduce a genetic mutation associated with a retinal degenerative disorder (retinopathy), inhibit expression of a gene, provide marker genes, etc. Vectors may be introduced that express an exogenous gene, reprogramming factors, CRISPR systems, antisense nucleic acids, or ribozymes. Various techniques known in the art may be used to introduce nucleic acids into the target cells, e.g., electroporation, calcium precipitated DNA, fusion, transfection, lipofection, infection and the like. The particular manner in which the DNA is introduced is not critical to the practice of the invention.

[0151] By " container" is meant a glass, plastic, or metal vessel that can provide an aseptic environment for culturing cells.

[0152] The term “postmitotic cell” refers to a non-dividing, mature, terminally differentiated cell that is no longer able to undergo mitosis. Examples of postmitotic cells include, without limitation, neurons, cardiomyocytes, skeletal muscle myofibers, retinal ganglion cells, cochlear hair cells, osteocytes, and adipocytes.

[0153] The term “retinal degeneration” or “retinopathy” refers to damage or deterioration of the retina, which leads to progressive death of retina cells. Retinal degeneration, may be caused by occlusion of an artery or vein, retinal detachment, tears, or holes, traumatic eye injuries, eye surgery, retrolental fibroplasia, or a disease such as, but not limited to, retinitis pigmentosa (e.g., autosomal dominant retinitis pigmentosa, autosomal recessive retinitis pigmentosa), age-related macular degeneration, lattice degeneration, diabetic retinopathy, proliferative vitreoretinopathy, uveitis, neovascular inflammatory vitreoretinopathy, glaucoma, Leber congenital amaurosis, cone dystrophy, Usher syndrome, Stargardt disease, retinoschisis, birdshot chorioretinopathy, or central serous retinopathy. Retinal degeneration may result in retinal ganglion cell loss, progressive loss of photoreceptors, degeneration of the retinal pigment epithelium (RPE), impaired vision, night blindness, retinal detachment, light sensitivity, tunnel vision, loss of peripheral vision, or total loss of vision.Recombinant Nucleic Acids and Vectors

[0154] Recombinant nucleic acids and vectors comprising coding sequences encoding ZIP7 and / or Rpnl l are provided. In certain embodiments, an RNA or DNA comprising a coding sequence encoding ZIP7 and / or Rpnl l is provided. In certain embodiments, a recombinant nucleic acid or a vector comprising a coding sequence encoding ZIP7 and / or a vector comprising a coding sequence encoding Rpnll is administered to a subject, wherein the ZIP7 and / or Rpnll is expressed in vivo in the subject in an effective amount sufficient to reduce protein misfolding, protein aggregation, or endoplasmic reticulum stress, increase survival or extend lifespan of the subject or to reverse, prevent, decrease, or delay senescence of a postmitotic cell. In certain embodiments, ZIP7 and Rpnl l are provided by a vector system, which is administered to a subject, wherein the ZIP7 and the Rpnll are co-expressed in vivo in the subject in effective amounts sufficient to reduce protein misfolding, protein aggregation, or endoplasmic reticulum stress, or increase survival or extend lifespan of the subject or to reverse, prevent, decrease, or delay senescence of a postmitotic cell. In some embodiments, the ZIP7 and Rpnl are provided bya vector system comprising a first vector comprising a first coding sequence encoding ZIP7 and a second vector comprising a second coding sequence encoding Rpnl 1 . Tn other embodiments, ZIP7 and the Rpnlf are co-expressed in vivo from a single vector.

[0155] Methods of introducing a nucleic acid (e.g., DNA or RNA) such as a recombinant nucleic acid comprising a coding sequence encoding ZIP7 and / or Rpnll or a recombinant expression vector comprising a coding sequence encoding ZIP7 and / or Rpnl 1 into a host cell are known in the art, and any convenient method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell. Suitable methods include e.g., nucleic acid delivery by encapsulation in lipid nanoparticles (LNPs), viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle- mediated nucleic acid delivery, and the like. Introducing a recombinant nucleic acid or expression vector into a cell or cells can occur in any culture media and under any culture conditions that promote the survival of the cells. Introducing the recombinant nucleic acid or expression vector into a target cell can be carried out in vivo, ex vivo or in vitro.

[0156] In some embodiments, a ZIP7 polypeptide-encoding nucleic acid or Rpnl l- polypeptide-encoding nucleic acid can be provided as RNA, such as a messenger RNA (mRNA), wherein translation of the mRNA results in production of the ZIP7 or Rpnl 1 polypeptide in the subject. The RNA can be provided by direct chemical synthesis or may be transcribed in vitro from a DNA (e.g., encoding the ZIP7 or Rpnl 1 polypeptide). Once synthesized, the RNA may be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., encapsulated in LNPs, microinjection, electroporation, transfection, etc.). Nucleic acids may be provided to the cells using well-developed transfection techniques; see, e.g., Angel and Yanik (2010) PLoS One 5(7): el 1756, and the commercially available TransMessenger® reagents from Qiagen, Stemfect™ RNA Transfection Kit from Stemgent, and TransIT®-mRNA Transfection Kit from Minis Bio LLC. See also Beumer et al. (2008) Proc. Natl. Acad. Sci. USA 105(50):1982L19826. In some embodiments, nucleic acids are introduced into cells by encapsulation in LNPs. For a description of LNP transfection techniques, see, e.g., Wang et al. (2023) J. Mater. Chem. B. 11 (23):5083-5093, Pozzi et al. (2023) ACS Pharmacol Transl Sci. 6(11): 1561-1573, and del Pozo-Rodriguez et al. (2011) Recent Pat Drug Deliv Formal . 5(3):214- 26. LNPs for nucleic acid delivery are commercially available, for example, from LipExoGenBiotech (Baltimore, MD), Avanti Polar Lipids, Inc. (Alabaster, AL), Lonza Biologies (Hayward, CA), and Exelead, Inc. (Indianapolis, IN).

[0157] A vector may be provided directly to a target host cell, for example, by contacting the host cell with the vector (e.g., a recombinant expression vector comprising a coding sequence encoding a ZIP7 or Rpnl 1 polypeptide) such that the vector is taken up by the cells. Methods of transfecting cells are well known in the art, and include, without limitation, electroporation, calcium chloride transfection, microinjection, and lipofection. For viral vector delivery, cells can be contacted with viral particles comprising viral expression vectors.

[0158] Nucleic acids encoding ZIP7 and / or Rpnll can be inserted into an expression vector to create an expression cassette capable of producing the ZIP7 and Rpnl 1 in a suitable host cell. The ability of constructs to produce the ZIP7 and Rpnll can be empirically determined. Expression cassettes typically include control elements operably linked to a coding sequence, which allow for the expression of a gene in vivo in the subject species. Depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector.

[0159] Promoters can be used to drive expression by an RNA polymerase (e.g., pol I, pol II, pol III). Suitable promoters can be derived from viruses (i.e., viral promoters) or an organism, including prokaryotic or eukaryotic organisms. Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), and human Hl promoter (Hl), and the like.

[0160] The promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active / “ON” state) or an inducible promoter (i.e., a promoter whose state, active / “ON” or inactive / “OFF” is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein). In some cases, a promoter is a spatially restricted promoter (e.g., tissue-specific promoter or cell type-specific promoter controlled by a transcriptional control element, enhancer, etc.). In some cases, a promoter is a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process).

[0161] Inducible promoters suitable for use include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically / biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)- responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid / retinoid / thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

[0162] In some cases, the promoter is a spatially restricted promoter (i.e., cell type-specific promoter, tissue-specific promoter, organ-specific, etc.) such that in a multi-cellular organism, the promoter is active (i.e., “ON”) in a subset of specific cells. Spatially restricted promoters may be regulated by enhancers, transcriptional control elements, control sequences, etc. Any convenient spatially restricted promoter may be used as long as the promoter is functional in the targeted host cell (e.g., eukaryotic cell). In some cases, the promoter is a tissue-specific promoter. In some cases, the promoter is a cell type-specific promoter. In some cases, the transcriptional control element (e.g., the promoter) is functional in a targeted cell type or targeted cell population. For example, in some cases, the transcriptional control element can be functional in a muscle cell (e.g., a cardiac muscle cell (cardiomyocyte), a skeletal muscle cell (skeletal myofiber), or a smooth muscle cell), a neuron, a retinal cell, a T cell, a B cell, a hematopoietic stem cell, a liver cell, a lung cell, or other targeted cell. In some cases, the transcriptional control element is functional in a postmitotic cell or non-dividing cell such as, but not limited to, a neuron, a cardiomyocyte, a skeletal muscle myofiber, a retinal ganglion cell, a cochlear hair cell, an osteocyte, or an adipocyte.

[0163] In some cases, the promoter is a reversible promoter. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from any of a variety of organisms. Modification of reversible promoters derived from a first organism for use in a second (different) organism is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g.,alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like. A suitable promoter can include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tetracycline-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, 1992, Proc. Natl. Acad. Sci. USA, 89:5547; Oligino et al., 1998, Gene Ther., 5:491-496; Wang et al., 1997, Gene Then, 4:432-441; Neering et al., 1996, Blood, 88:1147-55; and Rendahl et al., 1998, Nat. Biotechno]., 16:757-761 ).

[0164] For illustration purposes, examples of spatially restricted promoters include, but are not limited to, neuron- specific promoters, cardiomyocyte-specific promoters, skeletal musclespecific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, retinal ganglion cell-specific promoters, adipocyte-specific promoters, etc.

[0165] In some embodiments, the promoter is a neuron-specific promoter. Examples of neuron-specific promoters include, but are not limited to, a neuron-specific enolase (NSE) promoter (see, e.g., EMBL HSENO2, X51956; see also, e.g., U.S. Pat. No. 6,649,811, U.S. Pat. No. 5,387,742); an aromatic amino acid decarboxylase (AADC) promoter; a neurofilament promoter (see, e.g., GenBank HUMNFL, L04147); a synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-1 promoter (see, e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn et al. (2010) Nat. Med. 16:1161); a serotonin receptor promoter (see, e.g., GenBank S62283); a tyrosine hydroxylase promoter (TH) (see, e.g., Nucl. Acids. Res. 15:2363-2384 (1987) and Neuron 6:583-594 (1991)); a GnRH promoter (see, e.g., Radovick et al., Proc. Natl. Acad. Sci. USA 88:3402-3406 (1991)); an L7 promoter (see, e.g., Oberdick et al., Science 248:223-226 (1990)); a DNMT promoter (see, e.g., Bartge et al., Proc. Natl. Acad. Sci. USA 85:3648-3652 (1988)); an enkephalin promoter (see, e.g., Comb et al., EMBO J. 17:3793-3805 (1988)); a myelin basic protein (MBP) promoter; a CMV enhancer / platelet-derived growth factor-.beta. promoter (see, e.g., Liu et al. (2620) Gene Therapy 11:52-60); a motor neuron-specific gene Hb9 promoter(see, e.g., U.S. Pat. No. 7,632,679; and Lee et al. (2620) Development 131:3295-3306); an alpha subunit of Ca2+-calmodulin-dependent protein kinase TI (CaMKIT) promoter (see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93:13250), and a retinal ganglion cell Nefli promoter (see, e.g., Hanlon et al. (2017) Front Neurosci. 11:521). Other suitable promoters include elongation factor (EF) 1 and dopamine transporter (DAT) promoters, and the like.

[0166] In some embodiments, the promoter is a cardiomyocyte-specific promoter. Examples of cardiomyocyte-specific promoters include, but are not limited to, a cardiac musclespecific alpha myosin heavy chain (MHC) gene promoter (see, e.g., Gulick et al. (1991) J. Biol. Chem. 266:9180-9185, Aikawa et al. (2002) J. Biol. Chem. 277(21): 18979-18985). a ventriclespecific cardiac myosin light chain 2 (MLC-2v) promoter (see, e.g., Boecker et al. (2004) Mol. Imaging 3(2):69-75, Griscelli et al. (1997) C R Acad. Sci. Ill 320(2):103-12), a cardiac troponin T (cTNT) promoter (see, e.g., Ai et al. (2018) Cell Physiol. Biochem. 48(5):1894-1900), a troponin 2 (TNNT2) promoter (see, e.g., Fiedorowicz et al. (2020) Sci. Rep.10(1): 1895), an alpha cardiac actin (ACTC) promoter (see, e.g., Fiedorowicz et al., supra), and a cardiac ankyrin repeat protein gene (Carp / Ankrdl) promoter (see, e.g., Briegel et al. (2005) Development 132(14):3305- 16).

[0167] In some embodiments, the promoter is a skeletal muscle-specific promoter. Examples of skeletal muscle-specific promoters include, but are not limited to, a skeletal muscle a-actin promoter, creatine kinase promoter, desmin promoter, troponin promoter, myosin light chain promoter, myosin heavy chain promoter, dystrophin promoter, and Pitx3 promoter (see, e.g., (see, e.g., Skopenkova et al. (2021) Acta Naturae 13(1): 47-58, Coulon et al. (2007) J. Biol. Chem. 282(45):33192-33200, Sartorelli et al. (1993) Circ. Res. 72(5):925-931).

[0168] In some embodiments, cell subtype-specific expression of ZIP7 and / or Rpnll is achieved by using a recombination system, e.g., Cre-Lox recombination, Flp-FRT recombination, etc. Cell type-specific expression of genes using recombination has been described in, e.g., Fenno et al., Nat Methods, 2014 July; 11(7):763; Gompf et al., Front. Behav. Neurosci. 2015 Jul. 2;9: 152, and McCarthy et al. (2012) Skelet. Muscle. 2(1):8; which are herein incorporated by reference.

[0169] Typically, transcription termination and polyadenylation sequences will also be present, located 3' to the translation stop codon. Preferably, a sequence for optimization of initiation of translation, located 5' to the coding sequence, is also present. Examples of transcription terminator / polyadenylation signals include those derived from SV40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence.

[0170] Enhancer elements may also be used herein to increase expression levels of the mammalian constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMPO J. (1985) 4:761, the enhancer / promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and elements derived from human CMV, as described in Boshart et al., Cell (1985) 41:521, such as elements included in the CMV intron A sequence.

[0171] Additionally, 5'- UTR sequences can be placed adjacent to the coding sequence in order to enhance expression of the same. Such sequences may include UTRs comprising an internal ribosome entry site (IRES). Inclusion of an IRES permits the translation of one or more open reading frames from a vector. For example, ZIP7 and / or Rpnl 1 can be co-expressed from a multicistronic vector including an IRES element. The IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20: 102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques (1997) 22 150- 161. A multitude of IRES sequences are known and include sequences derived from a wide variety of viruses, such as from leader sequences of picornaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol. (1989) 63: 1651-1660), the polio leader sequence, the hepatitis A virus leader, the hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25): 15125-15130), an IRES element from the foot and mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), a giardiavirus IRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397), and the like. A variety of nonviral IRES sequences will also find use herein, including, but not limited to IRES sequences from yeast, as well as the human angiotensin II type 1 receptor IRES (Martin et al., Mol. Cell Endocrinol. (2003) 212:51-61), fibroblast growth factor IRESs (FGF-1 IRES and FGF-2 IRES, Martineau et al. (2004) Mol. Cell. Biol. 24(17):7622- 7635), vascular endothelial growth factor IRES (Baranick et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105(12):4733-4738, Stein et al. (1998) Mol. Cell. Biol. 18(6):3112-3119, Bert et al. (2006) RNA 12(6): 1074-1083), and insulin-like growth factor 2 IRES (Pedersen et al. (2002) Biochem. J. 363(Pt 1): 37-44). These elements are readily commercially available in plasmids sold, e.g., by Clontech (Mountain View, CA), Invivogen (San Diego, CA), Addgene (Cambridge, MA) and GeneCopoeia (Rockville, MD). See also IRESite: The database of experimentally verified IRES structures (iresite.org). An IRES sequence may be included in a vector, for example, to express multiple protein products in combination.

[0172] Alternatively, a polynucleotide encoding a viral T2A peptide can be used to allow production of multiple protein products (e.g., ZTP7 and Rpnl 1) from a single vector. 2A linker peptides are inserted between the coding sequences in the multicistronic construct. The 2A peptide, which is self-cleaving, allows co-expressed proteins from the multicistronic construct to be produced at equimolar levels. 2A peptides from various viruses may be used, including, but not limited to 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna vims, and porcine tescho virus- 1. See, e.g., Kim et al. (2011) PLoS One 6(4):el8556, Trichas et al. (2008) BMC Biol. 6:40, Provost et al. (2007) Genesis 45(10):625-629, Furler et al. (2001) Gene Ther. 8(11): 864-873; herein incorporated by reference in their entireties.

[0173] In certain embodiments, cells containing a construct encoding ZIP7 and / or Rpnl 1 are identified in vitro or in vivo by including a selection marker expression cassette in the construct. Selection markers confer an identifiable change to the cell permitting positive selection of cells having the construct. For example, fluorescent or bioluminescent markers (e.g., green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluoresecent protein, blue fluorescent protein, mCherry, mOrange, mPlum, Venus, YPet, phycoerythrin, or luciferase), cell surface markers, expression of a reporter gene (e.g., GFP, dsRed, GUS, lacZ, CAT), drug selection markers such as genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, or histidinol may be used to identify cells. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed. Any selectable marker may be used as long as it is capable of being expressed in the cell to allow identification of cells containing the construct. Further examples of selectable markers are well known to one of skill in the art.

[0174] In certain embodiments, the selection marker expression cassette encodes two or more selection markers. Selection markers may be used in combination, for example, a cell surface marker may be used with a fluorescent marker, or a drug resistance gene may be used with a suicide gene. In certain embodiments, the selection marker expression cassette is multicistronic to allow expression of multiple selection markers in combination. The multicistronic vector may include an IRES or viral 2A peptide to allow expression of more than one selection marker from a single vector.

[0175] In certain embodiments, a suicide marker is included as a negative selection marker to facilitate negative selection of cells. Suicide genes can be used to selectively kill cells by inducing apoptosis or converting a nontoxic drug to a toxic compound in genetically modified cells. Examples include suicide genes encoding thymidine kinases, cytosine deaminases,intracellular antibodies, telomerases, caspases, and DNases. In certain embodiments, a suicide gene is used in combination with one or more other selection markers, such as those described above for use in positive selection of cells. In addition, a suicide gene may be used in cells containing constructs expressing ZIP7 and / or Rpnl l, for example, to improve their safety by allowing their destruction at will. See, e.g., Jones et al. (2014) Front. Pharmocol. 5:254, Mitsui et al. (2017) Mol. Ther. Methods Clin. Dev. 5:51-58, Greco et al. (2015) Front. Pharmacol. 6:95; herein incorporated by reference.

[0176] Once complete, the constructs encoding ZIP7 and / or Rpnll can be administered to a subject using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466. Genes can be delivered either directly to a subject or, alternatively, delivered ex vivo, to cells derived from the subject and the cells reimplanted in the subject.

[0177] A number of viral based systems have been developed for gene transfer into mammalian cells. Suitable expression vectors include viral expression vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (AAV) (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63:38223828; Mendelson et al., Virol. (1988) 166:154165; and Flotte et al., PNAS (1993) 90:1061310617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:78127816, 1999); a retroviral vector (e.g., a lentivirus, ay-retrovirus such as murine leukemia virus and feline leukemia virus, an avian retrovirus such as spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. See also, e.g., Warnock et al. (2011) Methods Mol. Biol. 737:1-25; Walther et al. (2000) Drugs 60(2):249-271; and Lundstrom (2003) Trends Biotechnol. 21(3): 117- 122; herein incorporated by reference); and an anelloviral vector (Prince et al. (2024) biorxiv.org / content / 10.1101 / 2024.03.27.586964 vl).

[0178] For example, retroviruses provide a convenient platform for gene delivery systems.Selected sequences can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems have been described (U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109; and Ferry et al. (2011) Curr Pharm Des. 17(24):2516-2527). Lentiviruses are a class of retroviruses that are particularly useful for delivering polynucleotides to mammalian cells because they are able to infect both dividing and nondividing cells (see e.g., Lois et al (2002) Science 295:868-872; Durand et al. (2011) Viruses 3(2):132-159; herein incorporated by reference).

[0179] Commonly used retroviral vectors are “defective”, i.e., unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein (eco tropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells). The appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles. Methods of introducing subject vector expression vectors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art (see, e.g., Kafri et al. (2004) Methods Mol Biol. 246:367-390, herein incorporated by reference).

[0180] A number of adenovirus vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1:51- 58; Berkner, K. L. BioTechniques (1988) 6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476). Additionally, various adeno-associated virus (AAV) vector systems have been developed for gene delivery. AAV vectors can be readily constructed using techniques well knownin the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941 ; International Publication Nos. WO 92 / 01070 (published 23 January 1992) and WO 93 / 03769 (published 4 March 1993); Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in Microbiol, and Immunol. (1992) 158:97-129; Kotin, R. M. Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1: 165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

[0181] Another vector system useful for delivering nucleic acids encoding ZIP7 and / or Rpnl l is the enterically administered recombinant poxvirus vaccines described by Small, Jr., P. A., et al. (U.S. Pat. No. 5,676,950, issued Oct. 14, 1997, herein incorporated by reference).

[0182] Additional viral vectors which will find use for delivering the nucleic acid molecules encoding the ZIP7 and / or Rpnl 1 include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing the ZIP7 and / or Rpnl 1 can be constructed as follows. The DNA encoding the particular ZIP7 and Rpnl l coding sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the coding sequences of interest into the viral genome. The resulting TK- recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.

[0183] Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the genes. Recombinant avipox viruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with, respect to the production of vaccinia viruses. See, e.g., WO 91 / 12882; WO 89 / 03429; and WO 92 / 03545.

[0184] Molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.

[0185] Members of the Alphavirus genus, such as, but not limited to, vectors derived from the Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEE), will also find use as viral vectors for delivering the polynucleotides of the present invention. For a description of Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al. (1996) J. Virol. 70:508-519; and International Publication Nos. WO 95 / 07995, WO 96 / 17072; as well as Dubensky, Jr., T. W„ et al., U.S. Pat. No. 5,843,723, issued Dec. 1, 1998, and Dubensky, Jr., T. W., U.S. Patent No. 5,789,245, issued Aug. 4, 1998, both herein incorporated by reference. Particularly preferred are chimeric alphavirus vectors comprised of sequences derived from Sindbis virus and Venezuelan equine encephalitis virus. See, e.g., Perri et al. (2003) J. Virol. 77: 10394-10403 and International Publication Nos. WO 02 / 099035, WO 02 / 080982, WO 01 / 81609, and WO 00 / 61772; herein incorporated by reference in their entireties.

[0186] A vaccinia-based infection / transfection system can be conveniently used to provide for inducible, transient expression of the coding sequences of interest (for example, a ZIP7 expression cassette or an Rpn 11 expression cassette) in a host cell. In this system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

[0187] As an alternative approach to infection with vaccinia or avipox virus recombinants, or to the delivery of genes using other viral vectors, an amplification system can be used that will lead to high level expression following introduction into host cells. Specifically, a T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more template. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription of the desired gene. Because some T7 RNA polymerase is required to initiate the amplification, T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction. Thepolymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase. For a further discussion of T7 systems and their use for transforming cells, see, e.g., International Publication No. WO 94 / 26911; Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al., Biochem. Biophys. Res. Commun. (1994) 200:1201- 1206; Gao and Huang, Nuc. Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994) 22:2114-2120; and U.S. Pat. No. 5,135,855.

[0188] In addition, anelloviral vectors can be used to deliver genes. An anellovector, based on a virus of the Betatorquevirus genus, has been developed (see, e.g., Prince et al. (2024) (biorxiv.org / content / 10. 1101 / 2024.03.27.586964vl). The vector comprises a self-amplifying trans-complementation of a universal recombinant anellovector (SATURN) system, which relies on a self-replicating plasmid to provide viral proteins in trans that drive replication and capsiddependent packaging of vector genomes. The SATURN system uses Cre-lox-based recombination to generate single unit-sized circular genomes inside a MOLT-4 production cell line. Capsid protein-dependent particles that encapsidate single stranded DNA vector genomes can be produced using the SATURN system.

[0189] The synthetic expression cassette of interest can also be delivered without a viral vector. For example, the synthetic expression cassette can be packaged as DNA or RNA in liposomes prior to delivery to the subject or to cells derived therefrom. Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. The ratio of condensed DNA to lipid preparation can vary but will generally be around 1 : 1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991.) 1097:1-17; Straubinger et al., in Methods of Enzymology (1983), Vol. 101, pp. 512-527.

[0190] Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081); and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265:10189-10192), in functional form.

[0191] Cationic liposomes are readily available. For example, N[l-2,3- dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416). Other commercially available lipids include(DDAB / DOPE) and DOTAP / DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g., Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; PCT Publication No. WO 90 / 11092 for a description of the synthesis of DOTAP (l,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

[0192] Similarly, anionic and neutral liposomes are readily available, such as, from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

[0193] The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. See, e.g., Straubinger et al., in Methods of Immunology (1983), Vol. 101 , pp. 512-527; Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell (1979) 17:77); Deamer and Bangham, Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA (1979) 76:3348); Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA (1979) 76:145); Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA (1978) 75:145; and Schaefer-Ridder et al., Science (1982) 215:166.

[0194] The DNA and / or peptide(s) can also be delivered in cochleate lipid compositions similar to those described by Papahadjopoulos et al., Biochem. Biophys. Acta (1975) 394:483- 491. See, also, U.S. Pat. Nos. 4,663,161 and 4,871,488.

[0195] The expression cassette of interest may also be encapsulated, adsorbed to, or associated with, particulate carriers. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362- 368; McGee J. P., et al., J Microencapsul. 14(2): 197-210, 1997; O'Hagan D. T., et al., Vaccine 11(2): 149-54, 1993.

[0196] Furthermore, other particulate systems and polymers can be used for the in vivo or ex vivo delivery of the nucleic acid of interest. For example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules, areuseful for transferring a nucleic acid of interest. Similarly, DEAE dextran-mediated transfection, calcium phosphate precipitation or precipitation using other insoluble inorganic salts, such as strontium phosphate, aluminum silicates including bentonite and kaolin, chromic oxide, magnesium silicate, talc, and the like, will find use with the present methods. See, e.g., Feigner, P. L., Advanced Drug Delivery Reviews (1990) 5:163-187, for a review of delivery systems useful for gene transfer. Peptoids (Zuckerman, R. N., et al., U.S. Pat. No. 5,831,005, issued Nov. 3, 1998, herein incorporated by reference) may also be used for delivery of a construct of the present invention.

[0197] Additionally, biolistic delivery systems employing particulate carriers such as gold and tungsten, are especially useful for delivering synthetic expression cassettes encoding ZIP7 and / or Rpnl l. The particles are coated with the synthetic expression cassette(s) to be delivered and accelerated to high velocity, generally under a reduced atmosphere, using a gun powder discharge from a "gene gun." For a description of such techniques, and apparatuses useful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371,015; and 5,478,744. Also, needle-less injection systems can be used (Davis, H. L., et al, Vaccine 12:1503- 1509, 1994; Bioject, Inc., Portland, Oreg.).

[0198] Recombinant vectors carrying a synthetic expression cassette encoding ZIP7 and / or Rpnl l are formulated into compositions for delivery to a vertebrate subject. These compositions may either be prophylactic or therapeutic. The compositions will comprise a "therapeutically effective amount" of the nucleic acid of interest such that an amount of the ZIP7 and / or Rpnll protein (or a biologically active fragment thereof) can be produced in vivo in the individual to which it is administered that brings about a positive therapeutic response with respect to treatment of the individual for an aging-related disease or a disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress, and / or an amount that increases survival, increases lifespan, and / or prevents, decreases, delays, or reverses senescence. The exact amount necessary will vary depending on the subject being treated; the age and general condition of the subject to be treated; the degree of protection desired; the type of condition and severity of the condition being treated; the particular ZIP7 or Rpnl l protein produced and its mode of administration, among other factors. An appropriate effective amount can be readily determined by one of skill in the art. Thus, a "therapeutically effective amount" will fall in a relatively broad range that can be determined through routine trials.

[0199] The compositions will generally include one or more "pharmaceutically acceptable excipients or vehicles" such as water, saline, glycerol, polyethyleneglycol, hyaluronic acid,ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, surfactants and the like, may he present in such vehicles. Certain facilitators of nucleic acid uptake and / or expression can also be included in the compositions or coadministered.

[0200] Once formulated, the compositions can be administered directly to the subject (e.g., as described above) or, alternatively, delivered ex vivo, to cells derived from the subject, using methods such as those described above. For example, methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and can include, e.g., dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, lipofectamine and LT-1 mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

[0201] Direct delivery of synthetic expression cassette compositions in vivo will generally be accomplished with or without viral vectors, as described above, by injection using either a conventional syringe, needless devices such as Bioject™ or a gene gun, such as the Accell™ gene delivery system (PowderMed Ltd, Oxford, England).Genetically Modified Cells, Organoids, and Organisms

[0202] The genome of a cell, organoid, or organism may be genetically modified to express or increase expression of ZIP7 and / or Rpnl l. Various gene editing approaches can be used for this purpose, including, without limitation, the use of genome editing systems comprising clustered regularly interspaced short palindromic repeats (CRISPR)ZCRISPR-associated (Cas) nucleases, meganucleases, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). See, e.g., CRISPR Gene Editing: Methods and Protocols (edited by Luo, Humana, 2019), Genome Editing and Engineering: From TALENs, ZFNs and CRISPRs to Molecular Surgery (edited by Appasani and Church, Cambridge University Press, 2018); herein incorporated by reference in their entireties. These gene editing techniques involve creating a double-strand break (DSB) in the DNA at a target site of the intended gene edit. In some embodiments, the DSB is repaired by homology-directed repair (HDR) using a donor DNA template that is inserted into the genome at the target locus using homologous recombination to replace a portion of the genomic sequence with a modified sequence.

[0203] In some embodiments, the donor polynucleotide comprises a nucleotide sequence encoding ZIP7 or Rpnl 1, which is flanked by a pair of homology arms responsible for targeting the donor polynucleotide to a genomic locus (e.g., intron or exon) where the coding sequenceencoding the ZIP7 or Rpnl l is integrated into the genome. The donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence. The homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence encoding the ZIP7 or Rpnl l within the donor polynucleotide. The 5’ and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively.

[0204] The homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the target locus. For example, a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the nucleotide sequence encoding the Z1P7 or Rpnl 1 is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3’ homology arms.

[0205] In certain embodiments, the corresponding homologous nucleotide sequences in the genomic target sequence (i.e., the "5' target sequence" and "3' target sequence") flank a specific site for cleavage and / or a specific site for introducing the nucleotide sequence encoding the ZIP7 or Rpnll. The distance between the specific cleavage site and the homologous nucleotide sequences (e.g., each homology arm) can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides). In most cases, a smaller distance may give rise to a higher gene targeting rate. In a preferred embodiment, the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered.

[0206] A homology arm can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc. In some instances, the 5' and 3' homology arms are substantially equal in length to oneanother, e.g. one may be 30% shorter or less than the other homology arm, 20% shorter or less than the other homology arm, 10% shorter or less than the other homology arm, 5% shorter or less than the other homology arm, 2% shorter or less than the other homology arm, or only a few nucleotides less than the other homology arm. In other instances, the 5’ and 3' homology arms are substantially different in length from one another, e.g., one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm.

[0207] An RNA-guided nuclease can be targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by altering its guide RNA sequence. A target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site. For example, the gRNA can be designed with a sequence complementary to a sequence of the genomic target locus to target the nuclease-gRNA complex to a target site.

[0208] In certain embodiments, the RNA-guided nuclease used for genome modification is a clustered regularly interspersed short palindromic repeats (CRISPR) system Cas nuclease. Any RNA-guided Cas nuclease capable of catalyzing site-directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system type I, type II, or type III Cas nucleases. Examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof.

[0209] In certain embodiments, a type II CRISPR system Cas9 endonuclease is used. Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks) may be used to perform genome modification as described herein. The Cas9 need not be physically derived from an organism, but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP_002989955, WP_038434062, WP_011528583); Campylobacter jejuni (WP_022552435, YP_002344900),Campylobacter coli (WP_060786116); Campylobacter fetus (WP_059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP_033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychroflexus torquisl (NC_018721); Streptococcus thermophilus (YP_820832), Streptococcus mutans (WP_061046374, WP_024786433); Listeria innocua (NP_472073); Listeria monocytogenes (WP_061665472); Legionella pneumophila (WP_062726656); Staphylococcus aureus (WP_001573634); Francisella tularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP_033919308); Lactobacillus rhamnosus (WP_048482595, WP_032965177); and Neisseria meningitidis (WP_061704949, YP_002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol. 198(5):797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385-397, and Chylinski et al. (2014) Nucleic Acids Res. 42(10):6091-6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

[0210] The CRISPR-Cas system naturally occurs in bacteria and archaea where it plays a role in RNA-mediated adaptive immunity against foreign DNA. The bacterial type II CRISPR system uses the endonuclease, Cas9, which forms a complex with a guide RNA (gRNA) that specifically hybridizes to a complementary genomic target sequence, where the Cas9 endonuclease catalyzes cleavage to produce a double-stranded break. Targeting of Cas9 typically further relies on the presence of a 5' protospacer-adjacent motif (PAM) in the DNA at or near the gRNA-binding site.

[0211] The genomic target site will typically comprise a nucleotide sequence that is complementary to the gRNA, and may further comprise a protospacer adjacent motif (PAM). In certain embodiments, the target site comprises 20-30 base pairs in addition to a 3 base pair PAM. Typically, the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen. Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide. In certain embodiments, the intron sequence of the TCR gene targetedby a gRNA comprises a mutation that creates a PAM within the intron, wherein the PAM promotes binding of the Cas9-gRNA complex to the intron.

[0212] In certain embodiments, the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. The guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.

[0213] In another embodiment, the CRISPR nuclease from Prevotella and Francisella 1 (Cpfl) also referred to as CRISPR associated protein 12a (Casl2a) may be used. Casl2a is another class II CRISPR / Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Casl2a does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Casl2a for targeting than Cas9. Casl2a is capable of cleaving either DNA or RNA. The PAM sites recognized by Casl2a have the sequences 5'-YTN-3' (where "Y" is a pyrimidine and "N" is any nucleobase) or 5’-TTN-3', in contrast to the G-rich PAM site recognized by Cas9. Casl2a cleavage of DNA produces double-stranded breaks with sticky-ends having a 4 or 5 nucleotide overhang. For a discussion of Casl2a, see, e.g., Ledford et al. (2015) Nature. 526 (7571): 17-17 , Zetsche et al. (2015) Cell. 163 (3):759-771, Murovec et al. (2017) Plant Biotechnol. J. 15(8):917-926, Zhang et al. (2017) Front. Plant Sci. 8: 177, Fernandes et al. (2016) Postepy Biochem. 62(3):315-326; herein incorporated by reference.

[0214] C2clis another class II CRISPR / Cas system RNA-guided nuclease that may be used. C2cl, similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites. For a description of C2cl, see, e.g., Shmakov et al. (2015) Mol Cell. 60(3):385-397, Zhang et al. (2017) Front Plant Sci. 8:177; herein incorporated by reference.

[0215] In yet another embodiment, an engineered RNA-guided FokI nuclease may be used. RNA-guided FokI nucleases comprise fusions of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on FokI. For a description of engineered RNA-guided FokI nucleases, see, e.g., Havlicek et al. (2017) Mol. Ther. 25(2):342-355, Pan et al. (2016) Sci Rep. 6:35794, Tsai et al. (2014) Nat Biotechnol. 32(6):569-576; herein incorporated by reference.

[0216] The RNA-guided nuclease can be provided in the form of a protein, such as the nuclease complexed with a gRNA, or provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector such as a plasmid or viral vector). Codon usage may be optimized to improve production of an RNA-guided nuclease in a particular cell, organoid, or organism. For example, a nucleic acid encoding an RNA-guided nuclease can be modified to substitute codons having a higher frequency of usage in a human cell or a non-human mammalian cell, such as a non-human primate cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the gRNA and / or RNA-guided nuclease is introduced into cells, the gRNA and / or RNA-guided nuclease can be transiently, conditionally, or constitutively expressed in the cell. Recombinant nucleic acids encoding the gRNA, RNA-guided nuclease, and / or donor polynucleotide can be introduced into a cell using any suitable transfection technique such as, but not limited to electroporation, nucleofection, or lipofection. Alternatively, a ribonucleoprotein complex of the gRNA and the RNA-guided nuclease may be introduced into a cell by microinjection into the cytoplasm or nucleus.

[0217] In some embodiments, the CRISPR system is introduced into cells with a viral vector that encodes the RNA-guided nuclease and guide RNA (gRNA). Viral delivery of CRISPR components has been demonstrated using lentiviral, retroviral, adenovirus, and adeno-associated virus (AAV) vectors. For a description of methods of introducing a CRISPR system into cells with various viral vectors, see, e.g., Shalem et al. (2014) Science 343:84-87, Williams et al. (2016) Sci Rep. 6:25611, Ran et al. (2015) Nature 520: 186-191, Swiech et al. (2015) Nat Biotechnol. 33:102- 106; herein incorporated by reference.

[0218] Alternatively, a gRNA and a messenger RNA encoding the RNA-guided nuclease can be introduced into cells, wherein the RNA-guided nuclease is produced by translation of the mRNA in the cytoplasm. The gRNA and RNA-guided nuclease then form a complex in the cytoplasm and enter the nucleus. RNA transfection of cells can be performed using electroporation, cationic-lipid-mediated transfection, or using liposomes or lipid nanoparticles (LNPs) encapsulating the gRNA and mRNA. See, e.g., Billingsley et al. (2022) Nano Lett 22(l):533-542, Tchou et al. (2017) Cancer Immunol Res. 5(12): 1152- 1161 , Ye et al. (2022) ACS Biomater Sci Eng. 8(2):722-733, Guevara et al. (2020) Front. Chem. 8:589959; herein incorporated by reference.

[0219] Donor polynucleotides and gRNAs are readily synthesized by standard techniques, e.g., solid phase synthesis via phosphoramidite chemistry, as disclosed in U.S. Patent Nos.4,458,066 and 4,415,732, incorporated herein by reference; Beaucage et al., Tetrahedron (1992) 48:2223-231 1 ; and Applied Biosystems User Bulletin No. 13 (1 April 1987). Other chemical synthesis methods include, for example, the phosphotriester method described by Narang et al., Meth. Enzymol. (1979) 68:90 and the phosphodiester method disclosed by Brown et al., Meth. Enzymol. (1979) 68: 109. In view of the short lengths of gRNAs (typically about 20 nucleotides in length) and donor polynucleotides (typically about 100-150 nucleotides), gRNA-donor polynucleotide cassettes can be produced by standard oligonucleotide synthesis techniques and subsequently ligated into vectors.

[0220] Zinc-finger nucleases (ZFNs) are artificial DNA endonucleases generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be engineered to target desired DNA sequences, which enables zinc-finger nucleases to cleave unique target sequences. When introduced into a cell, ZFNs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. For more information on the use of ZFNs, see, for example: Asuri et al., Mol Ther. 2012 February; 20(2):329-38; Bibikova et al. Science. 2003 May 2; 300(5620):764; Wood et al. Science. 2011 Jul. 15; 333(6040):307; Ochiai et al. Genes Cells. 2010 August; 15(8):875-85; Takasu et. al., Insect Biochem Mol Biol. 2010 October; 40(10) :759-65 ; Ekker et al, Zebrafish 2008 Summer; 5(2): 121-3; Young et al, Proc Natl Acad Sci USA. 2011 Apr. 26; 108(17):7052-7; Goldberg et al, Cell. 2010 Mar. 5; 140(5):678-91; Geurts et al, Science. 2009 Jul. 24; 325(5939):433; Flisikowska et al, PLoS One. 2011; 6(6):e21045. doi: 10.1371 / journal.pone.0021045. Epub 2011 Jun. 13; Hauschild et al, Proc Natl Acad Sci USA. 2011 Jul. 19; 108(29): 12013-7; and Yu et al, Cell Res. 2011 November; 21(11): 1638-40; all of which are herein incorporated by reference for their teachings related to ZFNs. The term “ZFN agent” encompasses a zinc finger nuclease and / or a polynucleotide comprising a nucleotide sequence encoding a zinc finger nuclease.

[0221] Transcription activator-like effector nucleases (TALENs) are artificial DNA endonucleases generated by fusing a TAL (Transcription activator-like) effector DNA binding domain to a DNA cleavage domain. TALENS can be quickly engineered to bind practically any desired DNA sequence and when introduced into a cell, TALENs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. For more information on the use of TALENs, see, for example: Hockemeyer et al. Nat Biotechnol. 2011 Jul. 7; 29(8):731-4; Wood et al. Science. 2011 Jul. 15; 333(6040):307; Tesson et al. Nat Biotechnol. 2011 Aug. 5; 29(8):695-6; and Huang et. al., Nat Biotechnol. 2011 Aug. 5; 29(8):699-700; all of which are herein incorporated by reference for their teachings related to TALENs. The term “TALEN agent”encompasses a TALEN and / or a polynucleotide comprising a nucleotide sequence encoding a TALEN.Retinal Organoids

[0222] Methods are provided for generation of three-dimensional retinal organoids comprising retina-specific cell types such as bipolar cells, retina amacrine cells, horizontal cells, retinal ganglion cells, Muller glia, rod photoreceptor cells, and cone photoreceptor cells. In certain embodiments, the organoids are generated in part or whole using stem cells and / or progenitor cells. In some embodiments, the stem cells or progenitor cells are embryonic stem cells, embryonic germ cells, induced pluripotent stem cells, mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, bone marrow-derived mesenchymal stromal cells, tissue plasticadherent placental stem cells (PDACs), umbilical cord stem cells, amniotic fluid stem cells, amnion derived adherent cells (AMDACs), osteogenic placental adherent cells (OPACs), adipose stem cells, limbal stem cells, dental pulp stem cells, myoblasts, endothelial progenitor cells, neuronal stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells, amnion derived adherent cells, or side population stem cells.

[0223] In some embodiments, the organoids comprise retina- specific cells derived from induced pluripotent stem cells. Induced pluripotent stem cells can be generated by reprogramming somatic cells into pluripotent stem cells followed by redifferentiation into desired retina- specific cell types. Somatic cells can be induced into forming pluripotent stem cells, for example, by treating them with reprograming factors such as Yamanaka factors, including but not limited to, OCT3, OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28 (see, e.g., Takahashi et al. (2007) Cell. 131(5):861-872; herein incorporated by reference in its entirety). The types of somatic cells that may be converted into iPSCs include, without limitation, peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose derived stem cells, leukocytes, hematopoietic stem cells, bone marrow cells, and hepatocytes. Somatic cells are contacted with reprogramming factors in a combination and quantity sufficient to reprogram the cells to pluripotency. Reprogramming factors may be provided to the somatic cells individually or as a single composition, that is, as a premixed composition, of reprogramming factors. In some embodiments the reprogramming factors are provided as a plurality of coding sequences on a vector.

[0224] Methods for "introducing a cell reprogramming factor into somatic cells are not limited in particular, and known procedures can be selected and used as appropriate. For example, when a cell reprogramming factor as described above is introduced into somatic cells of the above- mentioned type in the form of proteins, such methods include ones using protein introducing reagents, fusion proteins with protein transfer domains (PTDs), electroporation, and microinjection. When a cell reprogramming factor as described above is introduced into somatic cells of the above-mentioned type in the form of nucleic acids encoding the cell reprogramming factor, a nucleic acid(s), such as cDNA(s), encoding the cell reprogramming factor can be inserted in an appropriate expression vector comprising a promoter that functions in somatic cells, which then can be introduced into somatic cells by procedures such as infection, lipofection, liposomes, electroporation, calcium phosphate coprecipitation, DEAE-dextran, microinjection, and electroporation. Examples of an "expression vector" include viral vectors, such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, and herpes viruses; and expression plasmids for animal cells. For example, retroviral or Sendai virus (SeV) vectors are commonly used to introduce a nucleic acid(s) encoding a cell reprogramming factor as described above into somatic cells. In some embodiments the iPSCs are derived from somatic cells obtained from neurologically normal individuals. In other embodiments the iPSCs are derived from somatic cells obtained from an individual comprising at least one allele encoding a mutation associated with a retinopathy.

[0225] The retinal organoid may comprise pluripotent stem cell (iPSC)-derived retinaspecific cell types, including, but not limited to, bipolar cells, retina amacrine cells, horizontal cells, retinal ganglion cells, Muller glia, rod photoreceptor cells and cone photoreceptor cells. The retinal organoid can be used to model retinal degeneration such as caused by occlusion of an artery or vein, retinal detachment, tears, or holes, traumatic eye injuries, surgery, retrolental fibroplasia, or a disease such as, but not limited to, retinitis pigmentosa (e.g., autosomal dominant retinitis pigmentosa, autosomal recessive retinitis pigmentosa), age-related macular degeneration, lattice degeneration, diabetic retinopathy, proliferative vitreoretinopathy, uveitis, neovascular inflammatory vitreoretinopathy, glaucoma, Leber congenital amaurosis, cone dystrophy, Usher syndrome, Stargardt disease, retinoschisis, birdshot chorioretinopathy, or central serous retinopathy. For example, a retinal organoid disease model can be generated by introducing a mutation linked to an inherited retinopathy into the genome of the retinal organoid or the iPSCs used to generate the retina- specific cells of the organoid. The iPSCs, used to generate the organoid disease model, may be derived from somatic cells of a patient who has an inherited retinopathy orgenetically modified to introduce a mutation associated with an inherited retinopathy. Such organoids mimic the pathology of the retinal degenerative disease, including the progressive loss of photoreceptors and degeneration of the retinal pigment epithelium (RPE) that may cause impaired vision and eventual blindness.

[0226] In some embodiments, a sample comprising somatic cells is obtained from a subject. The somatic cells may include, without limitation, peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose derived stem cells, leukocytes, hematopoietic stem cells, bone marrow cells, and hepatocytes, and other cell types capable of generating patient-derived iPSCs that can be differentiated into mature neurons or glia, The biological sample comprising somatic cells is typically whole blood, buffy coat, peripheral blood mononucleated cells (PBMCS), skin, fat, or a biopsy, but can be any sample from bodily fluids, tissue or cells that contain suitable somatic cells. A biological sample can be obtained from a subject by conventional techniques. For example, blood can be obtained by venipuncture, and solid tissue samples can be obtained by surgical techniques according to methods well known in the art.

[0227] Differentiation of IPSCs into retina-specific cell types such as bipolar cells, retina amacrine cells, horizontal cells, retinal ganglion cells, Muller glia, rod photoreceptor cells, and cone photoreceptor cells may be promoted by using lineage-determining transcription factors and various growth factors and other differentiation agents. For example, inhibitors of Wnt and bone morphogenetic protein (BMP) signaling in combination with insulin like growth factor 1 (IGF-1) promote differentiation of iPSCs into retinal neuronal cell types. BMP4 treatment promotes formation of neuroretinal epithelia, and IGF-1 promotes formation of three-dimensional (3D)- laminated retinal organoids when added to the media during the first 3 months of differentiation. Inhibition of Notch signaling with the gamma y-secretase inhibitor, DAPT ((2S)-N-[(3,5- Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine 1,1 -dimethylethyl ester), significantly increases the proportion of photoreceptor and retinal pigment epithelium (RPE). Rod- genesis factors such as retinoic acid (RA) and taurine increase the number of cells having photoreceptor markers. Use of 9-cis retinal, instead of all-trans RA improves rod photoreceptor differentiation in organoid cultures resulting in higher rhodopsin expression and more mature mitochondrial morphology. Neural induction media with heparin and chemically defined N2 supplement promote aggregation of iPSCs into embryoid bodies and neural retina differentiation. Fetal bovine serum (FBS) and the hedgehog agonist smoothened agonist (SAG) improve retinal differentiation for human stem cells into laminated retinas that express markers of all retinal cell types, includingganglion, amacrine, bipolar, horizontal, Muller, and photoreceptor cells. Thyroid hormone signaling regulation can be used to control the fate of cone subtypes in retinal organoids. Addition of triiodothyronine (T3) induces L and M opsins in nearly all cones. Non-adherent (3D) culturing protocols have been used to produce stratified neural retinas. Retinal ganglion cells typically appear 40 to 50 days after the start of differentiation. Ganglion cell development can be accelerated by encapsulating embryoid body-like aggregates in a 3D Matrigel drop instead of growing in suspension cultures. See, e.g., Example 3 and differentiation protocols described by Afanasyeva et al. (2021) Cellular and Molecular Life Sciences 78:6505-6532, Lamba et al. (2006) Proc Natl Acad Sci USA 103:12769-12774, Osakada et al. (2008) Nat Biotechnol 26:215-224, Nakano et al. (2012) Cell Stem Cell 10:771-785, Reichman et al. (2014) Proc Natl Acad Sci USA 111:8518-8523, Gonzalez-Cordero et al. (2017) Stem Cell Rep 9:820-837, Lowe et al. (2016) Stem Cell Reports 6:743-756, Haynes et al. (2007) Proc Natl Acad Sci 104:20380-20385, Huang et al. (2015) Dev Biol 402:119-126, Mellough et al. (2015) Stem Cells 33:2416-2430, Chichagova et al. (2020) Stem Cells 38: 195-201, and Kaya et al. (2019) Mol Vis 25:663-678; herein incorporated by reference in their entireties. However, any suitable method of inducing differentiation of iPSCs into retina-specific cell types may be used.

[0228] The iPSC-derived retina-specific cell types may be harvested at an appropriate stage of development, which may be determined based on the expression of photoreceptor markers and phenotypic characteristics of the desired mature differentiated cell type. Cultures may be empirically tested by staining for the presence of the markers of interest, by morphological determination, etc. for example, photoreceptor markers include, without limitation, RHO, 0PN1LW, 0PN1MW, OPN1SW, PNA, lectin, R0M1, RCVRN, RIBEYE, NRL, ARR3, PRPH2, SYNTAXIN3, NR2E3, and ARL13B. The mature IPSC-derived retina-specific cell types may be purified prior to assembly into organoids by positive selection for one or more markers expressed on mature retina-specific cell types. The cells are optionally enriched before or after the positive selection step by drug selection, panning, density gradient centrifugation, etc. In addition, a negative selection can be performed, where the selection is based on expression of one or more of the markers found on the somatic cells they are derived from (e.g., PBMCs, fibroblasts, epithelial cells, endothelial progenitor cells, leukocytes, hematopoietic stem cells, mesenchymal stem cells, bone marrow cells, hepatocytes), or retinal progenitor cells, and the like. Selection may utilize panning methods, magnetic particle selection, particle sorter selection, and the like.

[0229] The somatic cells, the IPSCs derived therefrom, or the mature IPSC-derived retinaspecific cell types may be genetically modified for a variety of purposes, e.g., to introduce agenetic mutation associated with a retinal degenerative disorder, inhibit expression of a gene, provide marker genes, etc. Vectors may be introduced that express an exogenous gene, reprogramming factors, CRISPR systems, antisense nucleic acids, or ribozymes. Various techniques known in the art may be used to introduce nucleic acids into the target cells, e.g., electroporation, calcium precipitated DNA, fusion, transfection, lipofection, infection and the like. The particular manner in which the DNA is introduced is not critical to the practice of the invention.Genome Modification to Introduce Disease-Relevant Genetic Changes

[0230] Disease-relevant mutations can be introduced into the genome of the mature iPSC- derived retina-specific cell types, or the iPSCs, progenitor cells, or somatic cells from which they are derived using any method known in the art to produce a three-dimensional organoid disease model. In some embodiments, a CRISPR / Cas system is used to make genetic changes to a gene of interest, for example, to introduce a mutation associated with a retinopathy to produce an organoid useful for disease modeling and drug screening. For example, a CRISPR / Cas system can be used to delete, inactivate, or mutate a gene, or eliminate or reduce gene expression or protein activity. Genome modification can be performed, for example, using homology directed repair (HDR) with a donor polynucleotide comprising a sequence comprising an intended genome edit flanked by a pair of homology arms responsible for targeting the donor polynucleotide to a target locus to be edited in a cell.

[0231] In certain embodiments, a CRISPR system is used to introduce one or more mutations linked to a retinopathy into the mature iPSC-derived retinal cells or the iPSCs, progenitor cells, or somatic cells they are derived from to produce an organoid that can be used as a disease model of a retinopathy. Exemplary mutations linked to autosomal dominant retinitis pigmentosa include, without limitation, mutations in RHO such as Pro23His, Argl35Leu, Argl35Trp, Pro347Leu, Pro347Ser, Pro347Arg, and Val345Met; mutations in HK1 such as Glu847Lys; mutations in IMPDH1 such as Asp226Asn and Asp311Asn; mutations in NR2E3 such as Gly56Arg; mutations in PRPF3 such as Thr494Met; mutations in PRPH2 such as 828+3A>T and Pro216Ser; mutations in RP1 such as Arg677X and Leu762Tyrfs*17; and mutations in RPGR such as Glu802Glyfs*32. See also, e.g., Daiger et al. (2014) Cold Spring Harb Perspect Med. 5(10):a017129 and Browne et al. (2011) Investigative Ophthalmology & Visual Science January 52:494-503; herein incorporated by reference in their entireties.Pharmaceutical Compositions

[0232] Any of the compositions described herein, including ZIP7, Rpnl 1 , vectors capable of expressing ZIP7 and / or Rpnll, viral particles comprising such vectors, or CRISPR systems or other genome modifying agents (e.g., ZNFs, TALENS) capable of expressing or increasing expression of ZIP7 and / or Rpnl 1 can be formulated into pharmaceutical compositions, optionally comprising one or more pharmaceutically acceptable excipients. Exemplary excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water, alcohols, polyols, glycerine, vegetable oils, phospholipids, and surfactants. A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and / or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[0233] A composition can also include an antimicrobial agent for preventing or deterring microbial growth. Nonlimiting examples of antimicrobial agents include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

[0234] An antioxidant can be present in the composition as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the ZIP7, Rpnl l, vectors capable of expressing ZIP7 and / or Rpnll, viral particles comprising such vectors, or CRISPR systems or other genome modifying agents, or other components of the preparation. Suitable antioxidants for use include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, mono thioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0235] A surfactant can be present as an excipient. Exemplary surfactants include: polysorbates, such as "Tween 20" and "Tween 80," and pluronics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form),fatty acids and fatty esters; steroids, such as cholesterol; chelating agents, such as EDTA; and zinc and other such suitable cations.

[0236] Acids or bases can be present as an excipient in the composition. Nonlimiting examples of acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.

[0237] The amount of the ZIP7, Rpnl 1, vectors capable of expressing ZIP7 and / or Rpnll, viral particles comprising such vectors, or CRISPR systems or other genome modifying agents (e.g., when contained in a drug delivery system) in the composition will vary depending on a number of factors, but will optimally be a therapeutically effective dose when the composition is in a unit dosage form or container (e.g., a vial). A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the composition in order to determine which amount produces a clinically desired endpoint.

[0238] The amount of any individual excipient in the composition will vary depending on the nature and function of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects. Generally, however, the excipient(s) will be present in the composition in an amount of about 1 % to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations less than 30% by weight most preferred. These foregoing pharmaceutical excipients along with other excipients are described in "Remington: The Science & Practice of Pharmacy", 19th ed., Williams & Williams, (1995), the "Physician’s Desk Reference", 52nd ed., Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, D.C., 2000.

[0239] The compositions encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted with a solvent prior to use, as well as ready for injection solutions or suspensions, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration. Examples of suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof. With respect to liquid pharmaceutical compositions, solutions and suspensions are envisioned. Additional compositions include those for oral, topical, transcutaneous, transdermal intratympanic, ocular, or localized delivery. Formulations suitable for topical, transcutaneous, transdermal, intratympanic, or ocular administration may be prepared through use of appropriate suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Such formulations may be utilized as liquid drops or with a means to provide continuous administration, for example, incorporation into slow-release pellets or controlled-release patches.

[0240] The pharmaceutical preparations herein can also be housed in a syringe, an implantation device, or the like, depending upon the intended mode of delivery and use. Preferably, the compositions comprising the ZIP7 and / or Rpnl 1 are in unit dosage form, meaning an amount of a composition appropriate for a single dose, in a premeasured or pre-packaged form.

[0241] The compositions herein may optionally include one or more additional agents, such as other drugs for treating an aging-related disease or a disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress, or other medications used to treat a subject for a condition or disease, and / or other medications used to treat a subject for a disease. Compounded preparations may include the ZIP7, Rpnll, vectors capable of expressing ZIP7 and / or Rpnl 1, viral particles comprising such vectors, or CRISPR systems or other genome modifying agents, and one or more other agents for treating an aging-associated disorder. Alternatively, such agents can be contained in a separate composition from the composition comprising the ZIP7, Rpnl l, vectors capable of expressing ZIP7 and / or Rpnl l, viral particles comprising such vectors, or CRISPR systems or other genome modifying agents, and coadministered concurrently, before, or after the composition comprising the ZIP7, Rpnl l, vectors capable of expressing ZIP7 and / or Rpnl l, viral particles comprising such vectors, or CRISPR systems or other genome modifying agents.Administration

[0242] At least one therapeutically effective dose of ZTP7 separately or in combination with a therapeutically effective dose of Rpnl 1 are administered. By “therapeutically effective dose or amount” of each of these agents is intended an amount that when administered either separately or in combination, brings about a positive therapeutic response with respect to treatment of an individual for an aging-related disease or a disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress, and / or an amount that increases survival, increases lifespan, and / or prevents, decreases, delays, or reverses senescence. Additionally, a therapeutically effective dose or amount may improve ERAD function, increase degradation of misfolded proteins, reduce aggregation of misfolded proteins, and / or reduce ER stress. As discussed above, the ZIP7 and / or Rpnl l may be provided as proteins or as vectors capable of expressing ZIP7 and / or Rpnl l proteins, or as viral particles comprising such vectors, or as CRISPR systems or other genome modifying agents (e.g., ZNFs, TALENS) capable of expressing or increasing expression of ZIP7 and / or Rpnl 1.

[0243] Aging-related diseases include any disease that occurs with increasing frequency in a population with individuals of increasing age. Examples of aging-related diseases include, but are not limited to, cardiovascular disease (e.g., atherosclerosis, heart attack, heart failure, coronary artery disease, and peripheral arterial disease), cerebrovascular diseases (e.g., ischemic stroke and hemorrhagic stroke), cancer, benign prostatic hyperplasia, hypertension, dementia (e.g., Alzheimer’s disease, vascular dementia, and Lewy body dementia), frontotemporal disorders, Huntington's disease, Parkinson's disease, chronic obstructive pulmonary disease, age-related macular degeneration, cataracts, age related endocrine disorders (e.g., type 2 diabetes mellitus, thyroid disorders, adrenal insufficiency, and parathyroid disorders), osteoporosis, osteoarthritis, abdominal obesity, hyperglycemia, dyslipidemia, and metabolic syndrome.

[0244] Disorders associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress include any disease or condition associated with pathological accumulation of misfolded proteins or protein aggregates, extracellular or cytoplasmic deposits of a fibrillary protein, and / or endoplasmic reticulum stress that causes organ damage and / or interferes with organ function. Disorders associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress include, but are not limited to, amyloidosis disorders, including transthyretin variant amyloidosis (ATTRv), transthyretin wild type amyloidosis (ATTRwt), immunoglobulin light chain amyloidosis (AL), immunoglobulin heavy chain amyloidosis (AH), leukocyte chemotactic factor 2 amyloidosis (ALECT2), fibrinogen alpha amyloidosis (AFib), apolipoproteinamyloidosis (various variants, including Al, All, CII, CIII, AIV), lysozyme amyloidosis (ALys), gelsolin amyloidosis (AGel), beta 2m macroglobulin variant amyloidosis (Beta- 2m v), beta 2m macroglobulin wild type amyloidosis (Beta- 2m), and amyloidosis involving viral proteins; amyloid beta aggregation-associated diseases such as Alzheimer’s disease, cerebral amyloid angiopathy, and Down syndrome; tauopathies such as Alzheimer's disease, Pick disease, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, primary age-related tauopathy, frontotemporal dementia and parkinsonism linked to chromosome 17, vacuolar tauopathy, lytico-bodig disease, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, pantothenate kinase-associated neurodegeneration, and lipofuscinosis; and synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA); polyQ aggregation-associated diseases, including Huntington's disease, dentatorubropallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA) also known as Kennedy's disease, and spinocerebellar ataxia (SC A) including SCA Type 1 (SCAl), SCA Type 2 (SCA2) , SCA Type 3 (SCA3) or Machado-Joseph disease, SCA Type 6 (SCA6), SCA Type 7 (SCA7), and SCA Type 17 (SCA17); prion diseases, including Creutzfeldt-Jakob disease (CJD), variant CJD, Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), and kuru; TAR DNA binding protein 43 aggregation diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, chronic traumatic encephalopathy (CTE), Lewy body disease (LBD), Huntington’s disease, argyrophilic grain disease (AGD), and hippocampal sclerosis; islet amyloid polypeptide aggregation diseases, including type 2 diabetes; diseases associated with repeat expansions in the C9orf72 gene, including ALS and frontotemporal dementia (FTD); diseases associated with mutations in the genes coding for superoxide dismutase-1 (SOD-1) and fused in sarcoma (FUS), including ALS; as well as diseases and conditions that are exacerbated by protein misfolding and / or protein aggregation and / or endoplasmic reticulum stress such as, but not limited to, diabetes, including type 1 and type 2 diabetes, and liver diseases, including non-alcoholic steatohepatitis (NASH), steatosis, nonalcoholic fatty liver disease, alcoholic liver disease, alpha- 1 antitrypsin (AAT) deficiency, fibrosis, cirrhosis, viral hepatitis, liver ischemia, drug toxicity, liver cancers such as hepatocellular carcinoma, intrahepatic cholangiocarcinoma, angiosarcoma, hemangiosarcoma, hepatoblastoma, and metastases to the liver. For a further description of disorders associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress, see, e.g., Ajoolabady et al. (2023) Hepatology 77(2): 619-639, Duwaerts et al. (2022) Front. Mol. Biosci. 8:804097,Hammarstrbm et al. (2023) Prion 17(1):82- 104, Kuscuoglu et al. (2021) J. Pathol. 254(l):80-91, Picken (2020) Acta Haematol. 143:322-334, Shreya et al. (2023) Tnt. J. Mol. Sci. 24(18):14066; and Wang et al. (2020) Cell Death and Disease 11:17; herein incorporated by reference in their entireties.

[0245] In certain embodiments, multiple therapeutically effective doses of ZIP7 or ZIP7 in combination with Rpnl l will be administered according to a daily dosing regimen, or intermittently. For example, a therapeutically effective dose can be administered, one day a week, two days a week, three days a week, four days a week, or five days a week, and so forth. By “intermittent” administration is intended the therapeutically effective dose can be administered, for example, every other day, every two days, every three days, and so forth. For example, in some embodiments, the ZIP7 and / or Rpnll will be administered twice-weekly or thrice-weekly for an extended period of time, such as for 1, 2, 3, 4, 5, 6, 7, 8...10...15. . .24 weeks, and so forth. By “twice-weekly” or “two times per week” is intended that two therapeutically effective doses of the agent in question is administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses. By “thrice weekly” or “three times per week” is intended that three therapeutically effective doses are administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present invention, this type of dosing is referred to as “intermittent” therapy. In accordance with the methods of the present invention, a subject can receive intermittent therapy (i.e., twice- weekly or thrice-weekly administration of a therapeutically effective dose) for one or more weekly cycles until the desired therapeutic response is achieved. The agents can be administered by any acceptable route of administration as noted herein below.

[0246] The ZIP7 can be administered prior to, concurrent with, or subsequent to the Rpnl l. If provided at the same time as the Rpnll, the ZIP7 can be provided in the same or in a different composition. Thus, the two agents can be presented to the individual by way of concurrent therapy. By “concurrent therapy” is intended administration to a human subject such that the therapeutic effect of the combination of the substances is caused in the subject undergoing therapy. For example, concurrent therapy may be achieved by administering at least one therapeutically effective dose of a pharmaceutical composition comprising ZIP7 and at least one therapeutically effective dose of a pharmaceutical composition comprising Rpnl l according to a particular dosing regimen. Administration of the separate pharmaceutical compositions can be at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on thesame day, or on different days), so long as the therapeutic effect of the combination of these substances is caused in the subject undergoing therapy.

[0247] In certain embodiments, the ZIP7 is administered for a brief period prior to administration of the Rpnf 1 and continued for a brief period after treatment with the Rpnf 1 is discontinued in order to ensure that the ZIP7 levels are adequate in the subject during therapy with the Rpnl l. For example, the ZIP7 can be administered starting one week before administration of the first dose of the Rpnl 1 and continued for one week after administration of the last dose of the Rpnl 1 to the subject.

[0248] In other embodiments, the pharmaceutical compositions comprising the agents, such as the ZIP7 and / or Rpnl l is a sustained-release formulation, or a formulation that is administered using a sustained-release device. Such devices are well known in the art, and include, for example, transdermal patches, and miniature implantable pumps that can provide for drug delivery over time in a continuous, steady-state fashion at a variety of doses to achieve a sustained- release effect with a non- sustained-release pharmaceutical composition.

[0249] The pharmaceutical compositions comprising ZIP7, Rpnl l , vectors capable of expressing ZIP7 and / or Rpnl l, viral particles comprising such vectors, or CRISPR systems or other genome modifying agents (e.g., ZNFs, TALENS) capable of expressing or increasing expression of ZIP7 and / or Rpnl l may be administered using the same or different routes of administration in accordance with any medically acceptable method known in the art. Suitable routes of administration include parenteral administration, such as subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), intravenous (IV), or infusion, oral and pulmonary, nasal, topical, transdermal, and suppositories. Where the composition is administered via pulmonary delivery, the therapeutically effective dose is adjusted such that the soluble level of the agent, such as the ZIP7 and / or Rpnl 1 in the bloodstream, is equivalent to that obtained with a therapeutically effective dose that is administered parenterally, for example SC, IP, IM, or IV. In some embodiments, a pharmaceutical composition comprising the ZIP7, Rpnll, vectors capable of expressing ZIP7 and / or Rpnl l, viral particles comprising such vectors, or CRISPR systems or other genome modifying agents is administered by IM or SC injection, particularly by IM or SC injection locally to a site of infection. In some embodiments, the pharmaceutical composition is administered topically as drops, on a patch, or in a gel.

[0250] Factors influencing the respective amount of the various compositions to be administered include, but are not limited to, the mode of administration, the frequency of administration (i.e., daily, or intermittent administration, such as twice- or thrice-weekly), theseverity of disease, the history of disease, whether the individual is undergoing concurrent therapy with another therapeutic agent, and the age, height, weight, health, and physical condition of the individual undergoing therapy. Generally, a higher dosage of this agent is preferred with increasing weight of the subject undergoing therapy.Production of ZIP7 and Rpnll

[0251] ZIP7 and Rpnl 1 proteins (or biologically active fragments thereof) can be prepared in any suitable manner (e.g., recombinant expression, purification from cell culture, chemical synthesis, etc.) and in various forms (e.g. native, fusions, labeled, lipidated, amidated, acetylated,PEGylated, etc.). The ZIP7 and Rpnl l proteins may include naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing proteins are well understood in the art. Proteins are preferably prepared in substantially pure form (i.e. substantially free from other host cell or non-host cell proteins).

[0252] ZIP7 nucleic acid and protein sequences may he derived from any source. A number of ZIP7 nucleic acid and protein sequences are known. Representative ZIP7 sequences are presented in SEQ ID NO:1 for ZIP7 from Drosophila melanogaster and SEQ ID NO:2 forZIP7 from Homo sapiens, and additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NM_001077516, NM_006979, NM_001288777, NM_130931, XM_015449564,XM_015449563, XM_005553337, XM_005553336, XM_040983171, XM_040983170,XM_040983169, XM_011976665, XM_011976664, XM_011976663, XM_017522703,XM_017522702, XM_017522700, XM_017522701, XM_021185695, XM_021185696,NM_001048100, XM_038682747, XM_038682746, XM_036766199, XM_036766198,XM_036766197, AQY77122, AQY77121, NP_001070984, NP_008910, and NP_001275706; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used to produce a ZIP7 protein or a recombinant polynucleotide comprising a coding sequence encoding a ZIP7 protein for use in the methods described herein. In certain embodiments, the ZIP7 protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:2, or a sequence displaying at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto, or a biologically active fragment thereof, wherein the ZIP7 is capable of increasing survival or extending lifespan, providing Zn2+to Rpnl l, improving ERAD function, increasing degradation of misfolded proteins, reducing aggregation of misfolded proteins, and / or reducing ER stress.

[0253] Rpnl l nucleic acid and protein sequences may be derived from any source. A number of Rpn 11 nucleic acid and protein sequences are known. Representative Rpn 11 sequences are presented in SEQ ID NO:3 for Rpnl l from Drosophila melanogaster and SEQ ID NO:4 for Rpnl l from Homo sapiens. Additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NM_135061, NM_OO58O5, XM_038584653, XM_019022073, NM_001025689, NM_021526, XM_029470729, XM_025471067, XM_019022073, XM_054679078, XM_045033869, XM_006935316, XM_051816741, NP_608905, NP_005796, NP_067501, NP_001020860, NP_001025636, NP_001077042, XP_003378024, XP_033766068, and XP_051669958; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used to produce a Rpnll protein or recombinant polynucleotide comprising a coding sequence encoding a Rpnll protein for use in the methods described herein. In certain embodiments, the Rpnl 1 protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4, or a sequence displaying at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto, or a biologically active fragment thereof, wherein the Rpnl l retains deubiquitinase activity and is capable of increasing survival or extending lifespan, improving ERAD function, increasing degradation of misfolded proteins, reducing aggregation of misfolded proteins, and / or reducing ER stress.

[0254] In one embodiment, ZIP7 and Rpnll proteins are generated using recombinant techniques. One of skill in the art can readily determine nucleotide sequences that encode the desired proteins using standard methodology and the teachings herein. Oligonucleotide probes can be devised based on the known sequences and used to probe genomic or cDNA libraries. The sequences can then be further isolated using standard techniques and, e.g., restriction enzymesemployed to truncate the gene at desired portions of the full-length sequence. Similarly, sequences of interest can be isolated directly from cells and tissues containing the same, using known techniques, such as phenol extraction and the sequence further manipulated to produce the desired truncations. See, e.g., Sambrook et al., supra, for a description of techniques used to obtain and isolate DNA.

[0255] The sequences encoding proteins can also be produced synthetically, for example, based on the known sequences. The nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired. The complete sequence is generally assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g.. Edge ( 1981) Atonn? 292:756; Nambairc / aZ. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem. 259:6311; Stemmer et al. (1995) Gene 164:49-53.

[0256] Recombinant techniques are readily used to clone sequences encoding proteins that can then be mutagenized in vitro by the replacement of the appropriate base pair(s) to result in the codon for the desired amino acid. Such a change can include as little as one base pair, effecting a change in a single amino acid, or can encompass several base pair changes. Alternatively, the mutations can be effected using a mismatched primer that hybridizes to the parent nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. See, e.g., Innis et al, (1990) PCR Applications: Protocols for Functional Genomics; Zoller and Smith, Methods Enzymol. (1983) 100:468. Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al. Proc. Natl. Acad. Sci USA (1982) 79:6409.

[0257] Once coding sequences have been isolated and / or synthesized, they can be cloned into any suitable vector or replicon for expression. (See, also, Examples). As will be apparent from the teachings herein, a wide variety of vectors encoding modified proteins can be generated by creating expression constructs which operably link, in various combinations, polynucleotides encoding proteins having deletions or mutations therein.

[0258] Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage A, (E. colt), pBR322 (E.coli), pACYC177 (E. coll), pKT230 (gram-negative bacteria), pGVl 106 (gram- negative bacteria), pLAFRl (gram-negative bacteria), pME290 (non-E coli gram-negative bacteria), pHV 14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCpl9 (Saccharomyces) and bovine papilloma virus (mammalian cells). See, generally, DNA Cloning; Vols. I & II, supra', Sambrook et al., supra', B. Perbal, supra.

[0259] Insect cell expression systems, such as baculovirus systems, can also be used and are known to those of skill in the art and described in, e.g., Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA ("MaxBac" kit).

[0260] Plant expression systems can also be used to produce the ZIP7 and Rpnl 1 proteins. Generally, such systems use virus-based vectors to transfect plant cells with heterologous genes. For a description of such systems, see, e.g., Porta et al., Mol. Biotech. (1996) 5:209-221 ; and Hackland et al., Arch. Virol. (1994) 139:1-22.

[0261] Viral systems, such as a vaccinia-based infection / transfection system, as described in Tomei et al., 7. Virol. (1993) 67:4017-4026 and Selby et al., J. Gen. Virol. (1993) 74: 1103-1113, will also find use with the present invention. In this system, cells are first transfected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the DNA of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA that is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation product(s).

[0262] The ZIP7 or Rpnl 1 gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the host cell transformed by a vector containing this expression construction. The coding sequence may or may not contain a signal peptide or leader sequence. With the present invention, both the naturally occurring signal peptides or heterologous sequences can be used. Leader sequences can be removed by the host in post-translational processing. See, e.g. , U.S. Patent Nos. 4,431,739; 4,425,437; 4,338,397. Such sequences include, but are not limited to, the TPA leader, as well as the honeybee mellitin signal sequence.

[0263] Other regulatory sequences may also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell. Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.

[0264] The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.

[0265] In some cases, it may be necessary to modify the coding sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the proper reading frame. Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and / or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, e.g., Sambrook et al., supra', DNA Cloning, Vols. I and II, supra', Nucleic Acid Hybridization, supra.

[0266] The expression vector is then used to transform an appropriate host cell. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Vero293 cells, as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.

[0267] Depending on the expression system and host selected, the fusion proteins of the present invention are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The selection of the appropriate growth conditions is within the skill of the art.

[0268] In one embodiment, the transformed cells secrete the ZIP7 or Rpnl l protein product into the surrounding media. Certain regulatory sequences can be included in the vector to enhance secretion of the protein product, for example using a tissue plasminogen activator (TP A) leader sequence, an interferon (y or a) signal sequence or other signal peptide sequences from known secretory proteins. The secreted ZIP7 or Rpnll protein product can then be isolated by various techniques described herein, for example, using standard purification techniques such as but not limited to, hydroxyapatite resins, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.

[0269] Alternatively, the transformed cells are disrupted, using chemical, physical or mechanical means, which lyse the cells yet keep the recombinant peptides or polypeptides substantially intact. Intracellular proteins can also be obtained by removing components from the cell wall or membrane, e.g., by the use of detergents or organic solvents, such that leakage of the polypeptides occurs. Such methods are known to those of skill in the art and are described in, e.g., Protein Purification Applications: A Practical Approach, (Simon Roe, Ed., 2001).

[0270] For example, methods of disrupting cells for use with the present invention include but are not limited to: sonication or ultrasonication; agitation; liquid or solid extrusion; heat treatment; freeze-thaw; desiccation; explosive decompression; osmotic shock; treatment with lytic enzymes including proteases such as trypsin, neuraminidase and lysozyme; alkali treatment; and the use of detergents and solvents such as bile salts, sodium dodecylsulphate, Triton, NP40 and CHAPS. The particular technique used to disrupt the cells is largely a matter of choice and will depend on the cell type in which the polypeptide is expressed, culture conditions and any pretreatment used.

[0271] Following disruption of the cells, cellular debris is removed, generally by centrifugation, and the intracellularly produced peptides or polypeptides are further purified, using standard purification techniques such as but not limited to, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.

[0272] For example, one method for obtaining the intracellular peptides or polypeptides of the present invention involves affinity purification, such as by immunoaffinity chromatography using antibodies (e.g., previously generated antibodies), or by lectin affinity chromatography. Particularly preferred lectin resins are those that recognize mannose moieties such as but not limited to resins derived from Galanthus nivalis agglutinin (GNA), Lens culinaris agglutinin(LCA or lentil lectin), Pisum sativum agglutinin (PSA or pea lectin), Narcissus pseudonarcissus agglutinin (NPA) and Allium ursinum agglutinin (AUA). The choice of a suitable affinity resin is within the skill in the art. After affinity purification, the peptides or polypeptides can be further purified using conventional techniques well known in the art, such as by any of the techniques described above.

[0273] The ZIP7 and Rpnl 1 proteins can be conveniently synthesized chemically, for example by any of several techniques that are known to those skilled in the peptide art. See, e.g., Fmoc Solid Phase Peptide Synthesis: A Practical Approach (W. C. Chan and Peter D. White eds., Oxford University Press, 1stedition, 2000) ; N. Leo Benoiton, Chemistry of Peptide Synthesis (CRC Press; 1stedition, 2005); Peptide Synthesis and Applications (Methods in Molecular Biology, John Howl ed., Humana Press, 1sted., 2005); and Pharmaceutical Formulation Development of Peptides and Proteins (The Taylor & Francis Series in Pharmaceutical Sciences, Lars Hovgaard, Sven Frokjaer, and Marco van de Weert eds., CRC Press; 1stedition, 1999); herein incorporated by reference.

[0274] In genera], these methods employ the sequential addition of one or more amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected, under conditions that allow for the formation of an amide linkage. The protecting group is then removed from the newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support, if solid phase synthesis techniques are used) are removed sequentially or concurrently, to render the final peptide or polypeptide. By simple modification of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under conditions which do not racemize chiral centers) a protected tripeptide with a properly protected dipeptide to form, after deprotection, a pentapeptide. See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis (Pierce Chemical Co., Rockford, IL 1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, (Academic Press, New York, 1980), pp. 3-254, for solid phase peptide synthesis techniques; and M. Bodansky, Principles of Peptide Synthesis. (Springer-Verlag, Berlin 1984) and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology. Vol. 1, for classical solution synthesis. These methods aretypically used for relatively small polypeptides, i.e., up to about 50-100 amino acids in length, but are also applicable to larger polypeptides.

[0275] Typical protecting groups include t-butyloxycarbonyl (Boc), 9- fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz); p-toluenesulfonyl (Tx); 2,4- dinitrophenyl; benzyl (Bzl); biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl, isobomyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl, isopropyl, acetyl, o- nitrophenylsulfonyl and the like.

[0276] Typical solid supports are cross-linked polymeric supports. These can include divinylbenzene cross-linked-styrene -based polymers, for example, divinylbenzene- hydroxymethylstyrene copolymers, divinylbenzene-chloromethylstyrene copolymers and divinylbenzene-benzhydrylaminopolystyrene copolymers.

[0277] The ZIP7 and Rpnll proteins can also be chemically prepared by other methods such as by the method of simultaneous multiple peptide synthesis. See, e.g., Houghten Proc. Nall. Acad. Sci. USA (1985) 82:5131-5135; U.S. Patent No. 4,631,211.Kits

[0278] Also provided are kits for treating a patient with a ZIP7 and / or Rpnl 1 protein, or a vector system, viral particles, or CRISPR system or other genome modifying agent (e.g., ZNFs, TALENS) capable of producing them, as described herein. The ZIP7 and Rpnl l proteins, vector system, viral particles, or genome modifying agent, and optionally other therapeutic agents may be contained in separate compositions or in the same composition. Kits may include unit doses of the formulations comprising the ZIP7 and / or Rpnl l (or vector system, viral particles, or genome modifying agents capable of producing them) suitable for use in the treatment methods described herein, e.g., in tablets or injectable dose(s). Transfection agents may also be included in the kit such as lipid nanoparticles (LNPs), calcium phosphate, polyethyleneimine (PEI), DEAE-dextran, liposomes, and the like.

[0279] Formulations suitable for intravenous administration are of particular interest, and in such embodiments the kit may further include a syringe or other device to accomplish such administration, which syringe or device may be pre-filled with a composition comprising the ZIP7 and / or Rpnl 1 proteins, or a vector system, viral particles, or genome modifying agents capable of producing them. The instructions can be printed on a label affixed to the container or can be a package insert that accompanies the container.

[0280] In certain embodiments, the kit comprises a ZIP7 protein comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and 2, or a sequence displaying at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto, or a biologically active fragment thereof, wherein the ZIP7 is capable of improving ERAD function, increasing degradation of misfolded proteins, reducing aggregation of misfolded proteins, and / or reducing ER stress, and / or increasing survival, increasing lifespan, or preventing, decreasing, delaying, or reversing senescence of a subject. In some embodiments, the kit comprises a vector system, viral particles, or genome modifying agents capable of producing the ZIP7 protein.

[0281] In certain embodiments, the kit comprises a Rpnl l protein comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:3 and 4, or a sequence displaying at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto, or a biologically active fragment thereof, wherein the Rpnl 1 has deubiquitinase activity and / or is capable of improving ERAD function, increasing degradation of misfolded proteins, reducing aggregation of misfolded proteins, and / or reducing ER stress, and / or increasing survival, increasing lifespan, or preventing, decreasing, delaying, or reversing senescence of a subject. In some embodiments, the kit comprises a vector system, viral particles, or genome modifying agents capable of producing the Rpnl l protein.

[0282] Kits may comprise one or more containers of the compositions described herein. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The kit can further comprise a container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery device. The kit may also provide a delivery device pre- filled with a solution comprising a unit dose of the ZIP7 and / or Rpnll proteins, or a vector system or viral particles or genome modifying agent capable of producing the ZIP7 and / or Rpnl 1 proteins, and a pharmaceutically acceptable excipient; and instructions to administer a unit doseaccording to a desired regimen or exemplary regimen dependent upon a patient’ s age, weight, gender, and the like.

[0283] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, Blu-ray, flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.Examples of Non-Limiting Aspects of the Disclosure

[0284] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-174 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:1. A method of treating an aging -related disease or a disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress of a subject, the method comprising administering a therapeutically effective amount of zinc transporter protein 7 (ZIP7) to the subject.2. The method of aspect 1 , wherein said treating reduces protein misfolding, protein aggregation, or endoplasmic reticulum stress, increases survival, increases lifespan, or prevents, decreases, delays, or reverses senescence of the subject.3. The method of aspect 1 or 2, further comprising administering a therapeutically effective amount of regulatory particle non-ATPase 11 (Rpnl 1 ) in combination with the therapeutically effective amount of the ZIP7 to the subject.4. The method of any one or aspects 1-3, wherein the ZIP7 and / or the Rpnll are provided by a recombinant nucleic acid or vector system.5. The method of aspect 4, wherein the recombinant nucleic acid is RNA or DNA.6. The method of aspect 5, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the ZIP7 or Rpnl l in the subject.7. The method of aspect 4, wherein the vector system comprises one or more viral vectors or plasmids.8. The method of aspect 7, wherein the one or more viral vectors comprise an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, anelloviral vector, or a retroviral vector.9. The method of aspect 7 or 8, wherein the vector system is administered intravenously.10. The method of aspect 7 or 8, wherein the vector system is administered locally to a target organ or tissue in need of treatment.11. The method of any one of aspects 7-10, wherein expression of the ZIP7 and / or the Rpnl 1 is inducible.12. The method of any one of aspects 7-11, wherein the vector system comprises a first vector comprising a first coding sequence encoding ZIP7 and a second vector comprising a second coding sequence encoding Rpnl l, wherein the ZIP7 and the Rpnl 1 are co-expressed in vivo in the subject in effective amounts sufficient to increase survival, increase lifespan, or prevent, decrease, or delay senescence of the subject.13. The method of aspect 12, wherein the first vector comprises a first promoter operably linked to the first coding sequence encoding the ZIP7.14. The method of aspect 13, wherein the first promoter is a tissue-specific promoter or cell type-specific promoter.15. The method of aspect 4, wherein the cell type-specific promoter is specific for a postmitotic cell type.16. The method of aspect 15, wherein the postmitotic cell is a neuron, a cardiomyocyte, a skeletal muscle myofiber, a retinal ganglion cell, a cochlear hair cell, an osteocyte, or an adipocyte.17. The method of aspect 12, wherein the first coding sequence encoding the ZIP7 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated first coding sequence encoding the ZIP7 at the chromosomal locus.18. The method of any one of aspects 12-17, wherein the second vector comprises a second promoter operably linked to the second coding sequence encoding the Rpnl 1.19. The method of aspect 18, wherein the second promoter is a tissue-specific promoter or cell type-specific promoter.20. The method of aspect 19, wherein the cell type-specific promoter is specific for a postmitotic cell type.21. The method of aspect 20, wherein the postmitotic cell is a neuron, a cardiomyocyte, a skeletal muscle myofiber, a retinal ganglion cell, a cochlear hair cell, an osteocyte, or an adipocyte.22. The method of aspect 12, wherein the second coding sequence encoding the Rpnl 1 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated second coding sequence encoding the Rpnll at the chromosomal locus.23. The method of any one of aspects 7-11, wherein the ZIP7 and the Rpnl 1 are provided by a multicistronic vector comprising a first coding sequence encoding ZIP7 and a second coding sequence encoding Rpnll, wherein the ZIP7 and the Rpnl l are co-expressed in vivo in the subject in effective amounts sufficient to increase survival, increase lifespan, or prevent, decrease, or delay senescence of the subject.24. The method of aspect 23, wherein the multicistronic vector further comprises a viral T2A peptide or internal ribosome entry site (IRES) sequence operably linked to the first coding sequence encoding the ZIP7 and the second coding sequence encoding the Rpnll to allow co-expression of the ZIP7 and the Rpnl l.25. The method of any one of aspects 1-24, wherein the ZIP7 is provided by a viral particle comprising a vector comprising a coding sequence encoding the ZIP7.26. The method of any one of aspects 3-25, wherein the Rpnl 1 is provided by a viral particle comprising a vector comprising a coding sequence encoding the Rpnl 1.27. The method of aspect 25 or 26, wherein the viral particle is administered intravenously.28. The method of aspect 25 or 26, wherein the viral particle is administered locally to a target organ or tissue in need of treatment.29. The method of aspect 1 or 2, wherein the ZIP7 and / or the Rpnl 1 are provided by genetically modifying the genome of the subject to express the ZIP7 and / or the Rpnll.30. The method of aspect 29, wherein the genome of the subject is genetically modified using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated(Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).31. The method of any one of aspects 1-30, wherein the ZIP7 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.32. The method of any one of aspects 3-31, wherein the Rpnl 1 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4.33. The method of any one of aspects 1-32, wherein the subject is a mammal.34. The method of aspect 33, wherein the mammal is human.35. A composition for use in a method of treating an aging-related disease and extending lifespan of a subject or treating a disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress, the composition comprising zinc transporter protein 7 (ZIP7) or a vector system encoding ZIP7.36. The composition of aspect 35, further comprising regulatory particle non-ATPase 11 (Rpnll) or a vector system encoding Rpnll.37. The composition of aspect 35 or 36, further comprising a pharmaceutically acceptable excipient.38. A method of increasing survival or extending lifespan of a cell, organoid, or organism, the method comprising introducing into the cell, organoid, or organism an effective amount of zinc transporter protein 7 (ZIP7).39. The method of aspect 38, further comprising introducing into the cell, organoid, or organism an effective amount of regulatory particle non-ATPase 11 (Rpnl l).40. The method of aspect 38 or 39, the cell is a postmitotic cell.41. The method of aspect 40, wherein the postmitotic cell is a neuron, a cardiomyocyte, a skeletal muscle myofiber, a retinal ganglion cell, a cochlear hair cell, an osteocyte, or an adipocyte.42. The method of any one of aspects 38-41, wherein the ZIP7 and / or the Rpnl 1 are provided by a recombinant nucleic acid or vector system.43. The method of aspect 42, wherein the recombinant nucleic acid is RNA or DNA.44. The method of aspect 43, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the ZIP7 or Rpnl 1 in the cell, organoid, or organism.45. The method of aspect 42, wherein the vector system comprises one or more viral vectors or plasmids.46. The method of aspect 45, wherein the one or more viral vectors comprise an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, anelloviral vector, or a retroviral vector.47. The method of aspect 45 or 46, wherein expression of the ZIP7 and / or the Rpn 11 is inducible.48. The method of any one of aspects 45-47, wherein the vector system comprises a first vector comprising a first coding sequence encoding ZIP7 and a second vector comprising a second coding sequence encoding Rpnll, wherein the ZIP7 and the Rpn 11 are co-expressed in the cell, organoid, or organism in effective amounts sufficient to increase survival or prevent, decrease, or delay senescence of the cell, organoid, or organism.49. The method of aspect 48, wherein the first vector comprises a first promoter operably linked to the first coding sequence encoding the ZIP7.50. The method of aspect 49, wherein the first promoter is a tissue-specific promoter or cell type-specific promoter.51. The method of aspect 48, wherein the first coding sequence encoding the ZIP7 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated first coding sequence encoding the ZIP7 at the chromosomal locus.52. The method of any one of aspects 48-51, wherein the second vector comprises a second promoter operably linked to the second coding sequence encoding the Rpnl l.53. The method of aspect 52, wherein the second promoter is a tissue-specific promoter or cell type-specific promoter.54. The method of any one of aspects 48-51 , wherein the second coding sequence encoding the Rpnl l is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated second coding sequence encoding the Rpnll at the chromosomal locus.55. The method of aspect 48, wherein the ZIP7 and the Rpnl 1 are provided by a multicistronic vector comprising a first coding sequence encoding ZIP7 and a second coding sequence encoding Rpnl l, wherein the ZIP7 and the Rpnl 1 are co-expressed in the cell, organoid, or organism in effective amounts sufficient to increase survival, increase lifespan, or prevent, decrease, or delay senescence of the cell, organoid, or organism.56. The method of aspect 55, wherein the multicistronic vector further comprises a viral T2A peptide or internal ribosome entry site (IRES) sequence operably linked to the first coding sequence encoding the ZIP7 and the second coding sequence encoding the Rpnl 1 to allow co-expression of the ZIP7 and the Rpnll.57. The method of any one of aspects 45-56, wherein the ZIP7 is provided by a viral particle comprising a vector comprising a coding sequence encoding the ZIP7.58. The method of any one of aspects 45-57, wherein the Rpnl 1 is provided by a viral particle comprising a vector comprising a coding sequence encoding the Rpn 1 1 .59. The method of aspect 57 or 58, wherein the viral particle is administered intravenously.60. The method of aspect 57 or 58, wherein the viral particle is administered locally to a target organ or tissue in need of treatment.61. The method of any one of aspects 38-41, wherein the ZIP7 and / or the Rpnl 1 are provided by genetically modifying the genome of the cell, organoid, or organism to express the ZIP7 and / or the Rpnl 1.62. The method of aspect 61, wherein the genome of the cell, organoid, or organism is genetically modified using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).63. The method of aspect 62, wherein the ZIP7 is introduced into the cell, organoid, or organism, by a method comprising: introducing a donor polynucleotide into the cell, organoid, or organism, wherein the donor polynucleotide comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3’ genomic target sequence flanking a nucleotide sequence encoding the ZIP7 ; introducing an RNA-guided nuclease into the cell, organoid, or organism; and introducing a guide RNA into the cell, organoid, or organism, wherein the guide RNA forms a complex with the RNA-guided nuclease such that the guide RNA directs the RNA- guided nuclease to a genomic target sequence at a genomic target locus to be modified, wherein the RNA-guided nuclease creates a double-stranded break in the genomic target sequence, wherein the donor polynucleotide is integrated at the genomic target locus recognized by its 5' homology arm and 3' homology arm by homology directed repair (HDR) such that a genetically modified cell, organoid, or organism is produced, wherein the survival or the lifespan of thegenetically modified cell, organoid, or organism is increased compared to the unmodified cell, organoid, or organism.64. The method of aspect 62 or 63, wherein the Rpnl 1 is introduced into the cell, organoid, or organism, by a method comprising: introducing a donor polynucleotide into the cell, organoid, or organism, wherein the donor polynucleotide comprises a 5' homology arm that hybridizes to a 5’ genomic target sequence and a 3’ homology arm that hybridizes to a 3’ genomic target sequence flanking a nucleotide sequence encoding the Rpnl 1; introducing an RNA-guided nuclease into the cell, organoid, or organism; and introducing a guide RNA into the cell, organoid, or organism, wherein the guide RNA forms a complex with the RNA-guided nuclease such that the guide RNA directs the RNA- guided nuclease to a genomic target sequence at a genomic target locus to be modified, wherein the RNA-guided nuclease creates a double-stranded break in the genomic target sequence, wherein the donor polynucleotide is integrated at the genomic target locus recognized by its 5' homology arm and 3' homology arm by homology directed repair (HDR) such that a genetically modified cell, organoid, or organism is produced, wherein the survival or the lifespan of the genetically modified cell, organoid, or organism is increased compared to the unmodified cell, organoid, or organism.65. The method of any one of aspects 63 or 64, wherein the donor polynucleotide, the RNA-guided nuclease, and the guide RNA are provided by one or more vectors.66. The method of aspect 65, wherein the one or more vectors are viral vectors or plasmids.67. The method of aspect 66, wherein the viral vectors are lentivirus vectors, retrovirus vectors, adenovirus vectors, or adeno-associated virus vectors68. The method of any one of aspects 65-67, wherein the one or more vectors are introduced into the cell, organoid, or organism by transient transfection or stable transfection.69. The method of any one of aspects 65-69, wherein expression of the RNA-guided nuclease or the guide RNA is inducible.70. The method of any one of aspects 63 or 64, wherein the RNA-guided nuclease is provided by a mRNA encoding the RNA-guided nuclease, wherein translation of the mRNA results in production of the RNA-guided nuclease in the cell, organoid, or organism.71. The method of aspect 63 or 64, wherein the RNA-guided nuclease and the guide RNA are provided as a ribonucleoprotein complex of the RNA-guided nuclease with the guide RNA, wherein the ribonucleoprotein complex is introduced into the cell, organoid, or organism by microinjection.72. The method of any one of aspects 63-71 , wherein the RNA-guided nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease.73. The method of aspect 72, wherein the Cas nuclease is Cas9 or Casl2a.74. The method of any one of aspects 38-73, wherein the organoid is a retinal organoid.75. The method of aspect 74, wherein the retinal organoid is used as a model of retinal degeneration.76. The method of aspect 75, wherein the retinal organoid comprises a mutation associated with an inherited retinopathy.77. The method of aspect 75 or 76, wherein the effective amount of the ZIP7 or the combination of the effective amount of the ZIP7 and the effective amount of the Rpnl 1 prevents, delays, decreases, or at least partially reverses the retinal degeneration in the retinal organoid.78. The method of any one of aspects 38-77, wherein the ZIP7 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.79. The method of any one of aspects 39-78, wherein the Rpnl 1 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4.80. A method of preventing, delaying, or reversing senescence of a postmitotic cell, the method comprising introducing into the postmitotic cell an effective amount of zinc transporter protein 7 (ZIP7).81. The method of aspect 80, further comprising introducing into the postmitotic cell, an effective amount of regulatory particle non-ATPase 11 (Rpnl 1).82. The method of aspect 80 or 81, wherein the postmitotic cell is a neuron, a cardiomyocyte, a skeletal muscle myofiber, a retinal ganglion cell, a cochlear hair cell, an osteocyte, or an adipocyte.83. The method of any one of aspects 80-82, wherein the ZIP7 and / or the Rpnl 1 are provided by a recombinant nucleic acid or vector system.84. The method of aspect 83, wherein the recombinant nucleic acid is RNA or DNA.85. The method of aspect 84, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the ZIP7 or Rpnl 1 in the postmitotic cell.86. The method of aspect 83, wherein the vector system comprises one or more viral vectors or plasmids.87. The method of aspect 86, wherein the one or more viral vectors comprise an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, anelloviral vector, or a retroviral vector.88. The method of aspect 86 or 87, wherein expression of the ZIP7 and / or the Rpnl 1 is inducible.89. The method of any one of aspects 86-88, wherein the vector system comprises a first vector comprising a first coding sequence encoding ZIP7 and a second vector comprising a second coding sequence encoding Rpnl 1, wherein the ZIP7 and the Rpnl 1 are co-expressed in the postmitotic cell in effective amounts sufficient to extend survival of the postmitotic cell.90. The method of aspect 89, wherein the first vector comprises a first promoter operably linked to the first coding sequence encoding the ZIP7.91. The method of aspect 89, wherein the first coding sequence encoding the ZIP7 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated first coding sequence encoding the ZIP7 at the chromosomal locus.92. The method of any one of aspects 89-91, wherein the second vector comprises a second promoter operably linked to the second coding sequence encoding the Rpnl 1.93. The method of any one of aspects 89-91, wherein the second coding sequence encoding the Rpnl l is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated second coding sequence encoding the Rpnll at the chromosomal locus.94. The method aspect 86 or 87, wherein the ZIP7 and the Rpnl 1 are provided by a multicistronic vector comprising a first coding sequence encoding ZIP7 and a second coding sequence encoding Rpnl l, wherein the ZIP7 and the Rpnl 1 are co-expressed in the postmitotic cell in effective amounts sufficient to extend survival of the postmitotic cell.95. The method of aspect 94, wherein the multicistronic vector further comprises a viral T2A peptide or internal ribosome entry site (IRES) sequence operably linked to the first coding sequence encoding the ZIP7 and the second coding sequence encoding the Rpnl 1 to allow co-expression of the ZIP7 and the Rpnll.96. The method of any one of aspects 80-95, wherein the ZIP7 is provided by a viral particle comprising a vector comprising a coding sequence encoding the ZIP7.97. The method of any one of aspects 80-96, wherein the Rpnl 1 is provided by a viral particle comprising a vector comprising a coding sequence encoding the Rpnl l.98. The method of any one of aspects 80-82, wherein the ZIP7 and / or the Rpnl 1 are provided by genetically modifying the genome of the postmitotic cell to express the ZIP7 and / or the Rpnl l.99. The method of aspect 98, wherein the genome of the postmitotic cell is genetically modified using a clustered regularly interspaced short palindromic repeats (CRIS PR) -associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).100. The method of aspect 99, wherein the ZIP7 is introduced into the postmitotic cell by a method comprising: introducing a donor polynucleotide into the postmitotic cell, wherein the donor polynucleotide comprises a 5' homology arm that hybridizes to a 5’ genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence encoding the ZIP7; introducing an RNA-guided nuclease into the postmitotic cell; and introducing a guide RNA into the postmitotic cell, wherein the guide RNA forms a complex with the RNA-guided nuclease such that the guide RNA directs the RNA-guided nuclease to a genomic target sequence at a genomic target locus to be modified, wherein the RNA-guided nuclease creates a double-stranded break in the genomic target sequence in the intron, wherein the donor polynucleotide is integrated at the genomic target locus recognized by its 5' homology arm and 3' homology arm by homology directed repair (HDR) such that agenetically modified postmitotic cell is produced, wherein the survival or the lifespan of the genetically modified postmitotic cell is increased compared to the unmodified postmitotic cell.101. The method of aspect 99 or 100, wherein the Rpnll is introduced into the postmitotic cell by a method comprising: introducing a donor polynucleotide into the postmitotic cell, wherein the donor polynucleotide comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence encoding the Rpnl l; introducing an RNA-guided nuclease into the postmitotic cell; and introducing a guide RNA into the postmitotic cell, wherein the guide RNA forms a complex with the RNA-guided nuclease such that the guide RNA directs the RNA-guided nuclease to a genomic target sequence at a genomic target locus to be modified, wherein the RNA-guided nuclease creates a double-stranded break in the genomic target sequence in the intron, wherein the donor polynucleotide is integrated at the genomic target locus recognized by its 5' homology arm and 3' homology arm by homology directed repair (HDR) such that a genetically modified postmitotic cell is produced, wherein the survival or the lifespan of the genetically modified postmitotic cell is increased compared to the unmodified postmitotic cell.102. The method of aspect 100 or 101, wherein the donor polynucleotide, the RNA- guided nuclease, and the guide RNA are provided by one or more vectors.103. The method of aspect 102, wherein the one or more vectors are viral vectors or plasmids.104. The method of aspect 103, wherein the viral vectors are lentivirus vectors, retrovirus vectors, adenovirus, or adeno-associated virus vectors105. The method of any one of aspects 102-104, wherein the one or more vectors are introduced into the cell, organoid, or organism by transient transfection or stable transfection.106. The method of any one of aspects 102-105, wherein expression of the RNA- guided nuclease or the guide RNA is inducible.107. The method of aspect 100 or 101 , wherein the RNA-guided nuclease is provided by a mRNA encoding the RNA-guided nuclease, wherein translation of the mRNA results in production of the RNA-guided nuclease in the postmitotic cell.108. The method of aspect 100 or 101, wherein the RNA-guided nuclease and the guide RNA are provided as a ribonucleoprotein complex of the RNA-guided nuclease with the guide RNA, wherein the ribonucleoprotein complex is introduced into the postmitotic cell by microinjection.109. The method of any one of aspects 100-108, wherein the RNA-guided nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease.110. The method of aspect 109, wherein the Cas nuclease is Cas9 or Cas 12a.111. The method of any one of aspects 80-110, wherein the organoid is a retinal organoid.112. The method of aspect 111, wherein the retinal organoid is used as a model of retinal degeneration.113. The method of aspect 111 or 112, wherein the retinal organoid comprises a mutation associated with an inherited retinopathy.114. The method of any one of aspects 80-113, wherein the ZIP7 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.115. The method of any one of aspects 80-114, wherein the Rpnll comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4.116. A method of treating a retinopathy in a subject, the method comprising administering a therapeutically effective amount of zinc transporter protein 7 (ZIP7) to the subject.117. The method of aspect 116, further comprising administering a therapeutically effective amount of regulatory particle non-ATPase 11 (Rpnl l) in combination with the therapeutically effective amount of the ZIP7 to the subject.118. The method of aspect 116 or 117, wherein the therapeutically effective amount of the ZIP7 or ZIP7 in combination with Rpnl 1 prevents, delays, decreases, or reverses retinal degeneration.119. The method of any one of aspects 116-118, wherein the ZIP7 and / or the Rpnl 1 are administered locally to the retina.120. The method of any one or aspects 116-119, wherein the ZIP7 and / or the Rpnl 1 are provided by a recombinant nucleic acid or vector system.121. The method of aspect 120, wherein the recombinant nucleic acid is RNA or DNA.122. The method of aspect 121, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the ZIP7 or Rpnl 1 in the subject.123. The method of aspect 120, wherein the vector system comprises one or more viral vectors or plasmids.124. The method of aspect 123, wherein the one or more viral vectors comprise an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, anelloviral vector, or a retroviral vector.125. The method of aspect 123 or 124, wherein the vector system is administered intravenously.126. The method of aspect 123 or 124, wherein the vector system is administered locally to the retina.127. The method of any one of aspects 123-126, wherein expression of the ZIP7 and / or the Rpnl 1 is inducible.128. The method of any one of aspects 123-127, wherein the vector system comprises a first vector comprising a first coding sequence encoding ZIP7 and a second vector comprising a second coding sequence encoding Rpnl 1, wherein the ZIP7 and the Rpnl l are co-expressed in vivo in the subject in effective amounts sufficient to prevent, delay, decrease, or reverse retinal degeneration in an eye of the subject.129. The method of aspect 128, wherein the first vector comprises a first promoter operably linked to the first coding sequence encoding the ZIP7.130. The method of aspect 129, wherein the first promoter is a retina- specific promoter.131. The method of aspect 130, wherein the retina-specific promoter is specific for a retinal-specific cell type.132. The method of aspect 131, wherein the retina-specific cell type is a bipolar cell, a retina amacrine cell, a horizontal cell, a retinal ganglion cell, Muller glia, a rod photoreceptor cell or a cone photoreceptor cell.133. The method of aspect 128, wherein the first coding sequence encoding the ZIP7 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated first coding sequence encoding the ZIP7 at the chromosomal locus.134. The method of aspect 128, wherein the second vector comprises a second promoter operably linked to the second coding sequence encoding the Rpnl l.135. The method of aspect 134, wherein the second promoter is a retina-specific promoter.136. The method of aspect 135, wherein the retina-specific promoter is specific for a retinal-specific cell type.137. The method of aspect 136, wherein the retina-specific cell type is a bipolar cell, a retina amacrine cell, a horizontal cell, a retinal ganglion cell, Muller glia, a rod photoreceptor cell or a cone photoreceptor cell.138. The method of aspect 128, wherein the second coding sequence encoding the Rpnll is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated second coding sequence encoding the Rpnll at the chromosomal locus.139. The method of any one of aspects 123-127, wherein the ZIP7 and the Rpnl 1 are provided by a multicistronic vector comprising a first coding sequence encoding ZIP7 and a second coding sequence encoding Rpnll, wherein the ZIP7 and the Rpnl l are co-expressed in vivo in the subject in effective amounts sufficient to prevent, delay, decrease, or reverse retinal degeneration in an eye of the subject.140. The method of aspect 139, wherein the multicistronic vector further comprises a viral T2A peptide or internal ribosome entry site (IRES) sequence operably linked to the first coding sequence encoding the ZIP7 and the second coding sequence encoding the Rpnl l to allow co-expression of the ZIP7 and the Rpnll.141. The method of any one of aspects 123-140, wherein the ZIP7 is provided by a viral particle comprising a vector comprising a coding sequence encoding the ZIP7.142. The method of any one of aspects 123-141, wherein the Rpnl 1 is provided by a viral particle comprising a vector comprising a coding sequence encoding the Rpnl l.143. The method of aspect 141 or 142, wherein the viral particle is administered intravenously.144. The method of aspect 141 or 142, wherein the viral particle is administered locally to the retina.145. The method of any one of aspects 116-118, wherein the ZIP7 and / or the Rpnl 1 are provided by genetically modifying the genome of the subject to express the ZIP7 and / or the Rpnll.146. The method of aspect 145, wherein the genome of the subject is genetically modified using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator- like effector nuclease (TALEN).147. The method of any one of aspects 116-146, wherein the ZIP7 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.148. The method of any one of aspects 116-147, wherein the Rpnl 1 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4.149. The method of any one of aspects 116-148, wherein the subject is a mammal.150. The method of aspect 149, wherein the mammal is human.151. A composition for use in a method of treating a retinopathy, the composition comprising zinc transporter protein 7 (ZIP7) or a vector system encoding ZIP7.152. The composition of aspect 151, further comprising regulatory particle non- ATPase 11 (Rpnl 1) or a vector system encoding Rpnl l.153. The composition of aspect 151 or 152, further comprising a pharmaceutically acceptable excipient.154. A method of increasing proteasomal degradation of poly-ubiquitinated proteins in a cell, the method comprising introducing an effective amount of zinc transporter protein 7 (ZIP7) into the cell.155. The method of aspect 154, further comprising introducing an effective amount of regulatory particle non-ATPase 11 (Rpnl 1) into the cell.156. The method of 154 or 155, wherein the ZIP7 and / or the Rpnll are provided by a recombinant nucleic acid or vector system.157. The method of aspect 156, wherein the recombinant nucleic acid is RNA or DNA.158. The method of aspect 157, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the ZIP7 or Rpnl l in the cell.159. The method of aspect 156, wherein the vector system comprises one or more viral vectors or plasmids.160. The method of aspect 159, wherein the one or more viral vectors comprise an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, anelloviral vector, or a retroviral vector.161. The method of aspect 159 or 160, wherein expression of the ZIP7 and / or the Rpnl 1 is inducible.162. The method of any one of aspects 159-161, wherein the vector system comprises a first vector comprising a first coding sequence encoding ZIP7 and a second vector comprising a second coding sequence encoding Rpnl l, wherein the ZIP7 and the Rpnl 1 are co-expressed in the cell in effective amounts sufficient to increase proteasomal degradation of poly-ubiquitinated proteins in the cell.163. The method of aspect 162, wherein the first vector comprises a first promoter operably linked to the first coding sequence encoding the ZIP7.164. The method of aspect 162, wherein the first coding sequence encoding the ZIP7 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated first coding sequence encoding the ZIP7 at the chromosomal locus.165. The method of any one of aspects 162-164, wherein the second vector comprises a second promoter operably linked to the second coding sequence encoding the Rpnll.166. The method of any one of aspects 162-164, wherein the second coding sequence encoding the Rpnl 1 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated second coding sequence encoding the Rpnll at the chromosomal locus.167. The method aspect 156, wherein the ZIP7 and the Rpnll are provided by a multicistronic vector comprising a first coding sequence encoding ZIP7 and a second coding sequence encoding Rpnl l, wherein the ZIP7 and the Rpnll are co-expressed in the cell in effective amounts sufficient to extend survival of the cell.168. The method of aspect 167, wherein the multicistronic vector further comprises a viral T2A peptide or internal ribosome entry site (IRES) sequence operably linked to the first coding sequence encoding the ZIP7 and the second coding sequence encoding the Rpnl l to allow co-expression of the ZIP7 and the Rpnll.169. The method of aspect 154 or 155, wherein the ZIP7 is provided by a viral particle comprising a vector comprising a coding sequence encoding the ZIP7.170. The method of aspect 155, wherein the Rpnl 1 is provided by a viral particle comprising a vector comprising a coding sequence encoding the Rpnl l.171. The method of aspect 154 or 155, wherein the ZIP7 and / or the Rpnll are provided by genetically modifying the genome of the cell to express the ZIP7 and / or the Rpnll.172. The method of aspect 171 , wherein the genome of the cell is genetically modified using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).173. The method of any one of aspects 154-172, wherein the ZIP7 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.174. The method of any one of aspects 155-172, wherein the Rpnl 1 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4.EXAMPLES

[0285] As can be appreciated from the disclosure provided above, the present disclosure has a wide variety of applications. Accordingly, the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, dimensions, etc.) but some experimental errors and deviations should be accounted for. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.EXAMPLE 1: THE ZN2+TRANSPORTER ZIP7 ENHANCES ERAD PROMOTING CELL MIGRATIONAND SUPPRESSES NEURODEGENERATION

[0286] ZIP7 is an evolutionarily conserved, ER Zn2+transporter that promotes cell survival and migration in diverse cell types and organisms8 11. ZIP7 promotes intestinal stem cell maintenance, is required for B cell differentiation, and is overexpressed in multiple cancers.8 12Loss of ZIP7 causes ER stress in a variety of cell types in organisms as diverse as plants and yeast to flies and humans. However, the mechanism(s) by which ZIP7 mitigates ER stress andcontributes to these diverse biological functions are unknown13, and the effects of ZIP7 overexpression are largely unexplored.

[0287] We previously identified the Drosophila ortholog of ZIP7 (dZIP7, aka Catsup) in a screen for mutations that disrupt border cell migration14and in a border cell gene expression profile15. Border cells in the Drosophila ovary provide an in vivo model of collective cell migration that is amenable to unbiased genetic screening16. Here we show that it is by mitigating ER stress that dZIP7 promotes border cell migration. Induction of ER stress by expressing a misfolded protein (RhlG69D)17 18blocks border cell migration. Remarkably, overexpression of dZIP7 is sufficient to degrade misfolded RhlG69D, prevent ER stress, and thereby rescue border cell migration. We further show that ER to cytosol Zn2+transport is rate-limiting for ERAD. dZIP7 overexpression in photoreceptor cells is sufficient to prevent RhlG69D-induced retinal degeneration. These results illuminate a previously unappreciated rate-limiting requirement for Zn2+in ERAD and suggest ZIP7 overexpression as a potential gene therapy for autosomal dominant retinitis pigmentosa and other degenerative diseases. dZIP7 promotes border cell migration and prevents ER stress

[0288] Drosophila ovaries are composed of ovarioles, which are strings of egg chambers (FIG. 1A) progressing through 14 stages of development, culminating with mature eggs. Each egg chamber is composed of 15 nurse cells and one oocyte (germ cells), surrounded by -850 epithelial follicle cells. At stage 9 (FIG. IB), 4-8 border cells round up at the anterior end of the egg chamber, delaminate from the follicular epithelium, and migrate posteriorly, reaching the anterior border of the oocyte by stage 10. Border cell clusters are composed of 4-6 migratory cells that surround and carry two non-migratory polar cells. Expression of dZIP7 RNAi in the outer, migratory border cells using fruitlessGal419inhibited migration (FIGS. 1C, 1G). The defect was rescued by coexpression of UAS-dZIP7::V5 (FIGS. ID, 1G). Reduction of dZIP::GFP confirmed the effectiveness of the RNAi (FIGS. IE, IE’ and IF, IF’). Border cell migration was also impaired when dZIP7 RNAi was driven by the c306Gal4 (FIG. 1G), which is expressed in both polar and migratory cells. FruitlessGal4-driven RNAi impaired border cell migration at least as much as c306Gal4, indicating that dZIP7 was primarily required in the outer, migratory cells (FIG. 1G). This was further supported by mosaic clone analysis, in which the severity of border cell migration defects was proportional to the number of outer, migratory cells that were homozygous mutant (FIGS. 7A-7C) and mutant cells were mostly excluded from leading positions (FIGS. 7D-7E).

[0289] dZIP7 is the ortholog of ZIP7, a Zn2+transporter that moves Zn2+from the ER to the cytoplasm and suppresses ER stress in many cell types including mammalian intestinal stem cells8 2°, cancer cells20, Drosophila imaginal discs 9 and even in plants13. We confirmed that dZIP7 localizes to the ER in border cells where it co-localized with the ER chaperone PDI much more significantly than with F-actin or the nucleus (FIG. 8). Additionally, homozygous mutant dZIP7 border cells expressed the ER stress reporter Xbpls::EGFP21whereas dZIP7+ / _and dZIP7+ / +cells did not (FIGS. 1H-1H” ). We also observed increased expression of the ER chaperone PDI in homozygous dZIP7 RNAi-expressing follicle cell clones (FIGS. 11-11”), a phenotype that we rescued with a wild type, V5-tagged dZIP7 transgene (FIGS. 1J-1J”). Accumulation of XBP1 and PDI are indicative of induction of an adaptive unfolded protein response (UPR)22. We conclude that cells lacking dZIP7 experience ER stress and impaired migration, raising the question as to whether ER stress inhibits motility. dZIP7 is limiting for ERAD

[0290] To address whether ER stress inhibits migration, we expressed a misfolded rhodopsin protein, RhlG69D, known to induce ER stress2324. Misfolded rhodopsin accumulates in the ER and causes retinal degeneration in flies and humans24-27. We found that RhlG69Daccumulated to high levels intracellularly in border cells, induced ER stress, and blocked their migration (FIGS. 2A-2E), demonstrating that ER stress is sufficient to impair motility.

[0291] Since loss of dZIP7 caused ER stress, we wondered if dZIP7 overexpression might suppress ER stress, so we co-expressed RhlG69Dand dZIP7::V5 in border cells. Interestingly, dZIP7::V5 expression restored normal border cell migration (FIGS. 2B, 2C), reduced ER stress (FIGS. 2B, 2D) and virtually eliminated RhlG69Dprotein (FIGS. 2B, 2E).

[0292] Together, these results suggest that dZIP7 is a limiting factor for degrading misfolded Rhl, which is known to be degraded by ERAD23-25, though a role for ZIP7 in ERAD has not previously been described.

[0293] If ZIP7 enhances ERAD, the proteasome inhibitor MG132 should block the ability of dZIP7 overexpression to promote RhlG69Ddegradation. In the absence of MG132, ZIP7 overexpression virtually eliminated RhlG69Dprotein (FIGS. 2B, 2E). In contrast, MG132 largely prevented the ability of ZIP7 to promote RhlG69Ddegradation (FIGS. 2F-2H). MG132 also prevented dZIP7-mediated rescue of the RhlG69Dborder cell migration defect (FIG. 2G compared to FIG. 2B).

[0294] Misfolded rhodopsin is ubiquitinated by ER membrane-localized ubiquitin ligases (HRD1 and SORDD1 / 2), extracted from the ER membrane via the p97 chaperone TER94, and degraded by the proteasome (FIG. 2I)35'37. Our data described above implicate ZIP7 and cytosolic Zn2+as limiting for this process. To investigate which step of the ERAD process requires dZIP7, we used an antibody that recognizes ubiquitinated proteins to compare wild type, dZIP7- overexpressing, and dZIP7 RNAi cells. One hypothesis was that dZIP7 might provide Zn2+to ubiquitin ligases such as Hrdl and SORDD1 / 2, which are Zn2+-binding proteins that reside in the ER membrane (FIG. 21). If this were true, we might expect dZIP7 overexpression to increase - and dZIP7 RNAi to decrease - the abundance of polyubiquitinated proteins (PUBs). However, we observed the opposite effect. dZIP7 RNAi increased polyubiquitinated proteins (FIGS. 2J, 2J’) compared to the lacZ control (FIGS. 2K, 2K’ and FIGS. 10A-10B’). Conversely, dZIP7 overexpression essentially eliminated detectable polyubiquitinated proteins (FIGS. 2L, 2M and FIGS. 10C, 10C’). We also found that RhlG69Dexpression increased the accumulation of polyubiquitinated proteins compared to the control (FIGS. 10D-10J’), and ZIP7 overexpression suppressed the effect (FIGS. 10F, 10F’, 10G and 10J, 101’). Since polyubiquitinated proteins accumulated in the absence of dZIP7, we conclude that ZIP7 functions downstream of ubiquitination to enhance ERAD.

[0295] The observation that expression of dZIP7 reduced the abundance of polyubiquitinated proteins suggested that it might be required instead for deubiquitination. There are many deubiquitinating enzymes (DUBs), but Rpnll stood out as a top candidate. Whereas deubiquitination by some DUBs can rescue proteins from degradation, deubiquitination by Rpnl 1 is an essential prerequisite for entry of client proteins into the proteasome core and thus, like dZIP7, is essential for misfolded protein degradation28(FIG. 3A). Furthermore, unlike most DUBs, Rpnll requires Zn2+for catalysis29-31. In fact, Rpnl l is the only Zn2+-requiring DUB implicated in ERAD32. In vitro, when ubiquitinated substrates and the 26S proteasome assemble into complexes chelating Zn2+inactivates Rpnll. Addition of Zn2+is sufficient to reactivate Rpnl l and stimulate deubiquitination29. Since dZIP7 transports Zn2+to the cytosol 20 and promotes loss of ubiquitinated proteins and degradation of RhlG69D, we hypothesized that dZIP7 might be limiting for Rpnll activity.

[0296] In vitro, when Rpnl l is activated by addition of Zn2+but the 20S core proteases are blocked, deubiquitinated substrates stall and accumulate29. So we tested the effect of ZIP7 overexpression in the presence of MG132. We observed that ZIP7 still stimulated deubiquitination (FIGS. 2N-2P, FIGS. 10K-10L’). Together with the observation that ZIP7 overexpression failedto degrade RhlG69Din the presence of MG132 (FIGS. 2F-2H), this result supports the idea that ZIP7 enhances deubiquitination of misfolded proteins upstream of 20S proteasomal degradation, possibly by providing Zn2+to Rpnl 1.

[0297] If ZIP7 promotes Rpnl 1 -mediated deubiquitination, an Rpnll inhibitor should block the ability of dZIP7 to enhance deubiquitination of misfolded proteins. To test this prediction, we used the potent and selective Rpnll inhibitor capzimin, which blocks the Zn2+within the Rpnl l catalytic site33. Capzimin largely prevented the effects of ZIP7 on deubiquitination (FIGS. 2Q-2S, FIGS. 10M-10N’). We conclude that dZIP7 enhances the obligatory Zn2+- and Rpnll -dependent deubiquitination of misfolded proteins prior to proteasomal degradation.ZIP7-mediated Zn2+transport is limiting for ERAD in Drosophila

[0298] ZIP7 resides in the ER membrane and transports Zn2+from the ER to the cytosol20,34. To test whether the Zn2+transporter activity of dZIP7 is important for border cell migration, we introduced point mutations, H315A and H344A, which replace histidine residues that are required for Zn2+transport35(FIG. 3 A, purple) and are conserved between ZIP7 and a more distant family member from Arabidopsis IRT1 (FIG. 11 A). As controls, we engineered dZIP7H187Aand dZIP7H1S3Amutants (FIG. 3A, green), which are not located in the ZIP7 core transmembrane domains involved in Zn2+transport and are predicted not to affect transport35. We generated transgenic flies expressing the mutants under Gal4 / UAS control and included a V5 tag so that we could monitor protein abundance and localization. We then co-expressed each of these RNAi- resistant transgenes with dZIP7 RNAi and evaluated protein expression and border cell migration. The point mutations predicted not to disrupt Zn2+transport, dZIP7H187Aand dZIP7H183A, rescued border cell migration to nearly wild type levels (FIGS. 3B, 3C) whereas neither dZIP7H344Anor dZIP7H315Aprovided significant rescue (FIGS. 3D, 3E), as quantified in FIG. 3F. All the proteins were stably expressed and correctly localized to the ER (FIGS. 3B-3E). Therefore, the lack of rescue was likely a consequence of impaired transporter activity rather than impaired expression or localization. Furthermore, dZIP7H187Aand dZIP7H183A, which rescued border cell migration and Notch and EGFR ER accumulation, also prevented ER stress (FIGS. 11F, 11G), whereas neither dZIP7H344Anor dZIP7H315Arescued ER stress (FIGS. 11D, HE). In ZIP7 knockdown cells, membrane proteins, such as Notch and EGFR, accumulate abnormally in the ER9, and we found this to be true in border cell clones expressing dZIP7 RNAi (FIG. 3G), demonstrating the generality of this effect. Furthermore, the Zn2+transport-deficient proteins (dZIP7H344AanddZIP7II315A) failed to rescue ER accumulation of Notch and EGFR, when clonally co-expressed with dZIP7 RNAi in a subset of border cells. Notch and EGFR accumulated abnormally in the cells co-expressing dZIP7H344Aor dZIP7H315Awith dZIP7 RNAi (FIGS. 3G-3J’, magenta nuclei) compared to neighboring control cells (blue nuclei).

[0299] In contrast, dZIP7 RNAi-expressing cells that co-expressed dZIP7H187Aor dZIP7H183Acells (magenta nuclei in FIGS. 3K-3N’) exhibited similar Notch and EGFR levels as the neighboring control cells (blue nuclei in FIGS. 3K-3N’). The results are quantified in FIGS. 30 and 3P. From these experiments, we conclude that Zn2+transport is an essential function of dZIP7 in promoting ERAD.Zn2+is limiting for deubiquitination of proteasome client proteins in human cells under proteotoxic stress

[0300] ZIP7 is nearly ubiquitously expressed in cells from organisms as diverse as plants and animals13. Our results, combined with the observations that the proteasome is highly abundant28whereas cytosolic [Zn2+]-free is extremely low (-100 pM - 1 nM)20, suggested that Zn2+might be rate limiting for proteasome activity, particularly when cells are stressed. So, we tested the ability of a Zn2+ionophore, pyrithione (an organic salt of zinc capable of permeating cell membranes)20to enhance deubiquitination of proteins in human cells.

[0301] This ionophore has previously been shown to rescue ER stress in ZIP7-deficient HeLa 211 cells20. So, we incubated HeLa cells with MG132 to induce ER stress and increase the abundance of polyubiquitinated proteins (FIG. 3Q). We then tested the effect of the Zn2+ionophore. The added Zn2+reduced the accumulation of polyubiquitinated proteins in the MG132-treated cells (FIG. 3Q). We conclude that cytosolic Zn2+is limiting for proteasomal degradation of misfolded proteins in cells with ER stress.ZIP7 is required to degrade misfolded Notch and EGFR whereas ER stress independently inhibits Notch transcriptional responses

[0302] Notch and EGFR accumulate abnormally in the ER in ZIP7-deficient fly wing disc cells9and human cancer cells36. We also observed abnormal accumulation of Notch (FIGS. 4A, 4A’ and FIGS. 9A, 9A’) and EGFR (FIGS. 4B, 4B’ and FIGS. 9B, 9B’) but not E-cadherin (FIGS. 9C, 9C’) in dZIP7 knockdown follicle cell clones, supporting the generality of the phenomenon. Accumulation of Notch and EGFR following inhibition of ZIP7 in flies and mammalian cells haspreviously been attributed to a defect in protein trafficking9’28. However, our results implicate defective ERAD as the likely cause of abnormal intracellular Notch and EGFR in ZIP7 k.d. cells.

[0303] Interestingly, neither Notch (FIGS. 4C, 4C’) nor EGFR (FIGS. 4D, 4D’) accumulated abnormally in RhlG69D-expressing follicle cells, including border cells. Yet, both ZIP7 knockdown cells (FIGS. 4E, 4E’) and RhlG69D-expressing cells (FIGS. 4G, 4G’) exhibited reduced Notch transcriptional responses (FIGS. 4F, 4H). We conclude that dZIP7 knockdown causes two independent effects on Notch: accumulation of misfolded protein in the ER due to reduced ERAD and inhibition of Notch transcriptional activity, presumably as a consequence of the ER stress response37-39. dZIP7 overexpression prevents retinal degeneration caused by RhlG6 l)

[0304] The observations that dZIP7 overexpression is sufficient to degrade RhlG69D, reduce ER stress, and rescue border cell migration and the ubiquity of ZIP7 and proteasomes suggests that dZIP7 overexpression might also be effective at suppressing retinal degeneration due to folding-defective rhodopsin. To test this hypothesis, we co-express UAS-dZIP7::V5 with UAS- RhlG69Din fly photoreceptor cells using GMR-Gal4. Eye morphology is normal in flies expressing dZIP7 alone (FIGS. 5A, 5B and 5G) whereas RhlG69Dcauses severe disruption of eye morphology compared to controls1723 25 27and (FIGS. 5C, 5D, and 5G). Co-expression of RhlG69Dand dZIP7 fully rescue eye morphology in the majority of flies examined (FIGS. 5E, 5F and 5G).

[0305] To test whether dZIP7 overexpression could restore visual function, we carry out electroretinogram (ERG) recordings, which measure the summed responses of all retinal cells to light. Control flies display a comeal negative receptor potential upon turning on a light stimulus, which quickly decays to baseline upon termination of the light (FIG. 5H). The large maintained component of the ERG results principally from activation of the phototransduction cascade. The on- and off-transient responses, which are nearly coincident with the initiation and cessation of the light stimulus (FIG. 5H), depend on synaptic transmission from the photoreceptor cells to postsynaptic cells in the optic lobes. Expression of RhlG69Din photoreceptor cells greatly diminishes the amplitude of the ERG, and eliminates the on- and off transients (FIGS. 51, 5L-5N).DiscussionA model for dZ!P7 function: Zn2+transport from the ER to the cytosol is limiting for ERAD and mitigation of ER stress

[0306] dZIP7 is a conserved protein that goes by names including ZRT1 in yeast, IRT1 in plants, dZIP7 or Catsup in Drosophila, and SLC39a7 / Zip7 / Ke4 in mammals. While many studies come to a common conclusion that loss or inhibition of ZIP7 disrupts ER homeostasis in cells from plants to flies and humans8, 1-20-36-40-41, the mechanism has been unclear13. Moreover, it has been unclear whether the disparate phenotypes caused by ZIP7 knockdown, which include defects in intestinal stem cell self-renewal, B cell differentiation, cell motility and survival amongst others, have a common underlying cause or represent pleiotropic activities of the ZIP7 protein. The data presented here provide evidence for an unanticipated and possibly unifying mechanism. Our data support a model in which dZIP7 promotes ERAD and prevents ER stress by providing free Zn2+to the catalytic site of the Rpnl 1 DUB in the proteasome lid (FIG.6A). This role for dZIP7 is critical since free Zn2+is present at exceedingly low intracellular levels20, and therefore could be limiting. In the absence of ZIP7, misfolded / unfolded proteins accumulate and cause ER stress (FIGS. 6B and 6C). Although Rpnl l is a top candidate for ZIP7-provided Zn2+, our experiments cannot rule out that other Zn2+binding proteins may contribute to the beneficial effects of ZIP7. For example, the chaperone p97 extracts misfolded and ubiquitinated proteins from the ER and transfers them to the 26S proteasome, and one subunit of p97, Npl4, is a zinc finger protein. Our experiments do not rule out the possibility that ZIP7 promotes deubiquitination by enhancing p97, in addition to, or instead of Rpnll activity.

[0307] It is striking that dZIP7 overexpression is sufficient to enhance proteasomal degradation of misfolded proteins, including RhlG69D, preventing the harmful effects of ER stress including blindness (FIGS. 6D and 6E).

[0308] In contrast to earlier work that suggested that ZIP7 primarily promotes trafficking of membrane proteins such as Notch and EGFR9>36, our results show that release of Zn2+from the ER to the cytosol via ZIP7 is limiting for ERAD. We favor the model that the step in ERAD that is most sensitive to [Zn2+] is deubiquitination of client proteins by Rpnl l and / or p97-mediated transfer of misfolded proteins to the proteasome,29,3°. Our in vivo genetic and pharmacological studies are concordant with in vitro biochemistry. Rpnll requires Zn2+to deubiquitinate client proteins29 31 33. This is an essential step so that the client protein can enter into the 20S proteasome for degradation by trypsin, chymotrypsin, and caspase-like endoproteases. Rpnl l also enhances proteasomal degradation by allowing ubiquitin to be recycled. Worden et al29were able toassemble a complex in vitro composed of a ubiquitinated substrate and the 26S proteasome, including Rpnl l . Tn the presence of a Zn2+chelator, the complex assembles but Rpn l l is catalytically inactive, so ubiquitinated substrate accumulates. Upon addition of Zn2+, Rpnll deubiquitinates the client protein. In the presence of a 20S protease inhibitor (epoxomicin), Rpnl 1 deubiquitinates the client but it is not degraded, so deubiquitinated protein accumulates.

[0309] We observe remarkably similar effects by manipulating dZIP7 in vivo as Worden et al. observed by manipulating Zn2+in vitro29. In the absence of dZIP7, ubiquitinated proteins accumulate, whereas upon overexpression of dZIP7 in the presence of MG132, deubiquitinated substrate proteins (e.g., RhlG69D) accumulate. It is reasonable to propose that cytosolic free Zn2+could be rate limiting because proteasomes are abundant, whereas cytosolic free Zn2+is 305 vanishingly rare at ~1 nM20, which is ~100-fold less than the typical free cytosolic [Ca2+]. We propose that ZIP7 provides rate -limiting Zn2+to p97 and / or Rpnll , and thus that the level of ZIP7 determines a cells’ capacity to degrade misfolded proteins. In support of this idea and the generality of the mechanism proposed here, we found that increasing intracellular Zn2+enhanced deubiquitination of proteins in a human cell line in the presence of MG132.

[0310] Free Zn2+is exceptionally rare in the cytosol despite the fact that Zn2+is the second most abundant divalent cation in cells because nearly all Zn2+is bound to proteins. While an essential trace element, excess cytosolic Zn2+can be toxic42, yet ZIP7 overexpression did not cause detectable harm either to follicle cells in the ovary or to photoreceptor cells in the eye. This suggests that the Zn2+transported to the cytosol via ZIP7 might predominantly exert its effects locally. ZIP7 may not directly bind to the ERAD machinery though because it was not detected in an extensive proteomic analysis.43The human genome encodes 24 Zn2+transporters, 14 of which belong to the ZIP family which move Zn2+into the cytoplasm from outside the cell or from inside an organelle while 10 are members of the ZnT family which transport Zn2+out of the cell or into organelles4445. The large sizes of these families are consistent with the idea that local Zn2+sources may be important for promoting necessary Zn2+-dependent processes without increasing global levels, which might be toxic.Biomedical implications of the role ofZIP7 in ERAD

[0311] The ability of dZIP7 overexpression to alleviate the ER stress and cellular defects due to RhlG69Dexpression has some general biomedical implications. Dominant mutations in rhodopsin that impair folding and cause accumulation in the ER cause retinal degeneration in human patients26, for which there is no effective prevention or therapy. Over-expression ofproteins that enhance ERAD is a promising therapeutic strategy. Additionally, toxic protein aggregates have been proposed to kill neurons by inhibiting ERAD in numerous neurodegenerative diseases including Huntington’s, Alzheimer’s, Parkinson’s, frontotemporal dementia, and others, even when the toxic protein is not localized in the ER4647. Thus, strategies to enhance ERAD may be useful in treating multiple degenerative diseases.

[0312] The suppression of ER stress and border cell migration by dZIP7 overexpression is consistent with the observation that ZIP7 is over-expressed in numerous cancers where it promotes survival, proliferation and migration and correlates with disease progression, invasion, and metastasis10'12,48. A ZIP7 inhibitor was identified in a screen for drugs to treat Notchdependent cancers, based on the model that ZIP7 is important for Notch trafficking36. We show that ER stress impairs Notch transcriptional activity independent of any trafficking defect because RhlG69Dinhibits Notch signaling without abnormal Notch or EGFR protein accumulation. How ER stress or the UPR inhibits Notch signaling is not clear, but the observation that a pharmacological inhibitor of ZIP7 was identified as a suppressor of Notch signaling by the Notch intracellular domain (NTCD) in cultured U2OS osteosarcoma cells36suggests that there is a deeply conserved requirement for ZIP7 for Notch transcriptional activity. Nolin et al36showed that ZIP7 inhibition causes accumulation of full-length Notch and a decrease in the NICD, and concluded that Notch activation by proteolysis was likely perturbed upon inhibition of ZIP7. An alternative interpretation is that full-length Notch accumulates in the ER lumen due to inhibition of ERAD, and that the NICD is affected by the global ER stress response.

[0313] Our results suggest that ZIP7 inhibitors might be effective against cancers that rely especially heavily on proteasomes. Proteasome inhibitors such as bortezomib are approved for the treatment of B cell malignancies including multiple myeloma and mantle cell lymphoma49. Our results suggest that ZIP7 inhibitors might be repurposed to treat those cancers as well, especially considering that resistance typically develops against a single therapeutic agent. Interestingly, hypomorphic mutations in ZIP7 cause a B cell deficiency due to defects in B cell differentiation in human patients50. Although the mechanism underlying this phenotype is unknown, our results implicate ZIP7 in the UPR, and mutations that compromise the UPR also cause B cell deficiency due to defective B cell differentiation. So, the results presented here suggest a possible link between these otherwise disparate observations. B cell development appears to depend upon a functional UPR and ER stress response, perhaps to ensure resilience to the natural ER stress B cells experience when they secrete large quantities of antibody.

[0314] Finally, the similarities in dZIP7 functions and phenotypes across disparate cells, tissues, and organisms suggests that the border cell system offers an excellent model for deciphering the fundamental and conserved effects of this protein in vivo.MethodsDrosophila genetics

[0315] The dZIP7 mutant was generated by ethyl methanesulfonate (EMS) mutagenesis 15. The mutation results in a glycine (G) to aspartic acid (D) substitution at amino acid 178. The FLP / FRT system was used to generate dZIP7G178D homozygous mutant clones by combining FRT40A-dZ / P7G178D with hsFLP12,yw;ubi:GFPnls, FRT40A or hsFLP12,yw;ubi:RFPnls, FRT40A / ( CyO). dZIP7: :GFP expression pattern was visualized using the line from VDRC 318542 in the fTRG stocks library. The UAS-dZIP7RNAi transgenic line is from VDRC 100095 P{KK103630]VIE-260B. Wild type rescue w[*]; sna[Sco] / CyO; P{w[+mC]=UAS-dZIP7.V5}6 Bloomington 63229. Additional transgenic Drosophila stocks used: UAS-white RNAi / Cyo is a lab stock 51, UAS-PleRNAi Bloomington 25796 yfl] v[lj; P{y[+t7.7] v[+tl.8]=TRiP.JF01813}attP2, UAS-Ple is Bloomington 37539 w[*]; Pfw[+mC]=UAS- ple.T}331f2, O-fucosyltransferasel Bloomington 9376 P{UAS-O-futl.O}ll.l , UAS- Notch.Intracellular.Domain on the third chromosome was a gift from Artavanis-Tsakonas lab 52. The following stocks were from the Bloomington Drosophila Stock Center: ER stress marker UAS-Xbpl-EGFP.HG (#60731, w[*]; P{w[+mC]=UAS-Xbpl.EGFP.HG]3) and UAS-HSC70-3 (#5843 w

[0126] ; P[w[+mC]=UAS-Hsc70-382 3.WT}B). dZIP7 point mutations were cloned into vector pUASt-attb with forward primer ctctgaatagggaattgggATGGCCAAACAAGTGGCTGA (SEQ ID NO:5_ and reverse primer ccgcagatctgttaacgtcaCGTAGAATCGAGACCGAGGAGAG (SEQ ID NO:6). The vector was injected into attp2 flies yl w67c23; P{CaryP}attP2 by BestGene Inc. UAS-RhlG69Dwas a gift from Dr. Hyung Don Ryoo lab25.Design of UAS-RNAi-resistant dZIP7 point mutations

[0316] When generating UAS-dZIP7-point-mutations, we designed the construct so it cannot be targeted by the dZIP7RNAi sequences by substituting redundant codons for the same amino acids within the region targeted by the RNAi. The RNAi resistant sequence is reported below.References

[0317] 1. Karagoz, G.E., Acosta- Alvear, D., and Walter, P. (2019). The UnfoldedProtein Response: Detecting and Responding to Fluctuations in the Protein-Folding Capacity of the Endoplasmic Reticulum. Cold Spring Harb. Perspect. Biol. 11. 10.1101 / cshperspect.a033886.

[0318] 2. Schubert, U., Anton, L.C., Gibbs, J., Norbury, C.C., Yewdell, J.W., andBennink, J.R. (2000). Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 404, 770-774. 10.1038 / 35008096.

[0319] 3. Tonoki, A., Kuranaga, E., Tomioka, T., Hamazaki, J., Murata, S., Tanaka,K., and Miura, M. (2009). Genetic evidence linking age-dependent attenuation of the 26S proteasome with the aging process. Mol. Cell. Biol. 29, 1095-1106. 10.1128 / MCB.01227 -08.

[0320] 4. G. Martinez, C. Duran-Aniotz, F. Cabral-Miranda, J. P. Vivar, C. Hetz,Endoplasmic reticulum proteostasis impairment in aging. Aging Cell. 16, 615-623 (2017).

[0321] 5. Hetz, C. (2012). The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat. Rev. Mol. Cell Biol. 13, 89-102. 10.1038 / nrm3270.

[0322] 6. Njomen, E., and Tepe, J.J. (2019). Proteasome activation as a new therapeutic approach to target proteotoxic disorders. J. Med. Chem. 62, 6469-6481. 10.1021 / acs.jmedchem.9b00101.

[0323] 7. George, D.E., and Tepe, J.J. (2021). Advances in proteasome enhancement by small molecules. Biomolecules 11. 10.3390 / bioml l 121789.

[0324] 8. Ohashi, W., Kimura, S., Iwanaga, T., Furusawa, Y., Irie, T., Izumi, H.,Watanabe, T., Hijikata, A., Hara, T., Ohara, O., et al. (2016). Zinc Transporter SLC39A7 / ZIP7 Promotes Intestinal Epithelial Self-Renewal by Resolving ER Stress. PLoS Genet. 12, el006349. 10. 1371 / journal.pgen.1006349.

[0325] 9. Groth, C., Sasamura, T., Khanna, M.R., Whitley, M., and Fortini, M.E.(2013). Protein trafficking abnormalities in Drosophila tissues with impaired activity of the ZIP7 zinc transporter Catsup. Development 140, 3018-3027. 10. 1242 / dev.088336.

[0326] 10. Sheng, N., Yan, L., You, W., Tan, G., Gong, J., Chen, H., Yang, Y., Hu,L., and Wang, Z. (2017). Knockdown of SLC39A7 inhibits cell growth and induces apoptosis in human colorectal cancer cells. Acta Biochim Biophys Sin (Shanghai) 49, 926-934. 10.1093 / abbs / gmx094.

[0327] 11. Wei, Y., Dong, J., Li, F., Wei, Z., and Tian, Y. (2017). Knockdown ofSLC39A7 suppresses cell proliferation, migration and invasion in cervical cancer. EXCLI J. 16, 1165-1176. 10.17179 / excli2017-690.I l l

[0328] 12. Hogstrand, C., Kille, P., Nicholson, R.I., and Taylor, K.M. (2009). Zinc transporters and cancer: a potential role for ZIP7 as a hub for tyrosine kinase activation. Trends Mol. Med. 15, 101-111. 10.1016 / j.molmed.2009.01.004.

[0329] 13. Adulcikas, J., Norouzi, S., Bretag, L., Sohal, S.S., and Myers, S. (2018).The zinc transporter SLC39A7 (ZIP7) harbours a highly-conserved histidine-rich N-terminal region that potentially contributes to zinc homeostasis in the endoplasmic reticulum. Comput. Biol. Med. 100, 196-202. 10.1016 / j.compbiomed.2018.07.007.

[0330] 14. Y. Liu, D. J. Montell, Identification of mutations that cause cell migration defects in mosaic clones. Development. 126, 1869-1878 (1999).

[0331] 15. Wang, X., Bo, J., Bridges, T„ Dugan, K.D., Pan, T„ Chodosh, L.A., andMontell, D.J. (2006). Analysis of cell migration using whole-genome expression profiling of migratory cells in the Drosophila ovary. Dev. Cell 10, 483-495. 10.1016 / j.devcel.2006.02.003.

[0332] 16. D. J. Montell, Border-cell migration: the race is on. Nat. Rev. Mol. CellBiol. 4, 13-24 (2003).

[0333] 17. Kurada, P., and O’Tousa, J.E. (1995). Retinal degeneration caused by dominant rhodopsin mutations in Drosophila. Neuron 14, 571-579. 10.1016 / 0896- 6273(95)90313-5.

[0334] 18. Colley, N.J., Cassill, J.A., Baker, E.K., and Zuker, C.S. (1995). Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration. Proc Natl Acad Sci USA 92, 3070-3074. 10.1073 / pnas.92.7.3070.

[0335] 19. Borensztejn, A., Boissoneau, E., Fernandez, G., Agnes, F., and Pret, A.-M.(2013). JAK / STAT autocontrol of ligand-producing cell number through apoptosis. Development 140, 195-204. 10.1242 / dev.079046.

[0336] 20. Woodruff, G., Bouwkamp, C.G., de Vrij, F.M., Lovenberg, T.,Bonaventure, P., Kushner, S.A., and Harrington, A.W. (2018). The zinc transporter SLC39A7 (ZIP7) is essential for regulation of cytosolic zinc levels. Mol. Pharmacol. 94, 1092-1100. 10.1124 / mol.ll8.112557.

[0337] 21. Sone, M., Zeng, X., Larese, J., and Ryoo, H.D. (2013). A modified UPR stress sensing system reveals a novel tissue distribution of IRE1 / XBP1 activity during normal Drosophila development. Cell Stress Chaperones 18, 307-319. 10.1007 / sl2192-012-0383-x.

[0338] 22. Karagoz, G.E., Aragon, T., and Acosta-Alvear, D. (2019). Recent advances in signal integration mechanisms in the unfolded protein response, [version 1; peer review: 2 approved]. FlOOORes. 8. 10.12688 / flOOOresearch. 19848.1.

[0339] 23. M.-J. Kang, H. D. Ryoo, Suppression of retinal degeneration in Drosophila by stimulation of ER-associated degradation. Proc Natl Acad Sci USA. 106, 17043-17048 (2009).

[0340] 24. Xu, J., Zhao, H., and Wang, T. (2020). Suppression of retinal degeneration by two novel ERAD ubiquitin E3 ligases SORDD1 / 2 in Drosophila. PLoS Genet. 16, el009172. 10.1371 / journal.pgen. 1009172.

[0341] 25. Ryoo, H.D., Domingos, P.M., Kang, M.-J., and Steller, H. (2007).Unfolded protein response in a Drosophila model for retinal degeneration. EMBO J. 26, 242-252. 10.1038 / sj.emboj.7601477.

[0342] 26. C. H. Sung, B. G. Schneider, N. Agarwal, D. S. Papermaster, J. Nathans,Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa. Proc Natl Acad Sci USA. 88, 8840-8844 (1991).

[0343] 27. Chow, C.Y., Kelsey, K.J.P., Wolfner, M.F., and Clark, A.G. (2016).Candidate genetic modifiers of retinitis pigmentosa identified by exploiting natural variation in Drosophila. Hum. Mol. Genet. 25, 651-659. 10.1093 / hmg / ddv502.

[0344] 28. Nolin, E., Gans, S., Llamas, L., Bandyopadhyay, S., Brittain, S.M.,Bemasconi-Elias, P., Carter, K.P., Loureiro, J.J., Thomas, J.R., Schirle, M., et al. (2019). Discovery of a ZIP7 inhibitor from a Notch pathway screen. Nat. Chem. Biol. 15, 179-188.

[0345] 29. Worden, E.J., Dong, K.C., and Martin, A. (2017). An AAA Motor-DrivenMechanical Switch in Rpnl 1 Controls Deubiquitination at the 26S Proteasome. Mol. Cell 67, 799- 811. e8. 10.1016 / j.molcel.2017.07.023.

[0346] 30. Yoshida, H., Matsui, T., Yamamoto, A., Okada, T., and Mori, K. (2001).XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107, 881-891.

[0347] 31. Pathare, G.R., Nagy, I., Sledz, P., Anderson, D.J., Zhou, H.-J., Pardon, E.,Steyaert, J., Forster, F., Bracher, A., and Baumeister, W. (2014). Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpnll. Proc Natl Acad Sci USA 111, 2984-2989. 10.1073 / pnas. 1400546111.

[0348] 32. Snyder, N.A., and Silva, G.M. (2021). Deubiquitinating enzymes (DUBs):Regulation, homeostasis, and oxidative stress response. J. Biol. Chem. 297, 101077. 10.1016 / j.jbc.2021.101077.

[0349] 33. J. Li, T. Yakushi, F. Parlati, A. L. Mackinnon, C. Perez, Y. Ma, K. P. Carter,S. Colayco, G. Magnuson, B. Brown, K. Nguyen, S. Vasile, E. Suyama, L. H. Smith, E. Sergienko, A. B. Pinkerton, T. D. Y. Chung, A. E. Palmer, I. Pass, S. Hess, R. J. Deshaies, Capzimin is apotent and specific inhibitor of proteasome isopeptidase Rpnll. Nat. Chem. Biol. 13, 486-493 (2017).

[0350] 34. Taylor, K.M., Hiscox, S., Nicholson, R.I., Hogstrand, C., and Kille, P.(2012). Protein kinase CK2 triggers cytosolic zinc signaling pathways by phosphorylation of zinc channel ZIP7. Sci. Signal. 5, rai l. 10.1126 / scisignal.2002585.

[0351] 35. Kambe, T., Tsuji, T., Hashimoto, A., and Itsumura, N. (2015). The physiological, biochemical, and molecular roles of zinc transporters in zinc homeostasis and metabolism. Physiol. Rev. 95, 749-784. 10.1152 / physrev.00035.2014.

[0352] 36. Nolin, E., Gans, S., Llamas, L., Bandyopadhyay, S., Brittain, S.M.,Bemasconi-Elias, P., Carter, K.P., Loureiro, J. J., Thomas, J.R., Schirle, M., et al. (2019). Discovery of a ZIP7 inhibitor from a Notch pathway screen. Nat. Chem. Biol. 15, 179-188. 10.1038 / s41589-018-0200-7.

[0353] 37. Yoshida, H., Matsui, T., Yamamoto, A., Okada, T., and Mori, K. (2001).XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107, 881 -891. 10.1016 / s0092-8674(01)0061 1-0.

[0354] 38. Arensdorf, A.M., Diedrichs, D., and Rutkowski, D.T. (2013). Regulation of the transcriptome by ER stress: non-canonical mechanisms and physiological consequences. Front. Genet. 4, 256. 10.3389 / fgene.2013.00256.

[0355] 39. Walter, P., and Ron, D. (2011). The unfolded protein response: from stress pathway to homeostatic regulation. Science 334, 1081-1086. 10.1126 / science.l209038.

[0356] 40. Tuncay, E., Bitirim, V.C., Durak, A., Carrat, G.R.J., Taylor, K.M., Rutter,G.A., and Turan, B. (2017). Hyperglycemia- Induced Changes in ZIP7 and ZnT7 Expression Cause Zn2+ Release From the Sarco(endo)plasmic Reticulum and Mediate ER Stress in the Heart. Diabetes 66, 1346-1358. 10.2337 / db 16- 1099.

[0357] 41. Tuncay, E., Bitirim, C.V., Olgar, Y., Durak, A., Rutter, G.A., and Turan,B. (2019). Zn2+- transporters ZIP7 and ZnT7 play important role in progression of cardiac dysfunction via affecting sarco(endo)plasmic reticulum-mitochondria coupling in hyperglycemic cardiomyocytes. Mitochondrion 44, 41-52. 10.1016 / j.mito.2017.12.011.

[0358] 42. Kukic, I., Kelleher, S.L., and Kiselyov, K. (2014). Zn2+ efflux through lysosomal exocytosis prevents Zn2+-induced toxicity. J. Cell Sci. 127, 3094-3103. 10.1242 / jcs. 145318.

[0359] 43. Christianson, J.C., Olzmann, J. A., Shaler, T.A., Sowa, M.E., Bennett, E.J.,Richter, C.M., Tyler, R.E., Greenblatt, E.J., Harper, J.W., and Kopito, R.R. (2011). Defininghuman ERAD networks through an integrative mapping strategy. Nat. Cell Biol. 14, 93-105. !0.1038 / ncb2383.

[0360] 44. Kambe, T., Hashimoto, A., and Fujimoto, S. (2014). Current understanding of ZIP and ZnT zinc transporters in human health and diseases. Cell. Mol. Life Sci. 71, 3281- 3295. 10.1007 / s00018-014-1617-0.

[0361] 45. Bin, B.-H., Seo, J., and Kim, S.T. (2018). Function, structure, and transport aspects of ZIP and znt zinc transporters in immune cells. J. Immunol. Res. 2018, 9365747. 10.1155 / 2018 / 9365747.

[0362] 46. Shacham, T., Sharma, N., and Lederkremer, G.Z. (2019). Protein misfolding and ER stress in huntington’s disease. Front. Mol. Biosci. 6, 20. 10.3389 / fmolb.2019.00020.

[0363] 47. S. A. Oakes, F. R. Papa, The role of endoplasmic reticulum stress in human pathology. Annu. Rev. Pathol. 10, 173-194 (2015).

[0364] 48. Taylor, K.M., Vichova, P.,...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of treating an aging -related disease or a disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress of a subject, the method comprising administering a therapeutically effective amount of zinc transporter protein 7 (ZIP7) to the subject.

2. The method of claim 1, wherein said treating reduces protein misfolding, protein aggregation, or endoplasmic reticulum stress, increases survival, increases lifespan, or prevents, decreases, delays, or reverses senescence of the subject.

3. The method of claim 1 or 2, further comprising administering a therapeutically effective amount of regulatory particle non-ATPase 11 (Rpnl 1) in combination with the therapeutically effective amount of the ZIP7 to the subject.

4. The method of any one or claims 1-3, wherein the ZIP7 and / or the Rpnl 1 are provided by a recombinant nucleic acid or vector system.

5. The method of claim 4, wherein the recombinant nucleic acid is RNA or DNA.

6. The method of claim 5, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the ZIP7 or Rpnl 1 in the subject.

7. The method of claim 4, wherein the vector system comprises one or more viral vectors or plasmids.

8. The method of claim 7, wherein the one or more viral vectors comprise an adeno- associated viral vector, an adenoviral vector, a lenti viral vector, anello viral vector, or a retroviral vector.

9. The method of claim 7 or 8, wherein the vector system is administered intravenously.

10. The method of claim 7 or 8, wherein the vector system is administered locally to a target organ or tissue in need of treatment.

11. The method of any one of claims 7-10, wherein expression of the ZIP7 and / or the Rpnll is inducible.

12. The method of any one of claims 7-11, wherein the vector system comprises a first vector comprising a first coding sequence encoding ZIP7 and a second vector comprising a second coding sequence encoding Rpnl l, wherein the ZIP7 and the Rpnl l are co-expressed in vivo in the subject in effective amounts sufficient to increase survival, increase lifespan, or prevent, decrease, or delay senescence of the subject.

13. The method of claim 12, wherein the first vector comprises a first promoter operably linked to the first coding sequence encoding the ZIP7.

14. The method of claim 13, wherein the first promoter is a tissue-specific promoter or cell type-specific promoter.

15. The method of claim 4, wherein the cell type-specific promoter is specific for a postmitotic cell type.

16. The method of claim 15, wherein the postmitotic cell is a neuron, a cardiomyocyte, a skeletal muscle myofiber, a retinal ganglion cell, a cochlear hair cell, an osteocyte, or an adipocyte.

17. The method of claim 12, wherein the first coding sequence encoding the ZIP7 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated first coding sequence encoding the ZIP7 at the chromosomal locus.

18. The method of any one of claims 12-17, wherein the second vector comprises a second promoter operably linked to the second coding sequence encoding the Rpnl l.

19. The method of claim 18, wherein the second promoter is a tissue-specific promoter or cell type-specific promoter.

20. The method of claim 19, wherein the cell type-specific promoter is specific for a postmitotic cell type.

21. The method of claim 20, wherein the postmitotic cell is a neuron, a cardiomyocyte, a skeletal muscle myofiber, a retinal ganglion cell, a cochlear hair cell, an osteocyte, or an adipocyte.

22. The method of claim 12, wherein the second coding sequence encoding the Rpnl 1 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated second coding sequence encoding the Rpnll at the chromosomal locus.

23. The method of any one of claims 7-1 1 , wherein the ZIP7 and the Rpnl 1 are provided by a multicistronic vector comprising a first coding sequence encoding ZIP7 and a second coding sequence encoding Rpnll, wherein the ZIP7 and the Rpnl l are co-expressed in vivo in the subject in effective amounts sufficient to increase survival, increase lifespan, or prevent, decrease, or delay senescence of the subject.

24. The method of claim 23, wherein the multicistronic vector further comprises a viral T2A peptide or internal ribosome entry site (IRES) sequence operably linked to the first coding sequence encoding the ZIP7 and the second coding sequence encoding the Rpnl l to allow co-expression of the ZIP7 and the Rpnll.

25. The method of any one of claims 1-24, wherein the ZIP7 is provided by a viral particle comprising a vector comprising a coding sequence encoding the ZIP7.

26. The method of any one of claims 3-25, wherein the Rpnll is provided by a viral particle comprising a vector comprising a coding sequence encoding the Rpnl l.

27. The method of claim 25 or 26, wherein the viral particle is administered intravenously.

28. The method of claim 25 or 26, wherein the viral particle is administered locally to a target organ or tissue in need of treatment.

29. The method of claim 1 or 2, wherein the ZIP7 and / or the Rpnl 1 are provided by genetically modifying the genome of the subject to express the ZIP7 and / or the Rpnll.

30. The method of claim 29, wherein the genome of the subject is genetically modified using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator- like effector nuclease (TALEN).

31. The method of any one of claims 1-30, wherein the ZIP7 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.

32. The method of any one of claims 3-31, wherein the Rpnl 1 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4.

33. The method of any one of claims 1-32, wherein the subject is a mammal.

34. The method of claim 33, wherein the mammal is human.

35. A composition for use in a method of treating an aging-related disease and extending lifespan of a subject or treating a disorder associated with protein misfolding, protein aggregation, or endoplasmic reticulum stress, the composition comprising zinc transporter protein 7 (ZIP7) or a vector system encoding ZIP7.

36. The composition of claim 35, further comprising regulatory particle non-ATPase 11 (Rpnl 1) or a vector system encoding Rpnll.

37. The composition of claim 35 or 36, further comprising a pharmaceutically acceptable excipient.

38. A method of increasing survival or extending lifespan of a cell, organoid, or organism, the method comprising introducing into the cell, organoid, or organism an effective amount of zinc transporter protein 7 (ZIP7).

39. The method of claim 38, further comprising introducing into the cell, organoid, or organism an effective amount of regulatory particle non-ATPase 11 (Rpnll).

40. The method of claim 38 or 39, the cell is a postmitotic cell.

41. The method of claim 40, wherein the postmitotic cell is a neuron, a cardiomyocyte, a skeletal muscle myofiber, a retinal ganglion cell, a cochlear hair cell, an osteocyte, or an adipocyte.

42. The method of any one of claims 38-41, wherein the ZIP7 and / or the Rpnl 1 are provided by a recombinant nucleic acid or vector system.

43. The method of claim 42, wherein the recombinant nucleic acid is RNA or DNA.

44. The method of claim 43, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the ZIP7 or Rpnl l in the cell, organoid, or organism.

45. The method of claim 42, wherein the vector system comprises one or more viral vectors or plasmids.

46. The method of claim 45, wherein the one or more viral vectors comprise an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, anelloviral vector, or a retroviral vector.

47. The method of claim 45 or 46, wherein expression of the ZIP7 and / or the Rpnl 1 is inducible.

48. The method of any one of claims 45-47, wherein the vector system comprises a first vector comprising a first coding sequence encoding ZIP7 and a second vector comprising a second coding sequence encoding Rpnl 1, wherein the ZIP7 and the Rpnl 1 are co-expressed in the cell, organoid, or organism in effective amounts sufficient to increase survival or prevent, decrease, or delay senescence of the cell, organoid, or organism.

49. The method of claim 48, wherein the first vector comprises a first promoter operably linked to the first coding sequence encoding the ZIP7.

50. The method of claim 49, wherein the first promoter is a tissue-specific promoter or cell type-specific promoter.

51. The method of claim 48, wherein the first coding sequence encoding the ZIP7 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated first coding sequence encoding the ZIP7 at the chromosomal locus.

52. The method of any one of claims 48-51, wherein the second vector comprises a second promoter operably linked to the second coding sequence encoding the Rpnl 1.

53. The method of claim 52, wherein the second promoter is a tissue-specific promoter or cell type-specific promoter.

54. The method of any one of claims 48-51, wherein the second coding sequence encoding the Rpnl 1 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated second coding sequence encoding the Rpnll at the chromosomal locus.

55. The method of claim 48, wherein the ZIP7 and the Rpnl 1 are provided by a multicistronic vector comprising a first coding sequence encoding ZIP7 and a second coding sequence encoding Rpnl l, wherein the ZIP7 and the Rpnl 1 are co-expressed in the cell,organoid, or organism in effective amounts sufficient to increase survival, increase lifespan, or prevent, decrease, or delay senescence of the cell, organoid, or organism.

56. The method of claim 55, wherein the multicistronic vector further comprises a viral T2A peptide or internal ribosome entry site (IRES) sequence operably linked to the first coding sequence encoding the ZIP7 and the second coding sequence encoding the Rpnl 1 to allow co-expression of the ZIP7 and the Rpnll.

57. The method of any one of claims 45-56, wherein the ZIP7 is provided by a viral particle comprising a vector comprising a coding sequence encoding the ZIP7.

58. The method of any one of claims 45-57, wherein the Rpnl 1 is provided by a viral particle comprising a vector comprising a coding sequence encoding the Rpnl l.

59. The method of claim 57 or 58, wherein the viral particle is administered intravenously.

60. The method of claim 57 or 58, wherein the viral particle is administered locally to a target organ or tissue in need of treatment.

61. The method of any one of claims 38-41, wherein the ZIP7 and / or the Rpnl 1 are provided by genetically modifying the genome of the cell, organoid, or organism to express the ZIP7 and / or the Rpnl l.

62. The method of claim 61, wherein the genome of the cell, organoid, or organism is genetically modified using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

63. The method of claim 62, wherein the ZIP7 is introduced into the cell, organoid, or organism, by a method comprising: introducing a donor polynucleotide into the cell, organoid, or organism, wherein the donor polynucleotide comprises a 5' homology arm that hybridizes to a 5' genomic targetsequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence encoding the ZIP7; introducing an RNA-guided nuclease into the cell, organoid, or organism; and introducing a guide RNA into the cell, organoid, or organism, wherein the guide RNA forms a complex with the RNA-guided nuclease such that the guide RNA directs the RNA- guided nuclease to a genomic target sequence at a genomic target locus to be modified, wherein the RNA-guided nuclease creates a double-stranded break in the genomic target sequence, wherein the donor polynucleotide is integrated at the genomic target locus recognized by its 5' homology arm and 3' homology arm by homology directed repair (HDR) such that a genetically modified cell, organoid, or organism is produced, wherein the survival or the lifespan of the genetically modified cell, organoid, or organism is increased compared to the unmodified cell, organoid, or organism.

64. The method of claim 62 or 63, wherein the Rpnll is introduced into the cell, organoid, or organism, by a method comprising: introducing a donor polynucleotide into the cell, organoid, or organism, wherein the donor polynucleotide comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence encoding the Rpnl l; introducing an RNA-guided nuclease into the cell, organoid, or organism; and introducing a guide RNA into the cell, organoid, or organism, wherein the guide RNA forms a complex with the RNA-guided nuclease such that the guide RNA directs the RNA- guided nuclease to a genomic target sequence at a genomic target locus to be modified, wherein the RNA-guided nuclease creates a double-stranded break in the genomic target sequence, wherein the donor polynucleotide is integrated at the genomic target locus recognized by its 5' homology arm and 3' homology arm by homology directed repair (HDR) such that a genetically modified cell, organoid, or organism is produced, wherein the survival or the lifespan of the genetically modified cell, organoid, or organism is increased compared to the unmodified cell, organoid, or organism.

65. The method of any one of claims 63 or 64, wherein the donor polynucleotide, the RNA-guided nuclease, and the guide RNA are provided by one or more vectors.

66. The method of claim 65, wherein the one or more vectors are viral vectors or plasmids.

67. The method of claim 66, wherein the viral vectors are lentivirus vectors, retrovirus vectors, adenovirus vectors, or adeno- associated virus vectors68. The method of any one of claims 65-67, wherein the one or more vectors are introduced into the cell, organoid, or organism by transient transfection or stable transfection.

69. The method of any one of claims 65-69, wherein expression of the RNA-guided nuclease or the guide RNA is inducible.

70. The method of any one of claims 63 or 64, wherein the RNA-guided nuclease is provided by a mRNA encoding the RNA-guided nuclease, wherein translation of the mRNA results in production of the RNA-guided nuclease in the cell, organoid, or organism.

71. The method of claim 63 or 64, wherein the RNA-guided nuclease and the guide RNA are provided as a ribonucleoprotein complex of the RNA-guided nuclease with the guide RNA, wherein the ribonucleoprotein complex is introduced into the cell, organoid, or organism by microinjection.

72. The method of any one of claims 63-71, wherein the RNA-guided nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease.

73. The method of claim 72, wherein the Cas nuclease is Cas9 or Casl2a.

74. The method of any one of claims 38-73, wherein the organoid is a retinal organoid.

75. The method of claim 74, wherein the retinal organoid is used as a model of retinal degeneration.

76. The method of claim 75, wherein the retinal organoid comprises a mutation associated with an inherited retinopathy.

77. The method of claim 75 or 76, wherein the effective amount of the ZIP7 or the combination of the effective amount of the ZIP7 and the effective amount of the Rpnll prevents, delays, decreases, or at least partially reverses the retinal degeneration in the retinal organoid.

78. The method of any one of claims 38-77, wherein the ZIP7 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.

79. The method of any one of claims 39-78, wherein the Rpnl 1 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4.

80. A method of preventing, delaying, or reversing senescence of a postmitotic cell, the method comprising introducing into the postmitotic cell an effective amount of zinc transporter protein 7 (ZIP7).

81. The method of claim 80, further comprising introducing into the postmitotic cell, an effective amount of regulatory particle non-ATPase 11 (Rpnl 1).

82. The method of claim 80 or 81, wherein the postmitotic cell is a neuron, a cardiomyocyte, a skeletal muscle myofiber, a retinal ganglion cell, a cochlear hair cell, an osteocyte, or an adipocyte.

83. The method of any one of claims 80-82, wherein the ZIP7 and / or the Rpnl 1 are provided by a recombinant nucleic acid or vector system.

84. The method of claim 83, wherein the recombinant nucleic acid is RNA or DNA.

85. The method of claim 84, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the ZIP7 or Rpnl 1 in the postmitotic cell.

86. The method of claim 83, wherein the vector system comprises one or more viral vectors or plasmids.

87. The method of claim 86, wherein the one or more viral vectors comprise an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, anelloviral vector, or a retroviral vector.

88. The method of claim 86 or 87, wherein expression of the ZIP7 and / or the Rpnl 1 is inducible.

89. The method of any one of claims 86-88, wherein the vector system comprises a first vector comprising a first coding sequence encoding ZIP7 and a second vector comprising a second coding sequence encoding Rpnll, wherein the ZIP7 and the Rpnl l are co-expressed in the postmitotic cell in effective amounts sufficient to extend survival of the postmitotic cell.

90. The method of claim 89, wherein the first vector comprises a first promoter operably linked to the first coding sequence encoding the ZIP7.

91. The method of claim 89, wherein the first coding sequence encoding the ZIP7 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated first coding sequence encoding the ZIP7 at the chromosomal locus.

92. The method of any one of claims 89-91, wherein the second vector comprises a second promoter operably linked to the second coding sequence encoding the Rpnl l.

93. The method of any one of claims 89-91, wherein the second coding sequence encoding the Rpnl 1 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated second coding sequence encoding the Rpnl 1 at the chromosomal locus.

94. The method claim 86 or 87, wherein the ZIP7 and the Rpnl 1 are provided hy a multicistronic vector comprising a first coding sequence encoding ZIP7 and a second coding sequence encoding Rpnl l, wherein the ZIP7 and the Rpnll are co-expressed in the postmitotic cell in effective amounts sufficient to extend survival of the postmitotic cell.

95. The method of claim 94, wherein the multicistronic vector further comprises a viral T2A peptide or internal ribosome entry site (IRES) sequence operably linked to the first coding sequence encoding the ZIP7 and the second coding sequence encoding the Rpnll to allow co-expression of the ZIP7 and the Rpnll.

96. The method of any one of claims 80-95, wherein the ZIP7 is provided by a viral particle comprising a vector comprising a coding sequence encoding the ZIP7.

97. The method of any one of claims 80-96, wherein the Rpnl 1 is provided by a viral particle comprising a vector comprising a coding sequence encoding the Rpnll.

98. The method of any one of claims 80-82, wherein the ZIP7 and / or the Rpnl 1 are provided by genetically modifying the genome of the postmitotic cell to express the ZIP7 and / or the Rpnll.

99. The method of claim 98, wherein the genome of the postmitotic cell is genetically modified using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator- like effector nuclease (TALEN).

100. The method of claim 99, wherein the ZIP7 is introduced into the postmitotic cell by a method comprising: introducing a donor polynucleotide into the postmitotic cell, wherein the donor polynucleotide comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence encoding the ZIP7; introducing an RNA-guided nuclease into the postmitotic cell; andintroducing a guide RNA into the postmitotic cell, wherein the guide RNA forms a complex with the RNA-guided nuclease such that the guide RNA directs the RNA-guided nuclease to a genomic target sequence at a genomic target locus to be modified, wherein the RNA-guided nuclease creates a double-stranded break in the genomic target sequence in the intron, wherein the donor polynucleotide is integrated at the genomic target locus recognized by its 5' homology arm and 3' homology arm by homology directed repair (HDR) such that a genetically modified postmitotic cell is produced, wherein the survival or the lifespan of the genetically modified postmitotic cell is increased compared to the unmodified postmitotic cell.

101. The method of claim 99 or 100, wherein the Rpnl 1 is introduced into the postmitotic cell by a method comprising: introducing a donor polynucleotide into the postmitotic cell, wherein the donor polynucleotide comprises a 5' homology arm that hybridizes to a 5’ genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence encoding the Rpnl 1 ; introducing an RNA-guided nuclease into the postmitotic cell; and introducing a guide RNA into the postmitotic cell, wherein the guide RNA forms a complex with the RNA-guided nuclease such that the guide RNA directs the RNA-guided nuclease to a genomic target sequence at a genomic target locus to be modified, wherein the RNA-guided nuclease creates a double-stranded break in the genomic target sequence in the intron, wherein the donor polynucleotide is integrated at the genomic target locus recognized by its 5’ homology arm and 3' homology arm by homology directed repair (HDR) such that a genetically modified postmitotic cell is produced, wherein the survival or the lifespan of the genetically modified postmitotic cell is increased compared to the unmodified postmitotic cell.

102. The method of claim 100 or 101, wherein the donor polynucleotide, the RNA- guided nuclease, and the guide RNA are provided by one or more vectors.

103. The method of claim 102, wherein the one or more vectors are viral vectors or plasmids.

104. The method of claim 103, wherein the viral vectors are lentivirus vectors, retrovirus vectors, adenovirus, or adeno-associated virus vectors105. The method of any one of claims 102- 104, wherein the one or more vectors are introduced into the cell, organoid, or organism by transient transfection or stable transfection.

106. The method of any one of claims 102-105, wherein expression of the RNA- guided nuclease or the guide RNA is inducible.

107. The method of claim 100 or 101, wherein the RNA-guided nuclease is provided by a mRNA encoding the RNA-guided nuclease, wherein translation of the mRNA results in production of the RNA-guided nuclease in the postmitotic cell.

108. The method of claim 100 or 101, wherein the RNA-guided nuclease and the guide RNA are provided as a ribonucleoprotein complex of the RNA-guided nuclease with the guide RNA, wherein the ribonucleoprotein complex is introduced into the postmitotic cell by microinjection.

109. The method of any one of claims 100-108, wherein the RNA-guided nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease.

110. The method of claim 109, wherein the Cas nuclease is Cas9 or Casl2a.

111. The method of any one of claims 80- 110, wherein the organoid is a retinal organoid.

112. The method of claim 111, wherein the retinal organoid is used as a model of retinal degeneration.

113. The method of claim 111 or 112, wherein the retinal organoid comprises a mutation associated with an inherited retinopathy.

114. The method of any one of claims 80-113, wherein the ZIP7 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.1 15. The method of any one of claims 80-1 14, wherein the Rpnl l comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4.

116. A method of treating a retinopathy in a subject, the method comprising administering a therapeutically effective amount of zinc transporter protein 7 (ZIP7) to the subject.

117. The method of claim 116, further comprising administering a therapeutically effective amount of regulatory particle non-ATPase 11 (Rpnl l) in combination with the therapeutically effective amount of the ZIP7 to the subject.

118. The method of claim 116 or 117, wherein the therapeutically effective amount of the ZIP7 or ZIP7 in combination with Rpnl 1 prevents, delays, decreases, or reverses retinal degeneration.

119. The method of any one of claims 116-118, wherein the ZIP7 and / or the Rpnl l are administered locally to the retina.

120. The method of any one or claims 116-119, wherein the ZIP7 and / or the Rpnll are provided by a recombinant nucleic acid or vector system.

121. The method of claim 120, wherein the recombinant nucleic acid is RNA or DNA.

122. The method of claim 121, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the ZIP7 or Rpnl 1 in the subject.

123. The method of claim 120, wherein the vector system comprises one or more viral vectors or plasmids.

124. The method of claim 123, wherein the one or more viral vectors comprise an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, anelloviral vector, or a retroviral vector.

125. The method of claim 123 or 124, wherein the vector system is administered intravenously.

126. The method of claim 123 or 124, wherein the vector system is administered locally to the retina.

127. The method of any one of claims 123-126, wherein expression of the ZIP7 and / or the Rpnl 1 is inducible.

128. The method of any one of claims 123-127, wherein the vector system comprises a first vector comprising a first coding sequence encoding ZIP7 and a second vector comprising a second coding sequence encoding Rpnll, wherein the ZIP7 and the Rpnl l are co-expressed in vivo in the subject in effective amounts sufficient to prevent, delay, decrease, or reverse retinal degeneration in an eye of the subject.

129. The method of claim 128, wherein the first vector comprises a first promoter operably linked to the first coding sequence encoding the ZIP7.

130. The method of claim 129, wherein the first promoter is a retina-specific promoter.

131. The method of claim 130, wherein the retina- specific promoter is specific for a retinal-specific cell type.

132. The method of claim 131, wherein the retina- specific cell type is a bipolar cell, a retina amacrine cell, a horizontal cell, a retinal ganglion cell, Muller glia, a rod photoreceptor cell or a cone photoreceptor cell.

133. The method of claim 128, wherein the first coding sequence encoding the ZIP7 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated first coding sequence encoding the ZIP7 at the chromosomal locus.

134. The method of claim 128, wherein the second vector comprises a second promoter operably linked to the second coding sequence encoding the Rpnl 1.

135. The method of claim 134, wherein the second promoter is a retina-specific promoter.

136. The method of claim 135, wherein the retina- specific promoter is specific for a retinal-specific cell type.

137. The method of claim 136, wherein the retina-specific cell type is a bipolar cell, a retina amacrine cell, a horizontal cell, a retinal ganglion cell, Muller glia, a rod photoreceptor cell or a cone photoreceptor cell.

138. The method of claim 128, wherein the second coding sequence encoding the Rpnl 1 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated second coding sequence encoding the Rpnll at the chromosomal locus.

139. The method of any one of claims 123-127, wherein the ZIP7 and the Rpnl 1 are provided by a multicistronic vector comprising a first coding sequence encoding ZIP7 and a second coding sequence encoding Rpnll, wherein the ZIP7 and the Rpnl l are co-expressed in vivo in the subject in effective amounts sufficient to prevent, delay, decrease, or reverse retinal degeneration in an eye of the subject.

140. The method of claim 139, wherein the multicistronic vector further comprises a viral T2A peptide or internal ribosome entry site (IRES) sequence operably linked to the first coding sequence encoding the ZIP7 and the second coding sequence encoding the Rpnll to allow co-expression of the ZIP7 and the Rpnl l.

141. The method of any one of claims 123-140, wherein the ZIP7 is provided by a viral particle comprising a vector comprising a coding sequence encoding the ZIP7.

142. The method of any one of claims 123-141, wherein the Rpnl 1 is provided by a viral particle comprising a vector comprising a coding sequence encoding the Rpnl 1.

143. The method of claim 141 or 142, wherein the viral particle is administered intravenously.

144. The method of claim 141 or 142, wherein the viral particle is administered locally to the retina.

145. The method of any one of claims 116-118, wherein the ZIP7 and / or the Rpnl 1 are provided by genetically modifying the genome of the subject to express the ZIP7 and / or the Rpnl l .

146. The method of claim 145, wherein the genome of the subject is genetically modified using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator- like effector nuclease (TALEN).

147. The method of any one of claims 116-146, wherein the ZIP7 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.

148. The method of any one of claims 116-147, wherein the Rpnl 1 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4.

149. The method of any one of claims 116-148, wherein the subject is a mammal.

150. The method of claim 149, wherein the mammal is human.

151. A composition for use in a method of treating a retinopathy, the composition comprising zinc transporter protein 7 (ZIP7) or a vector system encoding Z1P7.

152. The composition of claim 151, further comprising regulatory particle non- ATPase 11 (Rpnl 1) or a vector system encoding Rpnl 1.

153. The composition of claim 151 or 152, further comprising a pharmaceutically acceptable excipient.

154. A method of increasing proteasomal degradation of poly-ubiquitinated proteins in a cell, the method comprising introducing an effective amount of zinc transporter protein 7 (ZIP7) into the cell.

155. The method of claim 154, further comprising introducing an effective amount of regulatory particle non-ATPase 11 (Rpnl l) into the cell.

156. The method of 154 or 155, wherein the ZIP7 and / or the Rpnll are provided by a recombinant nucleic acid or vector system.

157. The method of claim 156, wherein the recombinant nucleic acid is RNA or DNA.

158. The method of claim 157, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the ZIP7 or Rpnl l in the cell.

159. The method of claim 156, wherein the vector system comprises one or more viral vectors or plasmids.

160. The method of claim 159, wherein the one or more viral vectors comprise an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, anelloviral vector, or a retroviral vector.

161. The method of claim 159 or 160, wherein expression of the ZIP7 and / or the Rpnl 1 is inducible.

162. The method of any one of claims 159- 161 , wherein the vector system comprises a first vector comprising a first coding sequence encoding ZIP7 and a second vector comprising a second coding sequence encoding Rpnll, wherein the ZIP7 and the Rpnl l are co-expressed in the cell in effective amounts sufficient to increase proteasomal degradation of poly-ubiquitinated proteins in the cell.

163. The method of claim 162, wherein the first vector comprises a first promoter operably linked to the first coding sequence encoding the ZIP7.

164. The method of claim 162, wherein the first coding sequence encoding the ZIP7 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated first coding sequence encoding the ZIP7 at the chromosomal locus.

165. The method of any one of claims 162- 164, wherein the second vector comprises a second promoter operably linked to the second coding sequence encoding the Rpnl l.

166. The method of any one of claims 162-164, wherein the second coding sequence encoding the Rpnl l is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated second coding sequence encoding the Rpnll at the chromosomal locus.

167. The method claim 156, wherein the ZIP7 and the Rpnl l are provided by a multicistronic vector comprising a first coding sequence encoding ZIP7 and a second coding sequence encoding Rpnl l, wherein the ZIP7 and the Rpnll are co-expressed in the cell in effective amounts sufficient to extend survival of the cell.

168. The method of claim 167, wherein the multicistronic vector further comprises a viral T2A peptide or internal ribosome entry site (IRES) sequence operably linked to the first coding sequence encoding the ZIP7 and the second coding sequence encoding the Rpnll to allow co-expression of the ZIP7 and the Rpnl l.

169. The method of claim 154 or 155, wherein the ZIP7 is provided by a viral particle comprising a vector comprising a coding sequence encoding the ZIP7.

170. The method of claim 155, wherein the Rpnll is provided by a viral particle comprising a vector comprising a coding sequence encoding the Rpnl 1.

171. The method of claim 154 or 155, wherein the ZIP7 and / or the Rpnl 1 are provided by genetically modifying the genome of the cell to express the ZIP7 and / or the Rpnll.

172. The method of claim 171, wherein the genome of the cell is genetically modified using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

173. The method of any one of claims 154- 172, wherein the ZIP7 comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.

174. The method of any one of claims 155-172, wherein the Rpnl l comprises or consists of an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4.

Images