Compositions and methods for using codon optimized ZNF865

Abstract

Disclosed are nucleic acid sequences comprising the sequence of SEQ ID NO: 5. Disclosed are nucleic acid sequences comprising the sequence of SEQ ID NO: 1 having one or more codon optimizing mutations. Disclosed are nucleic acid sequences comprising the sequence of SEQ ID NO:2. Disclosed are vectors comprising the nucleic acid sequence of SEQ ID NO: 5. Disclosed are vectors comprising the nucleic acid sequence of SEQ ID NO: 2. Disclosed are vectors comprising the nucleic acid sequence of SEQ ID NO:1 having one or more codon optimizing mutations. Disclosed are compositions comprising any of the disclosed nucleic acid sequences or vectors. Disclosed are methods of using the disclosed nucleic acid sequences, vectors or compositions.

Description

ATTORNEY DOCKET NO. 21101.0497P1COMPOSITIONS AND METHODS FOR USING CODON OPTIMIZED ZNF865CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No.63 / 755.800, filed on February 7, 2025, which is incorporated by reference herein in its entirety.REFERENCE TO SEQUENCE LISTINGThe Sequence Listing submitted February 6, 2026 as a xml file named “21101.0497Pl.xml,” created on February' 6, 2026, and having a size of 26,342 bytes is hereby incorporated by¬ reference pursuant to 37 C.F.R. § 1.52(e)(5).STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R01 AR083990 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Degenerative disc disease (DDD) is the leading cause of disability' worldwide, characterized by the breakdown of intervertebral discs (IVD). Stem cell therapies using adipose-derived stem cells (ASCs) are promising options to treat DDD because of stem cells’ ability to regenerate and restore functional tissue to the IVD.

[0004] Recently, a zine-finger (ZNF) protein, ZNF865 (also known as BLST), has been discovered that has shown potential as a tool to enhance cell and tissue engineering therapies. ZNFs represent the largest group of regulatory' proteins within eukary otic genomes and due to their extensive interactions, ZNFs regulate diverse cellular processes, such as transcription, cell migration, growth, proliferation, and differentiation. ZNF865 regulates cellular senescence, cell cycle, DNA replication, and protein processing and could be used as a gene therapy to rescue cell populations from senescence.

[0005] Previous studies used CRISPR-activation systems to upregulate native ZNF865. The current disclosure provides an improved gene therapy system with easier delivery and improved results.BRIEF SUMMARY

[0006] Disclosed are nucleic acid sequences comprising the sequence of SEQ ID NO: 1 having one or more codon optimizing mutations.ATTORNEY DOCKET NO. 2II0I.0497P1

[0007] Disclosed are nucleic acid sequences comprising the sequence of SEQ ID NO:2.

[0008] Disclosed are nucleic acid sequences comprising the sequence of SEQ ID NO:5.

[0009] Disclosed are vectors comprising the nucleic acid sequence disclosed herein.

[0010] Disclosed are vectors comprising the nucleic acid sequence of SEQ ID NO: 1 having one or more codon optimizing mutations.

[0011] Disclosed are vectors comprising the nucleic acid sequence of SEQ ID NO:2.

[0012] Disclosed are vectors comprising the nucleic acid sequence of SEQ ID NO:5.

[0013] Disclosed are compositions comprising any of the disclosed nucleic acid sequences, proteins, or vectors.

[0014] Disclosed are methods of increasing ZNF865 expression in a cell comprising contacting a cell with a composition comprising any of the disclosed nucleic acid sequences, proteins, or vectors.

[0015] Disclosed are methods of treating a subject in need thereof comprising administering to a subject in need thereof a composition compnsing one or more of the disclosed nucleic acid sequences, proteins, or vectors.

[0016] Disclosed are methods of increasing deposition of a target gene, such as aggrecan and / or collagen II, in the extracellular matrix of a cell using one or more of the nucleic acid sequences, proteins, or vectors disclosed herein.

[0017] Disclosed are methods of increasing deposition of a target gene, such as aggrecan and / or collagen II, in the extracellular matrix of a cell comprising contacting a cell with a composition comprising one or more of the nucleic acid sequences, proteins, or vectors disclosed herein.

[0018] Disclosed are methods of upregulating a target gene comprising contacting a cell with a composition comprising one or more of the nucleic acid sequences or vectors disclosed herein, wherein one or more of the nucleic acid sequences, proteins, or vectors disclosed herein upregulates the target gene.

[0019] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.BRIEF DESCRIPTION OF THE DRAWINGSATTORNEY DOCKET NO. 21101.0497P1

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

[0021] FIG. 1 shows qRT-PCR verifying upregulation of codon optimized ZNF865 with the EFS- codon optimized ZNF865 transgene lentiviral plasmid (bar on right) (n=3-4, *=p<0.05).

[0022] FIG. 2 shows quantified cell proliferation data showing that codon optimized ZNF865 expression using the promoters EFS (middle bar) and EFl A (far right bar) results in increased rates of proliferation compared to the control group (NTC, far left bar)). (n=8, *=p<0.05).

[0023] FIG. 3 shows a nucleic acid sequence alignment between wild type ZNF865 and codon optimized ZNF865. FIG. 3 shows about a 77% sequence similarity between the two.

[0024] FIG. 4 shows a percentage of EGFP expressing dNPs upon mRNA / LNPs treatment at increasing mRNA concentrations.

[0025] FIG. 5 shows fluorescence microscopy images of degenerative nucleus pulposus cells (dNPs) expressing EGFP. The dNPs were treated with LNPs at increasing ionizable lipid concentrations for 24 h, and cell viability was determined using the CCK-8 assay.

[0026] FIG. 6 shows viability of dNPs upon LNPs treatment for 24 h.

[0027] FIG. 7 shows a percent cell proliferation of untreated human dNPs and ZNF865 mRNA / LNPs treated human dNPs from 59-y ear-old female.

[0028] FIG. 8 shows doubling time of untreated human dNPs and ZNF865 mRNA / LNPs treated human dNPs from 59-year-old femaleDETAILED DESCRIPTION

[0029] The disclosed method and compositions may be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0030] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may 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.

[0031] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methodATTORNEY DOCKET NO. 2II0I.0497P1and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a peptide is disclosed and discussed and a number of modifications that can be made to a number of molecules including the amino acids are discussed, each and every combination and permutation of the peptide and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E. and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B. and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to. steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.A. Definitions

[0032] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary7. 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 limit the scope of the present invention which will be limited only by the appended claims.

[0033] It must be noted that as used herein and in the appended claims, the singular forms "a ", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid sequence" includes a plurality of such nucleic acid sequences, reference to "the vector” is a reference to one or more vectors and equivalents thereof known to those skilled in the art, and so forth.

[0034] The w ord “or” as used herein means any one member of a particular list and alsoATTORNEY DOCKET NO. 21101.0497P1includes any combination of members of that list.

[0035] As used herein, the term "wild-type" refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally-occurring source.

[0036] The terms "variant" and "mutant" are used interchangeably herein. As used herein, the term "mutant" refers to a modified nucleic acid or protein which displays the same characteristics when compared to a reference nucleic acid or protein sequence. A variant can be at least 65, 70, 75, 80, 85, 90, 95, or 99 percent homologous to a reference sequence. In some aspects, a reference sequence can be SEQ ID NO: 1 or SEQ ID NO: 421. Variants can also include nucleotide sequences that are substantially similar to sequences of miRNA disclosed herein. A ‘'variant” can mean a difference in some way from the reference sequence other than just a simple deletion of an N- and / or C-terminal nucleotide. Variants can also or alternatively include at least one substitution and / or at least one addition, there may also be at least one deletion. Alternatively, or in addition, variants can comprise modifications, such as non-natural residues at one or more positions with respect to a reference nucleic acid or protein.

[0037] Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative or variant. Generally, these changes are done on a few nucleotides to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances.

[0038] Generally, the nucleotide identity between individual variant sequences can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Thus, a “variant sequence” can be one with the specified identity to the parent or reference sequence (e.g. wildtype sequence) of the invention, and shares biological function, including, but not limited to. at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity7and / or activity of the parent sequence. For example, a “variant sequence” can be a sequence that contains 1, 2, or 3 4 nucleotide base changes as compared to the parent or reference sequence of the invention, and shares or improves biological function, specificity and / or activity of the parent sequence.

[0039] Thus, a “variant sequence” can be one with the specified identity to the parent sequence of the invention, and shares biological function, including, but not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%.96%. 97%. 98%. or 99% of the specificity and / or activity of the parent sequence. The variant sequence can also share at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity7and / or activity of a reference sequence (e.g. ZNF865 wild-ty pe sequence, SEQ ID NO:1 or SEQ ID NO:5 ).ATTORNEY DOCKET NO. 2II0I.0497P1

[0040] The phrase '‘nucleic acid” as used herein refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non-phosphodiester intemucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof

[0041] “Codon optimizing mutations” according to the present invention refers to one or more mutations as compared to a reference sequence to the coding sequence that do not result to a change of the amino acid sequence. For example, the use of the novel codon optimized ZNF865 sequences comprising codon optimizing mutations according to the present invention has the surprising technical advantage over prior art ZNF865 sequences that an improved expression and / or delivery in a given host cell organism is possible without negatively affecting ZNF865 activity by changing the nucleic acid sequence, thus, the structure or function of the resulting ZNF865 protein is preserved.

[0042] As used herein, the term “treat” or "treating" refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of. and / or reducing incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. For example, "treating" degenerative disc disease may refer to decreasing or preventing degeneration. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some aspects, “to treat” can mean to administer a nucleic acid, vector, or composition of the invention to a subject, such as a human or other mammal (for example, an animal model), that has an increased susceptibility for developing degenerative disc disease, or that has degenerative disc disease, in order to prevent or delay a worsening of the effects of the disease or condition, or to partially or fully reverse the effects of the disease.

[0043] In some aspects, "operably linked" as used herein refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid sequence capable of directing the transcription of a given gene (e.g., codon optimized ZNF865 nucleic acid) and / or the synthesis of a desired protein molecule is produced. Operably linked can mean that expression of a gene is under the control of a promoter (and / or enhancer) with which it is spatially connected. A promoter may be positioned 5' (upstream) or 3' (downstream) of the gene under its control. The distance betweenATTORNEY DOCKET NO. 2II0I.0497P1the promoter and the gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

[0044] As used herein, “subject” refers to the target of administration, e.g. an animal. Thus, the subject of the disclosed methods can be a vertebrate, such as a mammal. For example, the subject can be a human. In some aspects, the subject can be, but is not limited to, a human, a monkey, a dog, a cat, a horse, a pig, a goat, a rabbit, a rat, or a mouse. In some aspects, the subject can be a domesticated animal. The term does not denote a particular age or sex. Subject can be used interchangeably with “individual” or “patient”.

[0045] Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of theATTORNEY DOCKET NO. 2II0I.0497P1common general knowledge in the art.

[0047] Throughout the description and claims of this specification, the word '‘comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of’), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.B. Nucleic Acid Sequences

[0048] Disclosed are nucleic acid sequences capable of encoding wild type ZNF865.Disclosed are codon optimized nucleic acid sequences capable of encoding wild type ZNF865.

[0049] In some aspects, the disclosed nucleic acid sequences can be DNA or RNA sequences. In some aspects, the nucleic acid sequence can be mRNA.

[0050] Disclosed are nucleic acid sequences comprising the sequence of SEQ ID NO: 1, 2, 3, 4, or 5.

[0051] Thus, disclosed are nucleic acid sequences comprising the nucleic acid sequence of wild type (or native) ZNF865 comprising one or more codon optimizing mutations.

[0052] In some aspects, the wild type (native) nucleic acid sequence of ZNF865 is ATGGAGGCGAACCCAGCGGGCAGCGGCGCCGGGGGTGGCGGGAGCAGCGG CATCGGGGGCGAGGACGGGGTGCACTTCCAGAGCTACCCCTTCGACTTCC TGGAATTCCTCAACCACCAGCGCTTCGAGCCCATGGAACTGTATGGGGAA CACGCCAAGGCGGTGGCGGCCCTGCCCTGCGCCCCCGGCCCCCCGCCGCA GCCCCCGCCGCAGCCCCCTCCCCCGCAGTATGACTACCCGCCCCAGTCCA CCTTCAAGCCCAAGGCGGAGGTGCCCTCCTCGTCCTCGTCCTCGTCCTCC TCCTCCTCCTCTTCGTCCTCCTCGTCGTCATCTTCGTCCTCTTCCTCTTC CCAAGCCAAGAAGCCCGATCCGCCCCTGCCGCCCGCCTTCGGGGCGCCCC CTCCTCCCCTCTTTGACGCTGCTTTCCCCACTCCGCAGTGGGGCATCGTG GACCTCTCGGGGCACCAGCACTTGTTTGGGAACCTGAAGCGAGGAGGGCC CGCGTCCGGGCCGGGGGTGACGCCTGGGCTGGGCGCTCCCGCGGGGGCCC CAGGGCCGCTTCCTGCCCCCTCGCAGACCCCGCCAGGACCCCCCGCGGCG GCGGCCTGCGACCCCACCAAGGACGACAAGGGCTACTTCCGGAGACTGAA GTACCTGATGGAGCGGCGCTTCCCCTGCGGCGTGTGCCAGAAGTCCTTCA AGCAGTCCTCGCACCTGGTCCAGCACATGCTGGTGCACTCGGGGGAGAGG CCCTACGAATGCGGCGTCTGCGGCCGCACCTACAACCACGTGTCCAGCCTATTORNEY DOCKET NO. 21101.0497P1 CATCCGCCACCGCCGCTGCCACAAGGACGTGCCACCGGCCGCGGGGGGCC CGCCCCAGCCCGGCCCCCACCTCCCGCCGCTGGGCCTCCCAGCACCCGCT GCCAGCGCCGCCACCGCCGCCGCCCCCTCCACGGTGTCCTCGGGCCCTCC AGCCACGCCCGTGGCGCCTGCCCCCTCCGCAGACGGGAGCGCCGCCCCTG CTGGTGTTGGGGTGCCCCCTCCTGCCACCGGGGGTGGCGATGGCCCGTTC GCCTGCCCACTCTGCTGGAAGGTTTTCAAGAAGCCCAGTCACCTCCACCA GCACCAGATCATCCACACGGGCGAGAAGCCCTTCTCCTGCTCCGTGTGCA GCAAAAGCTTCAACCGCAGGGAGAGTCTGAAGCGCCACGTGAAGACGCAC TCGGCCGACCTCCTGCGCCTGCCCTGCGGCATCTGCGGGAAGGCCTTCCG CGACGCCTCCTACCTCCTCAAGCACCAGGCGGCCCACGCGGGGGCGGGCG CCGGGGGGCCTCGGCCCGTGTACCCCTGCGACCTGTGCGGCAAGTCCTAC TCGGCTCCGCAGAGCCTGCTCCGCCACAAGGCCGCCCACGCCCCGCCCGC TGCCGCTGCGGAGGCGCCCAAGGACGGGGCGGCCTCGGCCCCGCAGCCCC CGCCCACCTTCCCCCCGGGCCCGTACCTCCTGCCCCCCGACCCTCCCACC ACAGACAGCGAGAAGGCGGCGGCGGCCGCGGCGGCGGTGGTGTACGGCGC TGTGCCCGTCCCGCTCCTGGGCGCCCACCCGCTGCTGCTCGGCGGCGCGG GGACCAGCGGGGCGGGAGGCTCGGGCGCCAGCGTCCCAGGAAAGACGTTC TGCTGCGGCATCTGCGGGCGCGGCTTCGGGCGCCGCGAGACCCTGAAGCG CCATGAGCGCATCCACACGGGCGAGAAGCCCCACCAGTGCCCCGTGTGTG GGAAGCGCTTCCGCGAATCCTTCCACTTGAGCAAGCATCACGTGGTGCAC ACGCGCGAGCGGCCCTACAAGTGCGAGCTCTGCGGCAAGGTCTTCGGCTA CCCGCAGAGCCTCACCCGCCACCGCCAGGTGCACCGGCTCCAGCTGCCCT GCGCCCTGGCCGGGGCAGCCGGCCTCCCCTCCACCCAAGGCACACCGGGG GCCTGTGGGCCCGGGGCCTCGGGCACGTCTGCAGGGCCCACCGATGGGCT GAGCTACGCCTGCTCGGACTGCGGCGAGCACTTCCCGGATCTCTTTCACG TCATGAGTCACAAGGAGGTCCACATGGCAGAGAAGCCATACGGCTGCGAC GCCTGCGGCAAGACCTTCGGCTTCATCGAGAACCTCATGTGGCACAAGCT GGTCCACCAGGCCGCCCCCGAGCGCCTGCTCCCGCCCGCACCCGGCGGCC TGCAGCCCCCGGACGGCTCCAGCGGCACGGATGCGGCCAGCGTGCTGGAC AACGGGCTGGCGGGGGAGGTGGGGGCGGCCGTGGCGGCACTGGCAGGGGT GTCTGGGGGTGAGGACGCAGGCGGGGCGGCGGTGGCAGGTGCTGGCGGGG GTGCCAGTTCCGGCCCCGAGCGCTTCAGCTGTGCCACGTGCGGCCAGAGT TTCAAGCACTTCCTGGGCCTCGTGACTCACAAGTACGTGCACCTGGTGCG ACGGACCCTGGGCTGCGGCCTCTGCGGCCAGAGCTTCGCGGGCGCCTACGATTORNEY DOCKET NO. 2II0I.0497P1 ACTTGCTCCTACACCGCCGCAGCCATCGGCAGAAGCGGGGTTTCCGCTGC CCGGTGTGCGGGAAGCGCTTCTGGGAGGCGGCCCTGCTGATGCGCCACCA GCGCTGCCACACGGAACAGCGGCCGTACCGATGTGGCGTGTGCGGCCGAG GCTTCCTGCGCTCCTGGTACCTGCGGCAGCACCGCGTGGTGCACACTGGC GAGCGGGCCTTCAAGTGCGGCGTGTGCGCCAAGCGCTTCGCGCAGTCGTC CAGCCTGGCAGAGCACCGGCGGCTGCACGCTGTGGCCCGGCCCCAGCGCT GCAGCGCCTGTGGCAAGACCTTCCGCTACCGCTCCAACCTGCTGGAGCAC CAGCGGCTGCACCTGGGCGAGCGCGCCTACCGCTGTGAGCACTGCGGCAA GGGCTTCTTCTACCTGAGCTCCGTGCTGCGCCACCAGCGCGCCCATGAGC CGCCGCGGCCCGAGCTCCGCTGCCCCGCCTGCCTCAAGGCCTTCAAGGAT CCCGGCTACTTCCGTAAGCACCTGGCTGCCCACCAGGGCGGCCGGCCCTT CCGCTGCTCCTCCTGCGGCGAGGGCTTCGCCAACACCTACGGCCTCAAGA AACACCGCCTGGCGCACAAGGCCGAGAACCTCGGGGGGCCTGGAGCAGGG GCGGGCACCTTGGCCGGGAAGGATGCCTGA (SEQ ID NO: 1). Thus, in some aspects, disclosed are nucleic acid sequences comprising SEQ ID NO:1 having one or more codon optimizing mutations. In some aspects, a nucleic acid sequence comprising SEQ ID NO: 1 having one or more codon optimizing mutations can be referred to as a variant of SEQ ID NO:1. In some aspects, a nucleic acid sequence comprising SEQ ID NO: 1 having one or more codon optimizing mutations can be referred to as a “codon optimized ZNF865”.

[0053] In some aspects, disclosed are nucleic acid sequences comprising a variant of SEQ ID NO:1, wherein the variant is capable of encoding wild type ZNF865 protein. In some aspects, disclosed are variants of SEQ ID NO: 1. wherein at least 1%, 2%, 3%, 4%. 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the wild type ZNF865 nucleotide coding sequence according to SEQ ID NO: 1 are modified to codons most favored by the host cell. In some aspects, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or 59% of the codons of the wild type ZNF865 nucleotide coding sequence according to SEQ ID NO:1 are modified to codons most favored by the host cell. In some aspects. 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69% of the codons of the wild type ZNF865 nucleotide coding sequence according to SEQ ID NO: 1 are modified to codons most favored by the host cell. In some aspects, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79% of the codons of the wild type ZNF865 nucleotide coding sequence according to SEQ ID NO: 1 are modified to codons most favored by the host cell. In some aspects, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% of the codons of the wild type ZNF865 nucleotide coding sequence according to SEQ ID NO:1 are modified to codons most favored by the host cell. In some aspects, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,ATTORNEY DOCKET NO. 21101.0497P198%, or 99% of the codons of the wild type ZNF865 nucleotide coding sequence according to SEQ ID NO: 1 are modified to codons most favored by the host cell.

[0054] In some aspects, a variant of SEQ ID NO: 1 capable of encoding wild type ZNF865 comprises 1-10, 10-20, 20-30, 30-40, 40-50. 50-60, 60-70, 70-80. 80-90, 90-99% homology with SEQ ID NOT.

[0055] Disclosed are nucleic acid sequences comprising the sequence of ATGGAGGCCAACCCTGCTGGCAGCGGCGCTGGGGGGGGCGGCAGCAGCGGCATCG GTGGCGAGGATGGGGTGCACTTTCAGAGCTACCCATTCGATTTTCTGGAGTTCCTGA ACCACCAGAGATTCGAACCTATGGAACTGTACGGCGAGCATGCCAAGGCCGTGGCC GCCCTGCCCTGTGCTCCAGGCCCTCCCCCTCAGCCACCTCCCCAGCCTCCTCCTCCTC AGTATGATTACCCACCCCAGTCCACCTTCAAACCTAAAGCAGAGGTGCCATCTTCAA GTTCTTCCTCTTCCTCTAGCTCCAGCTCTAGCAGTAGTAGCTCCTCATCCTCCAGCTC CTCCAGCTCCCAGGCCAAGAAGCCCGACCCTCCACTTCCCCCTGCCTTCGGGGCCCC GCCCCCTCCCCTCTTTGATGCTGCCTTCCCAACCCCCCAGTGGGGGATCGTCGACCT GAGCGGCCACCAGCACCTGTTCGGCAATCTGAAAAGGGGCGGCCCCGCCTCCGGAC CTGGGGTGACCCCTGGCCTGGGAGCCCCAGCAGGGGCCCCGGGCCCCCTGCCAGCC CCCTCCCAGACCCCCCCTGGGCCCCCTGCCGCAGCTGCCTGTGATCCAACAAAGGA CGACAAAGGGTACTTTAGGCGCCTGAAGTACCTGATGGAAAGGAGATTCCCCTGTG GCGTCTGTCAGAAGAGCTTCAAGCAATCTAGCCACCTGGTGCAGCACATGCTGGTG CACTCTGGAGAGCGGCCGTATGAGTGCGGGGTGTGCGGCCGCACATACAACCATGT GTCAAGCCTGATTCGCCACAGGCGGTGTCACAAAGACGTGCCTCCCGCTGCCGGAG GGCCACCTCAGCCAGGCCCTCACTTACCCCCCCTGGGCCTCCCCGCTCCCGCCGCCT CCGCCGCCACGGCAGCTGCTCCCAGTACTGTGAGCTCTGGCCCCCCAGCCACTCCCG TGGCACCCGCACCCTCAGCAGACGGCTCTGCTGCCCCAGCCGGAGTGGGCGTGCCA CCACCAGCCACCGGCGGCGGCGACGGACCTTTCGCTTGTCCACTGTGCTGGAAAGT GTTTAAGAAGCCAAGCCATCTGCATCAGCATCAGATTATCCACACCGGCGAGAAGC CTTTCAGTTGTTCTGTGTGTTCTAAGTCTTTCAACAGGCGCGAATCTCTGAAACGGC ACGTGAAAACACATAGCGCGGATCTGCTGAGGTTGCCTTGTGGCATCTGCGGGAAG GCCTTTCGGGACGCATCTTACCTGCTGAAGCACCAGGCCGCCCACGCCGGTGCCGG TGCCGGCGGACCCCGCCCCGTCTACCCTTGTGATCTGTGTGGTAAAAGCTATAGCGC CCCTCAGAGCCTGCTGAGGCACAAGGCCGCACACGCCCCTCCTGCAGCCGCCGCCG AGGCTCCTAAGGACGGCGCCGCCAGCGCTCCCCAGCCCCCTCCCACCTTCCCACCTG GACCTTACCTGCTCCCTCCAGACCCCCCAACTACCGATAGCGAGAAAGCCGCTGCA GCCGCCGCTGCAGTGGTGTATGGCGCAGTGCCTGTGCCACTGCTGGGCGCACACCCATTORNEY DOCKET NO. 21101.0497P1 CCTGCTGCTGGGCGGCGCTGGCACCTCCGGCGCCGGCGGCTCAGGCGCCTCCGTCC CAGGCAAGACATTCTGTTGTGGAATTTGTGGAAGGGGCTTTGGCAGAAGAGAAACT CTGAAGAGACACGAGAGGATCCATACCGGCGAGAAGCCTCACCAGTGCCCAGTGTG CGGAAAGAGATTCAGAGAGTCCTTCCATCTGTCCAAGCACCACGTGGTGCACACAA GGGAGAGGCCTTATAAGTGCGAGCTCTGTGGAAAGGTTTTCGGCTATCCACAAAGC CTGACAAGGCACAGACAGGTGCACAGGCTGCAGCTCCCCTGCGCCCTGGCCGGAGC CGCCGGCCTGCCATCAACACAGGGGACCCCCGGAGCCTGTGGGCCTGGCGCCTCAG GGACCAGCGCCGGCCCAACAGATGGCCTGAGCTACGCATGTAGCGACTGCGGAGA ACACTTCCCTGACCTGTTTCACGTGATGTCCCATAAGGAGGTGCACATGGCCGAGA AGCCTTATGGCTGTGATGCCTGTGGGAAGACCTTTGGCTTCATAGAAAACCTGATGT GGCATAAGCTCGTACACCAGGCAGCCCCTGAGCGGCTCCTTCCACCTGCCCCTGGA GGGCTTCAGCCCCCTGACGGCTCCAGCGGTACCGACGCTGCTTCTGTGCTCGACAAC GGCCTGGCCGGTGAGGTGGGGGCCGCAGTGGCCGCACTGGCTGGAGTGTCCGGTGG AGAGGACGCTGGGGGCGCCGCCGTGGCCGGCGCTGGCGGCGGCGCCTCCAGCGGC CCCGAGAGATTTTCATGTGCCACCTGCGGGCAGTCCTTTAAGCACTTCCTGGGCCTT GTCACTCATAAATACGTGCACCTGGTGAGGAGGACTCTGGGTTGCGGCCTGTGTGG TCAGAGCTTCGCCGGCGCCTATGACCTGCTGCTGCACAGACGGAGCCACCGCCAGA AGCGCGGCTTCCGGTGTCCTGTGTGCGGGAAGAGGTTCTGGGAGGCAGCCCTGCTG ATGAGACACCAGAGATGCCACACCGAACAGAGACCATACCGCTGTGGCGTGTGCGG CAGAGGCTTTCTGCGCAGTTGGTACCTCAGGCAGCACAGAGTGGTGCATACCGGCG AAAGAGCCTTCAAGTGCGGCGTGTGCGCCAAGAGGTTCGCCCAGTCATCTTCTCTCG CTGAGCACCGGAGGTTGCATGCCGTGGCTAGACCCCAACGTTGCTCCGCCTGCGGC AAGACTTTCCGCTACCGGTCTAACCTTCTGGAGCACCAGAGGCTGCATCTGGGAGA ACGCGCCTACAGGTGCGAACACTGCGGTAAAGGATTCTTTTACCTGAGCAGCGTGC TGCGGCACCAGAGAGCTCACGAACCCCCACGGCCAGAGCTGAGGTGTCCCGCTTGT CTGAAAGCATTCAAGGACCCAGGATACTTTAGAAAGCATCTCGCGGCCCATCAAGG GGGAAGGCCTTTCAGATGCAGTTCCTGTGGTGAAGGGTTCGCAAACACCTACGGCC TCAAGAAACACAGATTGGCTCATAAAGCAGAGAACCTGGGCGGGCCTGGCGCCGG GGCTGGAACGCTGGCCGGCAAAGATGCC (SEQ ID N0:2).

[0056] In some aspects, the disclosed nucleic acid sequences comprise 1-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 450-500, 500-550. 550-600. 600-650. 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, or greater than 1000 codon optimizing mutations compared to SEQ ID NO: 1.

[0057] In some aspects, disclosed are nucleic acid constructs comprising a nucleic acidATTORNEY DOCKET NO. 2II0I.0497P1sequence comprising SEQ ID NO: 1 having one or more codon optimizing mutations or SEQ ID NO:2, and further comprising an expression control element, such as a promoter and / or enhancer.

[0058] Disclosed are nucleic acid constructs comprising the sequence of GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTT GGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAAC TGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACC GTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAG AACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGG CCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGA GCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTT CGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATC TGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATT TTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCC AAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGT GCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAAT CGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGTCTCGCGCCGC CGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGA GCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCG GCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCG TCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTC GATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTAT GCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCA CTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTC AAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACAAGTT TGTACAAAAAAGCAGGCTGCCACCATGGAGGCCAACCCTGCTGGCAGCGGCGCTGG GGGGGGCGGCAGCAGCGGCATCGGTGGCGAGGATGGGGTGCACTTTCAGAGCTAC CCATTCGATTTTCTGGAGTTCCTGAACCACCAGAGATTCGAACCTATGGAACTGTAC GGCGAGCATGCCAAGGCCGTGGCCGCCCTGCCCTGTGCTCCAGGCCCTCCCCCTCA GCCACCTCCCCAGCCTCCTCCTCCTCAGTATGATTACCCACCCCAGTCCACCTTCAA ACCTAAAGCAGAGGTGCCATCTTCAAGTTCTTCCTCTTCCTCTAGCTCCAGCTCTAG CAGTAGTAGCTCCTCATCCTCCAGCTCCTCCAGCTCCCAGGCCAAGAAGCCCGACCC TCCACTTCCCCCTGCCTTCGGGGCCCCGCCCCCTCCCCTCTTTGATGCTGCCTTCCCA ACCCCCCAGTGGGGGATCGTCGACCTGAGCGGCCACCAGCACCTGTTCGGCAATCTATTORNEY DOCKET NO. 21101.0497P1 GAAAAGGGGCGGCCCCGCCTCCGGACCTGGGGTGACCCCTGGCCTGGGAGCCCCAG CAGGGGCCCCGGGCCCCCTGCCAGCCCCCTCCCAGACCCCCCCTGGGCCCCCTGCC GCAGCTGCCTGTGATCCAACAAAGGACGACAAAGGGTACTTTAGGCGCCTGAAGTA CCTGATGGAAAGGAGATTCCCCTGTGGCGTCTGTCAGAAGAGCTTCAAGCAATCTA GCCACCTGGTGCAGCACATGCTGGTGCACTCTGGAGAGCGGCCGTATGAGTGCGGG GTGTGCGGCCGCACATACAACCATGTGTCAAGCCTGATTCGCCACAGGCGGTGTCA CAAAGACGTGCCTCCCGCTGCCGGAGGGCCACCTCAGCCAGGCCCTCACTTACCCC CCCTGGGCCTCCCCGCTCCCGCCGCCTCCGCCGCCACGGCAGCTGCTCCCAGTACTG TGAGCTCTGGCCCCCCAGCCACTCCCGTGGCACCCGCACCCTCAGCAGACGGCTCTG CTGCCCCAGCCGGAGTGGGCGTGCCACCACCAGCCACCGGCGGCGGCGACGGACCT TTCGCTTGTCCACTGTGCTGGAAAGTGTTTAAGAAGCCAAGCCATCTGCATCAGCAT CAGATTATCCACACCGGCGAGAAGCCTTTCAGTTGTTCTGTGTGTTCTAAGTCTTTC AACAGGCGCGAATCTCTGAAACGGCACGTGAAAACACATAGCGCGGATCTGCTGAG GTTGCCTTGTGGCATCTGCGGGAAGGCCTTTCGGGACGCATCTTACCTGCTGAAGCA CCAGGCCGCCCACGCCGGTGCCGGTGCCGGCGGACCCCGCCCCGTCTACCCTTGTG ATCTGTGTGGTAAAAGCTATAGCGCCCCTCAGAGCCTGCTGAGGCACAAGGCCGCA CACGCCCCTCCTGCAGCCGCCGCCGAGGCTCCTAAGGACGGCGCCGCCAGCGCTCC CCAGCCCCCTCCCACCTTCCCACCTGGACCTTACCTGCTCCCTCCAGACCCCCCAAC TACCGATAGCGAGAAAGCCGCTGCAGCCGCCGCTGCAGTGGTGTATGGCGCAGTGC CTGTGCCACTGCTGGGCGCACACCCCCTGCTGCTGGGCGGCGCTGGCACCTCCGGC GCCGGCGGCTCAGGCGCCTCCGTCCCAGGCAAGACATTCTGTTGTGGAATTTGTGG AAGGGGCTTTGGCAGAAGAGAAACTCTGAAGAGACACGAGAGGATCCATACCGGC GAGAAGCCTCACCAGTGCCCAGTGTGCGGAAAGAGATTCAGAGAGTCCTTCCATCT GTCCAAGCACCACGTGGTGCACACAAGGGAGAGGCCTTATAAGTGCGAGCTCTGTG GAAAGGTTTTCGGCTATCCACAAAGCCTGACAAGGCACAGACAGGTGCACAGGCTG CAGCTCCCCTGCGCCCTGGCCGGAGCCGCCGGCCTGCCATCAACACAGGGGACCCC CGGAGCCTGTGGGCCTGGCGCCTCAGGGACCAGCGCCGGCCCAACAGATGGCCTGA GCTACGCATGTAGCGACTGCGGAGAACACTTCCCTGACCTGTTTCACGTGATGTCCC ATAAGGAGGTGCACATGGCCGAGAAGCCTTATGGCTGTGATGCCTGTGGGAAGACC TTTGGCTTCATAGAAAACCTGATGTGGCATAAGCTCGTACACCAGGCAGCCCCTGA GCGGCTCCTTCCACCTGCCCCTGGAGGGCTTCAGCCCCCTGACGGCTCCAGCGGTAC CGACGCTGCTTCTGTGCTCGACAACGGCCTGGCCGGTGAGGTGGGGGCCGCAGTGG CCGCACTGGCTGGAGTGTCCGGTGGAGAGGACGCTGGGGGCGCCGCCGTGGCCGGC GCTGGCGGCGGCGCCTCCAGCGGCCCCGAGAGATTTTCATGTGCCACCTGCGGGCAATTORNEY DOCKET NO. 2II0I.0497P1 GTCCTTTAAGCACTTCCTGGGCCTTGTCACTCATAAATACGTGCACCTGGTGAGGAG GACTCTGGGTTGCGGCCTGTGTGGTCAGAGCTTCGCCGGCGCCTATGACCTGCTGCT GCACAGACGGAGCCACCGCCAGAAGCGCGGCTTCCGGTGTCCTGTGTGCGGGAAGA GGTTCTGGGAGGCAGCCCTGCTGATGAGACACCAGAGATGCCACACCGAACAGAG ACCATACCGCTGTGGCGTGTGCGGCAGAGGCTTTCTGCGCAGTTGGTACCTCAGGC AGCACAGAGTGGTGCATACCGGCGAAAGAGCCTTCAAGTGCGGCGTGTGCGCCAAG AGGTTCGCCCAGTCATCTTCTCTCGCTGAGCACCGGAGGTTGCATGCCGTGGCTAGA CCCCAACGTTGCTCCGCCTGCGGCAAGACTTTCCGCTACCGGTCTAACCTTCTGGAG CACCAGAGGCTGCATCTGGGAGAACGCGCCTACAGGTGCGAACACTGCGGTAAAG GATTCTTTTACCTGAGCAGCGTGCTGCGGCACCAGAGAGCTCACGAACCCCCACGG CCAGAGCTGAGGTGTCCCGCTTGTCTGAAAGCATTCAAGGACCCAGGATACTTTAG AAAGCATCTCGCGGCCCATCAAGGGGGAAGGCCTTTCAGATGCAGTTCCTGTGGTG AAGGGTTCGCAAACACCTACGGCCTCAAGAAACACAGATTGGCTCATAAAGCAGAG AACCTGGGCGGGCCTGGCGCCGGGGCTGGAACGCTGGCCGGCAAAGATGCC (SEQ ID N0:3). The underlined sequence of SEQ ID N0:3 is the EFl A promoter and the remaining portion of SEQ ID NO: 3 is a codon optimized ZNF865.

[0059] Disclosed are nucleic acid constructs comprising the sequence of GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTT GGGGGGAGGGGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAAC TGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACC GTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAG AACACAGGCAAGTTTGTACAAAAAAGCAGGCTGCCACCATGGAGGCCAACCCTGCT GGCAGCGGCGCTGGGGGGGGCGGCAGCAGCGGCATCGGTGGCGAGGATGGGGTGC ACTTTCAGAGCTACCCATTCGATTTTCTGGAGTTCCTGAACCACCAGAGATTCGAAC CTATGGAACTGTACGGCGAGCATGCCAAGGCCGTGGCCGCCCTGCCCTGTGCTCCA GGCCCTCCCCCTCAGCCACCTCCCCAGCCTCCTCCTCCTCAGTATGATTACCCACCC CAGTCCACCTTCAAACCTAAAGCAGAGGTGCCATCTTCAAGTTCTTCCTCTTCCTCT AGCTCCAGCTCTAGCAGTAGTAGCTCCTCATCCTCCAGCTCCTCCAGCTCCCAGGCC AAGAAGCCCGACCCTCCACTTCCCCCTGCCTTCGGGGCCCCGCCCCCTCCCCTCTTT GATGCTGCCTTCCCAACCCCCCAGTGGGGGATCGTCGACCTGAGCGGCCACCAGCA CCTGTTCGGCAATCTGAAAAGGGGCGGCCCCGCCTCCGGACCTGGGGTGACCCCTG GCCTGGGAGCCCCAGCAGGGGCCCCGGGCCCCCTGCCAGCCCCCTCCCAGACCCCC CCTGGGCCCCCTGCCGCAGCTGCCTGTGATCCAACAAAGGACGACAAAGGGTACTT TAGGCGCCTGAAGTACCTGATGGAAAGGAGATTCCCCTGTGGCGTCTGTCAGAAGAATTORNEY DOCKET NO. 21101.0497P1 GCTTCAAGCAATCTAGCCACCTGGTGCAGCACATGCTGGTGCACTCTGGAGAGCGG CCGTATGAGTGCGGGGTGTGCGGCCGCACATACAACCATGTGTCAAGCCTGATTCG CCACAGGCGGTGTCACAAAGACGTGCCTCCCGCTGCCGGAGGGCCACCTCAGCCAG GCCCTCACTTACCCCCCCTGGGCCTCCCCGCTCCCGCCGCCTCCGCCGCCACGGCAG CTGCTCCCAGTACTGTGAGCTCTGGCCCCCCAGCCACTCCCGTGGCACCCGCACCCT CAGCAGACGGCTCTGCTGCCCCAGCCGGAGTGGGCGTGCCACCACCAGCCACCGGC GGCGGCGACGGACCTTTCGCTTGTCCACTGTGCTGGAAAGTGTTTAAGAAGCCAAG CCATCTGCATCAGCATCAGATTATCCACACCGGCGAGAAGCCTTTCAGTTGTTCTGT GTGTTCTAAGTCTTTCAACAGGCGCGAATCTCTGAAACGGCACGTGAAAACACATA GCGCGGATCTGCTGAGGTTGCCTTGTGGCATCTGCGGGAAGGCCTTTCGGGACGCA TCTTACCTGCTGAAGCACCAGGCCGCCCACGCCGGTGCCGGTGCCGGCGGACCCCG CCCCGTCTACCCTTGTGATCTGTGTGGTAAAAGCTATAGCGCCCCTCAGAGCCTGCT GAGGCACAAGGCCGCACACGCCCCTCCTGCAGCCGCCGCCGAGGCTCCTAAGGACG GCGCCGCCAGCGCTCCCCAGCCCCCTCCCACCTTCCCACCTGGACCTTACCTGCTCC CTCCAGACCCCCCAACTACCGATAGCGAGAAAGCCGCTGCAGCCGCCGCTGCAGTG GTGTATGGCGCAGTGCCTGTGCCACTGCTGGGCGCACACCCCCTGCTGCTGGGCGG CGCTGGCACCTCCGGCGCCGGCGGCTCAGGCGCCTCCGTCCCAGGCAAGACATTCT GTTGTGGAATTTGTGGAAGGGGCTTTGGCAGAAGAGAAACTCTGAAGAGACACGAG AGGATCCATACCGGCGAGAAGCCTCACCAGTGCCCAGTGTGCGGAAAGAGATTCAG AGAGTCCTTCCATCTGTCCAAGCACCACGTGGTGCACACAAGGGAGAGGCCTTATA AGTGCGAGCTCTGTGGAAAGGTTTTCGGCTATCCACAAAGCCTGACAAGGCACAGA CAGGTGCACAGGCTGCAGCTCCCCTGCGCCCTGGCCGGAGCCGCCGGCCTGCCATC AACACAGGGGACCCCCGGAGCCTGTGGGCCTGGCGCCTCAGGGACCAGCGCCGGCC CAACAGATGGCCTGAGCTACGCATGTAGCGACTGCGGAGAACACTTCCCTGACCTG TTTCACGTGATGTCCCATAAGGAGGTGCACATGGCCGAGAAGCCTTATGGCTGTGA TGCCTGTGGGAAGACCTTTGGCTTCATAGAAAACCTGATGTGGCATAAGCTCGTAC ACCAGGCAGCCCCTGAGCGGCTCCTTCCACCTGCCCCTGGAGGGCTTCAGCCCCCTG ACGGCTCCAGCGGTACCGACGCTGCTTCTGTGCTCGACAACGGCCTGGCCGGTGAG GTGGGGGCCGCAGTGGCCGCACTGGCTGGAGTGTCCGGTGGAGAGGACGCTGGGG GCGCCGCCGTGGCCGGCGCTGGCGGCGGCGCCTCCAGCGGCCCCGAGAGATTTTCA TGTGCCACCTGCGGGCAGTCCTTTAAGCACTTCCTGGGCCTTGTCACTCATAAATAC GTGCACCTGGTGAGGAGGACTCTGGGTTGCGGCCTGTGTGGTCAGAGCTTCGCCGG CGCCTATGACCTGCTGCTGCACAGACGGAGCCACCGCCAGAAGCGCGGCTTCCGGT GTCCTGTGTGCGGGAAGAGGTTCTGGGAGGCAGCCCTGCTGATGAGACACCAGAGAATTORNEY DOCKET NO. 2II0I.0497P1 TGCCACACCGAACAGAGACCATACCGCTGTGGCGTGTGCGGCAGAGGCTTTCTGCG CAGTTGGTACCTCAGGCAGCACAGAGTGGTGCATACCGGCGAAAGAGCCTTCAAGT GCGGCGTGTGCGCCAAGAGGTTCGCCCAGTCATCTTCTCTCGCTGAGCACCGGAGG TTGCATGCCGTGGCTAGACCCCAACGTTGCTCCGCCTGCGGCAAGACTTTCCGCTAC CGGTCTAACCTTCTGGAGCACCAGAGGCTGCATCTGGGAGAACGCGCCTACAGGTG CGAACACTGCGGTAAAGGATTCTTTTACCTGAGCAGCGTGCTGCGGCACCAGAGAG CTCACGAACCCCCACGGCCAGAGCTGAGGTGTCCCGCTTGTCTGAAAGCATTCAAG GACCCAGGATACTTTAGAAAGCATCTCGCGGCCCATCAAGGGGGAAGGCCTTTCAG ATGCAGTTCCTGTGGTGAAGGGTTCGCAAACACCTACGGCCTCAAGAAACACAGAT TGGCTCATAAAGCAGAGAACCTGGGCGGGCCTGGCGCCGGGGCTGGAACGCTGGCC GGCAAAGATGCC (SEQ ID N0:4). The underlined sequence of SEQ ID N0:4 is the EFl A promoter and the remaining portion of SEQ ID NO:4 is a codon optimized ZNF865.

[0060] Disclosed are nucleic acid sequences comprising the mRNA sequence of AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGAGG CGAACCCAGCGGGCAGCGGCGCCGGGGGUGGCGGGAGCAGCGGCAUCGGGGGCG AGGACGGGGUGCACUUCCAGAGCUACCCCUUCGACUUCCUGGAAUUCCUCAACCA CCAGCGCUUCGAGCCCAUGGAACUGUAUGGGGAACACGCCAAGGCGGUGGCGGCC CUGCCCUGCGCCCCCGGCCCCCCGCCGCAGCCCCCGCCGCAGCCCCCUCCCCCGCA GUAUGACUACCCGCCCCAGUCCACCUUCAAGCCCAAGGCGGAGGUGCCCUCCUCG UCCUCGUCCUCGUCCUCCUCCUCCUCCUCUUCGUCCUCCUCGUCGUCAUCUUCGU CCUCUUCCUCUUCCCAAGCCAAGAAGCCCGAUCCGCCCCUGCCGCCCGCCUUCGG GGCGCCCCCUCCUCCCCUCUUUGACGCUGCUUUCCCCACUCCGCAGUGGGGCAUC GUGGACCUCUCGGGGCACCAGCACUUGUUUGGGAACCUGAAGCGAGGAGGGCCC GCGUCCGGGCCGGGGGUGACGCCUGGGCUGGGCGCUCCCGCGGGGGCCCCAGGGC CGCUUCCUGCCCCCUCGCAGACCCCGCCAGGACCCCCCGCGGCGGCGGCCUGCGAC CCCACCAAGGACGACAAGGGCUACUUCCGGAGACUGAAGUACCUGAUGGAGCGG CGCUUCCCCUGCGGCGUGUGCCAGAAGUCCUUCAAGCAGUCCUCGCACCUGGUCC AGCACAUGCUGGUGCACUCGGGGGAGAGGCCCUACGAAUGCGGCGUCUGCGGCC GCACCUACAACCACGUGUCCAGCCUCAUCCGCCACCGCCGCUGCCACAAGGACGU GCCACCGGCCGCGGGGGGCCCGCCCCAGCCCGGCCCCCACCUCCCGCCGCUGGGCC UCCCAGCACCCGCUGCCAGCGCCGCCACCGCCGCCGCCCCCUCCACGGUGUCCUCG GGCCCUCCAGCCACGCCCGUGGCGCCUGCCCCCUCCGCAGACGGGAGCGCCGCCCC UGCUGGUGUUGGGGUGCCCCCUCCUGCCACCGGGGGUGGCGAUGGCCCGUUCGCC UGCCCACUCUGCUGGAAGGUUUUCAAGAAGCCCAGUCACCUCCACCAGCACCAGAATTORNEY DOCKET NO. 21101.0497P1 UCAUCCACACGGGCGAGAAGCCCUUCUCCUGCUCCGUGUGCAGCAAAAGCUUCAA CCGCAGGGAGAGUCUGAAGCGCCACGUGAAGACGCACUCGGCCGACCUCCUGCGC CUGCCCUGCGGCAUCUGCGGGAAGGCCUUCCGCGACGCCUCCUACCUCCUCAAGC ACCAGGCGGCCCACGCGGGGGCGGGCGCCGGGGGGCCUCGGCCCGUGUACCCCUG CGACCUGUGCGGCAAGUCCUACLICGGCUCCGCAGAGCCLIGCUCCGCCACAAGGCC GCCCACGCCCCGCCCGCUGCCGCUGCGGAGGCGCCCAAGGACGGGGCGGCCUCGG CCCCGCAGCCCCCGCCCACCUUCCCCCCGGGCCCGUACCUCCUGCCCCCCGACCCU CCCACCACAGACAGCGAGAAGGCGGCGGCGGCCGCGGCGGCGGUGGUGUACGGCG CUGUGCCCGUCCCGCUCCUGGGCGCCCACCCGCUGCUGCUCGGCGGCGCGGGGAC CAGCGGGGCGGGAGGCUCGGGCGCCAGCGUCCCAGGAAAGACGUUCUGCUGCGGC AUCUGCGGGCGCGGCUUCGGGCGCCGCGAGACCCUGAAGCGCCAUGAGCGCAUCC ACACGGGCGAGAAGCCCCACCAGUGCCCCGUGUGUGGGAAGCGCUUCCGCGAAUC CUUCCACUUGAGCAAGCAUCACGUGGUGCACACGCGCGAGCGGCCCUACAAGUGC GAGCUCUGCGGCAAGGUCUUCGGCUACCCGCAGAGCCUCACCCGCCACCGCCAGG UGCACCGGCUCCAGCUGCCCUGCGCCCUGGCCGGGGCAGCCGGCCUCCCCUCCAC CCAAGGCACACCGGGGGCCUGUGGGCCCGGGGCCUCGGGCACGUCUGCAGGGCCC ACCGAUGGGCUGAGCUACGCCUGCUCGGACUGCGGCGAGCACUUCCCGGAUCUCU UUCACGUCAUGAGUCACAAGGAGGUCCACAUGGCAGAGAAGCCAUACGGCUGCG ACGCCUGCGGCAAGACCUUCGGCUUCAUCGAGAACCUCAUGUGGCACAAGCUGGU CCACCAGGCCGCCCCCGAGCGCCUGCUCCCGCCCGCACCCGGCGGCCUGCAGCCCC CGGACGGCUCCAGCGGCACGGAUGCGGCCAGCGUGCUGGACAACGGGCUGGCGGG GGAGGUGGGGGCGGCCGUGGCGGCACUGGCAGGGGUGUCUGGGGGUGAGGACGC AGGCGGGGCGGCGGUGGCAGGUGCUGGCGGGGGUGCCAGUUCCGGCCCCGAGCG CUUCAGCUGUGCCACGUGCGGCCAGAGUUUCAAGCACUUCCUGGGCCUCGUGACU CACAAGUACGUGCACCUGGUGCGACGGACCCUGGGCUGCGGCCUCUGCGGCCAGA GCUUCGCGGGCGCCUACGACUUGCUCCUACACCGCCGCAGCCAUCGGCAGAAGCG GGGUUUCCGCUGCCCGGUGUGCGGGAAGCGCUUCUGGGAGGCGGCCCUGCUGAU GCGCCACCAGCGCUGCCACACGGAACAGCGGCCGUACCGAUGUGGCGUGUGCGGC CGAGGCUUCCUGCGCUCCUGGUACCUGCGGCAGCACCGCGUGGUGCACACUGGCG AGCGGGCCUUCAAGUGCGGCGUGUGCGCCAAGCGCUUCGCGCAGUCGUCCAGCCU GGCAGAGCACCGGCGGCUGCACGCUGUGGCCCGGCCCCAGCGCUGCAGCGCCUGU GGCAAGACCUUCCGCUACCGCUCCAACCUGCUGGAGCACCAGCGGCUGCACCUGG GCGAGCGCGCCUACCGCUGUGAGCACUGCGGCAAGGGCUUCUUCUACCUGAGCUC CGUGCUGCGCCACCAGCGCGCCCAUGAGCCGCCGCGGCCCGAGCUCCGCUGCCCCATTORNEY DOCKET NO. 2II0I.0497P1 GCCUGCCUCAAGGCCUUCAAGGAUCCCGGCUACUUCCGUAAGCACCUGGCUGCCC ACCAGGGCGGCCGGCCCUUCCGCUGCUCCUCCUGCGGCGAGGGCUUCGCCAACAC CUACGGCCUCAAGAAACACCGCCUGGCGCACAAGGCCGAGAACCUCGGGGGGCCU GGAGCAGGGGCGGGCACCUUGGCCGGGAAGGAUGCCUGAGCUGCCUUCUGCGGG GCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCU UUGAAUAAAGCCUGAGUAGGAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID N0:5). In some aspects, SEQ ID NO:5 is an mRNA sequence based on the wild type ZNF865 DNA sequence (e.g., SEQ ID NO: 1). In some aspects, SEQ ID NO:5 is an mRNA sequence capable of ending the wild-type ZNF865 protein.

[0061] Disclosed herein are nucleic acid sequences capable of encoding wild type ZNF865 (i.e., SEQ ID NO:6). Thus, the nucleic acid sequence can be a wild type nucleic acid sequence or a codon optimized nucleic acid sequence.

[0062] In some aspects, disclosed are nucleic acid constructs comprising a nucleic acid sequence comprising SEQ ID NO:5 and further comprising an expression control element, such as a promoter and / or enhancer.

[0063] In some aspects, the 5’ untranslated region (UTR), the 3' UTR, the poly A, or the CAP of the disclosed nucleic acid sequence can be optimized from aZNF865 nucleic acid sequence.

[0064] In some aspects, one or more of the disclosed nucleic acid sequences can be modified. In some aspects, a modified nucleic acid sequence can comprise any of the disclosed nucleic acid sequences comprising a non-naturally occurring nucleotide or a nucleotide analog. In some aspects, a modified nucleic acid sequence can increase s tabi 1 i ty of the nucleic acid sequence, provide nuclease resistance, provide targeting capabilities, or provide a site for labeling.C. Proteins

[0065] Disclosed are proteins, polypeptides, or fragments thereof comprising a full length ZNF865, a fragment of ZNF865, or a variant thereof of a full length ZNF865 or a fragment of ZNF865.

[0066] In some aspects, ZNF865 can be a full length ZNF865 or can be a fragment thereof. In some aspects, the full length ZNF865 comprises the amino acid sequence:MEANPAGSGAGGGGSSGIGGEDGVHFQSYPFDFLEFLNHQRFEPMELYGEHAKAVAAL PCAPGPPPQPPPQPPPPQYDYPPQSTFKPKAEVPSSSSSSSSSSSSSSSSSSSSSSSSSQAKKPATTORNEY DOCKET NO. 21101.0497P1 DPPLPPAFGAPPPPLFDAAFPTPQWGIVDLSGHQHLFGNLKRGGPASGPGVTPGLGAPA GAPGPLPAPSQTPPGPPAAAACDPTKDDKGYFRRLKYLMERRFPCGVCQKSFKQSSHLV QHMLVHSGERPYECGVCGRTYNHVSSLIRHRRCHKDVPPAAGGPPQPGPHLPPLGLPAP AASAATAAAPSTVSSGPPATPVAPAPSADGSAAPAGVGVPPPATGGGDGPFACPLCWK VFKKPSHLHQHQ1IHTGEKPFSCSVCSKSFNRRESLKRHVKTHSADLLRLPCGICGKAFR DASYLLKHQAAHAGAGAGGPRPVYPCDLCGKSYSAPQSLLRHKAAHAPPAAAAEAPK DGAASAPQPPPTFPPGPYLLPPDPPTTDSEKAAAAAAAVVYGAVPVPLLGAHPLLLGGA GTSGAGGSGASVPGKTFCCGICGRGFGRRETLKRHERIHTGEKPHQCPVCGKRFRESFH LSKHHVVHTRERPYKCELCGKVFGYPQSLTRHRQVHRLQLPCALAGAAGLPSTQGTPG ACGPGASGTSAGPTDGLSYACSDCGEHFPDLFHVMSHKEVHMAEKPYGCDACGKTFG FIENL WHKLVHQAAPERLLPPAPGGLQPPDGSSGTDAASVLDNGLAGEVGAAVAAL AGVSGGEDAGGAAVAGAGGGASSGPERFSCATCGQSFKHFLGLVTHKYVHLVRRTLG CGLCGQSFAGAYDLLLHRRSHRQKRGFRCPVCGKRFWEAALLMRHQRCHTEQRPYRC GVCGRGFLRSWYLRQHRVVHTGERAFKCGVCAKRFAQSSSLAEHRRLHAVARPQRCS ACGKTFRYRSNLLEHQRLHLGERAYRCEHCGKGFFYLSSVLRHQRAHEPPRPELRCPAC LKAFKDPGYFRKHLAAHQGGRPFRCSSCGEGFANTYGLKKHRLAHKAENLGGPGAGA GTLAGKDA (SEQ ID N0:6). In some aspects, a fragment of ZNF865 is an amino acid sequence shorter than SEQ ID NO:6 that retains its transcription regulatory activity (e.g. ability to activate a gene of interest, such as Aggrecan or Collagen II).

[0067] In some aspects, a fragment of ZNF65 can comprise or consist of amino acids 1-769 of SEQ ID NO: 6. In some aspects, a fragment of ZNF865 can comprise or consist of the amino acid sequence:MEANPAGSGAGGGGSSGIGGEDGVHFQSYPFDFLEFLNHQRFEPMELYGEHAKAVAAL PCAPGPPPQPPPQPPPPQYDYPPQSTFKPKAEVPSSSSSSSSSSSSSSSSSSSSSSSSSQAKKP DPPLPPAFGAPPPPLFDAAFPTPQWGIVDLSGHQHLFGNLKRGGPASGPGVTPGLGAPA GAPGPLPAPSQTPPGPPAAAACDPTKDDKGYFRRLKYLMERRFPCGVCQKSFKQSSHLV QHMLVHSGERPYECGVCGRTYNHVSSL1RHRRCHKDVPPAAGGPPQPGPHLPPLGLPAP AASAATAAAPSTVSSGPPATPVAPAPSADGSAAPAGVGVPPPATGGGDGPFACPLCWK VFKKPSHLHQHQIIHTGEKPFSCSVCSKSFNRRESLKRHVKTHSADLLRLPCGICGKAFR DASYLLKHQAAHAGAGAGGPRPVYPCDLCGKSYSAPQSLLRHKAAHAPPAAAAEAPK DGAASAPQPPPTFPPGPYLLPPDPPTTDSEKAAAAAAAVVYGAVPVPLLGAHPLLLGGA GTSGAGGSGASVPGKTFCCGICGRGFGRRETLKRHERIHTGEKPHQCPVCGKRFRESFH LSKHHVVHTRERPYKCELCGKVFGYPQSLTRHRQVHRLQLPCALAGAAGLPSTQGTPG ACGPGASGTSAGPTDGLSYACSDCGEHFPDLFHVMSHKEVHMAEKPYGCDACGKTFGATTORNEY DOCKET NO. 21101.0497P1 FIENLMWHKLVHQAAPERLLPPAPGGLQPPDGSSGTDAASVLDNGLAGEVGAAVAAL AGVSG(SEQ ID N0:7).

[0068] In some aspects, a fragment of ZNF865 can comprise or consist of amino acids 770-1059 of SEQ ID NO:6. In some aspects, a fragment of ZNF65 can comprise or consist of the amino acid sequence:GEDAGGAAVAGAGGGASSGPERFSCATCGQSFKHFLGLVTHKYVHLVRRTLGCGLCG QSFAGAYDLLLHRRSHRQKRGFRCPVCGKRFWEAALLMRHQRCHTEQRPYRCGVCGR GFLRSWYLRQHRVVHTGERAFKCGVCAKRFAQSSSLAEHRRLHAVARPQRCSACGKT FRYRSNLLEHQRLHLGERAYRCEHCGKGFFYLSSVLRHQRAHEPPRPELRCPACLKAFK DPGYFRKHLAAHQGGRPFRCSSCGEGFANTYGLKKHRLAHKAENLGGPGAGAGTLAG KDA (SEQ ID NO: 8).

[0069] In some aspects, the disclosed proteins, polypeptides, or fragments thereof can be modified. In some aspects, a modified ZNF865 protein, polypeptide, or fragment thereof can be referred to as a variant of wild t pe ZNF865, wherein the modified ZNF865 protein, polypeptide, or fragment thereof retains its transcription regulatory activity (e.g. ability’ to activate a gene of interest, such as Aggrecan or Collagen II).

[0070] In some aspects, the disclosed ZNF865 proteins can be modified. In some aspects, the disclosed proteins can be modified to increase stability, increase half-life, improve bioavailability, increase efficacy, reduce off-target effects, reduce immunogenicity, improve solubility, enable conjugation or encapsulation, introduce imaging or reporter capabilities, or alter biological activity.

[0071] In some aspects, the disclosed ZNF865 proteins can be modified by pegylation. acylation, alkylation, oxidation, reduction, glycosylation, phosphorylation, methylation, acetylation, or lipidation. In some aspects, the disclosed ZNF865 proteins can be modified by conjugating to another entity, such as, but not limited to, an antibody, a drug, a nucleic acid sequence, a nanoparticle, or a lipid.D. Vectors

[0072] Disclosed are vectors comprising one or more of the disclosed nucleic acid sequences, proteins, or fragments thereof. As used herein, a vector can be any natural, synthetic, or engineered carrier capable of delivering, transferring, or facilitating the introduction of a cargo into a cell, tissue, organ, or organism. In some aspects, the cargo can be a nucleic acid sequence, a protein or fragment thereof, or other biologically active molecules. In some aspects, vectors include, without limitation, viral vectors, non-viral vectors, plasmids, expression constructs, nanoparticles, liposomes, lipid nanoparticles, inorganic nanoparticles (e.g., goldATTORNEY DOCKET NO. 2II0I.0497P1silica, iron oxide, quantum dots, silver, etc.), carbon-based nanoparticles, extracellular vesicles, polymers (e.g. dendrimers, micelles, peptidoglycans, chitosan, Poly(ethyleneimine) (PEI) and PLGA), conjugates, cell-based carriers, scaffold-based vehicle (e.g., hydrogel, polymer matrices, etc.) and combinations thereof. In some aspect, a vector is capable of transient or stable delivery, expression, localization, or functional activity of the nucleic acid sequence, protein or fragment thereof, or other biologically active molecule being carried.

[0073] Disclosed are vectors comprising the nucleic acid sequence of wild type ZNF865, the nucleic acid sequence of wild type ZNF865 comprising one or more codon optimizing mutations, or the ZNF865 protein. For example, disclosed are vectors comprising the nucleic acid sequence of SEQ ID NO:2. In some aspects, disclosed are vectors comprising the nucleic acid sequence of SEQ ID NO: 1 having one or more codon optimizing mutations. In some aspects, disclosed are vectors comprising the nucleic acid sequence of SEQ ID NO:5.

[0074] Disclosed are vectors that comprise a codon optimized ZNF865 nucleic acid sequence that encodes a wild type ZNF865 protein.

[0075] Disclosed are vectors that comprise a ZNF865 protein. Disclosed are vectors that comprise aZNF865 protein comprising the sequence of SEQ ID NO:6 or a fragment thereof.

[0076] In some aspects, the disclosed vectors can carry their cargo (i.e., one or more of the disclosed nucleic acid sequences, proteins, or fragments thereof) inside of the vector, on the surface of the vector, or a combination thereof.

[0077] In some aspects, the vector can be an expression vector. The term "expression vector" includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element). "Plasmid" and "vector" can be used interchangeably, as a plasmid is a commonly used form of vector. Moreover, the invention is intended to include other vectors which serve equivalent functions.

[0078] In some aspects, the vector can be a viral vector. For example, the viral vector can be a lentiviral vector. In some aspects, the vector can be a non-viral vector, such as a DNA based vector or lipid nanoparticle.i. Viral and Non- Viral Vectors

[0079] There are a number of compositions and methods which can be used to deliver the disclosed nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. For example, the nucleic acids can be delivered through a number of direct delivery' systems such as, electroporation, lipofection, calcium phosphate precipitation,ATTORNEY DOCKET NO. 21101.0497P1plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes. Appropriate means for transfection, including viral vectors, chemical transfectants, or physico-mechanical methods such as electroporation and direct diffusion of DNA, are described by, for example, Wolff. J. A., et al.. Science. 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352. 815-818. (1991). Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. In certain cases, the methods will be modified to specifically function with large DNA molecules. Further, these methods can be used to target certain diseases and cell populations by using the targeting characteristics of the carrier.

[0080] Expression vectors can be any nucleotide construction used to deliver genes or gene fragments into cells (e.g., a plasmid), or as part of a general strategy to deliver genes or gene fragments, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)). For example, disclosed herein are expression vectors comprising a nucleic acid sequence capable of encoding one or more of the disclosed CAR polypeptides.

[0081] The “control elements” or “expression control elements” present in an expression vector are those non-translated regions of the vector— enhancers, promoters, 5’ and 3’ untranslated regions— which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the pBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or pSPORTl plasmid (Gibco BRL, Gaithersburg, Md.) and the like may be used. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker. In some aspects, the promoter is EFl A promoter or EFS promoter. In some aspects, the expression control element can be operably linked to the nucleic acid sequences disclosed herein.

[0082] Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5’ (Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lusky, M L., et al., Mol. Cell Bio. 3: 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al.. Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al.. Mol. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elementsATTORNEY DOCKET NO. 2II0I.0497P1that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0083] The promoter or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs.

[0084] Optionally, the promoter or enhancer region can act as a constitutive promoter or enhancer to maximize expression of the polynucleotides of the invention. In certain constructs the promoter or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time.

[0085] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as poly adenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3’ untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contains a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases.

[0086] The expression vectors can include a nucleic acid sequence encoding a marker product. This marker product can be used to determine if the gene has been delivered to the cell and once delivered is being expressed. Marker genes can include, but are not limited to the E. coli lacZ gene, which encodes B-galactosidase, and the gene encoding the green fluorescent protein.

[0087] In some embodiments the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markersATTORNEY DOCKET NO. 21101.0497P1are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell’s metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. Two examples are CHO DHFR-cells and mouse LTK-cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary' for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.

[0088] Another ty pe of selection that can be used with the composition and methods disclosed herein is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells w hich have a novel gene would express a protein conveying drug resistance and w ould survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327 (1982)). mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employ bacterial genes under eukary otic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin.

[0089] As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as a nucleic acid sequence capable of encoding ZNF865 into the cell w ithout degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. In some embodiments the nucleic acid sequences disclosed herein are derived from either a virus or a retrovirus. Viral vectors are, for example, Adenovirus, Adeno-associated virus, Herpes virus. Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic pay load, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector. However, they are not as useful in non-proliferating cells. AdenovirusATTORNEY DOCKET NO. 2II0I.0497P1vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells. Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature. A preferred embodiment is a viral vector which has been engineered so as to suppress the immune response of the host organism, elicited by the viral antigens. Preferred vectors of this type will carry coding regions for Interleukin 8 or 10.

[0090] Viral vectors can have higher transaction abilities (i.e., ability to introduce genes) than chemical or physical methods of introducing genes into cells. Typically, viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene / promoter cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material. The necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans.

[0091] Retroviral vectors, in general, are described by Verma, I.M., Retroviral vectors for gene transfer. In Microbiology, Amer. Soc. for Microbiology, pp. 229-232. Washington. (1985). which is hereby incorporated by reference in its entirety. Examples of methods for using retroviral vectors for gene therapy are described in U.S. Patent Nos. 4,868,116 and 4,980,286; PCT applications WO 90 / 02806 and WO 89 / 07136; and Mulligan, (Science 260:926-932 (1993)); the teachings of which are incorporated herein by reference in their entirety for their teaching of methods for using retroviral vectors for gene therapy.

[0092] A retrovirus is essentially a package which has packed into it nucleic acid cargo. The nucleic acid cargo carries with it a packaging signal, which ensures that the replicated daughter molecules will be efficiently packaged within the package coat. In addition to the package signal, there are a number of molecules which are needed in cis, for the replication, and packaging of the replicated virus. Typically a retroviral genome contains the gag, pol, and env genes which are involved in the making of the protein coat. It is the gag, pol, and env genes which are typically replaced by the foreign DNA that it is to be transferred to the target cell. Retrovirus vectors typically contain a packaging signal for incorporation into the package coat, a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purineATTORNEY DOCKET NO. 21101.0497P1rich sequence 5' to the 3' LTR that serves as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTRs that enable the insertion of the DNA state of the retrovirus to insert into the host genome. This amount of nucleic acid is sufficient for the delivery of a one to many genes depending on the size of each transcript. It is preferable to include either positive or negative selectable markers along with other genes in the insert.

[0093] Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol, and env), the vectors are typically generated by placing them into a packaging cell line. A packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery but lacks any packaging signal. When the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.

[0094] The construction of replication-defective adenoviruses has been described (Berkner etal., J. Virology761:1213-1220 (1987); Massie etal., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology 57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987); Zhang "‘Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis” BioTechniques 15:868-872 (1993)). The benefit of the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell ty pes, since they can replicate within an initial infected cell but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites (Morsy, J. Clin. Invest.92:1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest.92:1085-1092 (1993); Moulher, Nature Genetics 4:154-159 (1993); La Salle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout, Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen. Virology 74:501-507 (1993)) the teachings of which are incorporated herein by reference in their entirety for their teaching of methods for using retroviral vectors for gene therapy. Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, in the same manner as wild type orATTORNEY DOCKET NO. 2I10I.0497P1replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, etal., J. Virol. 51:650-655 (1984); Seth, etal., Mol. Cell. Biol., 4:1528-1533 (1984); Varga etal., J. Virology 65:6061-6070 (1991); Wickham et al.. Cell 73:309-319 (1993)).

[0095] A viral vector can be one based on an adenovirus which has had the El gene removed and these virons are generated in a cell line such as the human 293 cell line.Optionally, both the El and E3 genes are removed from the adenovirus genome.

[0096] Another type of viral vector that can be used to introduce the polynucleotides of the invention into a cell is based on an adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site specific integration property are preferred. An especially preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, or a marker gene, such as the gene encoding the green fluorescent protein, GFP.

[0097] In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus. Typically the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and site-specific integration, but not cytotoxicity, and the promoter directs cell-specific expression. United States Patent No.6,261,834 is herein incorporated by reference in its entirety for material related to the AAV vector.

[0098] The inserted genes in viral and retroviral vectors usually contain promoters, or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

[0099] Other useful systems include, for example, replicating and host-restricted nonreplicating vaccinia virus vectors. In addition, the disclosed nucleic acid sequences can be delivered to a target cell in a non-nucleic acid based system. For example, the disclosedATTORNEY DOCKET NO. 2II0I.0497P1polynucleotides can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation. The delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery7is occurring for example in vivo or in vitro.

[0100] Thus, the compositions can comprise, in addition to the disclosed expression vectors, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a peptide and a cationic liposome can be administered to the blood, to a target organ, or inhaled into the respiratory tract to target cells of the respiratory tract. For example, a composition comprising a peptide or nucleic acid sequence described herein and a cationic liposome can be administered to a subjects lung cells. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Patent No. 4,897,355. Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.

[0101] In some aspects, the non-viral vector is a lipid nanoparticle. In some aspects, any known lipid nanoparticle can be used as a vector to deliver the disclosed nucleic acid sequences,proteins or fragments thereof.

[0102] In some aspects, a lipid nanoparticle can be formed using SM-102. In some aspects, lipid nanoparticle, such as those derived from SM-102, are proven to be effective at delivering mRNA. In some aspects, a lipid nanoparticle derived from SM-102 can be one or more of those described in International Application WO 2017049245, hereby incorporated by reference in its entirety7. In some aspects, a lipid nanoparticle compositions can include SMI 02 as well as additional lipids such as phospholipids, structural lipids, and PEG lipids.

[0103] Disclosed herein are lipid nanoparticles for use in the disclosed compositions and methods wherein the lipid nanoparticle comprises an ionizable lipid, cholesterol, a helper lipid, and a PEGylated lipid. In some aspects, the molar ratio of the ionizable lipid, cholesterol, a helper lipid, and a PEGylated lipid is 50:38.5:10:1.5, respectively. In some aspects, the ionizable lipid is SM-102. In some aspects, the helper lipid is l,2-distearoyl-sn-glycero-3-phosphatidylcholine (1,2-DSPC). In some aspects, the PEGylated lipid is PEGylated myristoyl diglyceride.

[0104] Disclosed herein are lipid nanoparticles for use in the disclosed compositions and methods wherein the lipid nanoparticles comprises 40-60% ionizable lipid (SM-102), 30-50% cholesterol, 5-15% helper lipid (DSPC) and 0.2-2.5% PEG-Lipid (DMG-PEG2000). DisclosedATTORNEY DOCKET NO. 2II0I.0497P1herein are lipid nanoparticles for use in the disclosed compositions and methods wherein the lipid nanoparticle comprises 45-55% ionizable lipid (SM-102), 35-30% cholesterol, 7-12% helper lipid (DSPC) and 1.0-2.0% PEG-Lipid (DMG-PEG2000). Disclosed herein are lipid nanoparticles for use in the disclosed compositions and methods wherein the lipid nanoparticle comprises 50% ionizable lipid (SM-102), 38.5% cholesterol, 10% helper lipid (DSPC) and 1.5% PEG-Lipid (DMG-PEG2000).E. Compositions

[0105] Disclosed are compositions comprising any of the disclosed nucleic acid sequences, proteins, or vectors. In some instances, disclosed are compositions comprising a nucleic acid sequence of SEQ ID NO:2 or a nucleic acid sequence of SEQ ID NO: 1 having one or more codon optimizing mutations. In some instances, disclosed are compositions comprising a nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:4. In some instances, disclosed are compositions comprising a nucleic acid sequence of SEQ ID NO:5. In some instances, disclosed are compositions comprising a codon optimized ZNF865. In some instances, disclosed are compositions comprising a protein having the sequence of SEQ ID NO: 6. In some instances, disclosed are compositions comprising a vector comprising one or more of the disclosed nucleic acid sequences or proteins.

[0106] In some instances, the compositions can further comprise a pharmaceutically acceptable carrier. By “pharmaceutically acceptable” is meant a material or carrier that would be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

[0107] Preparations of parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include w ater, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0108] The disclosed nucleic acid sequences or vectors can be formulated and / or administered in or with a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterileATTORNEY DOCKET NO. 21101.0497P1injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug (e.g. peptide) in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0109] Thus, the compositions disclosed herein can comprise lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a peptide and a cationic liposome can be administered to the blood, to a target organ, or inhaled into the respiratory tract to target cells of the respiratory tract. For example, a composition comprising a peptide or nucleic acid sequence described herein and a cationic liposome can be administered to a subject's lung cells. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95 100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413 7417 (1987); U.S. Patent No. 4,897,355. Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from theATTORNEY DOCKET NO. 2II0I.0497P1microcapsule is designed for a specific rate or dosage.

[0110] In some instances, disclosed are pharmaceutical compositions comprising any of the disclosed nucleic acid sequences or vectors described herein, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, buffer, or diluent. In various aspects, the peptide of the pharmaceutical composition is encapsulated in a delivery vehicle. In a further aspect, the delivery vehicle is a liposome, a microcapsule, or a nanoparticle. In a still further aspect, the delivery vehicle is PEG-ylated.

[0111] In the methods described herein, delivery' of the compositions to cells can be via a variety of mechanisms. As defined above, disclosed herein are compositions comprising any one or more of the peptides described herein and can also include a earner such as a pharmaceutically acceptable carrier. For example, disclosed are pharmaceutical compositions, comprising the peptides disclosed herein, and a pharmaceutically acceptable carrier. In one aspect, disclosed are pharmaceutical compositions comprising the disclosed peptides. That is, a pharmaceutical composition can be provided comprising a therapeutically effective amount of at least one disclosed peptide or at least one product of a disclosed method and a pharmaceutically acceptable carrier.

[0112] In certain aspects, the disclosed pharmaceutical compositions comprise the disclosed nucleic acid sequences or vectors as an active ingredient, a pharmaceutically acceptable carrier, and, optionally, other therapeutic ingredients or adjuvants. The instant compositions include those suitable for nasal, oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

[0113] In practice, the nucleic acid sequences or vectors described herein, or pharmaceutically acceptable salts thereof, of this invention can be combined as the active ingredient in intimate admixture yvith a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). Thus, the pharmaceutical compositions of the present invention can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. Further, the compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as aATTORNEY DOCKET NO. 21I01.0497P1non-aqueous liquid, as an oil-in-water emulsion or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the compounds of the invention, and / or pharmaceutically acceptable salt(s) thereof, can also be administered by controlled release (e.g., slow release or long-term release) means and / or delivery devices. The compositions can be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more necessary' ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.

[0114] The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen. Other examples of carriers include dimyristoylphosphatidyl (DMPC), phosphate buffered saline or a multivesicular liposome. For example, PG:PC:Cholesterol:peptide or PC:peptide can be used as carriers in this invention. Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Other examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution can be from about 5 to about 8, or from about 7 to about 7.5. Further carriers include sustained release preparations such as semi-permeable matrices of solid hydrophobic polymers containing the composition, which matrices are in the form of shaped articles, e.g., films, stents (which are implanted in vessels during an angioplasty' procedure), liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. These most typically' would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.

[0115] In order to enhance the solubility and / or the stability of the disclosed peptides in pharmaceutical compositions, it can be advantageous to employ a-. [3- or y-cyclodextrins or their derivatives, in particular hydroxyalkyl substituted cyclodextrins, e.g. 2-hydroxypropyl-|3-cyclodextrin or sulfobutyl-P-cyclodextrin. Also, co-solvents such as alcohols may improve the solubility and / or the stability of the compounds according to the invention in pharmaceuticalATTORNEY DOCKET NO. 21101.0497P1compositions.

[0116] Pharmaceutical compositions can also include carriers, thickeners, diluents, buffers, preservatives and the like, as long as the intended activity of the polypeptide, peptide, nucleic acid, vector of the invention is not compromised. Pharmaceutical compositions may also include one or more active ingredients (in addition to the composition of the invention) such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.

[0117] Because of the ease in administration, oral administration can be used, and tablets and capsules represent the most advantageous oral dosage unit forms in which case solid pharmaceutical carriers are obviously employed. In preparing the compositions for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by¬ standard aqueous or nonaqueous techniques.

[0118] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mon-, di-, trialkyd and ary 1 amines and substituted ethanolamines.

[0119] A tablet containing the compositions of the present invention can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants.Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding in aATTORNEY DOCKET NO. 2II0I.0497P1suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

[0120] The pharmaceutical compositions of the present invention comprise a disclosed nucleic acid sequence or vector as an active ingredient, a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents or adjuvants. The instant compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

[0121] Preparations of parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous earners include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0122] Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. Typically, the final injectable form should be sterile and should be effectively fluid for easy syringability. The pharmaceutical compositions should be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0123] Injectable solutions, for example, can be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations.

[0124] Preparations of parenteral administration include sterile aqueous or non-aqueousATTORNEY DOCKET NO. 2II0I.0497P1solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0125] Pharmaceutical compositions of the present invention can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles, and the like. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations can be prepared, utilizing a compound of the invention, or pharmaceutically acceptable salts thereof, via conventional processing methods. As an example, a cream or ointment is prepared by mixing hydrophilic material and water, together with about 5 wt% to about 10 wt% of the compound, to produce a cream or ointment having a desired consistency.

[0126] In the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and / or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin. Said additives may facilitate the administration to the skin and / or may be helpful for preparing the desired compositions. These compositions may be administered in various ways, e.g., as a transdermal patch, as a spot on. as an ointment.

[0127] Pharmaceutical compositions of this invention can be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in molds.

[0128] Formulations for optical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be desirable.

[0129] In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants canATTORNEY DOCKET NO. 21101.0497P1be included to render the formulation isotonic with the blood of the intended recipient.Compositions containing a disclosed peptide, and / or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.

[0130] The exact dosage and frequency of administration depends on the particular disclosed peptide, a product of a disclosed method of making, a pharmaceutically acceptable salt, solvate, or polymorph thereof, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof; the particular condition being treated and the severity of the condition being treated; various factors specific to the medical history' of the subject to whom the dosage is administered such as the age; weight, sex, extent of disorder and general physical condition of the particular subject, as well as other medication the individual may be taking; as is well known to those skilled in the art. Furthermore, it is evident that said effective daily amount may be lowered or increased depending on the response of the treated subject and / or depending on the evaluation of the physician prescribing the compositions.

[0131] Depending on the mode of administration, the pharmaceutical composition will comprise from 0.05 to 99 % by weight, preferably from 0.1 to 70 % by weight, more preferably from 0.1 to 50 % by w eight of the active ingredient, and, from 1 to 99.95 % by weight, preferably from 30 to 99.9 % by weight, more preferably from 50 to 99.9 % by w eight of a pharmaceutically acceptable earner, all percentages being based on the total weight of the composition.F. Administration

[0132] In some aspects, administration of the disclosed compositions can occur in a clinical, hospital, outpatient, or home setting and can include supervised or unsupervised administration, depending on the formulation, route of administration, and treatment regimen. In some aspects, one or more of the disclosed compositions can be administered by a healthcare professional or the subject (self-administration).1. Routes of administration

[0133] In some aspects, the disclosed compositions can be formulated for different routes of administration. In some aspects, the compositions described herein may be administered to a subject by any suitable route, mode, or technique known in the art, including systemic, local, regional, or targeted administration.

[0134] In some aspects, the disclosed compositions can be administered via parental, oral, mucosal, topical, transdermal, local / regional, or implantable administration.

[0135] In some aspects, parental administration includes, but is not limited to, intravenous, intra-arterial, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathecal,ATTORNEY DOCKET NO. 21101.0497P1intraventricular, or intracardiac administration. In some aspects, parenteral administration can be performed as a bolus injection, continuous infusion, or intermittent dosing.

[0136] In some aspects, mucosal administration can include administering via any mucosal surface. In some aspects, mucosal administration includes, but is not limited to, nasal (intranasal), pulmonary or inhalation, buccal, sublingual, rectal, vaginal, or ocular administration. In some aspects, mucosal administration can allow for local or systemic delivery.

[0137] In some aspects, local or regional administration includes, but is not limited to, intra-tumoral, intra-articular, intracranial, intraocular, intravesical, or intra-organ administration.

[0138] In some aspects, implantable administration includes, but is not limited to, biodegradable or non-biodegradable implants, hydrogels or polymer matrices, microspheres or microcapsules, or osmotic pumps.2. Dosing regimens

[0139] The disclosed compositions can be administered using one, or multiple, of several dosing regimens. The disclosed dosing regimens include using one or more of the routes of administration disclosed herein.

[0140] In some aspects, the disclosed compositions can be administered once in a subject’s lifetime. In some aspects, the disclosed compositions can be administered hourly. For example, a single dose, but not necessarily always the same dose, of the disclosed compositions can be administered every' hour for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some aspects, the disclosed compositions can be administered daily. For example, a single dose, but not necessarily always the same dose, of the disclosed compositions can be administered every day for 1. 2, 3, 4, 5, 6, or 7 days. In some aspects, the disclosed compositions can be administered weekly. For example, a single dose, but not necessarily always the same dose, of the disclosed compositions can be administered every7week for 1, 2, 3, or 4 weeks. In some aspects, the disclosed compositions can be administered monthly. For example, a single dose, but not necessarily always the same dose, of the disclosed compositions can be administered every month for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some aspects, the disclosed compositions can be administered yearly. For example, a single dose, but not necessarily always the same dose, of the disclosed compositions can be administered every7year for 1, 2, 3. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22.23, 24, or 25 years. In some aspects, yearly administration can occur every7year for as long as the subject either needs treatment and / or is alive.

[0141] Disclosed are dosing regimens comprising administering at least one, two, three, or four doses of one or more of the disclosed compositions to a subject in need thereof, whereinATTORNEY DOCKET NO. 2I10I.0497P1each dose is the same concentration. In some aspects, each dose after a first dose can be decreased. In some aspects, each dose after a first dose can be increased.

[0142] In some aspects, a single dose can be a continuous administration. In some aspects, a continuous administration can be hours, days, weeks, or months. In some aspects, there can be two or more doses. In some aspects, the two or more doses can be administered days, weeks, or months apart.3. Location

[0143] In some aspects, the disclosed compositions can be administered to one or more locations on or in a subject. In some aspects, the location can vary based on the disease or condition being treated.

[0144] In some aspects, the disclosed compositions can be administered to a cell, organ, or tissue (which includes blood).

[0145] In some aspects, the disclosed compositions can be administered to. but is not limited to administration to, a tumor, skin, a muscle, a tendon, a ligament, cartilage, blood, the brain, the cerebrospinal fluid, the synovium (e.g., a joint), a cell, fat, or bone. In some aspects, a cell can be, but is not limited to, a neural cell, a fibroblast, a synoviocyte, an adipocyte, a chondrocyte, or an osteoblast.4. Combinations

[0146] In some aspects, one or more of the disclosed compositions can be administered in combination with another therapeutic. In some aspects, the other therapeutic can be a known therapeutic based on the disease or condition being treated. For example, the other therapeutic can be an immunotherapy, chemotherapy, or anti-inflammatory.

[0147] In some aspects, the disclosed compositions can be administered simultaneously or consecutively with another therapeutic.G. Cells

[0148] Disclosed are cells comprising any of the compositions, nucleic acid sequences, proteins, protein fragments or vectors disclosed herein. In some aspects, the compositions, nucleic acid sequences, proteins, protein fragments or vectors are exogenous to the cell. In some aspects, the disclosed cells are recombinant cells.

[0149] Disclosed are cells comprising codon optimized ZNF865 as well as vectors comprising codon optimized ZNF865. These cells can be considered genetically modified. In some aspects, the cells can be a cell line.

[0150] Disclosed are cells comprising a nucleic acid sequence of SEQ ID NO:2. In some aspects, disclosed are cells comprising the nucleic acid sequence of SEQ ID NO: 1 having one orATTORNEY DOCKET NO. 2II0I.0497P1more codon optimizing mutations. In some aspects, disclosed are cells comprising the nucleic acid sequence of SEQ ID NO:1. In some aspects, disclosed are cells comprising the nucleic acid sequence of SEQ ID NO:5. In some aspects, disclosed are cells comprising the ZNF865 protein sequence of SEQ ID NO: 6. Also disclosed are cells comprising a vector comprising the nucleic acid sequence of SEQ ID NO: 1, the nucleic acid sequence of SEQ ID NO: 1 having one or more codon optimizing mutations, the nucleic acid sequence of SEQ ID NO:2, the nucleic acid sequence of SEQ ID NO:5, or the ZNF865 protein of SEQ ID NO:6.

[0151] In some aspects, the disclosed cell can be mammalian cells. In some aspects, the mammalian cell can be a human cell. In some aspects, the mammalian cell can be, but is not limited to, a monkey cell, a dog cell, a cat cell, a horse cell, a pig cell, a goat cell, a rabbit cell, a rat cell, or a mouse cell.

[0152] In some aspects, the cells can be nucleus pulposus cells. In some aspects, a cell can be, but is not limited to, a neural cell, a fibroblast, a synoviocyte, an adipocyte, a chondrocyte, or an osteoblast.H. Methods

[0153] Disclosed are methods of using the disclosed nucleic acid sequences, proteins, protein fragments, vectors or compositions. In some aspects, any use of wild type ZNF865 nucleic acid sequences can be replaced by using the disclosed codon optimized versions of ZNF865 nucleic acid sequences.I. Methods of Increasing ZNF865

[0154] Disclosed are methods of increasing ZNF865 in a cell or subject. In some aspects, an increase in ZNF865 refers to an increase in ZNF865 protein levels and / or an increase in ZNF865 protein activity.

[0155] In some aspects, an increase in ZNF865 can be from administering a ZNF865 protein to the cell. In some aspects, an increase in ZNF865 can be from administering a nucleic acid sequence that encodes ZNF865 in a cell. Thus, in some aspects, an increase in ZNF865 can be from an increase in expression of a ZNF865 nucleic acid sequence that encodes ZNF865.

[0156] Disclosed are methods of increasing ZNF865 expression in a cell comprising contacting a cell with a composition comprising any of the disclosed nucleic acid sequences. The increase in ZNF865 expression can be compared to a cell that is contacted with a wild type nucleic acid sequence, such as SEQ ID NO: 1. The increased ZNF865 expression can be due to the codon optimizing mutations maximizing expression of ZNF865. Thus, in some aspects, a nucleic acid sequence comprising SEQ ID NO: 1 having one or more codon optimizing mutations, a nucleic acid sequence comprising SEQ ID NO:2, or a nucleic acid sequenceATTORNEY DOCKET NO. 2II0I.0497P1comprising SEQ ID NO:5, increases the amount of ZNF865 protein produced.

[0157] In some aspects, the composition comprises a vector comprising one or more of the disclosed nucleic acid sequences, proteins, protein fragments, or vectors. In some aspects, the vector is viral or nonviral. In some aspects, the viral vector is a lentiviral, adenoviral or adeno-associated virus vector. In some aspects, the nonviral vector is a lipid nanoparticle. In some aspects, the vector further comprising one or more expression control elements. In some aspects, the expression control elements are one or more of a promoter and / or enhancer. For example, in some aspects, the promoter is EFl A promoter or EFS promoter. In some aspects, the expression control element is operably linked to the nucleic acid sequence. Thus, in some aspects, any of the vectors disclosed herein can be used.

[0158] In some aspects, an increase in ZNF865 expression can result in modulation of a target gene expression. In some aspects, the target gene is upregulated or downregulated by¬ increases transiently or permanently in ZNF865 protein. In some aspects, the target gene can be aggrecan or collagen II. In some aspects, an increase results in more extracellular matrix. In the disc space this can be especially beneficial to provide cushioning between the vertebrae. In some aspects, the target gene can be, but is not limited to, one or more inflammatory- genes (e.g. IL-1, IL-6, IL-8, TNF-a, TNF-beta, VEGF). In some aspects, the expression of these genes mimics pain responses. In some aspects, the target gene can be, but is not limited to. catabolic genes such as matrix metalloproteinases (e g. MMP-1, MMP-3, MMP-12), histone modifying genes (e.g., IDH-1), DNA repair genes (e.g. MSH2, MLH1, PMS2, XCCR1, RAD51, p53), acetylation genes, or senescence associated genes (e.g. p21, pl 6, or B-gal).

[0159] In some aspects, the disclosed methods of increasing ZNF865 in a cell can be performed in vitro, in vivo, or ex vivo. In some aspects, cells can be removed from a subject, treated with one or more of the disclosed compositions, nucleic acid sequences, proteins, or vectors in culture, and then returned to the subject.2. Method of Treating

[0160] Disclosed are methods of treating a subject in need thereof comprising administering to a subject in need thereof a composition comprising one or more of the disclosed compositions, nucleic acid sequences, proteins, protein fragments, or vectors. In some aspects, a subject in need thereof can be a subject with degenerative disc disease, a musculoskeletal disorder, neurodegenerative disorders, cancer, aging diseases / disorders, Parkinson’s.Alzheimer’s, osteoarthritis, or rare diseases caused by ZNF865 mutations. In some aspects, a subject in need thereof can be any subject that would benefit (become healthier, have reduced symptoms of a disease / disorder) from an increase in ZNF865.ATTORNEY DOCKET NO. 2II0I.0497P1

[0161] Disclosed are methods of treating a subject in need thereof comprising administering to a subject in need thereof a composition comprising a nucleic acid sequence comprising SEQ ID NO:1, SEQ ID NO:1 having one or more codon optimizing mutations, SEQ ID NO:2, or a nucleic acid sequence comprising SEQ ID NO:5. For example, disclosed are methods of treating a subject in need thereof comprising administering to a subject in need thereof a composition comprising nucleic acid sequences comprising SEQ ID NO:2. Also disclosed are methods of treating a subject in need thereof comprising administering to a subject in need thereof a composition comprising a vector, wherein the vector comprises a nucleic acid sequence comprising SEQ ID NO: 1. SEQ ID NO: 1 having one or more codon optimizing mutations, SEQ ID NO:2, or SEQ ID NO:5.

[0162] In some aspects, the compositions comprises a vector comprising one or more of the disclosed nucleic acid sequences. In some aspects, the vector is viral or nonviral. In some aspects, the viral vector is a lentiviral, adenoviral or adeno-associated virus vector. In some aspects, the nonviral vector is a lipid nanoparticle. In some aspects, the vector further comprising one or more expression control elements. In some aspects, the expression control elements are one or more of a promoter and / or enhancer. For example, in some aspects, the promoter is EFl A promoter or EFS promoter. In some aspects, the expression control element is operably linked to the nucleic acid sequence. Thus, in some aspects, any of the vectors disclosed herein can be used.

[0163] In some aspects, administering a composition, nucleic acid sequence, protein, protein fragments, or vector disclosed herein to a subject can result in a decrease in pain, increased tissue growth, increased flexibility, increased muscle growth, increased disc height or disc volume, increased cartilage, increased DNA repair, increased memory, increased focus and / or increased movement. In some aspects, the effects of the administration can depend on what disease or condition the subject has at the time of administration. In some aspects, these results can be increased by altering the route of administration, location of administration, or dosing regimen.

[0164] In some aspects, a depletion of ZNF865 can induce senescence. Thus, in some aspects, disclosed are methods of treating a subject comprising administering to the subject a composition comprising one or more of the disclosed compositions, nucleic acid sequences, proteins, protein fragments, or vectors, wherein the composition prevents or inhibits senescence of cells in the subject. In some aspects, removing cells from senescence can help treat pain in degenerative diseases (e.g. aging diseases) which have increased senescence.

[0165] In some aspects, the disclosed methods of treating can be performed in vitro, in vivo,ATTORNEY DOCKET NO. 2I10I.0497P1or ex vivo. In some aspects, cells can be removed from a subject, treated with one or more of the disclosed compositions, nucleic acid sequences, proteins, or vectors in culture, and then returned to the subject.

[0166] Disclosed are methods of treating a subject in need thereof comprising removing one or more cells from a subject in need thereof: contacting a composition comprising a nucleic acid sequence of SEQ ID NO:1, a nucleic acid sequence of SEQ ID NO:1 having one or more codon optimizing mutations, a nucleic acid sequence of SEQ ID NO:2, a nucleic acid sequence of SEQ ID NO:5. or a protein sequence of SEQ ID NO:6 or a fragment thereof to the one or more cells, forming one or more recombinant cells; and administering the one or more recombinant cells to the subject in need thereof, wherein the one or more recombinant cells provide a therapeutic effect to the subject in need thereof. In some aspects, the subject in need thereof is a subject having degenerative disc disease. In some aspects, the one or more cells are one or more nucleus pulposus cells.3. Method of Increasing Deposition of Aggrecan and / or Collagen II in the Extracellular Matrix of a Cell

[0167] In some aspects, ZNF865 increases deposition of a target gene, such as aggrecan and / or collagen II, in the extracellular matrix of a cell.

[0168] Disclosed are methods of increasing deposition of a target gene, such as aggrecan and / or collagen II, in the extracellular matrix of a cell using one or more of the compositions, nucleic acid sequences, proteins, protein fragments, or vectors disclosed herein.

[0169] Disclosed are methods of increasing deposition of aggrecan and / or collagen II in the extracellular matrix of a cell comprising administering to a cell a composition comprising one or more of the disclosed nucleic acid sequences or vectors, wherein ZNF865 increases deposition of aggrecan and / or collagen II in the extracellular matrix of the cell. In some aspects, the composition comprises a nucleic acid sequence comprising SEQ ID NO:1, a nucleic acid sequence comprising SEQ ID NO: 1 having one or more codon optimizing mutations, a nucleic acid sequence comprising SEQ ID NO:2, or a nucleic acid sequence of SEQ ID NO:5. In some aspects, the composition comprises a ZNF865 protein, such as SEQ ID NO: 6 or a fragment thereof

[0170] In some aspects, the cell is a eukaryotic cell. In some aspects, the eukaryotic cell is a mammalian cell. In some aspects, the mammalian cell is a human cell. In some aspects, the human cell is a stem cell.

[0171] In some aspects, the composition comprises a vector comprising nucleic acid sequences comprising SEQ ID NO:1 having one or more codon optimizing mutations, SEQ IDATTORNEY DOCKET NO. 2II0I.0497P1N0:2, or SEQ ID NO:5.

[0172] In some aspects, the compositions comprises a vector comprising one or more of the disclosed nucleic acid sequences. In some aspects, the vector is viral or nonviral. In some aspects, the viral vector is a lentiviral, adenoviral or adeno-associated virus vector. In some aspects, the nonviral vector is a lipid nanoparticle. In some aspects, the vector further comprising one or more expression control elements. In some aspects, the expression control elements are one or more of a promoter and / or enhancer. For example, in some aspects, the promoter is EFl A promoter or EFS promoter. In some aspects, the expression control element is operably linked to the nucleic acid sequence. Thus, in some aspects, any of the vectors disclosed herein can be used.4. Method of Upregulating a Target Gene

[0173] Disclosed are methods of upregulating a target gene comprising contacting a cell with a composition comprising one or more of the compositions, nucleic acid sequences, proteins, protein fragments, or vectors disclosed herein, wherein ZNF865 upregulates the target gene. Disclosed are methods of upregulating a target gene comprising contacting a cell with a composition comprising a codon optimized ZNF865.

[0174] In some aspects, the target gene is aggrecan or collagen II. In some aspects, the target gene can be, but is not limited to, one or more inflammatory genes, catabolic genes, anabolic genes, extracellular matrix genes, histone modifying genes, DNA repair genes, acetylation genes, or senescence associated genes.

[0175] In some aspects, the cell is a eukary otic cell. In some aspects, the eukaryotic cell is a mammalian cell. In some aspects, the mammalian cell is a human cell. In some aspects, the human cell is a stem cell.

[0176] In some aspects, the composition comprises a vector comprising nucleic acid sequences comprising SEQ ID NO: 1 having one or more codon optimizing mutations, SEQ ID NO:2, or SEQ ID NO:5.

[0177] In some aspects, the compositions comprises a vector comprising one or more of the disclosed nucleic acid sequences. In some aspects, the vector is viral or nonviral. In some aspects, the viral vector is a lentiviral, adenoviral or adeno-associated virus vector. In some aspects, the nonviral vector is a lipid nanoparticle. In some aspects, the vector further comprising one or more expression control elements. In some aspects, the expression control elements are one or more of a promoter and / or enhancer. For example, in some aspects, the promoter is EFl A promoter or EFS promoter. In some aspects, the expression control element is operably linked to the nucleic acid sequence. Thus, in some aspects, any of the vectorsATTORNEY DOCKET NO. 2I10I.0497P1disclosed herein can be used.

[0178] In some aspects, the disclosed methods of upregulating a target gene can be performed in vitro, in vivo, or ex vivo. In some aspects, cells can be removed from a subject, treated with one or more of the disclosed compositions, nucleic acid sequences, proteins, or vectors in culture, and then returned to the subject.5. Methods of Decreasing ZNF865

[0179] Disclosed are methods of decreasing ZNF865 in a cell or subject. In some aspects, decreasing ZNF865 can refer to decreasing ZNF865 protein activity' or decreasing ZNF865 protein levels.

[0180]

[0181] In some aspects, decreasing ZNF865 protein levels can be performed by knocking down, inhibiting or preventing ZNF865 expression using siRNA, antisense RNA, miRNA, IncRNA, or any other known technique for blocking gene expression. In some aspects, decreasing ZNF865 protein activity can be performed using antibodies or other proteins or compounds. In some aspects, any molecule that blocks the active site of the ZNF865 protein can be used to decrease ZNF865 protein activity.

[0182] In some aspects, the disclosed methods of decreasing ZNF865 in a cell can be performed in vitro, in vivo, or ex vivo. In some aspects, cells can be removed from a subject, treated with one or more of the disclosed compositions, nucleic acid sequences, proteins, or vectors in culture, and then returned to the subject.

[0183] In some aspects, the disclosed methods of decreasing ZNF865 can be performed by administering one or more of the disclosed compositions, in particular a lipid nanoparticle carrying an agent that decreases ZNF865.6. Methods of Reducing Senescence

[0184] Disclosed are methods of reducing senescence in a tissue or cell in a subject by administering to a subject in need thereof a composition comprising one or more of the disclosed compositions, nucleic acid sequences, proteins, protein fragments or vectors.

[0185] Disclosed are methods of reducing senescence in a tissue or cell in a subject by administering to a subject in need thereof a composition comprising a nucleic acid sequence comprising SEQ ID NO: 1, SEQ ID NO: 1 having one or more codon optimizing mutations, SEQ ID NO:2. or a nucleic acid sequence comprising SEQ ID NO:5. Also disclosed are methods of reducing senescence in a tissue or cell of a subject comprising administering to a subject in need thereof a composition comprising a vector, wherein the vector comprises a nucleic acid sequence comprising SEQ ID NO:1, SEQ ID NO:1 having one or more codon optimizing mutations, SEQATTORNEY DOCKET NO. 21101.0497P1ID N0:2, or SEQ ID NO:5.

[0186] The method of reducing senescence in a tissue or cell by treating one or more cells in a mammal with any one of the disclosed nucleic acids.

[0187] In some aspects, the compositions comprises a vector comprising one or more of the disclosed nucleic acid sequences. In some aspects, the vector is viral or nonviral. In some aspects, the viral vector is a lentiviral, adenoviral or adeno-associated virus vector. In some aspects, the nonviral vector is a lipid nanoparticle. In some aspects, the vector further comprising one or more expression control elements. In some aspects, the expression control elements are one or more of a promoter and / or enhancer. For example, in some aspects, the promoter is EFl A promoter or EFS promoter. In some aspects, the expression control element is operably linked to the nucleic acid sequence. Thus, in some aspects, any of the vectors disclosed herein can be used.

[0188] In some aspects, administering a composition, nucleic acid sequence, protein, protein fragments or vector disclosed herein to a subject can result in reduced senescence. In some aspects, these results can be increased by altering the route of administration, location of administration, or dosing regimen.

[0189] In some aspects, a depletion of ZNF865 induces senescence. Thus, in some aspects, disclosed are methods of reducing senescence in a tissue or cell of a subject comprising administering to the subject a composition comprising one or more of the disclosed compositions, nucleic acid sequences, proteins, or vectors, wherein the composition prevents or inhibits senescence of cells in the subject. In some aspects, removing cells from senescence can help treat pain in degenerative diseases (e.g. aging diseases) which have increased senescence.

[0190] In some aspects, the disclosed methods can be performed in vitro, in vivo, or ex vivo. In some aspects, cells can be removed from a subject, treated with one or more of the disclosed compositions, nucleic acid sequences, proteins, protein fragments or vectors in culture, and then returned to the subject.

[0191] Disclosed are methods of reducing senescence in a tissue or cell of a subject in need thereof comprising removing one or more cells from a subject in need thereof; contacting a composition comprising a nucleic acid sequence of SEQ ID NO: 1, a nucleic acid sequence of SEQ ID NO: 1 having one or more codon optimizing mutations, a nucleic acid sequence of SEQ ID NO:2. a nucleic acid sequence of SEQ ID NO:5. or a protein sequence of SEQ ID NO:6 to the one or more cells, forming one or more recombinant cells; and administering the one or more recombinant cells to the subject in need thereof, wherein the one or more recombinant cells provide a therapeutic effect to the subject in need thereof. In some aspects, the subject in needATTORNEY DOCKET NO. 2II0I.0497P1thereof is a subject having degenerative disc disease. In some aspects, the one or more cells are one or more nucleus pulposus cells. In some aspects, the therapeutic effect is reduced senescence.7. Method of Recovering Senescent Cells to Reproducing Cells

[0192] Disclosed are methods of recovering senescent cells to reproducing cells comprising administering to a subject in need thereof a composition comprising one or more of the disclosed compositions, nucleic acid sequences, proteins, protein fragments or vectors.

[0193] Disclosed are methods of recovering senescent cells to reproducing cells comprising administering to a subject in need thereof a composition comprising a nucleic acid sequence comprising SEQ ID NO: 1, SEQ ID NO: 1 having one or more codon optimizing mutations, SEQ ID NO:2, or a nucleic acid sequence comprising SEQ ID NO:5. Also disclosed are methods of recovering senescent cells to reproducing cells comprising administering to a subject in need thereof a composition comprising a vector, wherein the vector comprises a nucleic acid sequence comprising SEQ ID NO: 1, SEQ ID NO: 1 having one or more codon optimizing mutations, SEQ ID NO:2, or SEQ IDNO:5.

[0194] Disclosed are methods of recovering senescent cells to reproducing cells by treating one or more cells in a mammal with any of the disclosed nucleic acids.

[0195] In some aspects, the compositions comprises a vector comprising one or more of the disclosed nucleic acid sequences. In some aspects, the vector is viral or nonviral. In some aspects, the viral vector is a lentiviral, adenoviral or adeno-associated virus vector. In some aspects, the nonviral vector is a lipid nanoparticle. In some aspects, the vector further comprising one or more expression control elements. In some aspects, the expression control elements are one or more of a promoter and / or enhancer. For example, in some aspects, the promoter is EFl A promoter or EFS promoter. In some aspects, the expression control element is operably linked to the nucleic acid sequence. Thus, in some aspects, any of the vectors disclosed herein can be used.

[0196] In some aspects, administering a composition, nucleic acid sequence, protein, protein fragments or vector disclosed herein to a subject can result in reduced senescence. In some aspects, these results can be increased by altering the route of administration, location of administration, or dosing regimen.

[0197] In some aspects, a depletion of ZNF865 induces senescence. Thus, in some aspects, disclosed are methods of recovering senescent cells to reproducing cells comprising administering to a subject a composition comprising one or more of the disclosed compositions, nucleic acid sequences, proteins, protein fragments or vectors, wherein the composition preventsATTORNEY DOCKET NO. 2II0I.0497P1or inhibits senescence of cells in the subject. In some aspects, removing cells from senescence can help treat pain in degenerative diseases (e.g. aging diseases) which have increased senescence.

[0198] In some aspects, the disclosed methods can be performed in vitro, in vivo, or ex vivo. In some aspects, cells can be removed from a subject, treated with one or more of the disclosed compositions, nucleic acid sequences, proteins, protein fragments or vectors in culture, and then returned to the subject.

[0199] Disclosed are methods of recovering senescent cells to reproducing cells in need thereof comprising removing one or more cells from a subject in need thereof; contacting a composition comprising a nucleic acid sequence of SEQ ID NO: 1, a nucleic acid sequence of SEQ ID NO: 1 having one or more codon optimizing mutations, a nucleic acid sequence of SEQ ID NO:2, a nucleic acid sequence of SEQ ID NO:5, or a protein sequence of SEQ ID NO:6 to the one or more cells, forming one or more recombinant cells; and administering the one or more recombinant cells to the subject in need thereof, wherein the one or more recombinant cells provide a therapeutic effect to the subject in need thereof. In some aspects, the subject in need thereof is a subject having degenerative disc disease. In some aspects, the one or more cells are one or more nucleus pulposus cells. In some aspects, the therapeutic effect is an increase in reproducing cells.8. Methods of Remodeling Histones

[0200] Disclosed are methods of remodeling histones comprising contacting one or more cells with a composition comprising one or more of the disclosed compositions, nucleic acid sequences, proteins, protein fragments or vectors.

[0201] In some aspects, the disclosed methods of remodeling histones can be a remodeling of histones in cells in a subject or in culture.

[0202] Disclosed are methods of remodeling histones in a subject comprising administering to a subject in need thereof a composition comprising a nucleic acid sequence comprising SEQ ID NO:1. SEQ ID NO:1 having one or more codon optimizing mutations, SEQ ID NO:2, or a nucleic acid sequence comprising SEQ ID NO:5. Also disclosed are methods of remodeling histones in a subject comprising administering to a subject in need thereof a composition comprising a vector, wherein the vector comprises a nucleic acid sequence comprising SEQ ID NO: 1, SEQ ID NO: 1 having one or more codon optimizing mutations, SEQ ID NO:2, or SEQ ID NO:5.

[0203] Disclosed are methods for the remodeling of histones by treating one or more cells in a mammal with any of the disclosed nucleic acids.ATTORNEY DOCKET NO. 2II0I.0497P1

[0204] In some aspects, the compositions comprises a vector comprising one or more of the disclosed nucleic acid sequences. In some aspects, the vector is viral or nonviral. In some aspects, the viral vector is a lentiviral, adenoviral or adeno-associated virus vector. In some aspects, the nonviral vector is a lipid nanoparticle. In some aspects, the vector further comprising one or more expression control elements. In some aspects, the expression control elements are one or more of a promoter and / or enhancer. For example, in some aspects, the promoter is EFl A promoter or EFS promoter. In some aspects, the expression control element is operably linked to the nucleic acid sequence. Thus, in some aspects, any of the vectors disclosed herein can be used.

[0205] In some aspects, administering a composition, nucleic acid sequence, protein, or vector disclosed herein to a subject can result in reduced senescence. In some aspects, these results can be increased by altering the route of administration, location of administration, or dosing regimen.

[0206] In some aspects, the disclosed methods can be performed in vitro, in vivo, or ex vivo. In some aspects, cells can be removed from a subject, treated with one or more of the disclosed compositions, nucleic acid sequences, proteins, or vectors in culture, and then returned to the subject.

[0207] Disclosed are methods of remodeling histones in a subject need thereof comprising removing one or more cells from a subject in need thereof; contacting a composition comprising a nucleic acid sequence of SEQ ID NO: 1, a nucleic acid sequence of SEQ ID NO: 1 having one or more codon optimizing mutations, a nucleic acid sequence of SEQ ID NO:2, a nucleic acid sequence of SEQ ID NO:5, or a protein sequence of SEQ ID NO:6 to the one or more cells, forming one or more recombinant cells; and administering the one or more recombinant cells to the subject in need thereof, wherein the one or more recombinant cells provide a therapeutic effect to the subject in need thereof. In some aspects, the subject in need thereof is a subject having degenerative disc disease. In some aspects, the one or more cells are one or more nucleus pulposus cells.I. Kits

[0208] The materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method. For example disclosed are kits comprising one or more of the disclosed compositions, nucleic acid sequences, proteins, or vectors.

[0209] In some aspects, any of the disclosed kits can further comprise instructions for howATTORNEY DOCKET NO. 2II0I.0497P1and / or when to administer the compositions, nucleic acid sequences, proteins, protein fragments or vectors.Examples1. Example of Lipid Nanoparticle

[0210] The Example describes an example of a lipid nanoparticle (LNP) used to carry the disclosed ZNF865 mRNA is described herein.

[0211] Ionizable lipid 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1 -octylnonyl ester (SM-102), cholesterol, l,2-distearoyl-sn-glycero-3-phosphatidylcholine (1,2-DSPC), and PEGylated myristoyl diglyceride [DMG-PEG(2000)] were dissolved in absolute ethanol at a respective lipid molar ratio of 50:38.5: 10: 1.5. CleanCap M6® ZNF865 mRNA (N1 -Methylpseudouridine) (TriLink Biotechnologies) was dissolved in 10 mM sodium acetate (pH 5.0) and added to the ethanolic lipid mixture using a Herringbone microfluidics chip at an organic: aq (v / v) ratio of 1 :3 and flow rate ratio of 4 mL / min. The LNPs were diluted using equal volume of IX PBS, pH 7.4. Extrusion using a 100 nm membrane was performed to yield a narrow LNP size distribution. LNPs will then be dialyzed in IX PBS, pH 7.4, filter-sterilized, and stored at 4°C for up to 1 week.

[0212] Table 1: Components of the LNP and their molar ratio.

[0213] The mRNA used in these LNPs comprises SEQ ID NO: 5.

[0214] In some aspects, a similar LNP can be used with an ionizable lipid:cholesterol: helper lipid:PEG-lipid ratio of 50%±15%:38.5%±I5%:10%±10%:1.5%±l%, respectively.2. Proliferation of Patient degenerative nucleus pulposus cells (dNPs)

[0215] Degenerative nucleus pulposus cells (dNPs) were the target cells for mRNA delivery. For the following experiments, dNPs were cultured and treated w ith mRNA / LNPs. First, theATTORNEY DOCKET NO. 2II0I.0497P1cells were treated with enhanced green fluorescent protein (EGFP) mRNA / LNPs at 2, 3, 4, and 5 pg / mL mRNA concentrations, and the percentage of EGFP-expressing cells was determined by flow cytometry. No significant differences were observed between the tested concentrations. 2pg / mL mRNA concentration was chosen and used for further analysis. FIG. 4 shows the percentage of EGFP-positive cells. FIG. 5 shows the fluorescence microscopy images of dNPs after mRNA / LNPs treatment at 24 and 48 h time points.

[0216] The LNPs showed no significant toxicity on dNPs at the tested concentrations (FIG.6).

[0217] dNPs were treated with ZNF865 mRNA / LNPs (day 0) for 3 days and the LNPs were replaced with fresh media. The cells were cultured over the course of 11-70 days and images of the cells were taken each day. Cell counts were obtained by manually counting the cells. The percentage cell proliferation was determined compared to the beginning cell numbers. FIG. 7 shows a representative data from 59-year-ol female patient dNPs proliferation following ZNF865 mRNA / LNPs treatment and compared with untreated cells. FIG. 8 shows the doubling time of the human dNPs. The ZNF865 mRNA / LNPs treatment led the dNPs increased proliferation and lower doubling times on 5 different tested patient derived dNPs provided by increased ZNF865 expression through mRNA delivery.

[0218] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

ATTORNEY DOCKET NO. 2110E0497P1CLAIMSWe claim:

1. A nucleic acid sequence comprising the sequence of:ATGGAGGCCAACCCTGCTGGCAGCGGCGCTGGGGGGGGCGGCAGCAGCGGCATC GGTGGCGAGGATGGGGTGCACTTTCAGAGCTACCCATTCGATTTTCTGGAGTTCC TGAACCACCAGAGATTCGAACCTATGGAACTGTACGGCGAGCATGCCAAGGCCG TGGCCGCCCTGCCCTGTGCTCCAGGCCCTCCCCCTCAGCCACCTCCCCAGCCTCCT CCTCCTCAGTATGATTACCCACCCCAGTCCACCTTCAAACCTAAAGCAGAGGTGC CATCTTCAAGTTCTTCCTCTTCCTCTAGCTCCAGCTCTAGCAGTAGTAGCTCCTCA TCCTCCAGCTCCTCCAGCTCCCAGGCCAAGAAGCCCGACCCTCCACTTCCCCCTG CCTTCGGGGCCCCGCCCCCTCCCCTCTTTGATGCTGCCTTCCCAACCCCCCAGTGG GGGATCGTCGACCTGAGCGGCCACCAGCACCTGTTCGGCAATCTGAAAAGGGGC GGCCCCGCCTCCGGACCTGGGGTGACCCCTGGCCTGGGAGCCCCAGCAGGGGCC CCGGGCCCCCTGCCAGCCCCCTCCCAGACCCCCCCTGGGCCCCCTGCCGCAGCTG CCTGTGATCCAACAAAGGACGACAAAGGGTACTTTAGGCGCCTGAAGTACCTGA TGGAAAGGAGATTCCCCTGTGGCGTCTGTCAGAAGAGCTTCAAGCAATCTAGCC ACCTGGTGCAGCACATGCTGGTGCACTCTGGAGAGCGGCCGTATGAGTGCGGGG TGTGCGGCCGCACATACAACCATGTGTCAAGCCTGATTCGCCACAGGCGGTGTCA CAAAGACGTGCCTCCCGCTGCCGGAGGGCCACCTCAGCCAGGCCCTCACTTACCC CCCCTGGGCCTCCCCGCTCCCGCCGCCTCCGCCGCCACGGCAGCTGCTCCCAGTA CTGTGAGCTCTGGCCCCCCAGCCACTCCCGTGGCACCCGCACCCTCAGCAGACGG CTCTGCTGCCCCAGCCGGAGTGGGCGTGCCACCACCAGCCACCGGCGGCGGCGA CGGACCTTTCGCTTGTCCACTGTGCTGGAAAGTGTTTAAGAAGCCAAGCCATCTG CATCAGCATCAGATTATCCACACCGGCGAGAAGCCTTTCAGTTGTTCTGTGTGTT CTAAGTCTTTCAACAGGCGCGAATCTCTGAAACGGCACGTGAAAACACATAGCG CGGATCTGCTGAGGTTGCCTTGTGGCATCTGCGGGAAGGCCTTTCGGGACGCATC TTACCTGCTGAAGCACCAGGCCGCCCACGCCGGTGCCGGTGCCGGCGGACCCCG CCCCGTCTACCCTTGTGATCTGTGTGGTAAAAGCTATAGCGCCCCTCAGAGCCTG CTGAGGCACAAGGCCGCACACGCCCCTCCTGCAGCCGCCGCCGAGGCTCCTAAG GACGGCGCCGCCAGCGCTCCCCAGCCCCCTCCCACCTTCCCACCTGGACCTTACC TGCTCCCTCCAGACCCCCCAACTACCGATAGCGAGAAAGCCGCTGCAGCCGCCG CTGCAGTGGTGTATGGCGCAGTGCCTGTGCCACTGCTGGGCGCACACCCCCTGCT GCTGGGCGGCGCTGGCACCTCCGGCGCCGGCGGCTCAGGCGCCTCCGTCCCAGG415119095 52ATTORNEY DOCKET NO. 2110E0497P1CAAGACATTCTGTTGTGGAATTTGTGGAAGGGGCTTTGGCAGAAGAGAAACTCT GAAGAGACACGAGAGGATCCATACCGGCGAGAAGCCTCACCAGTGCCCAGTGTG CGGAAAGAGATTCAGAGAGTCCTTCCATCTGTCCAAGCACCACGTGGTGCACAC AAGGGAGAGGCCTTATAAGTGCGAGCTCTGTGGAAAGGTTTTCGGCTATCCACA AAGCCTGACAAGGCACAGACAGGTGCACAGGCTGCAGCTCCCCTGCGCCCTGGC CGGAGCCGCCGGCCTGCCATCAACACAGGGGACCCCCGGAGCCTGTGGGCCTGG CGCCTCAGGGACCAGCGCCGGCCCAACAGATGGCCTGAGCTACGCATGTAGCGA CTGCGGAGAACACTTCCCTGACCTGTTTCACGTGATGTCCCATAAGGAGGTGCAC ATGGCCGAGAAGCCTTATGGCTGTGATGCCTGTGGGAAGACCTTTGGCTTCATAG AAAACCTGATGTGGCATAAGCTCGTACACCAGGCAGCCCCTGAGCGGCTCCTTCC ACCTGCCCCTGGAGGGCTTCAGCCCCCTGACGGCTCCAGCGGTACCGACGCTGCT TCTGTGCTCGACAACGGCCTGGCCGGTGAGGTGGGGGCCGCAGTGGCCGCACTG GCTGGAGTGTCCGGTGGAGAGGACGCTGGGGGCGCCGCCGTGGCCGGCGCTGGC GGCGGCGCCTCCAGCGGCCCCGAGAGATTTTCATGTGCCACCTGCGGGCAGTCCT TTAAGCACTTCCTGGGCCTTGTCACTCATAAATACGTGCACCTGGTGAGGAGGAC TCTGGGTTGCGGCCTGTGTGGTCAGAGCTTCGCCGGCGCCTATGACCTGCTGCTG CACAGACGGAGCCACCGCCAGAAGCGCGGCTTCCGGTGTCCTGTGTGCGGGAAG AGGTTCTGGGAGGCAGCCCTGCTGATGAGACACCAGAGATGCCACACCGAACAG AGACCATACCGCTGTGGCGTGTGCGGCAGAGGCTTTCTGCGCAGTTGGTACCTCA GGCAGCACAGAGTGGTGCATACCGGCGAAAGAGCCTTCAAGTGCGGCGTGTGCG CCAAGAGGTTCGCCCAGTCATCTTCTCTCGCTGAGCACCGGAGGTTGCATGCCGT GGCTAGACCCCAACGTTGCTCCGCCTGCGGCAAGACTTTCCGCTACCGGTCTAAC CTTCTGGAGCACCAGAGGCTGCATCTGGGAGAACGCGCCTACAGGTGCGAACAC TGCGGTAAAGGATTCTTTTACCTGAGCAGCGTGCTGCGGCACCAGAGAGCTCACG AACCCCCACGGCCAGAGCTGAGGTGTCCCGCTTGTCTGAAAGCATTCAAGGACC CAGGATACTTTAGAAAGCATCTCGCGGCCCATCAAGGGGGAAGGCCTTTCAGAT GCAGTTCCTGTGGTGAAGGGTTCGCAAACACCTACGGCCTCAAGAAACACAGAT TGGCTCATAAAGCAGAGAACCTGGGCGGGCCTGGCGCCGGGGCTGGAACGCTGG CCGGCAAAGATGCC (SEQ ID NO: 2).

2. A nucleic acid sequence comprising the sequence of:ATGGAGGCGAACCCAGCGGGCAGCGGCGCCGGGGGTGGCGGGAGCAGCGG CATCGGGGGCGAGGACGGGGTGCACTTCCAGAGCTACCCCTTCGACTTCC TGGAATTCCTCAACCACCAGCGCTTCGAGCCCATGGAACTGTATGGGGAA415119095 53ATTORNEY DOCKET NO. 2110E0497P1CACGCCAAGGCGGTGGCGGCCCTGCCCTGCGCCCCCGGCCCCCCGCCGCA GCCCCCGCCGCAGCCCCCTCCCCCGCAGTATGACTACCCGCCCCAGTCCA CCTTCAAGCCCAAGGCGGAGGTGCCCTCCTCGTCCTCGTCCTCGTCCTCC TCCTCCTCCTCTTCGTCCTCCTCGTCGTCATCTTCGTCCTCTTCCTCTTC CCAAGCCAAGAAGCCCGATCCGCCCCTGCCGCCCGCCTTCGGGGCGCCCC CTCCTCCCCTCTTTGACGCTGCTTTCCCCACTCCGCAGTGGGGCATCGTG GACCTCTCGGGGCACCAGCACTTGTTTGGGAACCTGAAGCGAGGAGGGCC CGCGTCCGGGCCGGGGGTGACGCCTGGGCTGGGCGCTCCCGCGGGGGCCC CAGGGCCGCTTCCTGCCCCCTCGCAGACCCCGCCAGGACCCCCCGCGGCG GCGGCCTGCGACCCCACCAAGGACGACAAGGGCTACTTCCGGAGACTGAA GTACCTGATGGAGCGGCGCTTCCCCTGCGGCGTGTGCCAGAAGTCCTTCA AGCAGTCCTCGCACCTGGTCCAGCACATGCTGGTGCACTCGGGGGAGAGG CCCTACGAATGCGGCGTCTGCGGCCGCACCTACAACCACGTGTCCAGCCT CATCCGCCACCGCCGCTGCCACAAGGACGTGCCACCGGCCGCGGGGGGCC CGCCCCAGCCCGGCCCCCACCTCCCGCCGCTGGGCCTCCCAGCACCCGCT GCCAGCGCCGCCACCGCCGCCGCCCCCTCCACGGTGTCCTCGGGCCCTCC AGCCACGCCCGTGGCGCCTGCCCCCTCCGCAGACGGGAGCGCCGCCCCTG CTGGTGTTGGGGTGCCCCCTCCTGCCACCGGGGGTGGCGATGGCCCGTTC GCCTGCCCACTCTGCTGGAAGGTTTTCAAGAAGCCCAGTCACCTCCACCA GCACCAGATCATCCACACGGGCGAGAAGCCCTTCTCCTGCTCCGTGTGCA GCAAAAGCTTCAACCGCAGGGAGAGTCTGAAGCGCCACGTGAAGACGCAC TCGGCCGACCTCCTGCGCCTGCCCTGCGGCATCTGCGGGAAGGCCTTCCG CGACGCCTCCTACCTCCTCAAGCACCAGGCGGCCCACGCGGGGGCGGGCG CCGGGGGGCCTCGGCCCGTGTACCCCTGCGACCTGTGCGGCAAGTCCTAC TCGGCTCCGCAGAGCCTGCTCCGCCACAAGGCCGCCCACGCCCCGCCCGC TGCCGCTGCGGAGGCGCCCAAGGACGGGGCGGCCTCGGCCCCGCAGCCCC CGCCCACCTTCCCCCCGGGCCCGTACCTCCTGCCCCCCGACCCTCCCACC ACAGACAGCGAGAAGGCGGCGGCGGCCGCGGCGGCGGTGGTGTACGGCGC TGTGCCCGTCCCGCTCCTGGGCGCCCACCCGCTGCTGCTCGGCGGCGCGG GGACCAGCGGGGCGGGAGGCTCGGGCGCCAGCGTCCCAGGAAAGACGTTC TGCTGCGGCATCTGCGGGCGCGGCTTCGGGCGCCGCGAGACCCTGAAGCG CCATGAGCGCATCCACACGGGCGAGAAGCCCCACCAGTGCCCCGTGTGTG GGAAGCGCTTCCGCGAATCCTTCCACTTGAGCAAGCATCACGTGGTGCAC ACGCGCGAGCGGCCCTACAAGTGCGAGCTCTGCGGCAAGGTCTTCGGCTA415119095 54ATTORNEY DOCKET NO. 21101.0497P1CCCGCAGAGCCTCACCCGCCACCGCCAGGTGCACCGGCTCCAGCTGCCCT GCGCCCTGGCCGGGGCAGCCGGCCTCCCCTCCACCCAAGGCACACCGGGG GCCTGTGGGCCCGGGGCCTCGGGCACGTCTGCAGGGCCCACCGATGGGCT GAGCTACGCCTGCTCGGACTGCGGCGAGCACTTCCCGGATCTCTTTCACG TCATGAGTCACAAGGAGGTCCACATGGCAGAGAAGCCATACGGCTGCGAC GCCTGCGGCAAGACCTTCGGCTTCATCGAGAACCTCATGTGGCACAAGCT GGTCCACCAGGCCGCCCCCGAGCGCCTGCTCCCGCCCGCACCCGGCGGCC TGCAGCCCCCGGACGGCTCCAGCGGCACGGATGCGGCCAGCGTGCTGGAC AACGGGCTGGCGGGGGAGGTGGGGGCGGCCGTGGCGGCACTGGCAGGGGT GTCTGGGGGTGAGGACGCAGGCGGGGCGGCGGTGGCAGGTGCTGGCGGGG GTGCCAGTTCCGGCCCCGAGCGCTTCAGCTGTGCCACGTGCGGCCAGAGT TTCAAGCACTTCCTGGGCCTCGTGACTCACAAGTACGTGCACCTGGTGCG ACGGACCCTGGGCTGCGGCCTCTGCGGCCAGAGCTTCGCGGGCGCCTACG ACTTGCTCCTACACCGCCGCAGCCATCGGCAGAAGCGGGGTTTCCGCTGC CCGGTGTGCGGGAAGCGCTTCTGGGAGGCGGCCCTGCTGATGCGCCACCA GCGCTGCCACACGGAACAGCGGCCGTACCGATGTGGCGTGTGCGGCCGAG GCTTCCTGCGCTCCTGGTACCTGCGGCAGCACCGCGTGGTGCACACTGGC GAGCGGGCCTTCAAGTGCGGCGTGTGCGCCAAGCGCTTCGCGCAGTCGTC CAGCCTGGCAGAGCACCGGCGGCTGCACGCTGTGGCCCGGCCCCAGCGCT GCAGCGCCTGTGGCAAGACCTTCCGCTACCGCTCCAACCTGCTGGAGCAC CAGCGGCTGCACCTGGGCGAGCGCGCCTACCGCTGTGAGCACTGCGGCAA GGGCTTCTTCTACCTGAGCTCCGTGCTGCGCCACCAGCGCGCCCATGAGC CGCCGCGGCCCGAGCTCCGCTGCCCCGCCTGCCTCAAGGCCTTCAAGGAT CCCGGCTACTTCCGTAAGCACCTGGCTGCCCACCAGGGCGGCCGGCCCTT CCGCTGCTCCTCCTGCGGCGAGGGCTTCGCCAACACCTACGGCCTCAAGA AACACCGCCTGGCGCACAAGGCCGAGAACCTCGGGGGGCCTGGAGCAGGG GCGGGCACCTTGGCCGGGAAGGATGCCTGA (SEQ ID NO: 1) comprising one or more codon optimizing mutations.

3. A vector comprising the nucleic acid sequence of claim 1 or claim 2.

4. The vector of claim 3, wherein the vector is a viral vector or nonviral vector.

5. The vector of claim 4, wherein the viral vector is a lentiviral vector, adenoviral vector, or adeno-associated viral vector.415119095 55ATTORNEY DOCKET NO. 21101.0497P16. The vector of claim 4, wherein the nonviral vector is a lipid nanoparticle.

7. The vector of any one of claims 3-6, further comprising one or more expression control elements.

8. The vector of claim 7, wherein the expression control elements are one or more of a promoter and / or enhancer.

9. The vector of claim 8, wherein the promoter is EF 1 A promoter or EFS promoter.

10. The vector of any one of claims 7-9, wherein the expression control element is operably linked to the nucleic acid sequence of claim 1 or claim 2.

11. A composition comprising the nucleic acid sequence of any one of claims 1-2 or the vector of any one of claims 3-10.

12. The composition of claim 11. further comprising a pharmaceutically acceptable carrier.

13. A method of increasing ZNF865 expression in a cell comprising: contacting a cell with a composition comprising a nucleic acid sequence of any one of claims 1-2.

14. The method of claim 13, wherein the nucleic acid sequence is in a vector.

15. The method of claim 14, wherein the vector is viral or nonviral.

16. The method of claim 15, wherein the viral vector is a lentiviral, adenoviral or adeno-associated virus vector.

17. The method of claim 15, wherein the nonviral vector is a lipid nanoparticle.

18. The method of any one of claims 15-17, wherein the vector further comprising one or more expression control elements.

19. The method of claim 18, wherein the expression control elements are one or more of a promoter and / or enhancer.

20. The method of claim 19, wherein the promoter is EFl A promoter or EFS promoter.415119095 56ATTORNEY DOCKET NO. 2I10I.0497PI21. The method of any one of claims 19-20, wherein the expression control element is operably linked to the nucleic acid sequence.

22. A method of treating degenerative disc disease comprising administering to a subject a composition comprising a nucleic acid of any one of claims 1-2.

23. The method of claim 22, wherein the nucleic acid sequence is in a vector.

24. The method of claim 23, wherein the vector is viral or nonviral.

25. The method of claim 24, wherein the viral vector is a lentiviral, adenoviral or adeno-associated virus vector.

26. The method of claim 24, wherein the nonviral vector is a lipid nanoparticle.

27. The method of any one of claims 23-26, wherein the vector further comprising one or more expression control elements.

28. The method of claim 27, wherein the expression control elements are one or more of a promoter and / or enhancer.

29. The method of claim 28, wherein the promoter is EFl A promoter or EFS promoter.

30. The method of any one of claims 27-29, wherein the expression control element is operably linked to the nucleic acid sequence.

31. A method of increasing deposition of aggrecan and / or collagen II in the extracellular matrix of a cell comprising administering to a cell a composition comprising a nucleic acid of any one of claims 1-2,wherein ZNF865 increases deposition of aggrecan and / or collagen II in the extracellular matrix of the cell.

32. The method of claim 31, wherein the cell is a eukaryotic cell.

33. The method of claim 32, wherein the eukaryotic cell is a mammalian cell.

34. The method of claim 33, wherein the mammalian cell is a human cell.

35. The method of claim 34, wherein the human cell is a stem cell.415119095 57ATTORNEY DOCKET NO. 21101.0497P136. The method of any one of claims 31-35, wherein the nucleic acid sequence is in a vector.

37. The method of claim 36, wherein the vector is viral or nonviral.

38. The method of claim 37, wherein the viral vector is a lentiviral, adenoviral or adeno-associated virus vector.

39. The method of claim 37, wherein the nonviral vector is a lipid nanoparticle.

40. The method of any one of claims 36-39, wherein the vector further comprising one or more expression control elements.

41. The method of claim 40, wherein the expression control elements are one or more of a promoter and / or enhancer.

42. The method of claim 41, wherein the promoter is EFl A promoter or EFS promoter.

43. The method of any one of claims 41-42, wherein the expression control element is operably linked to the nucleic acid sequence.

44. A method of upregulating a target gene comprising contacting a cell with a composition comprising a nucleic acid sequence of any one of claims 1-2,wherein ZNF865 upregulates the target gene.

45. The method of claim 44, wherein the target gene is aggrecan or collagen II.

46. The method of any one of claims 43-44 wherein the cell is a eukaryotic cell.

47. The method of claim 46, wherein the eukaryotic cell is a mammalian cell.

48. The method of claim 47, wherein the mammalian cell is a human cell.

49. The method of claim 48, wherein the human cell is a stem cell.

50. The method of any one of claims 44-49, wherein the nucleic acid sequence is in a vector.

51. The method of claim 50, wherein the vector is viral or nonviral.415119095 58ATTORNEY DOCKET NO. 21101.0497P152. The method of claim 51, wherein the viral vector is a lentiviral, adenoviral or adeno-associated virus vector.

53. The method of claim 51 , wherein the nonviral vector is a lipid nanoparticle.

54. The method of any one of claims 50-53, wherein the vector further comprising one or more expression control elements.

55. The method of claim 54, wherein the expression control elements are one or more of a promoter and / or enhancer.

56. The method of claim 55, wherein the promoter is EFl A promoter or EFS promoter.

57. The method of any one of claims 54-56, wherein the expression control element is operably linked to the nucleic acid sequence.

58. The method of any one of claims 44-57, wherein contacting a cell comprises administering to a subject comprising the cell the composition comprising the nucleic acid sequence.

59. A vector comprising a nucleic acid sequence of SEQ ID NO: 1, a nucleic acid sequence of SEQ ID NO: 1 having one or more codon optimizing mutations, a nucleic acid sequence of SEQ ID NO:2, a nucleic acid sequence of SEQ ID NO:5, or a protein sequence of SEQ ID NO:6.

60. The vector of claim 59, wherein the vector is a lipid nanoparticle.

61. The vector of any one of claims 59-60, wherein the lipid nanoparticle comprises an ionizable lipid, cholesterol, a helper lipid, and a PEGylated lipid.

62. The vector of claim 61, wherein the ionizable lipid is SM-102.

63. The vector of any one of claims 61-62, wherein the helper lipid is 1.2-distearoyl-sn-glycero-3 -phosphatidylcholine (1,2-DSPC).

64. The vector of any one of claims 61-63, wherein the PEGylated lipid is PEGylated myristoyl diglyceride.415119095 59ATTORNEY DOCKET NO. 21101.0497P165. The vector of any one of claims 61-64. wherein the molar ratio of the ionizable lipid, cholesterol, a helper lipid, and a PEGylated lipid is 50:38.5:10:1.5, respectively.

66. A method of treating a subject in need thereof comprisingremoving one or more cells from a subject in need thereof;contacting a composition comprising a nucleic acid sequence of SEQ ID NO: 1, a nucleic acid sequence of SEQ ID NO: 1 having one or more codon optimizing mutations, a nucleic acid sequence of SEQ ID NO:2, a nucleic acid sequence of SEQ ID NO:5, or a protein sequence of SEQ ID NO:6 or a fragment thereof to the one or more cells, forming one or more recombinant cells; andadministering the one or more recombinant cells to the subject in need thereof, wherein the one or more recombinant cells provide a therapeutic effect to the subject in need thereof.

67. The method of claim 66, wherein the subject in need thereof is a subject having degenerative disc disease.

68. The method of any one of claims 66-67, wherein the one or more cells are one or more nucleus pulposus cells.

69. A nucleic acid sequence comprising the sequence of SEQ ID NO:5.

70. A method of reducing senescence in a tissue by treating one or more cells in a mammal with any one of the nucleic acid sequence of claims 1-2 or 69 or any one of the vectors of claims 3-10, 59-65 or the composition of claims 11-12.

71. A method of recovering senescent cells to reproducing cells by treating one or more cells in a mammal with any of claims 1 -4.

72. A method for the remodeling of histones by treating one or more cells in a mammal with any one of the nucleic acid sequence of claims 1-2 or 69 or any one of the vectors of claims 3-10, 59-65 or the composition of claims 11-12.415119095 60

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