Immortalized mesenchymal stromal cells and methods of making

Abstract

Provided herein is an immortalized MSC that heterologously expresses p53-upregulated-regulator-of-p53-levels (PURPL) lncRNA. Also provided is a method for making an immortalized MSC comprising heterologously expressing PURPL lncRNA in an MSC. Also provided are methods for treating a disease in a subject in need thereof comprising administering to the subject the immortalized MSC described herein or made by the methods described herein.

Description

PATENT Atorney Docket No. 081906-1542874-255610PC Client Ref. No. SF2024-208IMMORTALIZED MESENCHYMAL STROMAL CELLS AND METHODS OF MAKING CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present patent application claims benefit of priority to U.S. Provisional Patent Application No. 63 / 743,972, filed January 10, 2025, which is incorporated by reference in its entirety for all purposes.BACKGROUND OF THE INVENTION

[0002] Mesenchymal stromal cells (MSCs) have been investigated in well over 1,500 registered clinical trials worldwide. The interest in therapeutic applications of human MSCs stems from their immunomodulatory and trophic properties, along with their capacity to migrate to sites of tissue injury, inflammation, or tumor growth. The intrinsic ability of MSCs to migrate to metastatic sites and infiltrate solid tumors has prompted their utilization as living carriers for delivery of the anticancer therapeutic agents.

[0003] Clinical translation of MSCs necessitates a standardized manufacturing process for MSCs. While MSCs can self-renew, they have a limited replicative lifespan before reaching senescence, requiring constant re-derivation incurring recurring costs. Despite achievability, clinical trials have faced failures due to challenges like limited sources, donor variance, cell population heterogeneity, and inconsistent characteristics affecting potency and viability.BRIEF SUMMARY OF THE INVENTION

[0004] Provided herein is an immortalized MSC that heterologously expresses a p53-upregulated-regulator-of-p53-levels (PURPL) long non-coding RNA (IncRNA). In some embodiments, the PURPL IncRNA comprises one or more exon at least 95% identical to any one of SEQ ID NO: 3-8. In some embodiments, the PURPL IncRNA comprises exon 1, exon 2, exon 3, exon 3.5, exon 4, and exon 5, wherein exon 1 is at least 95% identical to SEQ ID NO: 3, exon2 is at least 95% identical to SEQ ID NO: 4, exon 3 is at least 95% identical to SEQ ID NO: 5, exon 3.5 is at least 95% identical to SEQ ID NO: 6, exon 4 is at least 95% identical to SEQ ID NO: 7, and exon 5 is at least 95% identical to SEQ ID NO: 8. In some embodiments, exon 1 comprises SEQ ID NO: 3, exon 2 comprises SEQ ID NO: 4, exon 3 comprises SEQ ID NO: 5, exon 3.5 comprises SEQ ID NO: 6, exon 4 comprises SEQ ID NO: 7, and exon 5 comprises SEQ ID NO: 8.

[0005] In some embodiments, the MSC further expresses a heterologous nucleotide sequence encoding an immortalization polypeptide selected from the group consisting of: MYC, SV40 Large T Antigen, STAT3, telomerase reverse transcriptase (TERT), E6 HPV, E7 HPV, Bmi-1, or NOTCH2 intracellular domain. In some embodiments, the MSC is a human MSC and TERT is human TERT (hTERT). In some embodiments, MYC is L-MYC, N-MYC, V-MYC, or C-MYC. In some embodiments, the MYC polypeptide is an L-MYC polypeptide at least 95% identical to SEQ ID NO: 1. In some embodiments, the TERT polypeptide is at least 95% identical to SEQ ID NO: 2. In some embodiments, SV40 Large T Antigen is at least 95% identical to SEQ ID NO: 12, STAT3 is at least 95% identical to SEQ ID NO: 13, E6 HPV is at least 95% identical to SEQ ID NO: 14, E7 HPV is at least 95% identical to SEQ ID NO: 15, Bmi-1 is at least 95% identical to SEQ ID NO: 16, and NOTCH2 intracellular domain is at least 95% identical to SEQ ID NO: 17. In some embodiments, the MSC comprises one or more expression cassettes comprising a promoter operably linked to a heterologous nucleotide sequence encoding the immortalization polypeptide and / or the PURPL IncRNA.

[0006] In some embodiments, the MSC further comprises an expression cassette comprising a promoter operably linked to a heterologous nucleotide sequence encoding a therapeutic payload. In some embodiments, the therapeutic payload is an antibody, for example a bispecific antibody or a bispecific T cell engager. In some embodiments, the therapeutic payload is a type VII collagen. In some embodiments, the therapeutic payload is a cytokine.

[0007] In some embodiments, the MSC comprises a CRISPR activation (CRISPRa) system effecting increased expression of the PURPL IncRNA by the MSC (e.g., by targeting the PURPL IncRNA promoter with a CRISPRa protein). In some embodiments, the MSC comprises a viral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the retroviral vector has independent replicative capacity. In some embodiments, the retroviral vectorlacks independent replicative capacity. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the lentiviral vector lacks independent replicative capacity.

[0008] In some embodiments, the MSC comprises a first expression cassette comprising a promoter operably linked to a nucleotide sequence encoding MYC; a second expression cassette comprising a promoter operably linked to a nucleotide sequence encoding TERT; and a third expression cassette comprising a promoter operably linked to a nucleotide sequence encoding the PURPL IncRNA. In some embodiments, one, two or all three of the first and second and third expression cassettes are in one of more vectors. In some embodiments, the MSC comprises a first expression cassette comprising a promoter operably linked to a nucleotide sequence encoding MYC and TERT; and a second expression cassette comprising a promoter operably linked to a nucleotide sequence encoding the PURPL IncRNA. In some embodiments, one or both of the first and second expression cassettes are in one or more vectors. In some embodiments, the vector is a viral vector.

[0009] Also provided is a method for making an immortalized MSC comprising heterologously expressing in an MSC a PURPL IncRNA, thereby immortalizing the MSC. Also provided is a method of treating a disease in a subject in need thereof comprising administering to the subject the immortalized MSC described above or elsewhere herein or the immortalized MSC made by the method described above or elsewhere herein. In some embodiments, the immortalized MSC used in method of treating a disease in a subject in need thereof delivers a therapeutic payload to the subject. In some embodiments, the disease is cancer. In some embodiments, the cancer is lung cancer, breast cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, kidney cancer, urinary tract cancer, oral cancer, head and neck cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, skin cancer, melanoma, sarcoma, lymphoma, leukemia, or brain cancer including glioblastoma, anaplastic astrocytoma, oligodendroglioma, and medulloblastoma. In some embodiments, the disease is a skin disorder. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is an autoinflammatory disease. In some embodiments, the disease is a neurodegenerative disease. In some embodiments, the disease is a monogenic hereditary disorder. In some embodiments, the disease is a chronic degenerative tissue disease.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1A shows schematics of vector configurations. The top panel shows vector configurations for generating clone LT 100 and the bottom panel shows vector configurations for generating clone ALT422. FIG. IB is a graph showing the growth rate as a function of population doubling time of naive WJ-MSC cells, and immortalized L-MYC and hTERT clones ALT422 and LT100.

[0011] FIG. 2 is a volcano plot showing overexpression of PURPL IncRNA in immortalized ALT422 clones versus naive MSCs.

[0012] FIG. 3 shows the PCR product of PURPL cDNA isolated from ALT422 clones at passage 21.

[0013] FIG. 4 is a schematic showing the longest PURPL splice variant (PCR product 4) with 6 exons and 1219 nt compared to another splice variant (PCR product 5) having 5 exons and 1118 nt and the Ensembl PURPL sequence having 5 exons and 1106 nt.

[0014] FIG. 5 shows schematics of two vector configurations for generating WPP immortalized clones WPP IB, WPPF72, and WPPC71.

[0015] FIG. 6 is a graph showing the growth rate as a function of population doubling time of naive WJ-MSC cells, immortalized L-MYC and hTERT clones ALT422 and LT100, and PURPL immortalized clones WPP IB, WPPF72, and WPPC71.

[0016] FIG. 7 shows schematics of three vector configurations for generating ELTP immortalized clones IF8, SC5, SF6, IB3, 2F11, IG8 and IC7.

[0017] FIG. 8 shows the growth rate as a function of population doubling time for naive WJ-MSCs, immortalized PURPL-overexpressing WPP and ELTP clones, and PURPL-non-overexpressing clones that did not achieve immortalization.

[0018] FIG. 9 is a graph showing PURPL expression in immortalized and non-immortalized clones. The graph is a result of RT-qPCR analysis of PURPL lincRNA expression in immortalized and non-immortalized MSCs relative to untransduced naive MSCs, normalized to RNaseP.

[0019] FIG. 10 is a graph showing L-MYC expression in immortalized and not growing cells. The graph is a result of RT-qPCR analysis of L-MYC expression in immortalized and nonimmortalized MSCs relative to untransduced naive MSCs, normalized to RNaseP.

[0020] FIG. 11 is a graph showing hTERT expression in immortalized and not growing cells. The graph is a result of RT-qPCR analysis of hTERT expression in immortalized and nonimmortalized MSCs relative to untransduced naive MSCs, normalized to RNaseP.

[0021] FIG. 12 is a graph showing the growth rate as a function of population doubling time of naive WJ-MSC cells, immortalized PURPL-positive WPPF72 clone versus the RRV infected counterparts (RRV-GFP or RRV-yCD, containing a cytosine deaminase payload).

[0022] FIG. 13 shows images of a 21 -day trilineage differentiation of MSCs into chondrogenic (left panel), osteogenic (middle panel), and adipogenic (right panel) lineages. The top row shows naive MSCs and the bottom row shows immortalized MSC clone ELTP12 IF8.

[0023] FIG. 14 shows images of immortalized clones in culture following assessment of cellular anchorage-independent growth in an in vitro soft agar colony formation and tumorigenicity assay.

[0024] FIG. 15 is a graph showing 6-month longitudinal body weight monitoring of mice injected with immortalized MSC clone ELTP12 IF8 (5*106cells) versus Hanks' Balanced Salt Solution (HBSS)-injected control mice.

[0025] FIG. 16 is a graph showing no qPCR detection of immortalized MSC clone ELTP12 IF8 in mouse tissues at 6-months post injection (5xl06cells; limit of detection = 10 / 2.5x 104mouse cells), absence of biodistribution and tumorigenic activity.

[0026] FIG. 17 shows karyotype results for immortalized MSC clone ELTP12 IF8 at passage 50.

[0027] FIG. 18 shows in vitro migration capacity of immortalized clones WPPF72, ELTP12 IF8, ELTP12 SC5, ELTP12 SF6, ELTP44 B3, and ELTP442F11 versus naive MSCs. The top panel is a graph showing the number of migrated cells across a 0.8-pm transmembrane toward serum-free control medium or serum-free conditioned medium from patient-derived NCT glioma cells. The bottom panels show images of migrated cells under the previous conditions.DEFINITIONS

[0028] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

[0029] “ About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.

[0030] The use of any and all examples or exemplary language (e g., “such as”) provided herein, is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0031] The terms “may,” “may be,” “can,” and “can be,” and related terms are intended to convey that the subject matter involved is optional (that is, the subject matter is present in some examples and is not present in other examples), not a reference to a capability of the subject matter or to a probability, unless the context clearly indicates otherwise.

[0032] The use herein of the terms "including," "comprising," or "having," and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as "including," "comprising,” or "having" certain elements are also contemplated as "consisting essentially of and "consisting of those certain elements. As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0033] The terms “nucleic acid” or “nucleotide” or “polynucleotide” are used interchangeably herein to refer to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. The nucleic acid molecule may be derived from a variety of sources, including DNA, cDNA, synthetic DNA, RNA, or combinations thereof. Such nucleic acid sequences may comprise genomic DNA which may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with promoter regions, introns, or poly A sequences.

[0034] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions),alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0035] The term “gene” or “transgene” can refer to the segment of DNA (e.g., a polynucleotide sequence) involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Alternatively, the term “gene” or “transgene” can refer to the segment of DNA involved in producing or encoding a non-translated RNA, such as a IncRNA, rRNA, tRNA, guide RNA (e.g., a single guide RNA), or micro RNA.

[0036] The term “exon” as used herein refers to a region of DNA that is transcribed to RNA and retained after introns are spliced out. In the context of a long noncoding (Inc) RNA, “exon” does not necessarily refer to protein coding regions (see, e.g., Apsden, Cell Genom. 3(4): 100296) (2023). For example, a IncRNA may have exons which are entirely non-coding, but transcribed into RNA and retained after introns are spliced out.

[0037] As used herein the phrase “heterologous” refers to what is not normally found in nature. The term "heterologous nucleotide sequence" refers to a nucleotide sequence not normally found in a given wild-type viral genome, or a cell in nature. As such, a heterologous nucleotide sequence may be: (a) foreign to its host cell (i.e., is exogenous to the cell); (b) naturally found in the host cell (i.e., endogenous) but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); or (c) be naturally found in the host cell but positioned outside of its natural locus. Relatedly, “heterologous expression” or “heterologously expresses” refers to expression of a nucleotide sequence at an unnatural level in the cell (i.e., a greater or lesser level than naturally found in the cell), or expression of a nucleotide sequence not normally found in the given cell in nature. By way of example, heterologous expression may occur due to: (a) introduction of an expression cassette (e.g., an expression cassette comprising a promoter operably linked to one or more nucleotide sequences encoding one or more immortalization polypeptides and / or PURPL IncRNA) into the cell thereby increasing expression of the nucleotide sequence; (b) modification of the promoter directing transcription of the nucleotide sequence (e.g., via anucleotide mutation) thereby increasing or decreasing expression of the nucleotide sequence; or (c) transactivation of the promoter directing transcription of the nucleotide sequence (e.g., using a CRISPR activation (CRISPRa) system) thereby increasing expression of the nucleotide sequence.

[0038] A “promoter” is defined as one or more a nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0039] A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

[0040] “Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0041] “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the amino acid sequence or polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (e.g., SEQ ID NO: 1 or 2), which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0042] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 95% identity, optionally96%, 97%, 98%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. For an amino acid sequence, optionally, identity exists over a region that is at least about 50 amino acids in length, or more preferably over a region that is 100 to 150 or 200 or more amino acids in length, or where not indicated over the entire length of the reference sequence.

[0043] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0044] A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 50 to 600, usually about 75 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art.

[0045] An algorithm for determining percent sequence identity and sequence similarity is the BLAST 2.0 algorithms, e.g., as described in, and Altschul et al. J. Mol. Biol. 215:403-410 (1990) (see also Altschul et al. Nuc. Acids Res. 25:3389-3402 (1977)). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M(reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, and N=-4.

[0046] As used herein, a “cell” can be in vivo, ex vivo or in vitro, and includes any eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0047] As used herein the term “mesenchymal stromal cell” or “MSC” refers to multipotent stromal cells with a mesodermal origin and a potential for differentiation into connective tissue cells. MSCs are able to replicate as undifferentiated cells a limited number of times, possess tissue repair and regenerative capabilities and immunomodulatory properties primarily through paracrine effects via secreted factors, such as cytokines and exosomes. MSC are located in perivascular and in bone marrow niches. In some embodiments, the cells are human mesenchymal stromal cells. In some cases, primary MSC cells are isolated from an organism, system, organ, or tissue, optionally sorted, and immortalized. In some embodiments, the primary MSC cells are immortalized by transducing the MSC with a PURPL IncRNA and / or a polynucleotide encoding an immortalization polypeptide as described herein.

[0048] In some cases, the primary cells are stimulated, activated, or differentiated prior to immortalization. As used herein, the phrase “primary” in the context of a primary cell is a cell that has not been transformed or immortalized. Such primary cells can be cultured, sub-cultured, or passaged a limited number of times (e.g., cultured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times). In contrast, immortalized cells can continuously replicate, allowingfor unlimited proliferation, and can be cultured, sub-cultured, or passaged at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 times or more. As used herein, the term “naive” refers to a cell, for example, an MSC, that has not been immortalized.

[0049] As used herein “introducing” refers to contacting of the cell with the expression cassette or nucleic acid or viral vector such that translocation of the expression cassette or nucleic acid sequence or viral vector from outside a cell to inside the cell occurs. In some cases, introducing refers to transducing or infecting a cell or a population of cells with a viral vector or viral particle carrying one or more non-viral nucleic acids. In some cases, translocation of the nucleic acid from outside the cell to inside the nucleus of the cell occurs. Various methods of such translocation are contemplated, including but not limited to, viral infection, electroporation, transfection, transduction, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome mediated translocation, and the like.

[0050] As used herein, "a viral vector" refers to a gene therapy vector used to deliver a polynucleotide construct to a cell. It is understood that the term viral vector encompasses recombinant vector particles or virions (i.e., viral particles comprising at least one capsid or envelope protein and an encapsidated recombinant viral vector) and recombinant vector plasmids.

[0051] As used herein, a "recombinant viral vector" refers to a viral vector, for example, retroviral vector comprising a nucleic acid sequence that is not normally present in the viral vector (i.e., a polynucleotide heterologous to the viral vector). In general, the heterologous nucleic acid, for example, a nucleic acid encoding a heterologous polypeptide, is flanked by at least one, and generally by two, long terminal repeat sequences (LTRs), for example, a 5’ LTR and a 3’LTR. As used herein, the retroviral vector can be a derivative of a murine, simian or human retrovirus. Examples of retroviral vectors in which a transgene (e.g., a heterologous polynucleotide sequence) can be inserted include, but are not limited to lentivirus, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), Rous Sarcoma Virus (RSV), murine leukemia virus (MLV), Gibbon ape leukemia virus (GALV), Feline Leukemia virus (FeLV), RD114, and xenotropic XMLV.DETAILED DESCRIPTION OF THE INVENTION

[0052] The following description recites various aspects and embodiments of the present compositions and methods. No particular embodiment is intended to define the scope of the compositions and methods. Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least included within the scope of the disclosed compositions and methods. The description is to be read from the perspective of one of ordinary skill in the art; therefore, information well known to the skilled artisan is not necessarily included.I. Introduction

[0053] Clinical translation of mesenchymal stromal cells (MSCs) can involve a standardized manufacturing process crucial for successful trials. While MSCs can self-renew, they have limited replicative lifespan before reaching senescence, requiring constant re-derivation incurring recurring costs. Despite achievability, clinical trials have faced failures due to challenges like limited sources, donor variance, cell population heterogeneity, and inconsistent characteristics affecting potency and viability. Immortalizing primary MSCs has been attempted to ensure a consistent production supply, however, sustainably and stably immortalizing MSCs has proven challenging. The inventors have discovered that p53-upregulated-regulator-of-p53-levels (PURPL) long non-coding RNA (IncRNA) can be used to stably immortalize MSCs to provide a biologic delivery product for use in cell and gene therapies. The immortalized MSCs provided herein significantly improves the non-tumorigenic immortalization efficacy by 10-fold. Further, the number of immortalized clones constituted 10-12% of the total proliferating clones initially isolated at an early passage.

[0054] Provided herein is an immortalized MSC that heterologously expresses PURPL IncRNA. Also provided is a method for making an immortalized MSC comprising heterologously expressing PURPL IncRNA in an MSC. Also provided are methods for treating a disease in a subject in need thereof comprising administering to the subject the immortalized MSC described herein or made by the methods described herein.II. Immortalized MSCs

[0055] An immortalized MSC that heterologously express PURPL IncRNA is provided herein. Populations of the immortalized MSCs described herein are also provided. MSCs, as used herein,refer to multipotent stromal cells which can differentiate into a number of cell types (e.g., osteoblasts, chondrocytes, adipocytes, etc.). MSCs are typically obtained from mesenchymal tissues, e.g., in placenta, adipose, lung, bone marrow, umbilical cord, and dental pulp. MSCs may be identified using cell surface markers, e.g. CD105, CD73, CD90, CD44, CD29 and CD166. The interest in therapeutic applications of human MSCs stems from their immunomodulatory and trophic properties, along with their capacity to migrate to sites of tissue injury, inflammation, or tumor growth.

[0056] MSCs for use herein can be obtained from any source e.g., primary cells from a mammal or group of mammals, or from a cell line, e.g., a stromal cell line. Typically, the source of the MSC is determined based on the intended use. In some embodiments, the MSCs are human MSCs. In some embodiments, the MSCs are derived from bone marrow, placenta, skin, adipose tissue, or umbilical cord (i.e., Wharton’s jelly). Methods for obtaining MSCs are described, e.g., in Nature Protocols 5:550 (2010).PURPL IncRNA

[0057] The immortalized MSCs provided herein heterologously express PURPL IncRNA. PURPL, also known as LINC01021, RNA 1020, LOC643401, orRPll-46C20.1, is a direct target gene of p53. PURPL downregulates basal p53 levels through negative feedback via MYBBP1A-mediated regulation of basal p53 protein levels and stability. Tumors and other disorders suppress p53 functions as a master regulatory transcription factor activated in cell response to stressors such as hypoxia, oncogene activation, DNA damage, replication stress, viral infection, or nutrient deprivation. Depending on the context and cellular conditions, p53 activation may trigger cellular pathways involved in DNA repair, cell cycle arrest, metabolism, cellular senescence, apoptosis, and autophagy, among others.

[0058] In some embodiments, the immortalized MSCs provided herein heterologously express a PURPL IncRNA comprising one or more exons at least 95% identical to any one or more of SEQ ID NOs: 3-8. In some embodiments, the PURPL IncRNA comprises SEQ ID NO: 3. In some embodiment, the PURPL IncRNA comprises SEQ ID NO: 4. In some embodiment, the PURPL IncRNA comprises SEQ ID NO: 5. In some embodiment, the PURPL IncRNA comprises SEQ ID NO: 6. In some embodiment, the PURPL IncRNA comprises SEQ ID NO: 7. In some embodiment, the PURPL IncRNA comprises SEQ ID NO: 8.

[0059] In some embodiments, the PURPL IncRNA comprises exon 1, exon 2, exon 3, exon 3.5, exon 4, and exon 5, wherein exon 1 is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 3, exon 2 is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 4, exon 3 is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 5, exon 3.5 is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 6, exon 4 is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 7, and exon 5 is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 8. In some embodiments, the PURPL IncRNA comprises exon 1, exon 2, exon 3, exon 3.5, exon 4, and exon 5, wherein exon 1 comprises SEQ ID NO: 3, exon 2 comprises SEQ ID NO: 4, exon 3 comprises SEQ ID NO: 5, exon 3.5 comprises SEQ ID NO: 6, exon 4 comprises SEQ ID NO: 7, and exon 5 comprises SEQ ID NO: 8. In some embodiments, the PURPL IncRNA comprises a nucleic acid sequence at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 9. In some embodiments, the PURPL IncRNA comprises a nucleic acid sequence comprising SEQ ID NO: 9.

[0060] In some embodiments, the PURPL IncRNA comprises exon 1, exon 2, exon 3, exon 3.5, and exon 5, wherein exon 1 is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 3, exon 2 is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 4, exon 3 is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 5, exon 3.5 is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 6, and exon 5 is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 8. In some embodiments, the PURPL IncRNA comprises exon 1, exon 2, exon 3, exon 3.5, and exon 5, wherein exon 1 comprises SEQ ID NO: 3, exon 2 comprises SEQ ID NO: 4, exon 3 comprises SEQ ID NO: 5, exon 3.5 comprises SEQ ID NO: 6, and exon 5 comprises SEQ ID NO: 8. In some embodiments, the PURPL IncRNA comprises a nucleic acid sequence atleast 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 10. In some embodiments, the PURPL IncRNA comprises a nucleic acid sequence comprising SEQ ID NO: 10.

[0061] In some embodiments, the PURPL IncRNA comprises exon 1, exon 2, exon 3, exon 4, and exon 5, wherein exon 1 is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 3, exon 2 is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 4, exon 3 is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 5, exon 4 is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 7, and exon 5 is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 8. In some embodiments, the PURPL IncRNA comprises exon 1, exon 2, exon 3, exon 4, and exon 5, wherein exon 1 comprises SEQ ID NO: 3, exon 2 comprises SEQ ID NO: 4, exon 3 comprises SEQ ID NO: 5, exon 4 comprises SEQ ID NO: 7, and exon 5 comprises SEQ ID NO: 8. In some embodiments, the PURPL IncRNA comprises a nucleic acid sequence at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 11. In some embodiments, the PURPL IncRNA comprises a nucleic acid sequence comprising SEQ ID NO: 11.

[0062] The PURPL IncRNA may be introduced to an MSC, such that the MSC heterologously expresses PURPL IncRNA using any suitable method known in the art. For example, viral infection, electroporation, transfection, transduction, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome mediated translocation, and the like may be used to introduce PURPL IncRNA into the MSC. In some embodiments, the PURPL IncRNA is introduced into the MSC using a viral vector, such as a lentiviral vector. In some embodiments, the PURPL IncRNA is introduced into the MSC an expression cassette comprising a promoter operably linked to a heterologous nucleotide sequences encoding the PURPL IncRNA, such that the immortalized MSC heterologously expresses the PURPL IncRNA. In some embodiments, the expression cassette comprising a promoter operablylinked to a heterologous nucleotide sequences encoding the PURPL IncRNA is in a viral vector and the viral vector is introduced to the MSC.Immortalization Polypeptides

[0063] In some embodiments, the immortalized MSCs further expresses (i.e., in addition to a PURPL IncRNA) a heterologous nucleotide sequence encoding an immortalization polypeptide. As used herein, immortalization polypeptide refers to a polypeptide which contributes to or causes immortalization in a target cell (e.g., a MSC) when expressed by the target cell. The immortalization polypeptide may be selected empirically by one skilled in the art based on the specific desired properties and use. In some embodiments, the immortalization polypeptide is selected from the group consisting of: MYC, SV40 Large T Antigen, STAT3, telomerase reverse transcriptase (TERT), E6 HPV, E7 HPV, or Bmi-1, or NOTCH2 intracellular domain.

[0064] In some embodiments, the immortalization polypeptide is MYC. In some embodiments, the MYC polypeptide is L-MYC, N-MYC, V-MYC, or V-MYC. In some embodiments, the MYC polypeptide is L-MYC. In some embodiments, the MYC polypeptide is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 1.

[0065] In some embodiments, the immortalization polypeptide is TERT. In some embodiments, the TERT polypeptide is a human TERT polypeptide. In some embodiments, the TERT polypeptide is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 2.

[0066] In some embodiments, the immortalization polypeptide is SV40 Large T Antigen. In some embodiments, the SV40 Large T Antigen polypeptide is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 12. In some embodiments, the immortalization polypeptide is STAT3. In some embodiments, the STAT3 polypeptide is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 13. In some embodiments, the immortalization polypeptide is E6 HPV. In some embodiments, the E6 HPV polypeptide is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 14. In some embodiments, the immortalization polypeptide is E7 HPV. In some embodiments, the E7 HPV polypeptide is at least85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 15. In some embodiments, the immortalization polypeptide is Bmi-1. In some embodiments, the Bmi-1 polypeptide is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 16. In some embodiments, the immortalization polypeptide is NOTCH2 intracellular domain. In some embodiments, the NOTCH2 intracellular domain polypeptide is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 17.Expression Cassettes

[0067] The immortalized MSCs provided herein may comprise one or more expression cassettes comprising a promoter operably linked to one or more heterologous nucleotide sequences encoding one or more immortalization polypeptides and / or the PURPL IncRNA, such that the immortalized MSC heterologously expresses the one or more immortalization polypeptides and / or the PURPL IncRNA. As described above, a promoter is “operably linked” to a polynucleotide when it is placed into a functional relationship with the polynucleotide sequence. Numerous promoters, described in further detail below, can be used in the constructs described herein. As described above, the term “promoter” as used herein refers to a nucleotide region or a sequence located upstream and / or downstream from the start of transcription that is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.

[0068] The one or more expression cassettes may comprise a promoter operably linked to one or more heterologous polynucleotide sequences encoding the one or more immortalization polypeptides and / or the PURPL IncRNA. In some embodiments, the one or more expression cassettes comprise a promoter operably linked to two heterologous polynucleotide sequences encoding two immortalization polypeptides (e g., MYC and TERT, or Bmi-1 and TERT, or SV40 Large T Antigen and TERT, or MYC and NOTCH2). In some embodiments, the one or more expression cassettes comprise a promoter operably linked to one heterologous polynucleotide sequence encoding one immortalization polypeptide (e.g., MYC, SV40 Large T Antigen, STAT3, TERT, E6 HPV, E7 HPV, or Bmi-1, or NOTCH2 intracellular domain). The one or more heterologous polynucleotide sequences encoding the one or more immortalization polypeptides may be under the control of the same promoter or a different promoter compared to the PURPLIncRNA. In some embodiments, the one or more heterologous polynucleotides sequences encoding the one or more immortalization polypeptides are under the control of a different promoter than the PURPL IncRNA.

[0069] In some embodiments, the immortalized MSC comprises a first expression cassette comprising a promoter operably linked to a nucleotide sequence encoding MYC; a second expression cassette comprising a promoter operably linked to a nucleotide sequence encoding TERT; and a third expression cassette comprising a promoter operably linked to a nucleotide sequence encoding the PURPL IncRNA. In some embodiments, the immortalized MSC comprises a first expression cassette comprising a promoter operably linked to a nucleotide sequence encoding MYC and TERT; and a second expression cassette comprising a promoter operably linked to a nucleotide sequence encoding the PURPL IncRNA. In some embodiments, the immortalized MSC comprises a first expression cassette comprising a promoter operably linked to a nucleotide sequence encoding, e.g., MYC and TERT, or Bmi-1 and TERT, or SV40 Large T Antigen and TERT, or MYC and NOTCH2; and a second expression cassette comprising a promoter operably linked to a nucleotide sequence encoding the PURPL IncRNA.

[0070] The choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a sequence by appropriately selecting and positioning promoters and other regulatory regions relative to that sequence. Exemplary promoters useful in the expression cassettes described herein include an EFla promoter, an EFS promoter (a short form of EFla promoter without introns), a PGK promoter, an Ubiquitin C (UbC) promoter, a REX promoter, an OCT4 promoter.

[0071] In some embodiments the promoter is tissue-specific (i.e., it directs transcription at high levels only in particular types of cells or tissues). Exemplary tissue-specific promoters include, inter alia, a synapsin, camKIIa, glial fibrillary acidic protein (GFAP), retinal pigment epithelium (RPE), albumin (ALB), thyroxine binding globulin (TBG), myelin basic proteins (MBP), muscle creatine kinase (MCK), cardiac troponin T (TnT), or alpha-myosin heavy chain (aMHC), CD68 gene promoter, P-globin locus control region and promoter, WASP gene promoter, muscle- and cardiac-specific synthetic promoter Spc-5-12, and the like.

[0072] In some embodiments, for example to make an immortalized MSC, the promoter is an MSC-preferential promoter (i.e., a promoter which is functional in MSCs). In some embodiments, the MSC-preferential promoter is a REX promoter or an OCT4 promoter. In some embodiments, the promoter is inducible (i.e., it directs transcription only under certain circumstances). For example, an inducible promoter used in the expression cassettes herein may be tetracycline inducible. In some embodiments, the promoter is constitutive (i.e., it directs transcription at relatively similar levels across all cell and tissue types). Exemplary constitutive promoters include, inter alia, a CMV promoter, CAG promoter, CBA promoter, EFla promoter, PGK promoter, UbC promoter, and the like.

[0073] The promoter can be a promoter native to or naturally occurring in the polynucleotide (e.g., PURPL IncRNA or the polynucleotide encoding an immortalization polypeptide) to which it is operably linked. The promoter can be heterologous to (i.e., not native to or naturally occurring in) the polynucleotide (e.g., PURPL IncRNA or the polynucleotide encoding an immortalization polypeptide) to which it is operably linked.

[0074] The one or more expression cassettes may be introduced into the MSC, such that the contents of the one or more expression cassettes are expressed by the MSC, using any suitable method in the art. As used herein “introducing” refers to contacting of the cell with the nucleic acid such that translocation of the nucleic acid sequence or viral vector from outside a cell to inside the cell occurs. Introducing may be in vivo, ex vivo, or in vitro. In some cases, translocation of the nucleic acid from outside the cell to inside the nucleus of the cell occurs. Various methods of such translocation are contemplated, including but not limited to, viral infection, electroporation, transfection, transduction, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome mediated translocation, and the like.

[0075] The one or more expression cassettes can be in one or more viral vectors. Thus, in some embodiments, the one or more expression cassettes in the vectors may be introduced into the MSC. In such cases, introducing refers to transducing or infecting a cell or a population of cells with a viral vector or viral particle carrying one or more non-viral nucleic acids. Exemplary viral vectors include but are not limited to adenovirus vectors (e.g., Ad2, Ad5, Ad7), adeno-associated viral vectors, herpes simplex viral vectors, retroviral vectors, pox viral vectors (such as vaccinia and avian poxvirus vectors, such as the fowlpox and canarypox vectors), lentiviral vectors, alphavirusvectors, poliovirus vectors, measles vectors, and other positive and negative stranded RNA viruses, viroids, and virusoids, or portions thereof. In some embodiments, the one or more expression cassettes are in a retroviral vector. In some embodiments, the one or more expression cassettes are in a lentiviral vector.

[0076] It is a routine matter for one of skill in the art to design viral vectors, e.g., by adjusting the orientation of the nucleic acid transgenes therein, such that transgenes are expressed as desired. In embodiments in which the one or more expression cassettes are in one or more viral vectors, the viral vectors are not limited to a certain configuration. In some embodiments, in particular in an expression cassette comprising PURPL IncRNA, the expression cassette may be inserted into the viral vector in a reverse orientation such that the polyadenylation signal (e.g., hGH) starts immediately downstream of the last PURPL IncRNA exon. Inserting the IncRNA expression cassette in a forward orientation within a viral vector (e.g., a lentiviral vector) may result in the presence of additional untranslated region vector downstream of the last PURPL IncRNA exon, which may hinder secondary structure formation of PURPL IncRNA and result in aberrant RNA-protein interaction and PURPL IncRNA function.CRISPR Activation (CRISPRa) Systems

[0077] In some embodiments, the immortalized MSC comprises a CRISPRa system effecting increased expression of PURPL IncRNA by the MSC. The CRISPRa system utilizes a catalytically-inactive CRISPR nuclease (e.g. dCas9) linked to one or more transcriptional activators to upregulate expression of the genes of interest. CRISPRa further includes a guide RNA (gRNA) targeting the area immediately upstream of the transcriptional start site (e.g., the promoter or enhancer sequence) of the gene (e.g., PURPL IncRNA) whose expression is to be upregulated. In some embodiments, transcriptional activators, fused to a catalytically-inactive nuclease or a gRNA, can recruit transcriptional factors close to the promoter or enhancer sequence of the gene of interest (e.g., the PURPL IncRNA). For example, the VP64 transcriptional activator or any other transcriptional activator can be fused to the catalytically-inactive nuclease to increase transcription of the target gene. Suitable transcriptional activators are described in Chen & Qi, Ad. Exp. Med. Bio., 983:147-157 (2023).

[0078] In some embodiments, PURPL IncRNA expression by the MSC is increased by introducing into the MSC CRISPRa system comprising a catalytically-inactive nuclease (e.g., a dCas9) linked to a transcriptional activator domain (e.g., VP64) and a gRNA that targets a portion of a promoter sequence operably linked to an exon of PURPL IncRNA. PURPL IncRNA has a 177 base pair long promoter sequence located in chromosome 5 (location 27472101-27472277), 14 base pairs upstream of the start of exon 1 (SEQ ID NO: 3) (chromosome 5, location 27472292). The PURPL IncRNA promoter sequence contains the p53 binding site sequence GGGCTTGTCTGGGCATGCCC (SEQ ID NO: 19). See, e g., Kaller et al., Oncotarget 8(61): 102783 (2017). In certain embodiments, the gRNA targets the promoter sequence comprising the nucleic acid sequences of SEQ ID NO: 18.Therapeutic Payloads

[0079] In some embodiments, the immortalized MSC may further comprise an expression cassette comprising a promoter operably linked to a heterologous nucleotide sequence encoding a therapeutic payload. In some embodiments, the immortalized MSC may further comprise an expression cassette comprising a promoter operably linked to a heterologous nucleotide sequence encoding one or more therapeutic payloads (e.g., two, three, four, or five therapeutic payloads). The choice of therapeutic payload depends upon the target disorder and the desired therapeutic effect. Generally, any gene encoding a protein involved in the target disease can be a therapeutic payload.A. Antibodies

[0080] In some embodiments, the therapeutic payload is an antibody. Antibodies recognize, based on their complementarity determining regions, a site (i.e., an epitope) on a target antigen. The antibody may target one epitope (i.e., a monospecific antibody) or two or more epitopes located on the same or different antigens (e.g., a bispecific antibody or a multispecific antibody). Antibodies useful as therapeutic payloads in the present compositions and methods include, but are not limited to, immune checkpoint inhibitor antibodies (e.g., anti-PDl or anti-PDLl antibodies), anti-immunosuppressive antibodies (e.g., anti-TGFp, anti-CD52, anti-mTOR, anti-KCNA3, or anti-IL2 receptor agonist (CD25) antibodies), anti-angiogenic antibodies (e.g., anti-VEGF, anti-ANG-2, anti-endostatin (COL18A1), anti-angiostatin, anti-thrombospondin (THS7DA or PLA2R), anti-PDGFR, anti-HGF, anti-c-MET, or anti-CLEC14a antibodies), antiinflammatory antibodies (e.g., anti-IL6, anti-ILl 1, anti-IL13, anti-IL17, anti-IL23 receptor agonistor anti-ILl receptor agonist antibodies). Other antibodies which may be used as therapeutic payloads in the present compositions and methods include, but are not limited to, anti-HER2 antibodies, anti-EGFR antibodies (including variants, e.g., EGFRvIII, anti-CEA antibodies, anti-CD19 antibodies, anti-GD2 antibodies, anti-CD147 antibodies, anti-SMARCALl antibodies, anti-Delta-Like Canonical Notch Ligand 3 (DLL3) antibodies,

[0081] Antibodies useful as therapeutic payloads in the compositions and methods herein may not have a conventional antibody structure (i.e., a tetramer composed of two heavy and two light chains, further divided into variable and constant regions). In some embodiments, the therapeutic payload may be an antigen binding fragment of an antibody. Antigen binding fragments are well known in the art, and include, for example, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv) (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); or (viii) a single domain antibody (i.,e., nanobody).

[0082] In some embodiments, the therapeutic payload is a small protein which may target an epitope on an antigen like an antibody, but is not classified as an antibody. For example, the therapeutic payload may be a Fynomer, a small (7 kDa) binding protein derived from the SH3 domain of human Fyn kinase (see, e.g., Sarvmeili et al., Sci. Rep., 14: 10297 (2024)).B. Bispecific T Cell Engagers

[0083] In some embodiments, the therapeutic payload is a bispecific T cell engager (BiTE). BiTES have two single-chain variable fragments antigen recognizing regions which target a T cellspecific antigen (e.g., CD3) and a tumor-associated antigen (e.g., CD 19 on a tumor cell). Unlike traditional bispecific antibodies, BiTES do not retain a constant (Fc) region to confer effector functions. Instead, BiTES link T cells to target cells and rely on the endogenous T cell effector function. BiTES useful as therapeutic payloads in the present compositions and methods may target CD3 and B-cell maturation antigen (BCMA), CD3 and CD33, CD3 and prostate-specific membrane antigen (PSMA).C. Cytokines

[0084] In some embodiments, the therapeutic payload is a cytokine. Cytokines which may be useful as therapeutic payloads in the present compositions and methods include, but are not limited to, immunostimulatory cytokines (e.g., ILip, IL2, IL6, IL12, IL15, IL16, IL17, IL36, TNFa, IFNy, IFNa, or IFNP) or immunosuppressive cytokines (e.g., IL4, IL10, IL13, IL33, IL35, IL37, TGF-P, CD25, CD52).D. Viral Vectors

[0085] In some embodiments, the therapeutic payload is a viral vector (e.g., a lentiviral vector or a retroviral vector). Thus, in some embodiments the heterologous nucleotide sequence encoding the therapeutic payload may encode constituent parts of a viral vector. By way of example, the nucleotide sequence may encode a vector backbone containing the heterologous nucleotide sequence encoding the desired therapeutic payload flanked by long terminal repeats (LTRs), a pol protein an env protein, and any other necessary viral protein (e.g., proteins necessary for viral replication such as Rev and Vpx). For retroviral vectors, the gag, pol, and env proteins may be derived from murine leukemia virus, Moloney murine leukemia virus, Gibbon ape leukemia virus, Feline Leukemia virus, RD114, xenotropic XMLV, and Foamy virus. In some embodiments for lentiviral vectors, the vector backbone, gag, pol, and env proteins may be derived from human immunodeficiency virus type 1, human immunodeficiency virus type 2, Feline immunodeficiency virus, Simian immunodeficiency virus, or Equine infectious anemia virus.

[0086] Lentiviral vectors may be pseudotyped with Vesicular stomatitis virus (VSV-G) envelope glycoprotein, amphotropic murine leukemia virus envelope glycoprotein, Gibbon ape leukemia virus envelope glycoprotein, Feline endogenous retrovirus RD114 envelope glycoprotein, Baboon endogenous retrovirus (BaEV) envelope glycoprotein, human immunodeficiency virus type 2 VCP envelope glycoprotein, human immunodeficiency virus type 2 ROD / B envelope glycoprotein, human immunodeficiency virus type 1 IIIBx envelope glycoprotein, Rabies virus envelope glycoprotein, baculovirus-derived GP64 envelope protein, Measles virus fusion (F) and hemagglutinin (H) glycoproteins, Sendai virus fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins, Nipah virus fusion (F) and G glycoproteins, receptor-targeted Measles virus H glycoprotein genetically fused to a single-chain antibody fragment (scFv) domain specific for CD8, CD19, CD20, CD30, CD133, or CD62L, receptor-targeted Measles virus H glycoprotein genetically fused to a designed ankyrin repeat molecule (DARPin) domain specific for CD4, CD8 or CD340 / HER-2 / neu, CD117 / c-kit-targeted Nipah virus G glycoprotein displaying a natural CD117 ligand stem cell factor (SCF), receptor-targeted Nipah virus G glycoprotein genetically fused to a scFv domain specific for CD8, or CD20, or receptor-targeted Nipah virus G glycoprotein genetically fused to a DARPin domain specific for CD340 / HER-2 / neu. For non-integrating lentiviral vectors, mutations are introduced in the viral integrase protein or in the integrase attachment sites (att sites) within the viral LTRs.

[0087] In some embodiments, the therapeutic payload may be delivered by a viral vector. Thus, in some embodiments the heterologous nucleotide sequence encoding the therapeutic payload may encode constituent parts of a viral vector and a heterologous therapeutic payload, which may be any therapeutic payload described above or elsewhere herein.

[0088] In instances in which the therapeutic payload is a retroviral vector or is delivered by a retroviral vector, the retroviral vector may be a replicating retroviral vector (i.e., a retroviral vector that has independent replicative capacity). Replicating retroviral vectors have viral genes necessary for replication. Thus, replicating retroviral vectors can replicate and spread in dividing host cells, leading to persistent expression of genetic information contained in the replicating retroviral vector. In some embodiments, the retroviral vector lacks independent replicative capacity, i.e., is a non-replicating retroviral vector. Non-replicating retroviral vectors lack necessary genes for replication, and thus can deliver a larger nucleotide sequence encoding a therapeutic payload. The same embodiments regarding replicating and non-replicating viral vectors apply when the therapeutic payload is another viral vector (e.g., a lentiviral vector) or is delivered by another viral vector (e.g., a lentiviral vector). Thus, in some embodiments, in instances in which the therapeutic payload is a lentiviral vector oris delivered by a lentiviral vector, the lentiviral vector lacks independent replicative capacity, i.e., is a replication-incompetent lentiviral vector.

[0089] In some instances, such as non-cancer applications or to treat benign disease (e.g., a monogenic hereditary disorder), a non-replicating viral vector may be advantageous as a therapeutic payload or to deliver a therapeutic payload. For example, in dystrophic epidermolysis bullosa, a disorder caused by a mutation in Collagen VII normally expressed in keratinocytes and fibroblasts a non-replicating viral (e.g., lentiviral or retroviral) vector may be advantageous suchthat immortalized MSCs either (1) express Collagen VTI themselves, which is only sustained as long as the MSCs are present, or (2) express a “one-shot” non-replicating viral (e.g., lentiviral or retroviral) vector that encodes Collagen VII, which would then transduce adjacent keratinocytes and fibroblasts, enabling those cells to also express Collagen VII. Thus, in some embodiments, a non-replicating viral vector can be used to deliver the correct copy of a gene affected in a genetic disorder.

[0090] A non-replicating lentiviral vector as a therapeutic payload constitutively produced by immortalized MSC may be advantageous in in vivo gene therapy applications. For example, immortalized MSC engineered as constitutive lentivirus producer cell lines are, unlike HEK-293T constitutive lentivirus producer cell lines, suitable for in vivo production of lentivirus in situ, such as for treatment of cystic fibrosis with MSC-produced lentivirus expressing CFTR transgene pseudotyped with Sendai virus F and hemagglutinin-neuraminidase (HN) envelope proteins targeting the lung epithelial cells; or, for treatment of Duchenne muscular dystrophy, with MSC-produced lentivirus expressing mini-dystrophin in muscles. Both diseases are characterized by chronic inflammation that promotes MSC homing to target tissues. Other examples include hematopoietic stem cell (HSC) gene therapy for treatment of hemoglobinopathies, immunodeficiencies, and storage diseases, in which MSCs could be a source of lentivirus produced in bone marrow in situ, such as for treatment of adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) with lentivirus -expressing ADA transgene pseudotyped with HSC-targeted envelope glycoproteins. In some instances, such as chimeric antigen receptor (CAR)-T cell therapy, the therapeutic payload may be lentivirus expressing a CAR pseudotyped with CD8- or CD62L-targeted envelope glycoprotein constitutively produced by MSC administered by intra-lymph node injection. In some embodiments, immortalized MSCs may be engineered to express Cas9 and a non-integrating lentivirus encoding guide RNA and donor DNA allowing for transient endonuclease function and inclusion of editing tools for HSC-targeted gene editing.E. Oncolytic Virus

[0091] In some embodiments, for instance when the target disorder is cancer, the therapeutic payload may be an oncolytic virus. Oncolytic viruses target cancer, selectively infecting and killing cancer cells. Oncolytic viruses may be naturally occurring or genetically modified to target cancercells. Oncolytic viruses useful as therapeutic payloads in the present compositions and methods include, but are not limited to, oncolytic herpes simplex virus, vesicular stomatitis virus, adenovirus, measles virus, vaccinia virus, Maraba virus, polio virus, picornavirus, Coxsackie virus, reovirus, Newcastle disease virus, parvovirus, Seneca Valley virus, Semliki Forest Virus, Foamy virus, and Sindbis virus. As oncolytic viruses are generally cytolytic, viral replication in MSCs may cause MSC lysis and death. Thus, it may be advantageous to select an oncolytic virus which only replicates in malignant cells (e.g., oncolytic adenovirus).III. Methods for Making Immortalized MSCs

[0092] Also provided herein are methods for making immortalized MSCs comprising heterologously expressing in MSCs (e.g., primary MSCs) PURPL IncRNA, thereby immortalizing the MSCs. In some embodiments, the method further comprises expressing in the MSC a heterologous nucleotide sequence encoding an immortalization polypeptide.

[0093] MSCs useful in the present methods for making immortalized MSCs may be isolated from any source as described above, including from any mammal or from a cell line. In some embodiments, the MSCs used in the present treatment methods are human MSCs. In the present methods for making immortalized MSCs, PURPL IncRNA and / or a heterologous nucleotide sequence encoding an immortalization polypeptide are introduced into primary MSCs (i.e., MSCs taken from tissue and optionally maintained for growth in vitro). Introduction may be any suitable method in the art, e.g., electroporation or via viral vectors. Introduction is described in detail above and elsewhere herein. The primary MSCs heterologously express the PURPL IncRNA and / or a heterologous nucleotide sequence encoding an immortalization polypeptide and become immortalized.

[0094] As discussed above, immortalizing allows MSCs to bypass senescence and to be cultured continuously while maintaining genomic stability, without the bias of cell malignancy or cell transformation. In some embodiments, immortalized MSCs can continuously replicate, allowing for unlimited proliferation, and can be cultured, sub-cultured, or passaged at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 times or more. In some embodiments, the immortalized MSCs can be passaged up to 100 times. Various methods for evaluating MSC immortalization are known in the art and can be usedfor validation of the present methods for making immortalized MSCs (see, e.g. Carvalho et al., Authorea (preprint) (2020)). For example, flow cytometry may be used to evaluate MSC markers; RT- and qRT-PCR analysis of pluripotency and immortalization genes, proliferation tests in late cell passages, karyotype, analysis of genomic copy number variation, genomic sequencing, fluorescence in situ hybridization, among others.

[0095] In some embodiments, MSCs produced by the present methods for making immortalized MSCs may be assessed for tumorigenicity. In some embodiments, MSCs produced by the present methods for making immortalized MSCs are non-tumorigenic MSCs. As used herein, non-tumorigenic MSCs refer to MSCs which do not have the ability or tendency to produce or develop tumors. Methods for assessing tumorigenicity are known in the art and include in vitro soft agar colony formation assay and in vivo assessments in immunocompromised murine models, for example (see, e.g., Sato et al., Cytotherapy, 21(11): 1095-1111 (2019)).

[0096] In some embodiments, the methods for making immortalized MSCs further comprise introducing into the MSCs one or more expression cassettes comprising a promoter operably linked to a heterologous nucleotide sequence encoding the immortalization polypeptide and / or PURPL IncRNA. In some embodiments, the method further comprises introducing into the MSCs one or more expression cassettes comprising a promoter operably linked to a heterologous nucleotide sequence encoding a therapeutic payload. The above discussion of MSCs, PURPL IncRNA, immortalization polypeptides, expression cassettes and vectors, and therapeutic payloads applies to the present methods for making immortalized MSCs. In some embodiments, for autologous uses, the individual providing the MSCs that are immortalized also receives the immortalized cells at a later point. In other embodiments, the cells are allogeneic, i.e., the individual from whom the cells are initially obtain and the individual receiving the immortalized cells, are different individuals.IV. Methods for Treatment Using Immortalized MSCs

[0097] Also provided herein are methods for treating a disease in a subject in need thereof comprising administering to the subject the immortalized MSC described herein or made by the methods described herein. In some embodiments, the immortalized MSCs used in the present treatment methods deliver a therapeutic payload to the subject. Thus, discussion above andelsewhere herein related to MSCs, PURPL IncRNA, immortalization polypeptides, expression cassettes and vectors, and therapeutic payloads applies to the present methods for treatment.

[0098] Immortalized MSCs useful in the present treatment methods for treating a disease in a subject in need thereof may be isolated from any source as described above, including from any mammal or from a cell line. In some embodiments, the immortalized MSCs used in the present treatment methods are human MSCs. The immortalized MSCs useful in the present treatment methods may be obtained from the same individual that is intended to receive the immortalized MSCs. That is, the initially harvested MSCs, and the immortalized MSCs, are autologous to the donor / recipient. In some embodiments, the MSC donor(s) is different than the recipient, such that the transplanted cells will be allogeneic to the recipient.

[0099] The immortalized MSCs used in the present treatment methods may be administered via any of several routes of administration, including parenterally, intramucosally, intravenously, intratumorally, intraperitoneally, intraventricularly, intramuscularly, subcutaneously, intracranially, intracavity, intra-bone marrow, intra-lymph node, or transdermally. Administration can be achieved by, e.g., topical administration, local infusion, injection, or by means of an implant. As discussed above, MSCs have homing capabilities to target injured or inflamed tissue. Thus, the immortalized MSCs used in the present treatment methods may be systemically administered (e.g., via intravenous infusion) and migrate in vivo to target areas (e.g., a tumor site or tissue in need of regeneration).

[0100] In any of the methods provided herein, the subject can be a subject diagnosed with or at risk for developing a disease, for example, cancer, a degenerative disease, or a monogenic hereditary disorder. A subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig). The term does not denote a particular age or sex. Thus, adult, newborn and pediatric subjects, whether male or female, are intended to be covered. As used herein, patient or subject may be used interchangeably and can refer to a subject with or at risk of developing a disorder. The term patient or subject includes human and veterinary subjects.

[0101] “Treating” refers to any indicia of success in the treatment or amelioration or prevention of the disease, condition, or disorder, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable tothe patient; slowing in the rate of degeneration or decline; preventing a relapse, or making the final point of degeneration less debilitating. For example, a method for treating cancer is considered to be a treatment if there is a 10% reduction in one or more symptoms of the cancer in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disorder or symptoms of the disorder.

[0102] The methods for treating a disease in a subject in need thereof comprising administering to the subject the immortalized MSC described herein or made by the methods described herein can be used to treat a subject having any disorder amendable to treatment using the immortalized MSCs described herein or made by the methods described herein. As described above, in instances where the immortalized MSCs comprise an expression cassette comprising a promoter operably linked to a heterologous nucleotide sequence encoding a therapeutic payload, the therapeutic payload may be selected based on the target disease and desired therapeutic effect.

[0103] In some embodiments, the disease is cancer. Cancer generally refers to a condition characterized by an abnormal cell proliferation. In some methods, the cancer disorder is selected from the group consisting of lung cancer, breast cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, kidney cancer, urinary tract cancer, oral cancer, head and neck cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, skin cancer, melanoma, sarcoma, lymphoma, leukemia, and brain cancer including glioblastoma, anaplastic astrocytoma, oligodendroglioma, medulloblastoma. In some methods, the cancer is glioblastoma.

[0104] In some embodiments, the disease is a skin disorder, such as a wound (e.g., an ulcer), atopic dermatitis, vitiligo, alopecia, epidermolysis bullosa, dermatomyositis, scleroderma, and psoriasis. In some embodiments, the disease is an autoimmune disease, such as systemic lupus erythematosus, systemic sclerosis, graft versus host disease, Crohn’s disease, multiple sclerosis, and arthritis. In some embodiments, the disease is an autoinflammatory disease, such as Familial Mediterranean Fever, Cryopyrin-associated periodic syndromes, Deficiency of IL-l-Receptor Antagonist, Hyper IgD Syndrome, Mevalonate kinase deficiency, TNF receptor associated periodic syndrome, or Muckle-Wells syndrome. In some embodiments, the disease is aneurodegenerative disease, such as Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease.

[0105] In some embodiments, the disease is a monogenic hereditary disorder. As used herein, monogenic hereditary disorder refers to a disorder caused by mutation of a single gene. Monogenic hereditary disorders, include, for example, hemoglobinopathies, immunodeficiencies, and storage diseases. By way of example, the monogenic hereditary disorder may be epidermolysis bullosa, adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID), X-linked SCID, P-thalassemia, sickle cell disease, Wiskott-Aldrich syndrome, hemophilia A, hemophilia B, Fanconi anemia, cerebral adrenoleukodystrophy, metachromatic leukodystrophy, cystic fibrosis, Duchenne muscular dystrophy, spinal muscular atrophy, phenylketonuria, Tay-Sachs disease, and Huntington's disease. In some embodiments, the disease is a chronic degenerative tissue disorder, such as Ehlers-Danlo syndrome, osteoarthritis, or chronic degenerative joint disease.

[0106] Any of the methods provided herein can further comprise administering a second therapeutic agent to the subject. For example, if the present methods are used to treat a subject having cancer, the second therapeutic agent can be selected from the group consisting of a chemotherapeutic agent, an adjuvant, an immunomodulatory agent, a vaccine, a tumor antigen, or a combination thereof. In cases, where the second therapeutic is a nucleic acid sequence encoding a therapeutic polypeptide, the second therapeutic agent can be delivered by viral or non-viral means, including any means described above and elsewhere herein.

[0107] It is understood that combinations, for example, a composition comprising immortalized MSCs and a second therapeutic agent, can be administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject), or sequentially (e.g., one of the compositions or agents is given first followed by the second). Any of the methods provided herein can further comprise additional treatment such as radiation therapy or surgery.

[0108] 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 methods and 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 specificreference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of modifications that can be made to a number of molecules including in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods 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 method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.

[0109] Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.EMBODIMENTSAl. An immortalized mesenchymal stromal cell (MSC) that heterologously expresses a p53-upregulated-regulator-of-p53-levels (PURPL) IncRNA.A2. The MSC of embodiment Al, wherein the MSC further expresses a heterologous nucleotide sequence encoding an immortalization polypeptide selected from the group consisting of: MYC, SV40 Large T Antigen, STAT3, telomerase reverse transcriptase (TERT), E6 HPV, E7 HPV, Bmi-1, or NOTCH2 intracellular domain.A3. The MSC of embodiment A2, wherein the MSC is a human MSC and TERT is human TERT (hTERT).A4. The MSC of embodiment A2, wherein MYC is L-MYC, N-MYC, V-MYC, or C-MYC. A5. The MSC of embodiment A2, comprising one or more expression cassettes comprising a promoter operably linked to a heterologous nucleotide sequence encoding the immortalization polypeptide and / or the PURPL IncRNA.A6. The MSC of embodiment A2, comprising an expression cassette comprising a promoter operably linked to a heterologous nucleotide sequence encoding a therapeutic payload.A7. The MSC of embodiment A6, wherein the therapeutic payload is an antibody.A8. The MSC of embodiment A7, wherein antibody is a bispecific antibody.A9. The MSC of embodiment A7, wherein the antibody is a bispecific T cell engager (BiTE). A10. The MSC of embodiment A6, wherein the therapeutic payload is a type VII collagen. All. The MSC of embodiment A6, wherein the therapeutic payload is a cytokine.A12. The MSC of embodiment Al or embodiment A2, comprising a CRISPR activation (CRISPRa) system effecting increased expression of the PURPL IncRNA by the MSC.Al 3. The MSC of embodiment Al or embodiment A2, wherein the MSC comprises a viral vector.A14. The MSC of embodiment A13, wherein the viral vector is a retroviral vector.Al 5. The MSC of embodiment A14, wherein the retroviral vector has independent replicative capacity.A16. The MSC of embodiment A14, wherein the retroviral vector lacks independent replicative capacity.A17. The MSC of embodiment A13, wherein the viral vector is a lentiviral vector.Al 8. The MSC of embodiment A2, comprising:a first expression cassette comprising a promoter operably linked to a nucleotide sequence encoding MYC;a second expression cassette comprising a promoter operably linked to a nucleotide sequence encoding TERT; anda third expression cassette comprising a promoter operably linked to a nucleotide sequence encoding the PURPL IncRNA.Al 9. The MSC of embodiment A2, comprising:a first expression cassette comprising a promoter operably linked to a nucleotide sequence encoding MYC and TERT; anda second expression cassette comprising a promoter operably linked to a nucleotide sequence encoding the PURPL IncRNA.A20. The MSC of embodiment Al 8, wherein one, two or all three of the first and second and third expression cassettes are in one of more vectors.A21. The MSC of embodiment Al 9, wherein one or both of the first and second expression cassettes are in one or more vectors.A22. The MSC of embodiment A20 or embodiment A21 , wherein the vector is a viral vector. A23. The MSC of embodiment A2, wherein the MYC polypeptide is an L-MYC polypeptide at least 95% identical to SEQ ID NO: 1.A24. The MSC of embodiment A2, wherein the TERT polypeptide is at least 95% identical to SEQ ID NO: 2A25. The MSC of embodiment A2, wherein SV40 Large T Antigen is at least 95% identical to SEQ ID NO: 12, wherein STAT3 is at least 95% identical to SEQ ID NO: 13, wherein E6 HPV is at least 95% identical to SEQ ID NO: 14, wherein E7 HPV is at least 95% identical to SEQ ID NO: 15, wherein Bmi-1 is at least 95% identical to SEQ ID NO: 16, and wherein N0TCH2 intracellular domain is at least 95% identical to SEQ ID NO: 17.A26. The MSC of embodiment Al, wherein the PURPL IncRNA comprises one or more exon at least 95% identical to any one of SEQ ID NO: 3-8.11. The MSC of embodiment Al, wherein the PURPL IncRNA is at least 95% identical to any one of SEQ ID NOs: 9-11.A28. The MSC of embodiment Al, wherein the PURPL IncRNA comprises exon 1, exon 2, exon 3, exon 3.5, exon 4, and exon 5, wherein exon 1 is at least 95% identical to SEQ ID NO: 3, exon 2 is at least 95% identical to SEQ ID NO: 4, exon 3 is at least 95% identical to SEQ ID NO: 5, exon 3.5 is at least 95% identical to SEQ ID NO: 6, exon 4 is at least 95% identical to SEQ ID NO: 7, and exon 5 is at least 95% identical to SEQ ID NO: 8.A29. The MSC of embodiment A28, wherein exon 1 comprises SEQ ID NO: 3, exon 2 comprises SEQ ID NO: 4, exon 3 comprises SEQ ID NO: 5, exon 3.5 comprises SEQ ID NO: 6, exon 4 comprises SEQ ID NO: 7, and exon 5 comprises SEQ ID NO: 8.Bl. A method for making an immortalized mesenchymal stromal cell (MSC) comprising heterologously expressing in a MSC a PURPL IncRNA, thereby immortalizing the MSC.B2. The method of embodiment Bl, further comprising expressing in the MSC a heterologous nucleotide sequence encoding an immortalization polypeptide selected from the group consisting of: MYC, SV40 Large T Antigen, STAT3, telomerase reverse transcriptase (TERT), E6 HPV, E7 HPV, Bmi-1, or NOTCH2 intracellular domain.B3. The method of embodiment B2, wherein the MSC is a human MSC and TERT is hTERT. B4. The method of embodiment Bl, wherein MYC is L-MYC, N-MYC, V-MYC, or C-MYC.B5. The method of embodiment Bl, comprising introducing into the MSC one or more expression cassettes comprising a promoter operably linked to a heterologous nucleotide sequence encoding the immortalization polypeptide and / or PURPL IncRNA.B6. The method of embodiment Bl, comprising introducing into the MSC an expression cassette comprising a promoter operably linked to a heterologous nucleotide sequence encoding a therapeutic payload.B7. The method of embodiment B6, wherein the therapeutic payload is an antibody.B8. The method of embodiment B7, wherein the antibody is a bispecific antibody.B9. The method of embodiment B7, wherein the antibody is a bispecific T cell engager (BiTE). BIO. The method of embodiment B6, wherein the therapeutic payload is a type VII collagen. B 11. The method of embodiment B6, wherein the therapeutic payload is a cytokine.Bl 2. The method of embodiments Bl -Bl 1, wherein the MSC comprises a CRISPR activation (CRISPRa) system effecting increased expression of the PURPL IncRNA by the MSC.B13. The method of embodiments B1-B12, wherein MSC comprises a viral vector.B 14. The method of embodiment B 13, wherein the viral vector is a retroviral vector.B15. The method of embodiment B 14, wherein the retroviral vector has independent replicative capacity.B16. The method of embodiment B14, wherein the retroviral vector lacks independent replicative capacity.B17. The method of embodiment B13, wherein the viral vector is a lentiviral vector.Bl 8. The method of embodiment B2, comprising:a first expression cassette comprising a promoter operably linked to a nucleotide sequence encoding MYC;a second expression cassette comprising a promoter operably linked to a nucleotide sequence encoding TERT; anda third expression cassette comprising a promoter operably linked to a nucleotide sequence encoding the PURPL IncRNA.Bl 9. The method of embodiment B2, comprising:a first expression cassette comprising a promoter operably linked to a nucleotide sequence encoding MYC and TERT; anda second expression cassette comprising a promoter operably linked to a nucleotide sequence encoding the PURPL IncRNA.B20. The method of embodiment B 18, wherein the expressing comprises providing to the MSC one, two, or all three of the first and second and third expression cassettes in one or more vectors, such that the PURPL, MYC and / or TERT are expressed in the MSC.B21. The method of embodiment Bl 9, wherein the expressing comprises providing to the MSC one or both of the first and second expression cassettes in one or more vectors, such that the PURPL, MYC, and / or TERT are expressed in the MSC.B22. The method of embodiment B20 or embodiment B21, wherein the vector is a viral vector. B23. The method of embodiment B2, wherein the MYC polypeptide is at least 95% identical to SEQ IDNO: 1.B24. The method of embodiment B2, wherein the TERT polypeptide is at least 95% identical to SEQ IDNO: 2B25. The method of embodiment B2, wherein SV40 Large T Antigen is at least 95% identical to SEQ ID NO: 12, wherein STAT3 is at least 95% identical to SEQ ID NO: 13, wherein E6 HPV is at least 95% identical to SEQ ID NO: 14, wherein E7 HPV is at least 95% identical to SEQ ID NO: 15, wherein Bmi-1 is at least 95% identical to SEQ ID NO: 16, and wherein N0TCH2 intracellular domain is at least 95% identical to SEQ ID NO: 17.B26. The method of embodiment Bl, wherein the PURPL IncRNA comprises one or more exon at least 95% identical to any one of SEQ ID NO: 3-8.B27. The method of embodiment Bl, wherein the PURPL IncRNA is at least 95% identical to any one of SEQ ID NOs: 9-11.B28. The method of embodiment Bl, wherein the PURPL IncRNA comprises exon 1, exon 2, exon 3, exon 3.5, exon 4, and exon 5, wherein exon 1 is at least 95% identical to SEQ ID NO: 3, exon 2 is at least 95% identical to SEQ ID NO: 4, exon 3 is at least 95% identical to SEQ ID NO: 5, exon 3.5 is at least 95% identical to SEQ ID NO: 6, exon 4 is at least 95% identical to SEQ ID NO: 7, and exon 5 is at least 95% identical to SEQ ID NO: 8.B29. The method of embodiment Bl, wherein exon 1 comprises SEQ ID NO: 3, exon 2 comprises SEQ ID NO: 4, exon 3 comprises SEQ ID NO: 5, exon 3.5 comprises SEQ ID NO: 6, exon 4 comprises SEQ ID NO: 7, and exon 5 comprises SEQ ID NO: 8.Cl . A method of treating a disease in a subject in need thereof comprising administering to the subject the immortalized MSC of embodiments Al or the immortalized MSC made by the method of embodiment Bl -B33.C2. The method of emobidment Cl, wherein the immortalized MSC of embodiment Al or the immortalized MSC made by the method of embodiment Bl delivers a therapeutic payload to the subject.C3. The method of embodiment C2, wherein the therapeutic payload is a viral vector.C4. The method of embodiment C3, wherein the viral vector is a retroviral vector.C5. The method of embodiment C4, wherein the retroviral vector has independent replicative capacity.C6. The method of embodiment C4, wherein the retroviral vector lacks independent replicative capacity.C7. The method of embodiment C3, wherein the viral vector is a lentiviral vector.C8. The method of embodiment C2, wherein the therapeutic payload is an antibody.C9. The method of embodiment C8, wherein the antibody is a bispecific antibody.CIO. The method of embodiment C8, wherein the antibody is abispecific T cell engager (BiTE). Cl 1. The method of embodiment C2, wherein the therapeutic payload is a type VII collagen. C12. The method of embodiment C2, wherein the therapeutic payload is a cytokine.C13. The method of embodiments C1-C12, wherein the disease is cancer.C14. The method of embodiment Cl 3, wherein the cancer is lung cancer, breast cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, kidney cancer, urinary tract cancer, oral cancer, head and neck cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, skin cancer, melanoma, sarcoma, lymphoma, leukemia, or brain cancer including glioblastoma, anaplastic astrocytoma, oligodendroglioma, and medulloblastoma.C15. The method of embodiments C1-C1269, wherein the disease is a skin disorder.C16. The method of embodiments C1-C1269, wherein the disease is an autoimmune disease. C17. The method of embodiments C1-C12, wherein the disease is a neurodegenerative disease. C18. The method of embodiments C1-C12, wherein the disease is a monogenic hereditary disorder.EXAMPLESExample 1. Immortalization using MYC and TERT

[0110] The immortalized human clonal cell line ALT422 was generated by infection with a replicating retroviral vector (RRV) carrying a GFP payload, RRV-GFP, followed by a combined lentiviral transduction of L-MYC and hTERT genes (FIG. 1A). Clone ALT422 represents one of only two clones obtained from primary human Wharton’s jelly MSCs (LifeLine Cell Technology FC-0020, LL-0034) that were immortalized using L-MYC + hTERT transgenes. ALT422 is considered immortalized due to its stable maintenance of self-renewal capabilities, sustained growth with 98% viability, and propagation to at least passage 53, reaching a cumulative population doubling level (PDL) of 144 at an average population doubling time (PDT) of 30.2 hr (FIG IB) (See, e.g., Li et al., Cell Rep., 20(10):2048 (2017)). In comparison, naive MSCs reached PDL 34.8 at passage 16, with an average PDT of 49.9 hr, and ceased proliferating thereafter. The LT100 clone, which was not infected with RRV-GFP (FIG. 1A), began to exhibit abnormal and decelerated growth with 94% viability from passage 46 and ceased proliferating by passage 50, with a PDT of 76.4 hr, indicating that it was not immortalized. At passage 50, LT100 reached a PDL of 128 with an average PDT of 37.2 hr (FIG. IB). Despite multiple efforts to derive additional immortalized clones from cells initially isolated at low passage, these attempts were unsuccessful. Only a single clone (ALT422) out of 104 clones transduced with L-MYC and hTERT achieved successful immortalization.Example 2. Identifying mutations which may contribute to immortalization[OHl] To identify the spontaneously occurring mutation(s) that enable RRV-infected cells transduced with LMYC + hTERT to bypass replicative crisis and achieve immortalization, total RNA from passage 22 of the immortalized ALT422 clone was subjected to RNA sequencing (RNA-seq) transcriptome analysis. Total RNA from passage 5 of the naive un-transduced MSCs was used for comparison. The RNA-seq data revealed the PURPL IncRNA gene as the 9th most upregulated, following the L-MYC and hTERT transgenes, with a 7.011 log2-fold change and anadjusted p-value (padj) of 1.92E-6, corresponding to a 129-fold upregulation in immortalized ALT422 cells compared to naive MSCs. Genes within the same cluster exhibit the same trends in expression levels. FIG. 2 illustrates the comparison of statistical significance in differential gene expression, represented by [-loglO(padj)] values (where padj < 0.05), relative to [log2(fold change)] > 1, highlighting the spontaneously upregulated PURPL gene and ectopically expressed L-MYC and hTERT transgenes within a volcano plot of genes in gene expression RNA-seq analysis. The reports of the PURPL IncRNA involvement in regulating the p53 pathway prompted its selection as a candidate gene to aid cells in evading replicative crisis (See, e.g., Li et al., Cell Rep., 20(10):2048 (2017) and Hunten, Mol. Cell Proteomics, 14(10):2609-29 (2015)).Example 3. Evaluating immortalization using PURPL

[0112] Next, it was determined whether PURPL could boost the effectiveness of MSC immortalization strategies. To isolate PURPL cDNA from the reverse-transcribed RNA extracted from ATL422 p21 cells, primers were designed based on the available PURPL sequence (NCBI Reference Sequence NR 038848.1). According to the Ensembl genome browser, PURPL gene (ID ENSG00000250337) encodes 126 alternatively spliced transcripts. The NR_038848.1 transcript is 1106nt long and contains 5 exons (SEQ ID NO: 11). Inconsistencies were found in the designation of the precise exon 1 start among available sequence information: the PURPL sequence at the Ensembl website revealed additional 9 nucleotides upstream of exon 1 (ID ENSE00003888082) compared to the 5’ end of the exon 1 in NR_038848.1, which was included in the forward primer (TGTGGTGACCTGGGAATCAATGTGTGAGG (SEQ ID NO: 17). The polyadenylation signal is embedded into the last exon 5 and the sequence of the reverse primer was TTAAGCCCCTCATTCGTTATATTTTATTTTAACTTCATAAGGTATGAC (SEQ ID NO: 18).

[0113] The PCR amplification was run in octuplicates, and only two samples (sample #4 and #5) yielded a PCR product, both of which had an expected length of approximately 1115nt (FIG.3), although the PCR product from sample #4 appeared to be slightly longer than PCR product from sample #5. The low efficiency of PURPL amplification from cDNA of ALT422 cells can be explained by a low abundance of the PURPL transcripts in the ALT422 samples, despite being upregulated 129-fold from undetectable levels in naive MSC cells. In general, the vast majority ofIncRNAs are expressed at lower levels compared to mRNAs (See, e.g., Grammatikakis & Lal, Mamm. Genome, 33(2):271-280 (2022)).

[0114] Two PCR fragments were isolated, re-amplified, and submitted for Sanger sequencing to confirm the authenticity of the PURPL sequence and its length. The two PCR fragments are of different length. The longer 1219nt fragment (SEQ ID NO: 9) from sample #4 has six exons and corresponds to the NR 038848.1 sequence, except for an additional 104nt long exon (ID ENSE00004014255) located between exons 3 and exon 4. This exon is referred to herein as “3.5” and its sequence is GAAACACTTTCTGAAAATGTCAGAACTCTTGAAAACATG GATGAGGAAAAGCATAAGTACTGCTTTATGCTTTTCTTATGAATTTTAAAAGATGTA ATTTAAAG (SEQ ID NO: 6) (FIG. 4). On the contrary, the shorter PCR fragment from sample #5 is missing exon 4 and contains exons 1, 2, 3, “3.5” and 5, resulting in 1118nt length containing five exons (SEQ ID NO: 10).

[0115] The longer 1219nt PURPL splice variant containing six exons was cloned into a lentiviral vector (LV) between EFl a promoter and a hGH polyadenylation signal. This expression cassette was inserted into a LV in the opposite orientation because the function of the IncRNA depends on its secondary structure, and it is essential that the polyadenylation tail starts immediately downstream of the last exon (FIG. 4). Inserting the IncRNA expression cassette in a forward orientation within a LV would result in the presence of additional 830nt-long 3’ untranslated region of LV vector downstream of the last PURPL exon. This sequence could impair the correct RNA secondary structure of PURPL IncRNA and consequently result in aberrant RNA-protein interaction and function of PURPL (See, e.g., Zhang et al., J. Nanobiotech. 19(l):303 (2021)). Therefore, inserting the IncRNA expression cassette in the opposite orientation ensures that the secondary structure of the IncRNA is maintained without interference from the vector's downstream sequences.

[0116] EFla promoter-PURPL-hGHpA expression cassette was inserted in the opposite orientation into a pRRL LV vector plasmid, upstream of PGK-Puro, which is in the forward orientation (FIG. 4). Lentiviral particles containing the PURPL payload were generated by cotransfecting HEK293T cells with plasmids PURPL-LV, pMD2.G, pMDL-G / P-RRE and pRSV-REV. Virus was concentrated by using the Amicon ultra-centrifugal fdter with a 100 kDa MWCO. The functional titer, indicative of the lentivirus' capacity to transduce naive MSCs, was determinedby quantifying the LV copy number per cell by quantitative (q)PCR (See Butler et al., Nat. Med.7:631-634 (2001)). Naive MSCs were infected with serial dilutions of the virus. Genomic DNA was extracted 10 days post-infection to reduce the likelihood of unintegrated DNA presence.

[0117] For immortalization studies, naive WJ-MSCs (at passage 4 or 5) were infected with L-MYC and hTERT lentiviral vectors at a multiplicity of infection (MOI) of 1 in presence or absence of PURPL-LV at MOI 1. Two days after infection, cells were split at a ratio of 1:4 and subjected to antibiotic selection. Resistant cells were then seeded into a 96-well plate at limited dilution to isolate all proliferating individual clones, which were subsequently expanded and monitored for growth rate. All proliferating clones were maintained in culture and passaged based on their individual growth rates, typically every 2 to 4 days. Clones surpassing passage 21 were deemed potentially immortalized and were passaged every 72 hours, with medium replenished within 36 hours of plating. Population doubling time (PDT) was calculated for each passage.

[0118] In an experiment where L-MYC and hTERT were expressed from a single lentiviral vector, PLTIN, and PURPL was introduced through a simultaneous second lentiviral transduction, the combination of PURPL with PLTIN vectors proved highly efficient in generating long-term immortalized clones (FIG. 5). Specifically, out of the 25 clones originally isolated at early passage 8, three clones (WPPF72, WPP1B, and WPPC71) were immortalized and exhibited robust and sustained growth, with an average population doubling time (PDT) of 25.6 hr. To demonstrate immortalization, clone WPPF72 was cultured for 70 passages, reaching a cumulative population doubling level (PDL) of 212 with sustained growth and an average population doubling time (PDT) of 24.5 hr. Clone WPP1B was cultured up to passage 65, reaching PDL 174 with an average PDT of 27.4 hr. Clone WPPC71 was maintained in culture until passage 46, reaching PDL 135 with an average PDT of 25.0 hr (FIG. 6). RT-qPCR expression analysis confirmed the heterologous expression of PURPL in all three immortalized clones, which were also overexpressing L-MYC and hTERT (FIGs. 9-11). In contrast, in the absence of PURPL, when using PLTIN alone, no immortalized clones were generated, despite initially isolating 48 clones that were proliferating at early passage. These results demonstrate that PURPL overexpression, in combination with L-MYC and hTERT expressed from the PLTIN vector, significantly enhances the immortalization rate from 0% to 12%, resulting in the generation of three immortalized cell lines out of 25 initially isolated clones.

[0119] Furthermore, PURPL extends the lifespan of the clonal lines, persisting up to at least passage 70 with PDL 212. Moreover, PURPL transgene improves the cell doubling time by almost 5 hours, reducing the average doubling time from PDT of 30.2hr (clone ALT422) to 25.6hr (average for three WPP clones) (FIG. 6). Nonetheless, while PURPL overexpression contributes to the efficacy of immortalization, simply overexpressing PURPL does not inherently lead to cellular immortalization in PURPL-transduced cells.Example 4. Assessing the relationship of PURPL, MYC, and TERT in immortalization

[0120] To validate and further substantiate the evidence that PURPL IncRNA boosts the L-MYC + hTERT immortalization efficacy of MSC cells, another MSC immortalization experiment was designed using three lentiviral vectors, wherein two hTERT and PURPL vectors share the same antibiotic resistance gene while the third, L-MYC, vector uses another (FIG. 7). This vector configuration allowed us to evaluate the contribution of PURPL (and hTERT) to MSC immortalization by comparing the proportion of PURPL-overexpressing versus nonoverexpressing immortalized clones. Using this approach, we found that all clones considered immortalized based on sustained proliferation at passage 2 land beyond, were overexpressing all three transgenes: PURPL, L-MYC, and hTERT (FIGs. 8-11).

[0121] Intriguingly, no immortalized clones were generated in the absence of PURPL expression. The clones generated from this experiment were ELTP clones, with a total of 67 proliferating clones initially isolated from 96-well plates at a low passage number. However, by passage 20, only nine of these clones continued to proliferate. Surprisingly, two clones, ELTP14 F7 and ELTP14 G8, abruptly ceased to proliferate when they reached passage 21 (FIG. 8). Subsequent RT-PCR analysis revealed that these two clones did not overexpress the PURPL transgene (FIG.9), while overexpressing L-MYC (FIG. 10) and hTERT (FIG. 11). Therefore, these clones were re-labeled ELT(P)14 F7 and ELT(P)14 G8 to indicate that they did not acquire PURPL transgene during the transduction with all three LV vectors. Observing that clones not overexpressing PURPL ceased proliferation abruptly at passage 21, we defined a high probability of immortalization as the ability of clones to sustain growth at passage 21 and beyond. The remaining seven proliferating clones out of the nine ELTPs growing at passage 20, gave rise to theimmortalized clones ELTP12 IF8, ELTP12 SC5, ELTP12 SF6, ELTP44 IB3, ELTP44 2F11, ELTP12 IG8, and ELTP12 IC7 (FIG. 8) and overexpressed PURPL (FIG. 9), L-MYC (FIG. 10), and hTERT (FIG. 11). Six of these seven ELTP clones showed robust growth and maintained a population doubling time (PDT) of <20 (Fig. 8). Four clones — ELTP12 IF8, ELTP12 SC5, ELTP44 IB3, and ELTP12 IG8 — were cultured long-term to assess sustained growth. ELTP12 IF8 was propagated up to passage 62 with stable population doubling times, while the other three clones were maintained at least until passage 50, demonstrating robust and sustained proliferative capacity. Clones ELTP12 IF8, ELTP12 SC5, and ELTP12 IG8 exhibited steady growth with PDTs of <16, whereas clone ELTP44 IB3 grew with a PDT of <20. At passage 50, ELTP12 IF8 had reached a cumulative population doubling level (PDL) of 198, while ELTP12 SC5, ELTP12 IG8, and ELTP44 IB3 reached PDLs of 170, 166.2, and 160.7, respectively. Clones ELTP12 SF6 and ELTP442F11 were maintained in culture up to passage 40 and exhibited PDTs of <20, while clone ELTP12 IC7 grew with a PDT of <32. Thus, successful immortalization of seven clones out of the original 67 clones in the ELTP series was demonstrated, corresponding to an immortalization efficiency of 10.4%.

[0122] On the contrary, using the same L-MYC and hTERT LV vectors without the third LV delivering PURPL yielded only one immortalized clone (ALT422) out of 104 clones, resulting in a 0.96% efficacy. Thus, the inclusion of PURPL IncRNA alongside L-MYC and hTERT transgenes leads to a 10.8-fold enhancement in MSC immortalization efficacy, underscoring the essential role of PURPLoverexpression in the immortalization process. No immortalized clones lacking PURPL overexpression were generated. All eleven immortalized clones (ALT422, three WPP clones, and seven ELTP clones) have either spontaneous or vector-mediated upregulation of PURPL expression (FIG. 9). Furthermore, clones that did not overexpress PURPL, which initially exhibited signs of immortalization, such as clone LT100, or clones ELT(P)14 F7 and ELT(P)14 G8, eventually ceased to proliferate.Example 5. Characterizing immortalized MSCs

[0123] In addition to long-term proliferation studies and transgene expression assays, the immortalized clonal lines were characterized for surface immunomarkers, lack of turn origeni city, karyotype stability at late passage (P50), RRV production, and migration capacity. Three WPPclones (WPPF72, WPP IB, and WPPC71) and three ELTP clones (ELTP12 IF8, ELTP12 SC5, and ELTP44 IB3) were evaluated for the expression of characteristic MSC surface markers CD 105, CD73, CD90, CD44, CD29, and CD166, in comparison with naive MSC at passage 8, to validate maintenance of the MSC phenotype. All six immortalized clones and the naive MSC cells were also negative for the tested hematopoietic markers CD45, CD34, CD14, HLA II-DRDP DQ, and CD79b.

[0124] WPPF72 cells infected at passage 38 with either RRV-GFP or a therapeutic variant comprising a cytosine deaminase payload (RRV-yCD) were permissive to RRV infection and maintained proliferation rates comparable to uninfected WPPF72 cells (Fig. 12). The infected cells expressed MSC markers and lacked expression of hematopoietic lineage markers. The infected WPPF72 clones produced high levels of viral particles, with titers of 1.81E+07 TU / ml for RRV-GFP and 1.46E+07 TU / ml for RRV-yCD, corresponding to approximately 10 TU / cell within 24 hours.

[0125] To further confirm its mesenchymal identity, the immortalized clone ELTP12 IF8 was evaluated for tri-lineage differentiation potential. Under lineage-specific induction conditions, the cells successfully differentiated into adipogenic, osteogenic, and chondrogenic lineages (Fig. 13). Adipogenic differentiation was confirmed by the accumulation of intracellular lipid droplets visualized by Oil Red O staining. Osteogenic differentiation was demonstrated by robust extracellular matrix mineralization detected by Alizarin Red staining. Chondrogenic differentiation, performed in pellet culture, resulted in compact spheroidal pellets exhibiting uniform Alcian Blue staining, indicative of sulfated glycosaminoglycan-rich cartilage-like extracellular matrix. Collectively, these results confirm that the immortalized MSC clone ELTP12 IF8 retain multipotent differentiation capacity consistent with a bona fide MSC phenotype.

[0126] The non-transformed phenotype of the immortalized clones with PURPL transgene was confirmed by assessing their cellular anchorage-independent growth in an in vitro soft agar colony formation and tumorigenicity assay. After a 14-day culture period, only the control U87 glioblastoma cell line demonstrated colony growth in 1% soft agar. In contrast, all assayed immortalized clones, including the RRV-infected WPPF72 clones, did not form colonies but remained viable as single cells (Fig. 14). The lack of tumorigenicity was further confirmed in vivo using an immunodeficient NSG mouse model. ELTP12 IF8 cells (5 x 106) were injectedsubcutaneously into NSG mice, which were monitored over six months. No tumor formation or adverse clinical signs were observed. Body weight was regularly monitored and remained comparable to control mice, with all animals surviving the six-month study period (Fig. 15). At the six-month endpoint, comprehensive necropsy and qPCR-based biodistribution analysis of twelve tissues per animal revealed no detectable human DNA, with a sensitivity of 10 human cells per 25,000 murine cells, indicating absence of biodistribution and tumorigenic activity (Fig. 16). Consistent with these in vivo safety findings, ELTP12 IF8 cells maintained a normal human karyotype at passage 50 (Fig. 17).

[0127] The selected immortalized clones also exhibited a significant in vitro migration capacity toward serum-free conditioned media from patient-derived NCT glioma cells compared to serum-free unconditioned medium (SF) (Fig. 18). MSCs were allowed to migrate across membranes with an 8 pm pore size for 24 h. In conclusion, these data demonstrate that incorporation of PURPL IncRNA alongside L-MYC and hTERT transgenes successfully generated multiple immortalized, non-transformed human MSC clones while preserving their identity and biological function. The immortalized ELTP12 IF8 clone maintained MSC phenotypic markers, normal karyotype at passage 50, and multipotent differentiation capacity into osteogenic, adipogenic, and chondrogenic lineages. In vivo, six months after subcutaneous injection, no tumor formation or adverse clinical signs were observed. Comprehensive necropsy revealed no macroscopic abnormalities, and qPCR analysis detected no human cells in any tissues, indicating the absence of biodistribution and tumorigenic activity under the tested conditions. Collectively, these results confirm that immortalized ELTP12 IF8 MSCs retain MSC identity, phenotype, and multipotency without acquiring tumorigenic properties.

[0128] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.INFORMAL SEQUENCE LISTINGSEQ ID NO: I L -MYC amino acid sequence MDYDSYQHYFYDYDCGEDFYRSTAPSEDIWKKFELVPSPPTSPPWGLGPGAGDPAPGIG PPEPWPGGCTGDEAESRGHSKGWGRNYASIIRRDCMWSGFSARERLERAVSDRLAPGA PRGNPPKASAAPDCTPSLEAGNPAPAAPCPLGEPKTQACSGSESPSDSENEEIDVVTVEK RQSLGIRKPVTITVRADPLDPCMKHFHISIHQQQHNYAARFPPESCSQEEASERGPQEEVL ERDAAGEKEDEEDEEIVSPPPVESEAAQSCHPKPVSSDTEDVTKRKNHNFLERKRRNDL RSRFLALRDQVPTLASCSKAPKVVILSKALEYLQALVGAEKRMATEKRQLRCRQQQLQ KRIAYLTGY SEQ ID NO: 2 hTERT amino acid sequence MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCV PWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTT SVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPL YQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLP LPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGT RHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPS LTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTH CPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLR RLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGC VPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSI GIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRR EK R A ER LT SRVK ALF S VLNYERARRPGLLGAS VLGLDDIHRAWRTF VLRVRAQDPPPEL YFVKVDVTGAYDTIPQDRLTEV1AS11KPQNTYCVRRYAVVQKAAHGHVRKAFKSHVST LTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKS YVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFL RTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEV Q SD YS S YARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQ VNSLQTVC TNI YKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKG AAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAA NPALPSDFKTILD SEQ ID NO: 3 PURPL Exonl (length 228nt) RNA sequence CUGGGAAUCAAUGUGUGAGGUGGUGCUAUGUAGCAGGAACCCCUCUUGCUUUGC AAAUAGUUUUUUGUUUGUUUUCCUUUUUGCCCAAUAGAGCCCUGCUCUACUGAC CCUUCAAUGUGCCCGUGUGCCUAAAUAUUCCUGGUCGUGUGAAAAGAACCCAGGU AUUAGCUGAACUAAGGAGCACAAUUCUGCAACAUUUUGGCACCCAAACACGGGGC UUGAGAAAUG SEQ ID NO: 4 PURPL Exon 2 (length 161 nt) RNA sequenceAAUGCAAUUGGAGAAACUGGUUGUUUUACCAGGCGUUGAUUGGAAAUGUGUGCU UCCCUUUAAGCAGUCAAGCUCAACUUGCAGAACUGAUGGGAACCCCUUGGGAAAA CUGGCCUCAAAUGUUUGUCUACACAGUCCACAUACAGGGUUCUUAACCUGCG SEQ ID NO: 5 PURPL Exon 3 (length 163 nt) RNA sequence AUGAGGGAAACUUCCCAGGGCUUGUCUGGGUAUGCCCACAGUGGACUGGAGCCCA AAAUGCACACUGGAAGAAGUGGAUGGAGCCACGUGGAUGUCAUGCCUUAUGCAG GGGAGGAGCCUGCUCUCUUCAGCUCCUGUGGUAAUGUGGGAAUCGAUCUGUGAG SEQ ID NO: 6 PURPL Exon “3.5” (length 104 nt) RNA sequence GAAACACUUUCUGAAAAUGUCAGAACUCUUGAAAACAUGGAUGAGGAAAAGCAU AAGUACUGCUUUAUGCUUUUCUUAUGAAUUUUAAAAGAUGUAAUUUAAAG SEQ ID NO: 7 PURPL Exon 4 (length 101 nt) RNA sequence CAUAUUUAACUAUCCGGCUAUUUUGGAGAUUGAAAAAAGAAGACCUGAUUUAUC AUGUGCAAUAUCUCACACAUCUGUCAUUUCUAUUCUACCGCAAUUCG SEQ ID NO: 8 PURPL Exon 5 (length 453 nt) RNA sequence AUGGAGUCUUGUUCUGUGGUCCAGGCUGGAGGGCAGUGGCAUGAUCUCGGCUCA CUACAAGCUCUGCCUCCCGGGUUCACGCCAUUCUCCUGCCUCAGCCUCUUGAGUA GCUGGGACUACAGGCGCCCGCCACCACGCCUGGCUAAUUUUUUGUAUAUUUACUA GAGAUGGCUAGUGUUAGCCAAGAUGGUCUCGAUCUCCUGACCUCGUGAUCUGCCC ACCUCAGCCUGCCAAAGUGCUGGGAUUACUGGCGUGAGCCACCGUGCCUGGCCAG CAUCCUCUAAUUAUUAUUCAUAGUUUGUGAAUUUAGGCCUACAUGAAUAAUAAU AUAAUAUUUACUAUGAUGUUACUAUCUACCAACCUUAUUUUUCUUUUCUAUAAA AUAGGAGCCAAAAUAAUAUUUAUGUCAUACCUUAUGAAGUUAAAAUAAAAUAUA ACGAAUGAGGGGCUUAA SEQ ID NO: 91219 nt PURPL IncRNA fragment RNA sequence; has exons: 1, 2, 3, 3.5, 4, and 5 UGUGGUGACCUGGGAAUCAAUGUGUGAGGUGGUGCUAUGUAGCAGGAACCCCUC UUGCUUUGCAAAUAGUUUUUUGUUUGUUUUCCUUUUUGCCCAAUAGAGCCCUGC UCUACUGACCCUUCAAUGUGCCCGUGUGCCUAAAUAUUCCUGGUCGUGUGAAAAG AACCCAGGUAUUAGCUGAACUAAGGAGCACAAUUCUGCAACAUUUUGGCACCCAA ACACGGGGCUUGAGAAAUGAAUGCAAUUGGAGAAACUGGUUGUUUUACCAGGCG UUGAUUGGAAAUGUGUGCUUCCCUUUAAGCAGUCAAGCUCAACUUGCAGAACUG AUGGGAACCCCUUGGGAAAACUGGCCUCAAAUGUUUGUCUACACAGUCCACAUAC AGGGUUCUUAACCUGCGAUGAGGGAAACUUCCCAGGGCUUGUCUGGGUAUGCCC ACAGUGGACUGGAGCCCAAAAUGCACACUGGAAGAAGUGGAUGGAGCCACGUGG AUGUCAUGCCUUAUGCAGGGGAGGAGCCUGCUCUCUUCAGCUCCUGUGGUAAUG UGGGAAUCGAUCUGUGAGGAAACACUUUCUGAAAAUGUCAGAACUCUUGAAAACAUGGAUGAGGAAAAGCAUAAGUACUGCUUUAUGCUUUUCUUAUGAAUUUUAAAA GAUGUAAUUUAAAGCAUAUUUAACUAUCCGGCUAUUUUGGAGAUUGAAAAAAGA AGACCUGAUUUAUCAUGUGCAAUAUCUCACACAUCUGUCAUUUCUAUUCUACCGC AAUUCGAUGGAGUCUUGUUCUGUGGUCCAGGCUGGAGGGCAGUGGCAUGAUCUC GGCUCACUACAAGCUCUGCCUCCCGGGUUCACGCCAUUCUCCUGCCUCAGCCUCU UGAGUAGCUGGGACUACAGGCGCCCGCCACCACGCCUGGCUAAUUUUUUGUAUAU UUACUAGAGAUGGCUAGUGUUAGCCAAGAUGGUCUCGAUCUCCUGACCUCGUGA UCUGCCCACCUCAGCCUGCCAAAGUGCUGGGAUUACUGGCGUGAGCCACCGUGCC UGGCCAGCAUCCUCUAAUUAUUAUUCAUAGUUUGUGAAUUUAGGCCUACAUGAA UAAUAAUAUAAUAUUUACUAUGAUGUUACUAUCUACCAACCUUAUUUUUCUUUU CUAUAAAAUAGGAGCCAAAAUAAUAUUUAUGUCAUACCUUAUGAAGUUAAAAUA AAAUAUAACGAAUGAGGGGCUUAA SEQ ID NO: 101118 nt PURPL IncRNA fragment RNA sequence; has exons: 1, 2, 3, 3.5, and 5 (missing 4) UGUGGUGACCUGGGAAUCAAUGUGUGAGGUGGUGCUAUGUAGCAGGAACCCCUCLrUGCLrUUGCAAAUAGUUUUUUGUUUGUUUUCCUUUUUGCCCAAUAGAGCCCUGC UCUACUGACCCUUCAAUGUGCCCGUGUGCCUAAAUAUUCCUGGUCGUGUGAAAAG AACCCAGGUAUUAGCUGAACUAAGGAGCACAAUUCUGCAACAUUUUGGCACCCAA ACACGGGGCUUGAGAAAUGAAUGCAAUUGGAGAAACUGGUUGUUUUACCAGGCG UUGAUUGGAAAUGUGUGCUUCCCUUUAAGCAGUCAAGCUCAACUUGCAGAACUG AUGGGAACCCCUUGGGAAAACUGGCCUCAAAUGUUUGUCUACACAGUCCACAUAC AGGGUUCUUAACCUGCGAUGAGGGAAACUUCCCAGGGCUUGUCUGGGUAUGCCC ACAGUGGACUGGAGCCCAAAAUGCACACUGGAAGAAGUGGAUGGAGCCACGUGG AUGUCAUGCCUUAUGCAGGGGAGGAGCCUGCUCUCUUCAGCUCCUGUGGUAAUG UGGGAAUCGAUCUGUGAGGAAACACUUUCUGAAAAUGUCAGAACUCUUGAAAAC AUGGAUGAGGAAAAGCAUAAGUACUGCUUUAUGCUUUUCUUAUGAAUUUUAAAA GAUGUAAUUUAAAGAUGGAGUCUUGUUCUGUGGUCCAGGCUGGAGGGCAGUGGC AUGAUCUCGGCUCACUACAAGCUCUGCCUCCCGGGUUCACGCCAUUCUCCUGCCU CAGCCUCUUGAGUAGCUGGGACUACAGGCGCCCGCCACCACGCCUGGCUAAUUUU UUGUAUAUUUACUAGAGAUGGCUAGUGUUAGCCAAGAUGGUCUCGAUCUCCUGA CCUCGUGAUCUGCCCACCUCAGCCUGCCAAAGUGCUGGGAUUACUGGCGUGAGCC ACCGUGCCUGGCCAGCAUCCUCUAAUUAUUAUUCAUAGUUUGUGAAUUUAGGCC UACAUGAAUAAUAAUAUAAUAUUUACUAUGAUGUUACUAUCUACCAACCUUAUU UUUCUUUUCUAUAAAAUAGGAGCCAAAAUAAUAUUUAUGUCAUACCUUAUGAAG UUAAAAUAAAAUAUAACGAAUGAGGGGCUUAA SEQ ID NO: 11: 1106 nt PURPL IncRNA fragment RNA sequence; has exons: 1, 2, 3, 4, and 5 CUGGGAAUCAAUGUGUGAGGUGGUGCUAUGUAGCAGGAACCCCUCUUGCUUUGC AAAUAGUUUUUUGUUUGUUUUCCUUUUUGCCCAAUAGAGCCCUGCUCUACUGAC CCUUCAAUGUGCCCGUGUGCCUAAAUAUUCCUGGUCGUGUGAAAAGAACCCAGGU AUUAGCUGAACUAAGGAGCACAAUUCUGCAACAUUUUGGCACCCAAACACGGGGC UUGAGAAAUGAAUGCAAUUGGAGAAACUGGUUGUUUUACCAGGCGUUGAUUGGA AAUGUGUGCUUCCCUUUAAGCAGUCAAGCUCAACUUGCAGAACUGAUGGGAACCCCUUGGGAAAACUGGCCUCAAAUGUUUGUCUACACAGUCCACAUACAGGGUUCUU AACCUGCGAUGAGGGAAACUUCCCAGGGCUUGUCUGGGUAUGCCCACAGUGGACU GGAGCCCAAAAUGCACACUGGAAGAAGUGGAUGGAGCCACGUGGAUGUCAUGCC UUAUGCAGGGGAGGAGCCUGCUCUCUUCAGCUCCUGUGGUAAUGUGGGAAUCGA UCUGUGAGCAUAUUUAACUAUCCGGCUAUUUUGGAGAUUGAAAAAAGAAGACCU GAUUUAUCAUGUGCAAUAUCUCACACAUCUGUCAUUUCUAUUCUACCGCAAUUCG AUGGAGUCUUGUUCUGUGGUCCAGGCUGGAGGGCAGUGGCAUGAUCUCGGCUCA CUACAAGCUCUGCCUCCCGGGUUCACGCCAUUCUCCUGCCUCAGCCUCUUGAGUA GCUGGGACUACAGGCGCCCGCCACCACGCCUGGCUAAUUUUUUGUAUAUUUACUA GAGAUGGCUAGUGUUAGCCAAGAUGGUCUCGAUCUCCUGACCUCGUGAUCUGCCC ACCUCAGCCUGCCAAAGUGCUGGGAUUACUGGCGUGAGCCACCGUGCCUGGCCAG CAUCCUCUAAUUAUUAUUCAUAGUUUGUGAAUUUAGGCCUACAUGAAUAAUAAU AUAAUAUUUACUAUGAUGUUACUAUCUACCAACCUUAUUUUUCUUUUCUAUAAA AUAGGAGCCAAAAUAAUAUUUAUGUCAUACCUUAUGAAGUUAAAAUAAAAUAUA ACGAAUGAGGGGCUUAA SEQ ID NO: 12 SV40 Large T Antigen amino acid sequence MDKVLNREESLQLMDLLGLERSAWGNIPLMRKAYLKKCKEFHPDKGGDEEKMKKMN TLYKKMEDGVKYAHQPDFGGFWDATETPTYGTDEWEQWWNAFNEENLFCSEEMPSSD DEATAD SQHSTPPKKKRKVEDPKDFPSELL SFL SHAVF SNRTLACF AIYTTKEKAALLYK KIMEKYSVTFISRHNSYNHNILFFLTPHRHRVSAINNYAQKLCTFSFLICKGVNKEYLMY SALTRDPFSVIEESLPGGLKEHDFNPEEAEETKQVSWKLVTEYAMETKCDDVLLLLGMY LEFQYSFEMCLKCIKKEQPSHYKYHEKHYANAAIFADSKNQKTICQQAVDTVLAKKRV DSLQLTREQMLTNRFNDLLDRMDIMFGSTGSADIEEWMAGVAWLHCLLPKMDSVVYD FLKCMVYNIPKKRYWLFKGPIDSGKTTLAAALLELCGGKALNVNLPLDRLNFELGVAID QFLVVFEDVKGTGGESRDLPSGQGINNLDNLRDYLDGSVKVNLEKKHLNKRTQIFPPGI VTMNEYSVPKTLQARFVKQIDFRPKDYLKHCLERSEFLLEKRIIQSGIALLLMLIWYRPV AEF AQ SIQ SRIVEWKERLDKEF SLS VYQKMKFNVAMGIGVLDWLRNSDDDDED SQENA DKNEDGGEKNMEDSGHETGIDSQSQGSFQAPQSSQSVHDHNQPYHICRGFTCFKKPPTP PPEPET SEQ ID NO: 13 STAT3 amino acid sequence MAQWNQLQQLDTRYLEQLHQLYSDSFPMELRQFLAPWIESQDWAYAASKESHATLVF HNLLGEIDQQYSRFLQESNVLYQHNLRRIKQFLQSRYLEKPMEIARIVARCLWEESRLLQ TAATAAQQGGQANHPTAAVVTEKQQMLEQHLQDVRKRVQDLEQKMKVVENLQDDFD FNYKTLKSQGDMQDLNGNNQSVTRQKMQQLEQMLTALDQMRRSIVSELAGLLSAMEY VQKTLTDEELADWKRRQQIACIGGPPNICLDRLENWITSLAESQLQTRQQIKKLEELQQK VSYKGDPIVQHRPMLEERIVELFRNLMKSAFVVERQPCMPMHPDRPLVIKTGVQFTTKV RLLVKFPELNYQLKIKVCIDKDSGDVAALRGSRKFNILGTNTKVMNMEESNNGSLSAEF KHLTLREQRCGNGGRANCDASLIVTEELHLITFETEVYHQGLKIDLETHSLPVVVISNICQ MPNAW ASILWYNMLTNNPKNVNFFTKPPIGTWDQ VAEVL SWQF S STTKRGL SIEQLTTL AEKLLGPGVNYSGCQ1TWAKFCKENMAGKGFSFWVWLDN11DLVKKY1LALWNEGY1M GFISKERERAILSTKPPGTFLLRFSESSKEGGVTFTWVEKDISGKTQIQSVEPYTKQQLNN MSFAEIIMGYKIMDATNILVSPLVYLYPDIPKEEAFGKYCRPESQEHPEADPGSAAPYLKTKFTCVTPTTCSNTTDLPMSPRTLDSLMQFGNNGEGAEPSAGGQFESLTFDMELTSECATS PM SEQ ID NO: 14 E6 HPV amino acid sequence MHQKRTAMFQDPQERPRKLPQLCTELQTT1HD11LECVYCKQQLLRREVYDFAFRDLC1V YRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPE EKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL SEQ ID NO: 15 E7 HPV amino acid sequence MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCC KCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP SEQ ID NO: 16 Bmi-1 amino acid sequence MHRTTRIKITELNPHLMCVLCGGYFIDATTIIECLHSFCKTCIVRYLETSKYCPICDVQVH KTRPLLNIRSDKTLQDIVYKLVPGLFKNEMKRRRDFYAAHPSADAANGSNEDRGEVAD EEKRIITDDEIISLSIEFFDQSRLDRKVNKEKPKEEVNDKRYLRCPAAMTVMHLRKFLRSK MDIPNTFQIDVMYEEEPLKDYYTLMDTAYIYTWRRNGPLPLKYRVRPTCKRMKMSHQR DGLTNAGELESDSGSDKANSPAGGVPSTSSCLPSPSTPVQSPHPQFPHISSTMNGTSNSPS ANHQSSFASRPRKSSLNGSSATSSGSEQ ID NO: 17 NOTCH2 intracellular domain amino acid sequence MLACLQVDSRMAKRKRKHGFLWLPEGFTLRRDSSNHKRREPVGQDAVGLKNLSVQVS EANLIGSGTSEHWVDDEGPQPKKAKAEDEALLSEDDPIDRRPWTQQHLEAADIRHTPSL ALTPPQAEQEVDVLDVNVRGPDGCTPLMLASLRGGSSDLSDEDEDAEDSSANIITDLVY QGASLQAQTDRTGEMALHLAARYSRADAAKRLLDAGADANAQDNMGRCPLHAAVAA DAQGVFQILIRNRVTDLDARMNDGTTPLILAARLAVEGMVAELINCQADVNAVDDHGK SALHWAAAVNNVEATLLLLKNGANRDMQDNKEETPLFLAAREGSYEAAKILLDHFAN RDITDHMDRLPRD VARDRMHHDIVRLLDEYNVTP SPPGTVLTS AL SP VLCGPNRSFL SL KHTPMGKKARRPNTKSTMPTSLPNLAKEAKDAKGSRRKKCLNEKVQLSESSVTLSPVD SLESPHTYVSDATSSPMITSPGILQASPTPLLAAAAPAAPVHTQHALSFSNLHDMQPLAP GASTVLPSVSQLLSHHHIAPPGSSSAGSLGRLHPVPVPADWMNRVEMNETQYSEMFGM VLAPAEGAHPGIAAPQSRPPEGKHMSTQREPLPPIVTFQLIPKGSIAQAAGAPQTQSSCPP AVAGPLPSMYQIPEMPRLPSVAFPPTMMPQQEGQVAQTIVPTYHPFPASVGKYPTPPSQH SYASSNAAERTPSHGGHLQGEHPYLTPSPESPDQWSSSSPHSASDWSDVTTSPTPGGGGG GQRGPGTHMSEPPHSNMQVYA SEQ ID NO: 18 ATTTCTGGCCTTCTTCACAAGTGTTTACATTAGATGCTTTTGTGCAGATGAGGGAACC TGCCCAGGGCTTGTCTGGGCATGCCCACAGTGGACTGGAGCCTGACATGCTCACTGG GGCAAGTGGGTGGAGCCATGAGGAATTCATGCCTTGCAAAGGGGAGGAGCCTGCCC TCTTGA

Claims

1. WHAT IS CLAIMED IS:

1. An immortalized mesenchymal stromal cell (MSC) that heterologously expresses a p53-upregulated-regulator-of-p53-levels (PURPL) IncRNA.

2. The MSC of claim 1, wherein the MSC further expresses a heterologous nucleotide sequence encoding an immortalization polypeptide selected from the group consisting of: MYC, SV40 Large T Antigen, ST AT3, telomerase reverse transcriptase (TERT), E6 HPV, E7 HPV, Bmi-1, or N0TCH2 intracellular domain.

3. The MSC of claim 2, wherein the MSC is a human MSC and TERT is human TERT (hTERT).

4. The MSC of claim 2, wherein MYC is L-MYC, N-MYC, V-MYC, or C-MYC.

5. The MSC of claim 2, comprising one or more expression cassettes comprising a promoter operably linked to a heterologous nucleotide sequence encoding the immortalization polypeptide and / or the PURPL IncRNA.

6. The MSC of claim 2, comprising an expression cassette comprising a promoter operably linked to a heterologous nucleotide sequence encoding a therapeutic payload.

7. The MSC of claim 6, wherein the therapeutic payload is an antibody.

8. The MSC of claim 7, wherein antibody is a bispecific antibody.

9. The MSC of claim 7, wherein the antibody is a bispecific T cell engager (BiTE).

10. The MSC of claim 6, wherein the therapeutic payload is a type VII collagen.

11. The MSC of claim 6, wherein the therapeutic payload is a cytokine.

12. The MSC of claim 1 or claim 2, comprising a CRISPR activation (CRISPRa) system effecting increased expression of the PURPL IncRNA by the MSC.

13. The MSC of claim 1 or claim 2, wherein the MSC comprises a viral vector.

14. The MSC of claim 13, wherein the viral vector is a retroviral vector.

15. The MSC of claim 14, wherein the retroviral vector has independent replicative capacity.

16. The MSC of claim 14, wherein the retroviral vector lacks independent replicative capacity.

17. The MSC of claim 13, wherein the viral vector is a lentiviral vector.

18. The MSC of claim 2, comprising:a first expression cassette comprising a promoter operably linked to a nucleotide sequence encoding MYC;a second expression cassette comprising a promoter operably linked to a nucleotide sequence encoding TERT; anda third expression cassette comprising a promoter operably linked to a nucleotide sequence encoding the PURPL IncRNA.

19. The MSC of claim 2, comprising:a first expression cassette comprising a promoter operably linked to a nucleotide sequence encoding MYC and TERT; anda second expression cassette comprising a promoter operably linked to a nucleotide sequence encoding the PURPL IncRNA.

20. The MSC of claim 18, wherein one, two or all three of the first and second and third expression cassettes are in one of more vectors.

21. The MSC of claim 19, wherein one or both of the first and second expression cassettes are in one or more vectors.

22. The MSC of claim 20 or 21, wherein the vector is a viral vector.

23. The MSC of claim 2, wherein the MYC polypeptide is an L-MYC polypeptide at least 95% identical to SEQ ID NO: 1.

24. The MSC of claim 2, wherein the TERT polypeptide is at least 95% identical to SEQ ID25. The MSC of claim 2, wherein SV40 Large T Antigen is at least 95% identical to SEQ ID NO: 12, wherein STAT3 is at least 95% identical to SEQ ID NO: 13, wherein E6 HPV is at least 95% identical to SEQ ID NO: 14, wherein E7 HPV is at least 95% identical to SEQ ID NO: 15, wherein Bmi-1 is at least 95% identical to SEQ ID NO: 16, and wherein N0TCH2 intracellular domain is at least 95% identical to SEQ ID NO: 17.

26. The MSC of claim 1, wherein the PURPL IncRNA comprises one or more exon at least 95% identical to any one of SEQ ID NO: 3-8.

27. The MSC of claim 1, wherein the PURPL IncRNA is at least 95% identical to any one of SEQ IDNOs: 9-11.

28. The MSC of claim 1, wherein the PURPL IncRNA comprises exon 1, exon 2, exon 3, exon 3.5, exon 4, and exon 5, wherein exon 1 is at least 95% identical to SEQ ID NO: 3, exon 2 is at least 95% identical to SEQ ID NO: 4, exon 3 is at least 95% identical to SEQ ID NO: 5, exon 3.5 is at least 95% identical to SEQ ID NO: 6, exon 4 is at least 95% identical to SEQ ID NO: 7, and exon 5 is at least 95% identical to SEQ ID NO: 8.

29. The MSC of claim 28, wherein exon 1 comprises SEQ ID NO: 3, exon 2 comprises SEQ ID NO: 4, exon 3 comprises SEQ ID NO: 5, exon 3.5 comprises SEQ ID NO: 6, exon 4 comprises SEQ ID NO: 7, and exon 5 comprises SEQ ID NO: 8.

30. A method for making an immortalized mesenchymal stromal cell (MSC) comprising heterologously expressing in a MSC a PURPL IncRNA, thereby immortalizing the MSC.

31. The method of claim 30, further comprising expressing in the MSC a heterologous nucleotide sequence encoding an immortalization polypeptide selected from the group consisting of: MYC, SV40 Large T Antigen, STAT3, telomerase reverse transcriptase (TERT), E6 HPV, E7 HPV, Bmi-1, or NOTCH2 intracellular domain.

32. The method of claim 30 or 31, wherein the MSC is a human MSC and TERT is hTERT.

33. The method of claim 31, wherein MYC is L-MYC, N-MYC, V-MYC, or C-MYC.

34. The method of claim 31, comprising introducing into the MSC one or more expression cassettes comprising a promoter operably linked to a heterologous nucleotide sequence encoding the immortalization polypeptide and / or PURPL IncRNA.

35. The method of claim 31, comprising introducing into the MSC an expression cassette comprising a promoter operably linked to a heterologous nucleotide sequence encoding a therapeutic payload.

36. The method of claim 35, wherein the therapeutic payload is an antibody.

37. The method of claim 36, wherein the antibody is a bispecific antibody.

38. The method of claim 36, wherein the antibody is a bispecific T cell engager (BiTE).

39. The method of claim 35, wherein the therapeutic payload is a type VII collagen.

40. The method of claim 35, wherein the therapeutic payload is a cytokine.

41. The method of claim 30 or claim 31, wherein the MSC comprises a CRISPR activation (CRISPRa) system effecting increased expression of the PURPL IncRNA by the MSC.

42. The method of claim 30 or claim 31, wherein MSC comprises a viral vector.

43. The method of claim 42, wherein the viral vector is a retroviral vector.

44. The method of claim 43, wherein the retroviral vector has independent replicative capacity.

45. The method of claim 43, wherein the retroviral vector lacks independent replicative capacity.

46. The method of claim 42, wherein the viral vector is a lentiviral vector.

47. The method of claim 31, comprising:a first expression cassette comprising a promoter operably linked to a nucleotide sequence encoding MYC;a second expression cassette comprising a promoter operably linked to a nucleotide sequence encoding TERT; anda third expression cassette comprising a promoter operably linked to a nucleotide sequence encoding the PURPL IncRNA.

48. The method of claim 31 , comprising:a first expression cassette comprising a promoter operably linked to a nucleotide sequence encoding MYC and TERT; anda second expression cassette comprising a promoter operably linked to a nucleotide sequence encoding the PURPL IncRNA.

49. The method of claim 47, wherein the expressing comprises providing to the MSC one, two, or all three of the first and second and third expression cassettes in one or more vectors, such that the PURPL, MYC and / or TERT are expressed in the MSC.

50. The method of claim 48, wherein the expressing comprises providing to the MSC one or both of the first and second expression cassettes in one or more vectors, such that the PURPL, MYC, and / or TERT are expressed in the MSC.

51. The method of claim 49 or 50, wherein the vector is a viral vector.

52. The method of claim 31, wherein the MYC polypeptide is at least 95% identical to SEQ ID NO: 1.

53. The method of claim 31, wherein the TERT polypeptide is at least 95% identical to SEQ ID NO:

254. The method of claim 31, wherein SV40 Large T Antigen is at least 95% identical to SEQ ID NO: 12, wherein STAT3 is at least 95% identical to SEQ ID NO: 13, wherein E6 HPV is at least 95% identical to SEQ ID NO: 14, wherein E7 HPV is at least 95% identical to SEQ ID NO: 15, wherein Bmi-1 is at least 95% identical to SEQ ID NO: 16, and wherein NOTCH2 intracellular domain is at least 95% identical to SEQ ID NO: 17.

55. The method of claim 30, wherein the PURPL IncRNA comprises one or more exon at least 95% identical to any one of SEQ ID NO: 3-8.

56. The method of claim 30, wherein the PURPL IncRNA is at least 95% identical to any one of SEQ IDNOs: 9-11.

57. The method of claim 30, wherein the PURPL IncRNA comprises exon 1, exon 2, exon 3, exon 3.5, exon 4, and exon 5, wherein exon 1 is at least 95% identical to SEQ ID NO: 3, exon 2 is at least 95% identical to SEQ ID NO: 4, exon 3 is at least 95% identical to SEQ ID NO: 5, exon 3.5 is at least 95% identical to SEQ ID NO: 6, exon 4 is at least 95% identical to SEQ ID NO: 7, and exon 5 is at least 95% identical to SEQ ID NO: 8.

58. The method of claim 30, wherein exon 1 comprises SEQ ID NO: 3, exon 2 comprises SEQ ID NO: 4, exon 3 comprises SEQ ID NO: 5, exon 3.5 comprises SEQ ID NO: 6, exon 4 comprises SEQ ID NO: 7, and exon 5 comprises SEQ ID NO: 8.

59. A method of treating a disease in a subject in need thereof comprising administering to the subject the immortalized MSC of claim 1 or the immortalized MSC made by the method of claim 30.

60. The method of claim 59, wherein the immortalized MSC of claim 1 or the immortalized MSC made by the method of claim 30 delivers a therapeutic payload to the subject.

61. The method of claim 60, wherein the therapeutic payload is a viral vector.

62. The method of claim 61, wherein the viral vector is a retroviral vector.

63. The method of claim 62, wherein the retroviral vector has independent replicative capacity.

64. The method of claim 62, wherein the retroviral vector lacks independent replicative capacity.

65. The method of claim 61, wherein the viral vector is a lentiviral vector.

66. The method of claim 60, wherein the therapeutic payload is an antibody.

67. The method of claim 66, wherein the antibody is a bispecific antibody.

68. The method of claim 66, wherein the antibody is a bispecific T cell engager (BiTE).

69. The method of claim 60, wherein the therapeutic payload is a type VTI collagen.

70. The method of claim 60, wherein the therapeutic payload is a cytokine.

71. The method of claim 59, wherein the disease is cancer.

72. The method of claim 71, wherein the cancer is lung cancer, breast cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, kidney cancer, urinary tract cancer, oral cancer, head and neck cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, skin cancer, melanoma, sarcoma, lymphoma, leukemia, or brain cancer including glioblastoma, anaplastic astrocytoma, oligodendroglioma, and medulloblastoma.

73. The method of claim 5969, wherein the disease is a skin disorder.

74. The method of claim 5969, wherein the disease is an autoimmune disease.

75. The method of claim 59, wherein the disease is a neurodegenerative disease.

76. The method of claim 59, wherein the disease is a monogenic hereditary disorder.

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