Temperature-based transient delivery of ZSCAN4 nucleic acids and proteins into cells and tissues

Abstract

The present disclosure relates to methods of transiently activating a temperature-sensitive agent in one or more cells, for example, by contacting one or more cells with a temperature-sensitive agent and transiently incubating the cells at a permissive temperature to induce activity of the temperature-sensitive agent in the cells. [0003] Additionally, the present disclosure relates to methods for contacting one or more cells of a subject with a temperature-sensitive agent and then lowering the subject's body temperature to a permissive temperature to induce activity of the temperature-sensitive agent in the cells. The present disclosure also relates to methods for treating a subject with a temperature-sensitive therapeutic agent. In particular, the present disclosure provides tools for temperature-sensitive delivery of ZSCAN4 nucleic acids and proteins to cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 992,745, filed March 20, 2020, and U.S. Provisional Patent Application No. 62 / 955,820, filed December 31, 2019, the entire disclosures of which are incorporated herein by reference. Submission of sequence listing as an ASCII text file

[0002] The entire contents of the following submission as an ASCII text file are incorporated herein by reference: Sequence Listing in Computer Readable Format (CRF) (Filename: 699442001340SEQLIST.TXT, Recorded: December 23, 2020, Size: 25KB). Field

[0003] The present disclosure relates to methods of transiently activating a temperature-sensitive agent (ts agent) in one or more cells, for example, by contacting one or more cells with the ts agent and transiently incubating the cells at a permissive temperature that induces activity of the ts agent in the cells. For ex vivo therapeutic strategies, one or more cells are treated with a therapeutic ts agent ex vivo at a permissive temperature, and the cells are subsequently transplanted into a subject at a non-permissive temperature (e.g., the subject's normal core body temperature). For in vivo therapeutic strategies, a therapeutic ts agent is delivered to a subject, i.e., maintained at a permissive temperature, and when the subject's core body temperature returns to normal or the subject's surface temperature increases (e.g., to a non-permissive temperature), the therapeutic ts agent is allowed to function in vivo for a limited period of time before the ts agent permanently ceases to function. Alternatively, a therapeutic ts agent is delivered to a subject, and subsequently the ts agent is transiently activated by lowering the subject's core body temperature to a permissive temperature that induces activity of the therapeutic ts agent in the subject's cells. In particular, the present disclosure provides tools for temperature-sensitive delivery of ZSCAN4 nucleic acids and proteins to cells. [Background technology]

[0004] background The delivery of therapeutic gene products to human cells, tissues, and organs presents a significant challenge. For traditional gene therapy (which requires continuous expression of a gene to compensate for a patient's genetic defect), this has been achieved by using viral vectors such as retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses. However, equally important gene therapy strategies involve transient, short-term gene expression. For such applications, sustained expression of the gene is not necessary and may actually be harmful to the cell.

[0005] For example, CAS9 is a bacterial enzyme that cleaves DNA. It is a key component of CRISPR / CAS9-based gene editing complexes and has been investigated for gene therapy. Both guide RNA and CAS9 can be encoded by genes on a single Sendai virus vector (Park et al., 2016). To use a gene editing system therapeutically, a vector containing CRISPR-CAS9 must be introduced into human cells or the human body. However, continuous expression of CAS9 can induce DNA breaks and the introduction of mutations. Therefore, it is desirable to express CAS9 for a short period of time, e.g., on the order of a few hours or days, rather than for more than a week.

[0006] Another application of short-term gene expression is for cell reprogramming. Recently, ectopic expression of a set of transcription factors has been shown to convert cells into therapeutically effective cell types. For example, a set of three transcription factors can convert pancreatic ductal cells into insulin-secreting pancreatic β cells (Zhou et al., 2008). Another set of transcription factors can convert fibroblasts into cardiomyocytes (Ieda et al., 2010). In vivo delivery of these transcription factors into the human body could be used as a type of regenerative medicine. However, because continuous expression of these powerful transcription factors can cause harm, it is desirable to express these powerful cell identity-altering transcription factors only transiently.

[0007] Given the above-mentioned examples, traditional gene therapy using viral vectors to achieve continuous gene expression may become undesirable. For time-limited expression of gene products, delivery of synthetic or in vitro transcribed mRNA into cells has begun to be used (Warren et al., 2010). However, there are several problems with these methodologies. For example, the amount of mRNA delivered to cells, tissues, and organs is limited, which may result in an insufficient amount of protein product for biologically significant effects in vivo.

[0008] Furthermore, due to the rapid turnover of RNA, which typically lasts for only a maximum of 12 hours (Warren et al., 2010; Goparaju et al., 2017), synthetic RNA must be transfected into cells multiple times. For forced differentiation of human pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, transfection is required twice daily for several days (Akiyama et al., 2016; Goparaju et al. 2017). To generate iPS cells from human fibroblasts, daily transfection with synthetic RNA cocktails must be continued for more than two weeks (Warren et al., 2010). This is not only tedious but also inefficient.

[0009] For the generation of iPS cells, this problem has been addressed by using self-replicating RNA (which allows for long-term expression even after only one delivery) (Yoshioka et al., 2013). Self-replicating RNA is a single-stranded RNA typically produced by alphaviruses such as Venezuelan equine encephalitis virus (VEEV), Sindbis virus (SINV), and Semliki Forest virus (SFV) (Jose et al., 2009) by removing DNA encoding structural proteins required for viral particle formation (Petrakova et al., 2005). Self-replicating RNA encodes nonstructural proteins (nsPs), and it functions as an RNA-dependent RNA polymerase to replicate itself and produce transcripts for translation. Self-replicating RNAs can also contain a gene of interest (GOI) encoding a protein of interest and other genetic elements. Due to their positive feedback production of RNA, self-replicating RNAs can express the GOI at high levels. Self-replicating RNA can be delivered to mammalian cells as naked RNA (i.e., synthetic RNA) or as viral particles, which can be produced by packaging helper cells to complement viral structural proteins.

[0010] The advantage of self-replicating RNA vectors is their self-replicating feature, which leads to enhanced expression levels of the GOI. However, one of the drawbacks of self-replicating RNA vectors for delivering RNA / proteins to mammalian cells is their sustained expression. Usually, a positive feedback loop between RNA-dependent RNA polymerase and the GOI ensues, which can lead to the death of cells transfected with naked RNA forms of the self-replicating RNA or infected with viral forms of the self-replicating RNA.

[0011] Thus, what is needed in the art of gene therapy are tools for the transient expression of GOIs encoding proteins of interest, such as therapeutic agents (e.g., human ZSCAN4). In particular, controlled transcription and translation of RNA vectors and self-replicating RNAs are desirable. Summary of the Invention

[0012] overview Based on the need to have time-limited expression of a gene of interest (GOI), there is a need for a transient gene product delivery system in which a nucleic acid or protein can be delivered to or expressed in specific cells in vitro or in vivo, the amount of nucleic acid / protein is sufficient to have a biologically significant effect, and the transient expression can be permanently terminated after achieving the biologically significant effect. To meet these and other needs, the present disclosure relates to tools that transiently induce the activity of a temperature-sensitive agent (ts agent), such as a therapeutic ts agent, in a subject (in vivo) or cells in culture (ex vivo). In some embodiments, the therapeutic ts agent is used in combination with mild therapeutic hypothermia. In other embodiments, the therapeutic ts agent is used in combination with mild therapeutic hyperthermia or local application of heat. In some embodiments, the ts agent is a ts-RNA molecule or a ts-protein molecule. In some embodiments, the ts agent is encoded by a heterologous nucleic acid or a self-replicating RNA inserted into a temperature-sensitive viral vector. In some embodiments, the viral vector is selected from, but is not limited to, a Sendai virus vector, a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, and an alphavirus vector. In some embodiments, the self-replicating RNA comprises an alphavirus replicon lacking viral structural protein coding regions. In some embodiments, the alphavirus is selected from, but is not limited to, Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. A particular gene product of interest is ZSCAN4, particularly human ZSCAN4.

[0013] The above and other objects and features of the present disclosure will become more apparent from the following detailed description taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0014] [Figures 1A-1D]Figures 1A-1D show the structure of the Venezuelan equine encephalitis virus (VEEV) genome and the location of the mutated regions. Figure 1A shows a schematic diagram of the wild-type VEEV genome (TC-83 strain: complete genome of 11,446 bp linear RNA; NCBI accession: L01443.1 GI:323714). The genes for nonstructural proteins (nsP1, nsP2, nsP3, and nsP4) encode the RNA-dependent RNA polymerase, and the genes for structural proteins encode the viral envelope proteins (C, E1, and E2), 5'-UTR (5'-untranslated region), and 3'-UTR (3'-untranslated region). The gene for the nsP2 protein, represented as a bold box, was mutated to confer temperature sensitivity. Figure 1B shows a schematic diagram of nsP2 with mutation 1 (temperature-sensitive mutant 1: ts1). Five amino acids were inserted between amino acids 439 and 440. Figure 1C shows a schematic diagram of nsP2 with mutation 2 (ts2). Five amino acids were inserted between amino acids 586 and 587. Figure 1D shows a schematic diagram of nsP2 with mutation 3 (ts3). Five amino acids were inserted between amino acids 594 and 595.

[0015] [Figures 2A-2C] Figures 2A-2C show partial sequences of VEEV nsP2 corresponding to the regions mutated to ts1, ts2, and ts3. Figure 2A shows the wild-type sequence compared to mutant 1 (ts1), which contains a 15-nucleotide insertion resulting in a five-amino acid insertion. Figure 2B shows the wild-type sequence compared to mutant 2 (ts2), which contains a 15-nucleotide insertion resulting in a five-amino acid insertion. Figure 2C shows the wild-type sequence compared to mutant 3 (ts3), which contains a 15-nucleotide insertion resulting in a five-amino acid insertion.

[0016] [Figure 3] Figure 3 shows the partial nucleotide sequences of wild-type VEEV nsP1 (strain TC-83) and mutant 4 (ts4), set forth as SEQ ID NO:19 and SEQ ID NO:20, respectively. The 5'-UTR and 51-nt CSE (conserved sequence element) are shown in bold. The mutated nucleotides in ts4 are underlined.

[0017] [Figure 4A-4B] Figures 4A and 4B show the temperature sensitivity of srRNA1ts2 and srRNA1ts3 at 30°C, 32°C, and 37°C. Wild-type (srRNA1wt-GFP) and mutant (srRNA1ts2-GFP, srRNA1ts3-GFP) self-replicating RNA (srRNA) vectors were generated. RNAs produced by in vitro transcription were transfected into human induced pluripotent stem cells (ADSC-iPSC lines). Cells were cultured in a CO2 incubator maintained at 30°C, 32°C, and 37°C, respectively. Images of the cells were taken at 20 and 48 hours, respectively. The upper panel shows phase-contrast images, and the lower panel shows fluorescent images detecting green fluorescent protein (GFP) expression. Figure 4A shows the results of cell transfection with srRNA1wt-GFP, srRNA1ts2-GFP, and srRNA1ts3-GFP RNA. FIG. 4B shows the results of transfection of cells with synthetic mRNA encoding GFP (synRNA-GFP).

[0018] [Figure 5] Figure 5 shows the temperature sensitivity of srRNA1ts1 and srRNA1ts2 at 32°C. Wild-type (srRNA1wt-GFP) and mutant (srRNA1ts2-GFP and srRNA1ts1-GFP) self-replicating RNA (srRNA) vectors were generated. RNAs produced by in vitro transcription were transfected into human induced pluripotent stem cells (ADSC-iPSC lines). Cells were cultured in a CO2 incubator maintained at 32°C. Images of the cells were obtained at 24, 48, 72, 96, 120, 144, 168, 192, 240, and 288 hours, respectively. For srRNA1ts1-GFP, only images were taken at 24 and 168 hours. The upper panel shows phase-contrast images, and the lower panel shows fluorescent images to detect GFP expression.

[0019] [Figure 6]Figure 6 shows the temperature sensitivity of srRNA1ts2 and srRNA1ts4 at 32°C, 33°C, and 37°C. Mutant (srRNA1ts2-GFP and srRNA1ts4-GFP) self-replicating RNA (srRNA) vectors were generated. RNAs produced by in vitro transcription were transfected into human induced pluripotent stem cells (ADSC-iPSC lines). Cells were cultured in a CO2 incubator maintained at 32°C, 33°C, and 37°C, respectively. Images of the cells were obtained at 20, 48, and 96 hours, respectively. The upper panel shows phase-contrast images, and the lower panel shows fluorescent images detecting green fluorescent protein (GFP) expression.

[0020] [Figure 7] Figure 7 shows the temperature sensitivity of mutant srRNA1ts2-GFP at 32°C. RNA produced by in vitro transcription of the mutant vector (srRNA1ts2-GFP) was transfected into human induced pluripotent stem cells (ADSC-iPSC line). Cells were cultured in a CO2 incubator maintained at 32°C. The srRNA1ts2-GFP vector contains a puromycin N-acetyltransferase (pac) selection gene inserted immediately after the "IRES" sequence, allowing transfected cells to be selected using puromycin. Experiments were performed in the absence (upper panel) or presence (lower panel) of 1 μg / ml puromycin. Images of cells were taken at 24, 48, 72, 96, 144, 168, and 192 h, respectively. For srRNA1ts1-GFP, only images were taken at 24 and 168 h. The upper panel shows a phase contrast image, and the lower panel shows a fluorescent image detecting the expression of GFP.

[0021] [Figure 8]Figure 8 shows the temperature sensitivity of mutant srRNA1ts2-GFP, tested using a temperature shift from 32°C to 37°C over 24 hours. RNA produced by in vitro transcription of the mutant vector (srRNA1ts2-GFP) was transfected into human induced pluripotent stem cells (ADSC-iPSC line). Cells were cultured in a CO2 incubator maintained at 32°C. At 24 hours, cells were transferred to a CO2 incubator maintained at 37°C. The srRNA1ts2-GFP vector contains a puromycin N-acetyltransferase (pac) selection gene inserted immediately after the "IRES" sequence, allowing transfected cells to be selected using puromycin. Experiments were performed in the absence (upper panel) or presence (lower panel) of 1 μg / ml puromycin. Images of cells were taken at 24, 48, 72, 96, 144, 168, and 192 hours, respectively. For srRNA1ts1-GFP, only images were taken at 24 and 168 hours. The upper panel shows a phase-contrast image, and the lower panel shows a fluorescent image detecting GFP expression.

[0022] [Figure 9]Figure 9 shows the temperature sensitivity of mutant srRNA1ts2-GFP, tested using a temperature switch from 32°C to 37°C over 48 hours. RNA produced by in vitro transcription of the mutant vector (srRNA1ts2-GFP) was transfected into human induced pluripotent stem cells (ADSC-iPSC line). Cells were cultured in a CO2 incubator maintained at 32°C. At 48 hours, cells were transferred to a CO2 incubator maintained at 37°C. The srRNA1ts2-GFP vector contains a puromycin N-acetyltransferase (pac) selection gene inserted immediately after the "IRES" sequence, allowing transfected cells to be selected using puromycin. Experiments were performed in the absence (upper panel) or presence (lower panel) of 1 μg / ml puromycin. Images of cells were taken at 24, 48, 72, 96, 144, 168, and 192 hours, respectively. For srRNA1ts1-GFP, only images were taken at 24 and 168 hours. The upper panel shows a phase-contrast image, and the lower panel shows a fluorescent image detecting GFP expression.

[0023] [Figure 10]Figure 10 shows the temperature sensitivity of mutant srRNA1ts2-GFP tested using a temperature switch from 32°C to 37°C over 72 hours. RNA produced by in vitro transcription of the mutant vector (srRNA1ts2-GFP) was transfected into human induced pluripotent stem cells (ADSC-iPSC line). Cells were cultured in a CO2 incubator maintained at 32°C. At 72 hours, cells were transferred to a CO2 incubator maintained at 37°C. The srRNA1ts2-GFP vector contains a puromycin N-acetyltransferase (pac) selection gene inserted immediately after the "IRES" sequence, allowing selection using puromycin. Experiments were performed in the absence (upper panel) or presence (lower panel) of 1 μg / ml puromycin. Images of cells were taken at 24, 48, 72, 96, 144, 168, and 192 hours, respectively. For srRNA1ts1-GFP, only images were taken at 24 and 168 hours. The upper panel shows a phase-contrast image, and the lower panel shows a fluorescent image detecting GFP expression.

[0024] [Figures 11A-11D] Figures 11A-11D show the temperature sensitivity of mutant srRNA1ts2-GFP in fibroblasts. RNA produced by in vitro transcription of the mutant vector (srRNA1ts2-GFP) was transfected into human neonatal dermal fibroblasts (HDFn strain). Cells were cultured in a CO2 incubator maintained at 32°C. Images of the cells were taken at 24, 48, and 96 hours. The upper panel shows a phase-contrast image, and the lower panel shows a fluorescent image detecting GFP expression. Figures 11A and 11B show transfections performed using JetMessenger (Polyplus). Cells were cultured in standard medium alone (Figure 11A) or standard medium supplemented with 200 ng / ml B18R (Figure 11B). Figures 11C and 11D show transfections performed using MessengerMax (ThermoFisher). Cells were cultured in standard medium alone (Fig. 11C) or standard medium supplemented with 200 ng / ml B18R (Fig. 11D).

[0025] [Figure 12] Figure 12 shows an alignment of amino acid sequences corresponding to nsP2 variant 2 (ts2) of various alphavirus family members. The left panel shows an alignment of wild-type sequences set forth as SEQ ID NOS: 21-28 (partially reproduced from Figure 1 in Russo et al., 2006), while the right panel shows an alignment of variants set forth as SEQ ID NOS: 29-36, which contain a five amino acid insertion between "β5" and "β6" (the fifth and sixth β-strands) of the nsP2 secondary structure. VEEV (Venezuelan equine encephalitis virus), Aura (Aura virus), WEEV (Western equine encephalitis virus), BFV (Barmah Forest virus), ONNV (Onyong-Nyong virus), RRV (Ross River virus), SFV (Semliki Forest virus), and SINV (Sindbis virus).

[0026] [Figure 13] Figure 13 illustrates a schematic diagram showing typical ex vivo treatment of cells with a temperature-sensitive agent (ts agent). ts agents, such as srRNAs or Sendai virus vectors, are functional at permissive temperatures (e.g., 33°C) but not at nonpermissive temperatures (e.g., 37°C). Target cells treated with the ts agent are cultured at the permissive temperature for a specific duration (e.g., 3 days) and then cultured at the nonpermissive temperature for a specific duration (e.g., 10 days). The expected level of RNA (or protein translated from RNA) of the gene of interest (GOI) increases at the permissive temperature and reaches a high level. After switching to the nonpermissive temperature, the expected level of RNA (or protein) gradually decreases as transcription and translation cease.

[0027] [Figure 14]Figure 14 illustrates a schematic diagram showing an exemplary ex vivo therapeutic approach. Temperature-sensitive agents (ts agents), such as srRNAs or Sendai virus vectors, are functional at permissive temperatures (e.g., 33°C) but not at non-permissive temperatures (e.g., 37°C). Target cells are harvested from a patient's body (natural transplant) and incubated with the ts agent ex vivo at a permissive temperature, e.g., 33°C, for a specific duration, e.g., 24 hours. The target cells with the ts agent are then transplanted into the patient. At the non-permissive temperature of 37°C, the ts agent is not functional in the patient's body.

[0028] [Figure 15] Figure 15 illustrates a schematic diagram showing another exemplary ex vivo therapeutic approach. Temperature-sensitive agents (ts agents), such as srRNAs or Sendai virus vectors, are functional at permissive temperatures (e.g., 33°C) but not at non-permissive temperatures (e.g., 37°C). Target cells are harvested from a donor's body (allograft) and incubated with the ts agent ex vivo at a permissive temperature, e.g., 33°C, for a specific duration, e.g., 24 hours. The target cells with the ts agent are then transplanted into a patient. At the non-permissive temperature of 37°C, the ts agent is not functional in the patient's body.

[0029] [Figure 16]Figure 16 illustrates a schematic diagram showing an exemplary semi-in vivo therapeutic approach. Temperature-sensitive agents (ts agents), such as srRNAs or Sendai virus vectors, are functional at permissive temperatures (e.g., 33°C) but not at non-permissive temperatures (e.g., 37°C). A patient undergoes therapeutic hypothermia, and the patient's core body temperature is maintained at a low temperature (e.g., 33°C) (lower than normothermia (e.g., 37°C)). Target cells (either autologous or allogeneic) are treated with the ts agent ex vivo and immediately infused into the patient's circulation or injected into the patient's organs. While the patient is maintained at a low temperature (e.g., 33°C) for a period of time (e.g., 24 hours), the ts agent remains functional. Subsequently, the patient's core body temperature returns to normothermia (37°C), at which point the ts agent no longer functions.

[0030] [Figure 17] FIG. 17 illustrates a schematic diagram showing an exemplary semi-in vivo therapeutic approach. Temperature-sensitive agents (ts agents), such as srRNAs or Sendai virus vectors, are functional at permissive temperatures (e.g., 33°C) but not at non-permissive temperatures (e.g., 37°C). A patient undergoes therapeutic hypothermia, and the patient's core body temperature is maintained at a low temperature (e.g., 33°C) (below normothermia (e.g., 37°C)). The ts agent is delivered systemically or to a specific organ, tissue, or cell type. While the patient is maintained at the low temperature (e.g., 33°C) for a period of time (e.g., 24 hours), the ts agent is functional. Subsequently, the patient's core body temperature returns to normothermia (37°C), at which point the ts agent no longer functions.

[0031] [Figure 18]Figure 18 shows a schematic diagram of an exemplary temperature-sensitive Sendai virus vector containing the coding region (open reading frame) of human ZSCAN4 (SeV18+hZscan4 / TS15ΔF; also known as "SeVts-ZSCAN4"). The vector backbone (described as TS15 (Ban et al., 2011)) lacks the F (fusion) gene, which is necessary for reproduction of infectious progeny virus. Thus, this vector does not transmit virus from infected to uninfected cells. This vector encodes two RNA polymerase genes (P and L) and three structural protein genes (NP, M, and HN) and contains point mutations in the M, HN, P, and L genes, which render the vector temperature-sensitive: permissive at 33°C; nonpermissive above 37°C. To construct SeVts-ZSCAN4, the coding region of the human ZSCAN4 gene was inserted upstream of the NP gene in the TS15 vector backbone, a position that provides the highest expression of any gene in the Sendai virus genome.

[0032] [Figure 19] FIG. 19 shows that the majority of human CD34+ cells contacted with SeVts-ZSCAN4 and incubated at a permissive temperature (eg, 33° C.) for 16 or 24 hours express ZSCAN4 protein.

[0033] [Figures 20A-20B] Figures 20A and 20B show that human CD34+ cells contacted with SeVts-ZSCAN4 and incubated at a permissive temperature (e.g., 33°C) express ZSCAN4 protein, but begin to lose ZSCAN4 protein expression when subsequently incubated at a non-permissive temperature (e.g., 37°C).

[0034] [Figure 21] FIG. 21 shows that telomeres of human CD34+ cells contacted with SeVts-ZSCAN4 and incubated at permissive temperatures (eg, 33° C.) for a minimum of 24 hours are elongated.

[0035] [Figure 22] Figure 22 illustrates a schematic diagram showing the ex vivo procedure of Example 14 in which human CD34+ cells are contacted with a ZSCAN4 therapeutic agent at a permissive temperature (e.g., 33°C) and subsequently injected into immunodeficient mice with a non-permissive normothermia (e.g., 37°C) to assess the safety and efficacy of CD34+ cell transplantation.

[0036] [Figure 23] FIG. 23 shows that SeVts-ZSCAN4 treatment for 24 hours at permissive temperatures (eg, 33° C.) was effective in elongating telomeres of human CD34+ cells in vitro.

[0037] [Figure 24] Figure 24 shows that the telomeres of human cells transplanted into immunodeficient mice were longer when human CD34+ cells injected into the mice in vitro were first contacted with SeVts-ZSCAN4 and then incubated at a permissive temperature (e.g., 33°C). Mice 492 and 493 received SeVts-ZSCAN4-contacted CD34+ cells, while mouse 496 received CD34+ cells that had not been contacted with SeVts-ZSCAN4.

[0038] [Figure 25] Figure 25 illustrates a schematic diagram showing the ex vivo treatment procedure of Example 15, in which autologous CD34+ cells are contacted with a ZSCAN4 therapeutic ts agent at a permissive temperature (e.g., 33°C) and subsequently infused into a patient with a non-permissive normothermia (e.g., 37°C).

[0039] [Figure 26] FIG. 26 shows a flow chart of the clinical diagnostic method of Example 15 for evaluation of autologous CD34+ cells contacted with SeVts-ZSCAN4 in human patients suffering from telomere biology disorders and bone marrow failure.

[0040] [Figure 27] FIG. 27 illustrates another schematic diagram showing the ex vivo treatment protocol of Example 15 for evaluation of autologous CD34+ cells contacted with SeVts-ZSCAN4 in human patients suffering from telomere biology disorders and bone marrow failure.

[0041] [Figure 28] Figure 28 illustrates a schematic diagram showing an exemplary in vivo therapeutic approach. Temperature-sensitive agents (ts agents), such as srRNAs or Sendai virus vectors, are functional at permissive temperatures (e.g., 31-34°C) but non-functional at non-permissive temperatures (e.g., >37°C). The temperature at or just below the patient's body surface (surface temperature) (which is approximately 31-34°C) is lower than the patient's core body temperature (which is approximately 37°C). ts agents that are functional at the patient's surface temperature are delivered directly to the patient by intradermal, subcutaneous, or intramuscular administration. No additional activity is required. Alternatively, when the function of the ts agent is no longer needed, the ts agent can be transiently rendered non-functional by elevating the patient's surface temperature.

[0042] [Figure 29] Figure 29 illustrates a schematic diagram showing an exemplary in vivo therapeutic approach. Temperature-sensitive agents (ts agents), such as srRNAs or Sendai virus vectors, are functional at permissive temperatures (e.g., 31-35°C) but are non-functional at non-permissive temperatures (e.g., >37°C). The temperature of a patient's body's airways (airway temperature) (approximately 32°C for the nasal passages and upper trachea and 35°C for the subsegmental bronchi (McFadden et al., 1985)) is lower than the patient's core body temperature (approximately 37°C). ts agents that are functional at a patient's airway temperature are delivered directly to the patient by intranasal administration (e.g., insufflation, inhalation, or infusion). The absence of additional activity is necessary. When the function of the ts agent is no longer required, the ts agent is rendered transiently inoperable by elevating the patient's airway temperature. DETAILED DESCRIPTION OF THE INVENTION

[0043] Detailed Description Review Applicant has demonstrated that cells can be cultured at a permissive temperature to induce the activity of a temperature-sensitive therapeutic agent, and that this activity can lead to intracellular therapeutic effects. Furthermore, the activity of a temperature-sensitive therapeutic agent can be subsequently reduced or inhibited by incubating the cells at a non-permissive temperature. Applicant has also provided, for the first time, methods for using temperature-sensitive agents (ts agents) in vivo. The same types of ts agents used in vivo can also be used in vitro. For example, after administration of a ts agent to the trunk of a subject, the subject's core body temperature can be lowered to a permissive temperature to induce the activity of the ts agent. Alternatively, after administration of a ts agent to the surface of a subject (epidermis, dermis, subcutaneous tissue, or skeletal muscle), the subject's surface body temperature can be maintained at a permissive temperature to induce the activity of the ts agent. The subject's surface body temperature can be maintained naturally or artificially. These methods provide new ways to deliver and transiently activate therapeutic agents, such as nucleic acids and polypeptides. In particular, the present disclosure provides tools for temperature-sensitive delivery of ZSCAN4 nucleic acids and proteins into cells.

[0044] Accordingly, the present disclosure generally relates to methods for transiently inducing the activity of a temperature-sensitive agent (e.g., a temperature-sensitive therapeutic agent) in vitro. In some embodiments, one or more cells containing a temperature-sensitive therapeutic agent are cultured at a permissive temperature to induce activity of the temperature-sensitive therapeutic agent. The cells are cultured at the permissive temperature for a period of time sufficient for the temperature-sensitive therapeutic agent to induce a therapeutic effect in the cells. The cells are then returned to a non-permissive temperature, where the non-permissive temperature reduces or inhibits the activity of the temperature-sensitive therapeutic agent. In another embodiment, the one or more cells do not previously contain a temperature-sensitive therapeutic agent and are contacted with the temperature-sensitive therapeutic agent for the first time. In some embodiments, after inducing a therapeutic effect in one or more cells, the cells are administered to a subject in need thereof. In some embodiments, one or more cells are isolated from a subject in need of treatment and, after treatment with a temperature-sensitive therapeutic agent, the cells are returned to the subject.

[0045] In another aspect, the present disclosure relates to methods for transiently inducing the activity of a temperature-sensitive agent (e.g., a temperature-sensitive therapeutic agent) in vivo. In some embodiments, one or more cells of a subject contain a temperature-sensitive therapeutic agent, the subject's body temperature is lowered to a permissive temperature for a period of time sufficient for the temperature-sensitive therapeutic agent to induce a therapeutic effect in the cells, and then the subject's body temperature is returned to normothermia. In another embodiment, the temperature-sensitive therapeutic agent is administered to the subject either before or after the subject's body temperature has been lowered to a permissive temperature.

[0046] Another aspect of the present disclosure relates to treating a disease or condition by mobilizing CD34+ cells from the bone marrow of a subject suffering from (or in need of) the disease or condition, comprising isolating the mobilized cells from the subject, incubating the isolated cells at a temperature of about 33°C ± 0.5°C, contacting the cells with a temperature-sensitive viral vector, such as a Sendai virus vector, or a temperature-sensitive self-replicating RNA (srRNA), wherein the viral vector or srRNA comprises a heterologous nucleic acid molecule encoding a protein of interest, maintaining the contacted cells at about 33°C ± 0.5°C for a sufficient period of time, wherein the viral vector or srRNA is capable of replicating at 33°C ± 0.5°C, and wherein replication of the viral vector or srRNA leads to increased expression of the heterologous nucleic acid molecule, and injecting the contacted cells into the subject, thereby engrafting the contacted cells and treating the disease or condition. Alternatively, after isolating mobilized cells from a subject, the isolated cells are contacted with a temperature-sensitive viral vector, such as a Sendai virus vector, or a temperature-sensitive srRNA, and then the cells are incubated at a temperature of about 33° C.±0.5° C. In some embodiments, the disease or condition is a telomere biology disorder and the protein of interest is ZSCAN4, such as human ZSCAN4.

[0047] In another aspect, the present disclosure relates to treating a disease or condition by administering a temperature-sensitive viral vector, such as a Sendai virus vector, or a temperature-sensitive self-replicating RNA (srRNA) to a subject suffering from the disease or condition (in need thereof), wherein the viral vector or srRNA contains a heterologous nucleic acid encoding a protein of interest; lowering the subject's core body temperature to about 33°C ± 0.5°C; maintaining the subject's core body temperature at about 33°C ± 0.5°C for a sufficient period of time, wherein the viral vector or srRNA is capable of replicating at 33°C ± 0.5°C, and replication of the viral vector or srRNA leads to increased expression of the heterologous nucleic acid; and returning the subject's core body temperature to normal. Alternatively, lowering the subject's core body temperature to about 33°C ± 0.5°C is performed before administering the temperature-sensitive viral vector, such as a Sendai virus vector, or the temperature-sensitive srRNA. In some embodiments, the disease or condition is a telomere biology disorder, and the protein of interest is ZSCAN4, such as human ZSCAN4.

[0048] References and claims to methods of treating a disease or condition by administering to a subject a ts agent, or cells containing a ts agent, in their general and specific forms, may also include the following: a) use of a ts agent or a cell containing a ts agent for the manufacture of an agent for the treatment of a disease or condition; and b) a pharmaceutical composition comprising a ts agent or cells comprising a ts agent for the treatment of a disease or condition; Regarding.

[0049] In some embodiments of the procedural and method section, the heterologous nucleic acid comprises a gene of interest (GOI) or otherwise encodes a protein of interest. In other methods, the heterologous nucleic acid comprises a coding region for a protein of interest. In preferred embodiments, the protein of interest is ZSCAN4, such as human ZSCAN4 or a variant thereof. definition

[0050] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless otherwise indicated. For example, "a polynucleotide" includes one or more polynucleotides.

[0051] As used herein, the phrase "comprising" is open-ended and indicates that such embodiment may include additional elements. In contrast, the phrase "consisting of" is closed and indicates that such embodiment does not include additional elements (except for trace impurities). The phrase "consisting essentially of" is part-closed and indicates that such embodiment may include additional elements that do not materially alter the basic characteristics of such embodiment. Aspects and embodiments described herein as "comprising" are understood to include "consisting of" and "consisting essentially of" embodiments.

[0052] As used herein, with the exception of temperature, the term "about" in reference to a value includes 90% to 110% of that value unless otherwise indicated (e.g., about 30 minutes refers to 27 minutes to 33 minutes). When used with respect to temperature in degrees Celsius, about includes -1°C to +1°C of that value unless otherwise indicated (e.g., about 37°C refers to 36°C to 38°C). In contrast, the use of plus and minus without other indications delineates the indicated range (e.g., 33°C ± 0.5°C refers to 32.5°C to 33.5°C).

[0053] As used herein, numerical ranges are inclusive of the numbers defining the range (eg, 12 to 18 nucleotides includes 12, 13, 14, 15, 16, 17, and 18 nucleotides).

[0054] The terms "isolated" and "purified," as used herein, refer to objects (e.g., cells) that have been removed (e.g., separated) from their environment (e.g., cell culture, biological sample, etc.). "Isolated" objects are at least 50% free, preferably 75% free, more preferably at least 90% free, and most preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) free from other components with which they are associated.

[0055] The terms "individual" and "subject" refer to mammals, including, but not limited to, humans, non-human primates (e.g., monkeys), farm animals, sport animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats).

[0056] The term "dose" as used herein with respect to a pharmaceutical composition refers to the metered portion of the composition taken by (administered to or given to) a subject at any one time.

[0057] The term "treating" a disease or condition refers to the implementation of a protocol that may include administering one or more pharmaceutical compositions to an individual (human or other animal) in an attempt to alleviate the signs or symptoms of the disease. Thus, "treating" or "treatment" specifically includes protocols that have only a palliative effect on an individual, without requiring complete relief of signs or symptoms, and without requiring a cure. As used herein, and as well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, relief or amelioration of one or more symptoms, reduction in the extent of disease, a stabilized (i.e., non-progressing) state of disease, prevention of disease spread, delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or complete). Temperature sensitive agents

[0058] Certain aspects of the present disclosure relate to transiently inducing the activity of a temperature-sensitive agent (e.g., a temperature-sensitive therapeutic agent) in one or more cells. Temperature-sensitive agent activity refers to any desired activation, replication, or increased expression of the agent. As used herein, the term "temperature-sensitive agent" refers to any nucleic acid or polypeptide that has different levels of functionality at different temperatures. Exemplary temperature-sensitive agents include, but are not limited to, temperature-sensitive viral vectors, temperature-sensitive self-replicating RNA, and temperature-sensitive polypeptides.

[0059] As used herein, the term "permissive temperature" refers to any temperature at which activity of a temperature-sensitive agent of the present disclosure is elicited. Typically, the permissive temperature is not the subject's normal body temperature. Normal body temperature for a human subject is approximately 37°C ± 0.5°C. Depending on the temperature-sensitive agent, the permissive temperature may be higher or lower than the subject's normal body temperature. In some aspects, the permissive temperature of a temperature-sensitive agent is in the range of 30°C to 36°C. In some embodiments, the permissive temperature is about 31°C to about 35°C, or 32°C to 34°C (33°C ± 1.0°C). In some preferred embodiments, the permissive temperature is 33°C ± 0.5°C. Consequently, in some embodiments, the non-permissive temperature of a temperature-sensitive self-replicating RNA of the present disclosure is above 36°C. In some preferred embodiments, the non-permissive temperature is 37°C ± 0.5°C.

[0060] In some embodiments, the activity of a temperature-sensitive agent induced at a permissive temperature is reduced or inhibited at a non-permissive temperature. The term "non-permissive temperature," as used herein, refers to any temperature at which the activity of a temperature-sensitive agent of the present disclosure is not induced. A temperature-sensitive agent is not induced when its activity is at least 95% lower, at least 90% lower, at least 85% lower, at least 80% lower, at least 75% lower, or at least 50% lower than the activity level at the optimal permissive temperature. Typically, the non-permissive temperature is the subject's normal body temperature. Depending on the temperature-sensitive agent, the non-permissive temperature may also be higher or lower than the subject's normal body temperature. Temperature-sensitive viral vectors

[0061] In certain embodiments, the temperature-sensitive therapeutic agent of the present disclosure may comprise a temperature-sensitive viral vector. In some embodiments, the activity of a temperature-sensitive viral vector induced at a permissive temperature may include vector replication. As used herein, the term "temperature-sensitive viral vector" refers to any viral vector that has different levels of functionality at different temperatures. Exemplary temperature-sensitive viral vectors include, but are not limited to, Sendai virus vectors, adeno-associated virus vectors, retrovirus vectors, or alphavirus vectors. Exemplary temperature-sensitive alphavirus vectors include, but are not limited to, Venezuelan equine encephalitis virus vectors, Sindbis virus vectors, and Semliki Forest virus vectors.

[0062] In some embodiments of the present disclosure, the temperature-sensitive viral vector comprises a heterologous nucleic acid (e.g., a foreign nucleic acid associated with the viral vector). The heterologous nucleic acid may comprise one or more additional genetic elements, such as a promoter, operably associated with the coding region. In a preferred embodiment, the protein of interest is ZSCAN4, such as human ZSCAN4 or a variant thereof.

[0063] The permissive temperature for a temperature-sensitive viral vector of the present disclosure is typically in the range of 30°C to 36°C or 38°C to 50°C. In some embodiments, the permissive temperature is about 31°C to about 35°C, or 32°C to 34°C (33°C ± 1.0°C). In some preferred embodiments, the permissive temperature is 33°C ± 0.5°C. Consequently, in some embodiments, the non-permissive temperature for a temperature-sensitive viral vector of the present disclosure is greater than 36°C and less than 38°C. In some preferred embodiments, the non-permissive temperature is 37°C ± 0.5°C.

[0064] As disclosed herein, the cells may be maintained at the permissive temperature for a period of time sufficient for the temperature-sensitive agent to induce an effect. In some embodiments, the temperature-sensitive viral vector comprises a genetic element, and the effect comprises increased expression of the genetic element, where expression of the genetic element results in the production of an RNA or polypeptide that produces a biological effect in the cell. In some preferred embodiments, the effect is a therapeutic effect. Temperature-sensitive self-replicating RNA

[0065] In certain embodiments, a temperature-sensitive therapeutic agent of the present disclosure may comprise a temperature-sensitive self-replicating RNA. As used herein, the term "temperature-sensitive self-replicating RNA" refers to any self-replicating RNA that has different levels of functionality at different temperatures.

[0066] In some embodiments, temperature-sensitive self-replicating RNAs are generated by engineering self-replicating RNAs, which are typically single-stranded RNAs produced by alphaviruses such as Venezuelan equine encephalitis virus (VEEV), Sindbis virus (SINV), and Semliki Forest virus (SFV), by removing DNA encoding structural proteins necessary for viral particle formation (Petrakova et al., 2005). In some embodiments, the self-replicating RNA encodes nonstructural proteins (nsPs), which function as an RNA-dependent RNA polymerase to replicate the self-replicating RNA itself and produce transcripts for translation. In some embodiments, the self-replicating RNA may also contain a gene of interest (GOI) that includes the coding region for the protein of interest. The gene of interest may also include one or more additional genetic elements, such as a promoter, operably associated with the coding region. In a preferred embodiment, the protein of interest is ZSCAN4, such as human ZSCAN4 or a variant thereof. Without wishing to be bound by any theory, in some embodiments, the self-replicating RNA may express the GOI at high levels due to positive feedback production of that RNA. In some embodiments, the temperature-sensitive self-replicating RNA may be generated by mutating genes encoding nsPs.

[0067] In some embodiments, the temperature-sensitive self-replicating RNA can be delivered to mammalian cells as naked RNA (i.e., synthetic RNA). In some embodiments, the temperature-sensitive self-replicating RNA can be delivered to mammalian cells as naked RNA (i.e., synthetic RNA) encapsulated in nanoparticles. In some embodiments, the nanoparticles are engineered to target specific cell types, tissues, organs, cancers, tumors, or diseased cells. In some embodiments, the temperature-sensitive self-replicating RNA can be delivered to mammalian cells as viral particles, which are produced by packaging helper cells to complement missing viral structural proteins. In some embodiments, the viral particles are engineered to target specific cell types, tissues, organs, cancers, tumors, or diseased cells.

[0068] When the temperature-sensitive agent is a temperature-sensitive self-replicating RNA, the activity of the temperature-sensitive self-replicating RNA induced at the permissive temperature may include replication of the RNA.

[0069] In some aspects, the permissive temperature of a temperature-sensitive self-replicating RNA of the present disclosure is typically in the range of 30°C to 36°C. In some embodiments, the permissive temperature is about 31°C to about 35°C, or 32°C to 34°C (33°C ± 1.0°C). In some preferred embodiments, the permissive temperature is 33°C ± 0.5°C. Consequently, in some embodiments, the non-permissive temperature of a temperature-sensitive self-replicating RNA of the present disclosure is above 36°C. In some preferred embodiments, the non-permissive temperature is 37°C ± 0.5°C.

[0070] In other aspects, the permissive temperature of a temperature-sensitive self-replicating RNA of the present disclosure is typically in the range of 38° C. to 50° C. Consequently, in some embodiments, the non-permissive temperature of a temperature-sensitive self-replicating RNA of the present disclosure is above 36° C. and below 38° C. In some preferred embodiments, the non-permissive temperature is 37° C.±0.5° C. Temperature-sensitive polypeptides

[0071] In certain embodiments, the temperature-sensitive therapeutic agent of the present disclosure may comprise a temperature-sensitive polypeptide. As used herein, the term "temperature-sensitive polypeptide" refers to any temperature-sensitive polypeptide that has different levels of functionality at different temperatures. In some embodiments, the temperature-sensitive polypeptide may be a temperature-sensitive ZSCAN4. In other embodiments, the temperature-sensitive polypeptide is selected from, but is not limited to, a transcription factor for the ZSCAN4 gene.

[0072] When the temperature-sensitive agent is a temperature-sensitive polypeptide, the activity of the temperature-sensitive protein induced at the permissive temperature may include a conformational change (eg, an alteration to the structure or shape) of the protein.

[0073] The permissive temperature for a temperature-sensitive polypeptide of the present disclosure is typically in the range of 30°C to 36°C or 38°C to 50°C. In some embodiments, the permissive temperature is about 31°C to about 35°C, or 32°C to 34°C (33°C ± 1.0°C). In some preferred embodiments, the permissive temperature is 33°C ± 0.5°C. Consequently, in some embodiments, the non-permissive temperature for a temperature-sensitive self-replicating polypeptide of the present disclosure is above 36°C and below 38°C. In some preferred embodiments, the non-permissive temperature is 37°C ± 0.5°C.

[0074] Various aspects of the present disclosure relate to substantially purified polypeptides. A substantially purified polypeptide may refer to a polypeptide that is substantially free from other polypeptides, lipids, carbohydrates, or other substances with which it is naturally associated. In one embodiment, the polypeptide is at least 50%, e.g., at least 80%, free from other polypeptides, lipids, carbohydrates, or other substances with which it is naturally associated. In another embodiment, the polypeptide is at least 90% free from other polypeptides, lipids, carbohydrates, or other substances with which it is naturally associated. In yet another embodiment, the polypeptide is at least 95% free from other polypeptides, lipids, carbohydrates, or other substances with which it is naturally associated. Nucleic acids and polypeptides

[0075] Certain aspects of the present disclosure relate to transiently inducing the activity of a temperature-sensitive therapeutic agent in one or more cells, where the activity leads to increased expression of a nucleic acid molecule. In some embodiments, the nucleic acid is a polynucleotide. A polynucleotide can refer to a nucleic acid sequence of any length (e.g., a linear sequence). Thus, a polynucleotide includes oligonucleotides and also includes gene sequences found in chromosomes. An oligonucleotide is a plurality of linked nucleotides joined by natural phosphodiester bonds. An oligonucleotide is a polynucleotide between 6 and 300 nucleotides in length. An oligonucleotide analog refers to a moiety that functions similarly to an oligonucleotide but has non-naturally occurring portions. For example, an oligonucleotide analog can contain non-naturally occurring portions, altered sugar moieties or inter-sugar linkages, e.g., phosphorothioate oligodeoxynucleotides. Functional analogs of natural polynucleotides can bind to RNA or DNA and include peptide nucleic acid molecules.

[0076] In certain embodiments, the nucleic acid molecule or polynucleotide encodes a genetic element. These polynucleotides include DNA (encoding a gene of interest), cDNA, and RNA sequences, such as mRNA sequences. A coding sequence can be operably linked to a heterologous promoter to direct transcription of the genetic element. A promoter can refer to a nucleic acid control sequence that drives transcription of a nucleic acid. A promoter contains essential nucleic acid sequences near the transcription start site. A promoter may also contain distal enhancer or repressor sequences. A constitutive promoter is a promoter that is constantly active and is not subject to regulation by external signals or molecules. In contrast, the activity of an inducible promoter is regulated by external signals or molecules (e.g., transcription factors). A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Typically, operably linked nucleic acid sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. A heterologous polypeptide or heterologous polynucleotide refers to a polypeptide or polynucleotide derived from a different source or species. A promoter includes essential nucleic acid sequences near the transcription start site, such as a TATA sequence in the case of a polymerase II type promoter. A promoter may also include distal enhancer or repressor sequences, which may be located as far as several thousand base pairs from the transcription start site. In one example, the promoter is a constitutive promoter, such as a CAG promoter (Niwa et al., Gene 108(2):193-9, 1991) or a phosphoglycerate kinase (PGK) promoter. In some embodiments, the promoter is an inducible promoter, such as a tetracycline-inducible promoter (Masui et al., Nucleic Acids Res. 33:e43, 2005).Other exemplary promoters that can be used to drive expression of genetic elements include, but are not limited to, the lac system, the trp system, the tac system, the trc system, the lambda phage major operator and promoter region, the fd coat protein control region, the SV40 early and late promoters; promoters from polyoma virus, adenovirus, retrovirus, baculovirus, and simian virus; the promoter for 3-phosphoglycerate kinase, the promoter for yeast acid phosphatase, and the promoter for yeast alpha mating factor. Genetic elements of the present disclosure can be under the control of a constitutive promoter, an inducible promoter, or other suitable promoters described herein or readily recognized by one of skill in the art.

[0077] In some embodiments, eliciting activity of a temperature sensitive agent leads to increased expression of a nucleic acid or polypeptide, and can be, for example, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2.0-fold, at least 2.1-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3.1-fold, at least 3.2-fold, at least 3.3-fold, at least 3.4-fold, at least 3.5-fold, at least 3.6-fold, at least 3.7-fold, at least 3.8-fold, at least 3.9-fold, at least 4.0-fold, at least 4.1-fold, at least 4.2-fold, at least 4.3-fold, at least 4.4-fold, at least 4.5-fold, at least 4.6-fold, at least 4.7-fold, at least 4.8-fold, at least 4.9-fold, at least 5.0-fold, at least 5.1-fold, at least 5.2-fold, at least 5.3-fold, at least 5.4-fold, at least 5.5-fold, at least 5.6-fold, at least 5.7-fold, at least 5.8-fold, at least 5.9-fold, at least 6.0-fold, at least 6.1-fold, at least 6.2-fold, at least 6.3-fold, at least 6.4-fold, at least 6.5-fold, at least 6.6-fold, at least 6.7-fold, at least At least 2.7 times, at least 2.8 times, at least 2.9 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4.5 times, at least 5.0 times, at least 5.5 times, at least 6.0 times, at least 6.5 times, at least 7.0 times, at least 7.5 times, at least 8.0 times, at least 8.5 times, at least 9.0 times, at least 9.5 times, at least 10 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1,000 times, at least 2,000 times, at least 3,000 times, at least 4,000 times, at least 5,000 times, at least 6,000 times, at least 7,000 times, at least 8,000 times, at least 9,000 times, at least 10,000 times, at least 25,000 times, at least 50,000 times, at least 75,000 times, at least 100,000 times, at least 125,000 times, at least 150,000 times , at least 175,000 fold, at least 200,000 fold, at least 225,000 fold, at least 250,000 fold, at least 275,000 fold, at least 300,000 fold, at least 325,000 fold, at least 350,000 fold, at least 375,000 fold, at least 400,000 fold, at least 425,000 fold, at least 450,000 fold, at least 475,000 fold, at least 500,000 fold, at least 750,000 fold, or at least 1,000,000 fold increase in expression.

[0078] Various aspects of the present disclosure relate to isolated entities, such as isolated nucleic acids or synthetic mRNA molecules. It has been substantially separated or purified from the cells of an organism in which other nucleic acid sequences and nucleic acids, i.e., other chromosomal and extrachromosomal DNA and RNA, naturally occur. Thus, the term "isolated" encompasses nucleic acids purified by standard nucleic acid purification methods. The term also encompasses nucleic acids prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids. Similarly, an isolated polypeptide has been substantially separated or purified from other polypeptides of the cells of an organism in which the protein naturally occurs, including polypeptides prepared by recombinant expression in a host cell and chemically synthesized polypeptides. Similarly, an isolated cell is substantially separated from other cell types. Method for introducing temperature-sensitive agents into cells

[0079] In some embodiments, one or more cells are contacted with a temperature-sensitive agent. Contacting refers to placement in direct physical association, including both solid and liquid. "Contacting" may be used interchangeably with "exposing." In some cases, "contacting" includes transfection, such as transfection of a nucleic acid molecule into a cell. In some cases, "contacting" includes introduction of a temperature-sensitive agent into one or more cells.

[0080] In some embodiments, the temperature-sensitive agent is a polynucleotide (e.g., a self-replicating RNA), and the polynucleotide is introduced into the cell. Introduction of a nucleic acid molecule or protein into the cell encompasses any means of delivering a nucleic acid molecule or protein into the cell. For example, the nucleic acid molecule can be transfected, transduced, or electroporated into the cell. In some embodiments, the temperature-sensitive agent is a polypeptide (e.g., a temperature-sensitive polypeptide), and the polypeptide is introduced into the cell. Delivery of the polypeptide into the cell can be achieved by fusing the protein to a cellular peptide, such as a peptide having a protein transduction domain (e.g., HIV-1 Tat) or a poly-arginine peptide tag (Fuchs and Raines, Protein Science 14:1538-1544, 2005). A protein transduction domain may refer to a small cationic peptide that facilitates entry of larger molecules (e.g., proteins, nucleic acid molecules) into the cell by a mechanism independent of classical endocytosis. A poly-arginine peptide tag may refer to a short peptide (generally 7-11 residues) consisting of arginine residues that facilitates the delivery of larger molecules (such as proteins or nucleic acid molecules) into cells (see, e.g., Fuchs and Raines, Protein Science 14:1538-1544, 2005).

[0081] Introduction of nucleic acids into cells using temperature-sensitive agents may involve the use of temperature-sensitive viral vectors (such as integrative or non-integrative viral vectors) or temperature-sensitive plasmid vectors. Each of these methods has been described in the art and is therefore within the capabilities of one of ordinary skill in the art. A brief overview of each method that can be used to deliver nucleic acid molecules to one or more host cells (e.g., preferred mammalian host cells, such as human host cells) is provided herein. A vector may refer to a nucleic acid molecule when introduced into a host cell, resulting in a transformed host cell. A vector may contain a nucleic acid sequence that enables its replication in a host cell, such as an origin of replication (a DNA sequence involved in the initiation of DNA synthesis). For example, an expression vector contains the necessary regulatory sequences to enable the transcription and translation of the inserted gene(s). A vector may also contain one or more selectable marker genes and other genetic elements known in the art. A vector may include, for example, a viral vector or a plasmid vector. Allowable temperature Incubation of one or more cells at a permissive temperature

[0082] Certain aspects of the present disclosure relate to transiently inducing the activity of a temperature-sensitive agent in one or more cells by incubating the cells at a permissive temperature for inducing activity of the temperature-sensitive agent. In some embodiments, the permissive temperature may be higher or lower than standard cell culture temperatures. For example, human and rodent cells are typically cultured at a temperature of about 37°C. Thus, in some embodiments, the permissive temperature may be lower than about 36.5°C. For example, in some embodiments, cells are cultured at a permissive temperature of 36°C, 35.5°C, 35°C, 34.5°C, 34°C, 33.5°C, 33°C, 32.5°C, 32°C, 31.5°C, 31°C, 30.5°C, or 30°C. In some preferred embodiments, the permissive temperature is 30°C to 36°C, 31°C to 35°C, 32°C to 34°C, or 32.5°C to 33.5°C. In some embodiments, the acceptable temperature is (lower limit) 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C or higher, and (upper limit) 36°C, 35°C, 34°C, 33°C, 32°C, or 31°C or lower.

[0083] In other embodiments, the permissive temperature may be greater than about 37.5° C. For example, in some embodiments, cells are cultured at a permissive temperature of 38° C., 38.5° C., 39° C., 39.5° C., 40° C., 40.5° C., 41° C., 41.5° C., 42° C., 42.5° C., 43° C., 43.5° C., 44° C., 44.5° C., 45° C., 45.5° C., 46° C., 46.5° C., 47° C., 47.5° C., 48° C., 48.5° C., 49° C., 49.5° C., or 50° C.

[0084] In some embodiments, after incubation at the permissive temperature, one or more cells are cultured at a non-permissive temperature, where the activity of the temperature-sensitive agent is reduced or inhibited. For example, replication of a temperature-sensitive viral vector may be inhibited, replication of a temperature-sensitive self-replicating RNA may be inhibited, and conformational changes to temperature-sensitive polypeptides may be inhibited. This temperature shift allows the activity of the temperature-sensitive agent to be transiently induced and then inhibited. In other embodiments, one or more cells are administered to a subject after being cultured at the permissive temperature. One or more cells may be administered to a subject directly from culture at the permissive temperature, or may be first shifted from the permissive temperature to a non-permissive temperature during culture and then administered to a subject. In certain embodiments, the temperature-sensitive agent is subsequently degraded. For example, non-integrated temperature-sensitive viral vectors, RNA, and polypeptides are degraded. Lowers the subject's core body temperature to an acceptable level

[0085] Certain aspects of the present disclosure relate to transiently inducing the activity of a temperature-sensitive therapeutic agent in cells of a subject by lowering the subject's core body temperature to a permissive temperature to induce activity of the temperature-sensitive agent. In some embodiments, the subject's core body temperature is lowered using a targeted temperature management (TTM) procedure. TTM procedures are designed to achieve and maintain a specific body temperature in a subject for a sustained period of time. Such procedures have previously been used therapeutically to reduce the negative effects resulting from various acute health problems, such as heart attack and stroke. Devices and general methods for using them are known in the art and can be used in the methods described herein. The procedures can be performed using a number of methods, including cooling catheters, cooling blankets, and the application of ice around the body.

[0086] After lowering the subject's core body temperature to a permissive temperature, the subject's core body temperature is maintained at the permissive temperature for a time sufficient to induce activity of the temperature-sensitive agent. The subject's core body temperature is then returned to a normal core body temperature (a non-permissive temperature), where the activity of the temperature-sensitive agent is reduced or inhibited. In certain embodiments, the temperature-sensitive agent is subsequently degraded. For example, non-integrated temperature-sensitive viral vectors, RNA, and polypeptides are degraded at non-permissive temperatures. As used herein, the term "body temperature" refers to "core body temperature" unless otherwise clearly indicated. Maintains the subject's surface body temperature at an acceptable level

[0087] Certain aspects of the present disclosure relate to utilizing normal temperature differences in regions of a subject's body. For example, the temperature at or near the surface of a human subject's body (surface body temperature) is approximately 31-34°C, which is lower than the human subject's core body temperature (which is approximately 37°C). As used herein, the "surface" of a subject's body refers to one or more of the epidermis, dermis, subcutaneous tissue, or muscle. The "skin" of a subject's body refers to one or both of the epidermis and dermis. Thus, suitable routes of administration to the epidermis, dermis, or subcutaneous tissue of a subject's body include intradermal and subcutaneous administration. A suitable route of administration to muscle near the surface of a subject's body is intramuscular administration.

[0088] For example, ts agents are delivered directly to a specific area of ​​a subject's skin (in the case of vaccination) or to a larger area of ​​the subject's skin (in the case of treating a skin disease). Skin temperature (approximately 31-34°C) is the tolerant temperature for ts agents and allows them to function. No additional activity is required for long-term expression of the GOI. When cessation of ts agent function is required or desired, the temperature of the treated skin is elevated to a non-permissive temperature (>37°C) and transiently maintained by local application of heat (e.g., a heating patch or heating blanket) or by mild therapeutic hyperthermia (e.g., a hot bath or hot sauna). Because core body temperature is non-permissive (approximately 37°C), this therapeutic approach is safe, i.e., the ts agent functions only in the intended area of ​​the body. In some embodiments, if the subject's surface temperature must be higher than normal, the surface temperature is lowered to match the tolerant temperature for the ts agent. Maintaining the subject's upper airway temperature at an acceptable level

[0089] Like the surface body temperature of a human subject, the temperature of the upper respiratory tract and upper trachea of ​​a human subject is a tolerant temperature for ts agonists and allows them to function. That is, the temperature of the nasal cavity and upper trachea of ​​a human subject is approximately 32°C, and the temperature of the subsegmental bronchi of a human subject is approximately 35°C (McFadden et al., 1985). Thus, ts agonists administered intranasally to cells of the upper respiratory tract (nasal cavity, pharynx, and / or larynx) and / or upper trachea of ​​a human patient are functional without lowering the core body temperature of the human patient. Intranasal administration may be by insufflation, inhalation, or instillation. Unacceptable Temperature Incubating one or more cells at a non-permissive temperature

[0090] Typically, in vitro culture of cells is performed at the normal body temperature of the subject from which the cells are obtained. For example, mammalian cells, such as human or mouse cells, are typically cultured at about 37°C. Certain aspects of the present disclosure relate to temperature-sensitive agents that do not function (e.g., do not replicate or express genes) at the normal body temperature of the subject. Thus, the normal body temperature of the subject is the non-permissive temperature for the temperature-sensitive agent. In some preferred embodiments, the non-permissive temperature is 37°C ± 0.5°C. Subject's normal core body temperature

[0091] In some embodiments, a temperature-sensitive agent, a cell contacted with a temperature-sensitive agent, or a cell carrying a temperature-sensitive agent is introduced into a subject maintained at normal core body temperature. Certain aspects of the present disclosure relate to temperature-sensitive agents that do not function, e.g., replicate or express genes, at this normal (non-permissive) body temperature of the organism. This characteristic provides a safety mechanism to prevent undesired effects or reactivation of the temperature-sensitive agent throughout the life of the subject. human cells

[0092] Certain aspects of the present disclosure relate to transiently inducing the activity of a temperature-sensitive therapeutic agent in one or more human cells, including, but not limited to, adult human cells, hi certain embodiments, the one or more human cells are in need of treatment with the therapeutic agent in a subject.

[0093] Various human cells are useful in the methods described herein. As disclosed herein, the term "human cell" refers to any cell found in the human body during and after embryonic development, such as human embryonic cells, stem cells, pluripotent cells, differentiated cells, adult cells, somatic cells, and adult cells. In some embodiments, the human cells of the present disclosure are human adult cells. As disclosed herein, the term "human adult cell" refers to any cell found in the human body after embryonic development (i.e., a non-embryonic cell). Human cells of the present disclosure include, but are not limited to, sperm cells, oocytes, fertilized oocytes (i.e., zygotes), embryonic cells, adult cells, differentiated cells, somatic cells, primordial cells, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, adult stem cells, somatic stem cells, and tissue stem cells. Adult stem cells, also known as somatic stem cells or tissue stem cells, can refer to undifferentiated cells found in the body after embryonic development, which proliferate by cell division to replenish dead cells and regenerate damaged tissues. Progenitor cells can refer to oligopotent or unipotent cells that differentiate into specific cell types or cell lineages. Progenitor cells are similar to stem cells but are more differentiated and exhibit limited self-renewal. Exemplary adult stem cells, tissue stem cells, and / or progenitor cells can include, but are not limited to, hematopoietic stem cells, mesenchymal stem cells, adipose stem cells, neural stem cells, intestinal stem cells, skin stem cells, and germ cells (e.g., sperm cells and oocytes).

[0094] Human cells may include, but are not limited to, somatic cells, mature cells, and differentiated cells. Somatic cells can refer to any cell in the body, including, but not limited to, germ cells, tissue stem cells, progenitor cells, induced pluripotent stem (iPS) cells, and differentiated cells. Exemplary somatic, mature, and / or differentiated cells can include, but are not limited to, epidermal cells, fibroblasts, lymphocytes, hepatocytes, epithelial cells, myocytes, chondrocytes, osteocytes, adipocytes, cardiac myocytes, pancreatic beta cells, keratinocytes, erythrocytes, peripheral blood cells, bone marrow cells, neurons, astrocytes, and germ cells. Germ cells can refer to cells that give rise to gametes (i.e., eggs and sperm) in sexually reproducing organisms. In certain embodiments, germ cells include, but are not limited to, oocytes and sperm cells. In some embodiments, somatic, mature, and / or differentiated cells of the present disclosure also include, but are not limited to, preimplantation embryos.

[0095] Human cells may also include, but are not limited to, cells obtained from umbilical cord blood, hematopoietic stem cells, CD34+ cells, mesenchymal stem cells, vascular endothelial stem cells, tissue stem cells, granulocytes, lymphocytes, T cells, B cells, monocytes, macrophages, dendritic cells, erythrocytes, reticulocytes, and megakaryocytes. Human cells may also include, but are not limited to, abnormal cells of human origin, such as cancer cells, tumor cells, malignant cells, benign cells, hyperplastic cells, dysplastic cells, and atypical cells. Human cells may also include, but are not limited to, diploid cells, haploid cells, tetraploid cells, polyploid cells, cells with karyotypic abnormalities, cells with chromosomal abnormalities, cells with mutated genes, cells with abnormal telomere length, cells with short telomeres, and cells with long telomeres. Human cells may also include cells with epigenetic abnormalities, such as, but not limited to, cells with hypomethylated genomic regions, cells with hypermethylated genomic regions, and cells with abnormal histone modifications such as acetylation or methylation.

[0096] In some embodiments, the subject of the present disclosure is a non-human animal. Non-human animals may refer to all animals other than humans. Non-human animals include, but are not limited to, non-human primates, farm animals such as pigs, cows, and poultry, sport animals or pets such as dogs, cats, horses, and hamsters, rodents such as mice, or zoo animals such as lions, tigers, or bears. In one embodiment, the non-human animal is a mouse. Therapeutic Uses of Temperature-Sensitive Agents

[0097] The temperature-sensitive agents of the present disclosure may be administered by any suitable method known in the art, including, but not limited to, oral administration, sublingual administration, buccal administration, topical administration, rectal administration, via inhalation, transdermal administration, subcutaneous injection, intravenous injection, intraarterial injection, intramuscular injection, intracardiac injection, intraosseous injection, intradermal injection, intraperitoneal injection, transmucosal administration, intravaginal administration, intravitreal administration, intraarticular administration, periarticular administration, topical administration, epicutaneous administration, or any combination thereof. In some embodiments, the compositions are administered by subcutaneous and / or intravenous injection.

[0098] In some embodiments, the methods of the present disclosure involve the use of a therapeutically effective amount of a temperature-sensitive agent. A therapeutically effective amount of an agent can refer to the amount of a therapeutic agent sufficient to achieve its intended purpose. For example, a therapeutically effective amount of a temperature-sensitive agent for treating a disease or condition is an amount sufficient to alleviate the disease or condition, or one or more symptoms of the disease or condition. In some instances, a therapeutically effective amount may not treat 100% of the disease or condition, or symptoms of the disease or condition. However, a reduction in any known characteristic or symptom of the disease or condition, e.g., at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, can be therapeutic.

[0099] The therapeutically effective amount of a given therapeutic agent will vary depending on factors such as the nature of the agent, the route of administration, the size and / or age of the subject receiving the therapeutic agent, and the purpose of the administration. The therapeutically effective amount in each individual case can be determined empirically by one of ordinary skill in the art, without undue experimentation, according to methods established in the art.

[0100] A subject may refer to living multi-cellular vertebrate organisms, a category that includes humans and non-human mammals. In some embodiments, the subject is a human. Subjects that can be treated using the methods provided herein can include mammalian subjects, e.g., veterinary subjects or human subjects. Subjects can include fertilized eggs, zygotes, preimplantation embryos, embryos, fetuses, newborns, infants, children, and / or adults. In some embodiments, the subject to be treated is selected, e.g., by selecting a subject that would benefit from a treatment, particularly a treatment that includes administration of a temperature-sensitive agent of the present disclosure.

[0101] Pharmaceutical compositions of the present disclosure include a ts agent, such as a therapeutic ts agent, and one or more additional compounds. As used herein, the terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable vehicle" refer to one or more additional compounds (i.e., compounds other than a ts agent). Pharmaceutically acceptable carriers suitable for use in the present disclosure are conventional. In particular, compositions and formulations suitable for pharmaceutical delivery of compositions containing temperature-sensitive agents are as previously described (see, e.g., Gennaro, AR (editor) Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th edition (1990); and Felton, LA (editor) Remington Essentials of Pharmaceutics, Pharmaceutical Press, London, United Kingdom, 1st edition, (2013)).

[0102] Generally, the nature of the carrier will depend on the particular mode of administration being used. For example, parenteral formulations usually comprise an injectable liquid containing a carrier such as a pharmaceutically and physiologically acceptable liquid, such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, or the like. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the administered pharmaceutical composition can also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, e.g., sodium acetate or sorbitan monolaurate. In some embodiments, the pharmaceutical compositions of the present disclosure comprise a ts agent, such as a therapeutic ts agent, and one or more additional compounds (which facilitate uptake of the ts agent into cells). In the case of RNA-based ts agents, the ts agent is encapsulated within nanoparticles. In some cases, the nanoparticles are lipid-based (e.g., lipofectamine).

[0103] The most appropriate therapeutic dose and treatment regimen for treating a patient will vary depending on the disease or condition being treated and on the patient's weight and other parameters. Effective dosages and treatment protocols can be determined by conventional methods, such as starting with low doses in experimental animals and then monitoring the effects while increasing the dose and systematically modifying the dosing regimen. When determining the optimal dosage for a given subject, a physician can take into account many factors, including the patient's size, the patient's age, the patient's general condition, the particular disease being treated, the severity of the disease, and the presence of other medications in the patient. Test dosages are selected after consideration of the results of animal studies and the clinical literature. Bone marrow cell mobilization

[0104] In some embodiments, the methods include mobilizing bone marrow cells (including but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells) into the spleen and peripheral blood of the subject. In some embodiments, the methods include administering a therapeutically effective amount of a temperature-sensitive agent (e.g., a temperature-sensitive therapeutic agent) of the present disclosure under conditions suitable for the temperature-sensitive agent to deliver nucleic acid to one or more bone marrow cells (including but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells) in the spleen.

[0105] In some embodiments of the methods disclosed herein, mobilizing bone marrow cells (including but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) to the spleen and peripheral blood comprises administering to the subject a therapeutically effective amount of a cytokine and / or a chemotherapeutic agent. In some embodiments, mobilizing bone marrow cells (including but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) to the spleen and peripheral blood comprises administering to the subject a therapeutically effective amount of a cytokine. In some embodiments, mobilizing bone marrow cells (including but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) to the spleen comprises administering to the subject a therapeutically effective amount of a chemotherapeutic agent. In some embodiments, mobilizing bone marrow cells (including but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) to the spleen comprises administering to the subject a therapeutically effective amount of a cytokine and a chemotherapeutic agent. The cytokines and / or chemotherapeutic agents may be administered by any suitable method known in the art, including, but not limited to, oral administration, sublingual administration, buccal administration, topical administration, rectal administration, via inhalation, transdermal administration, subcutaneous injection, intravenous injection, intraarterial injection, intramuscular injection, intracardiac injection, intraosseous injection, intradermal injection, intraperitoneal injection, transmucosal administration, intravaginal administration, intravitreal administration, intraarticular administration, periarticular administration, topical administration, epicutaneous administration, or any combination thereof. In some embodiments, the cytokines and / or chemokines are administered by subcutaneous and / or intravenous injection.

[0106] In some embodiments, the subject's bone marrow cells (including but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells) are mobilized at least 4 weeks, at least 3 weeks, at least 2 weeks, at least 1 week, at least 6 days, at least 5 days, at least 4 days, at least 3 days, at least 2 days, at least 1 day, less than 1 day, at least 18 hours, at least 16 hours, at least 12 hours, at least 8 hours, at least 6 hours, or at least 1 hour prior to administration of the composition (e.g., any nanoparticle composition described herein). In some embodiments, the subject's bone marrow cells (including but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells) are mobilized for 7 consecutive days, 5 consecutive days, 4 consecutive days, 3 consecutive days, 2 consecutive days, or 1 day prior to administration of the composition. In some embodiments, the subject's bone marrow cells (including but not limited to CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) are mobilized in parallel with administration of the composition.

[0107] Any cytokine known in the art that can mobilize bone marrow cells (including, but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) can be used, including, but not limited to, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO), thrombopoietin (TPO), stem cell factor (SCF), parathyroid hormone (PTH), and any combination thereof. In some embodiments, the cytokine is G-CSF.

[0108] In some embodiments, G-CSF is administered to a subject at a concentration of about 0.1 μg / kg to about 100 μg / kg or about 1.0 μg / kg to about 10 μg / kg. In some embodiments, G-CSF is administered to a subject at a concentration of about 2.5 μg / kg. In some embodiments, G-CSF is administered to a subject at a concentration of about 10 μg / kg.

[0109] Any chemotherapeutic agent known in the art that can mobilize bone marrow cells (including but not limited to CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) can be used, including but not limited to, plerixafor, cyclophosphamide (CY), paclitaxel, etoposide, POL6326, BKT-140, TG-0054, NOX-A12, SEW2871, BIO5192, bortezomib, SB-251353, FG-4497, and any combination thereof. In some embodiments, the chemotherapeutic agent is plerixafor.

[0110] In some embodiments, plerixafor is administered to a subject at a concentration of about 1 μg / kg to about 1000 μg / kg or about 75 μg / kg to about 500 μg / kg. In some embodiments, plerixafor is administered to a subject at a concentration of about 150 μg / kg. In some embodiments, plerixafor is administered to a subject at a concentration of about 240 μg / kg.

[0111] In some embodiments, mobilization of bone marrow cells (including, but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) to the spleen and peripheral blood comprises administering a therapeutically effective amount of G-CSF and a therapeutically effective amount of plerixafor. In some embodiments, G-CSF and plerixafor are co-administered to the subject. In some embodiments, G-CSF and plerixafor are co-administered to the subject for 1, 2, 3, 4, or more days. In some embodiments, G-CSF is administered to the subject prior to plerixafor. In some embodiments, G-CSF is administered to the subject for 1, 2, 3, 4, or more days prior to plerixafor. In some embodiments, G-CSF is administered to the subject 1, 2, 3, 4, or more days prior to plerixafor, and then G-CSF and plerixafor are co-administered to the subject for 1, 2, 3, 4, or more days. In some embodiments, plerixafor is administered to the subject before G-CSF. In some embodiments, plerixafor is administered to the subject 1, 2, 3, 4, or more days before G-CSF. In some embodiments, plerixafor is administered to the subject 1, 2, 3, 4, or more days before G-CSF, and then G-CSF and plerixafor are co-administered to the subject for 1, 2, 3, 4, or more days.

[0112] In some embodiments, one or more human cells are contacted with a temperature-sensitive agent (e.g., a temperature-sensitive therapeutic agent) that delivers a nucleic acid to one or more human cells. In some embodiments, the nucleic acid comprises a gene of interest or encodes a protein of interest.

[0113] In some embodiments, the methods of the present disclosure involve the use of a therapeutic amount of a temperature-sensitive agent (e.g., a temperature-sensitive therapeutic agent) to deliver nucleic acids to cells of a subject in vitro or in vivo. A therapeutically effective amount of an agent can refer to the amount of a therapeutic agent sufficient to achieve an intended purpose. For example, a therapeutically effective amount of a temperature-sensitive agent (e.g., a temperature-sensitive therapeutic agent) to deliver nucleic acids to human cells to treat a disease or condition is an amount sufficient to alleviate the disease or condition or one or more symptoms of the disease or condition. In some examples, a therapeutically effective amount may not treat 100% of the disease or condition or symptoms of the disease or condition. However, a reduction in any known characteristic or symptom of the disease or condition, e.g., at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, can be therapeutic.

[0114] In another example, a therapeutically effective amount of a cytokine and / or chemokine capable of mobilizing bone marrow cells (including, but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) in a subject is an amount sufficient to induce mobilization of one or more bone marrow cells (including, but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) from the bone marrow into the peripheral blood.

[0115] The therapeutically effective amount of a given temperature-sensitive agent (e.g., a temperature-sensitive therapeutic agent) will vary depending on factors such as the nature of the agent, the route of administration, the size and / or age of the subject receiving the therapeutic agent, and the purpose of administration. The therapeutically effective amount in each individual case can be determined empirically by one of ordinary skill in the art, without undue experimentation, according to methods established in the art.

[0116] A subject may refer to living multi-cellular vertebrate organisms, a category that includes humans and non-human mammals. In some embodiments, the subject is a human. Subjects that may be treated using the methods provided herein may include mammalian subjects, e.g., veterinary subjects or human subjects. Subjects may include fetuses, neonates, infants, children, and / or adults. In some embodiments, the subject to be treated is selected, such as by selecting a subject that would benefit from the treatment.

[0117] Examples of disorders or diseases that may benefit from the administration of a temperature-sensitive agent (e.g., a temperature-sensitive therapeutic agent) include disorders or diseases associated with genetic mutation(s), abnormal telomere length, or abnormal epigenetic modification(s). In some embodiments, the disease or disorder is a telomere biology disorder. Further examples of disorders or diseases that may benefit from the administration of a temperature-sensitive agent (e.g., a temperature-sensitive therapeutic agent) include, but are not limited to, cancer, autoimmune diseases, and neurological or neurodegenerative disorders, as well as diseases in which cellular regeneration is beneficial, such as blindness and hearing loss. In some embodiments, the disease or disorder is a disease of the blood or blood-forming organs.

[0118] Cancer includes malignant tumors characterized by abnormal or uncontrolled cell proliferation. Cancer is often associated with genetic mutations and abnormal telomere regulation. Exemplary cancers that may benefit from treatment with ts agents include, but are not limited to, cancers of the heart (e.g., sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma); lung cancer (e.g., bronchogenic carcinoma (lepidic cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar epithelial (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondroitin hamartoma, mesothelioma); gastrointestinal cancer (e.g., esophageal (squamous) Cancer, adenocarcinoma, leiomyosarcoma, lymphoma); gastric cancer (epithelial carcinoma, lymphoma, leiomyosarcoma); pancreatic cancer (pancreatic ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma); small intestine cancer (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma); colorectal cancer (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); genitourinary tract cancer (e.g., kidney (adenocarcinoma, Wilms' tumor, nephroblastoma, lymphoma, leukemia); Bladder and urethral cancer (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma); prostate cancer (adenocarcinoma, sarcoma); testicular cancer (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenoid tumor, lipoma); liver cancer (e.g., hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma); bone cancer (e.g., osteogenic sarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant Giant cell tumor, chordoma, osteochondroma (osteochondroma exostosis), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumor; cancers of the nervous system (e.g., skull (osteoma, hemangioma, granuloma, xanthomas, osteitis deformans), meninges (meningioma, meningeal sarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma > pinealoma!, glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord (neurofibroma, meningioma, glioma, sarcoma));Gynecological cancers (e.g., uterus (endometrial carcinoma), cervix (cervical carcinoma, preneoplastic cervical dysplasia), ovary (ovarian carcinoma, serous cystadenocarcinoma, mucinous cystadenocarcinoma, endometrioid tumor, Brenner tumor, clear cell carcinoma, unclassified carcinoma, granulosa / theca cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma, embryonal rhabdomyosarcoma, fallopian tube (cancer) )); blood cancers (e.g., blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndromes), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma)); skin cancers (e.g., malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, lentigines, atypical nevi, lipoma, hemangioma, dermatofibroma, keloids, psoriasis); and adrenal gland cancers (e.g., neuroblastoma);

[0119] Autoimmune diseases result in an abnormal immune response, such as the production of antibodies or cytotoxic T cells that are specific for self-antigens or the subject's own cells or tissues. In some instances, autoimmune diseases are restricted to a particular organ (e.g., in thyroiditis) or can affect specific tissues in various locations (e.g., Goodpasture's disease). Exemplary autoimmune diseases that can benefit from treatment with a ts agonist include, but are not limited to, rheumatoid arthritis, juvenile oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo vulgaris, type 1 diabetes, non-obese diabetes, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemic lupus erythematosus, autoimmune thrombocytopenic purpura, Goodpasture's syndrome, Addison's disease, systemic sclerosis, polymyositis, dermatomyositis, autoimmune hemolytic anemia, and pernicious anemia.

[0120] In some embodiments, the subject is one who has experienced nerve injury or is suffering from a neurodegenerative disorder. Nerve injury can refer to trauma to the nervous system (e.g., to the brain or spinal cord or specific nerve cells) that adversely affects the injured patient's movement and / or memory. For example, such patients may suffer from dysarthria (a speech impediment), hemiparesis, or hemiplegia. Nerve injury can result from trauma to the nervous system (e.g., to the brain or spinal cord or specific nerve cells) that adversely affects the injured patient's movement and / or memory. Such trauma can be caused by infectious agents (e.g., bacteria or viruses), toxins, injuries resulting from falls or other types of accidents, genetic disorders, or for other unknown reasons. Thus, in some embodiments, a temperature-sensitive agent (e.g., a temperature-sensitive therapeutic agent) of the present disclosure that is temperature-sensitive can be used to treat nerve injury in a subject who has suffered nerve injury by modifying tissue stem cells in the nervous system of the patient, thereby producing neurons and glial cells through the modification of the tissue stem cells in the nervous system, and thereby repairing the nervous system defect. In some embodiments, the patient may have suffered a neurological injury, such as a brain or spinal cord injury resulting from an accident, such as a car accident or diving accident, or resulting from a stroke.

[0121] Neurodegenerative diseases are conditions that result in the loss of cells in the brain and / or spinal cord. Neurodegenerative diseases result from the deterioration of nerve cells or the myelin sheath of nerve cells, which over time leads to dysfunction and disability. The resulting condition can cause movement problems (e.g., ataxia) and memory problems (e.g., dementia). Thus, in some embodiments, a temperature-sensitive agent (e.g., a temperature-sensitive therapeutic agent) of the present disclosure can be used to treat a neurodegenerative disease in a subject by modifying tissue stem cells in the nervous system of a patient suffering from a neurodegenerative disease, thereby producing neurons and glial cells, thereby repairing the nervous system defects. In some embodiments, the agent modifies the subject's nervous system and reverses the degenerative state of the disease. Exemplary neurodegenerative diseases include, but are not limited to, adrenoleukodystrophy (ALD), alcoholism, Alexander disease, Alpers disease, Alzheimer's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), ataxia-telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), bovine spongiform encephalopathy (BSE), Canavan disease, cerebral palsy, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, fatal familial insomnia, frontotemporal lobar degeneration, Huntington's disease, HIV-associated dementia, Kennedy disease, Krabbe disease, and others. Neuropathy, dementia with Lewy bodies, neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type 3), multiple system atrophy, multiple sclerosis, narcolepsy, Niemann-Pick disease, Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis, prion diseases, progressive supranuclear palsy, Refsum disease, Sandhoff disease, Schilder's disease, subacute combined spinal cord degeneration complicated with pernicious anemia, Spielmeyer-Vogt-Sjögren-Batten disease (also known as Batten disease), spinocerebellar ataxia, spinal muscular atrophy, Steele-Richardson-Olszewski disease, tabes dorsalis, and toxic encephalopathy.

[0122] Thus, a temperature-sensitive agent (e.g., a temperature-sensitive therapeutic agent) is administered to a subject to reduce or ameliorate symptoms associated with a particular disorder. Treatment endpoints for cancer treatment can include a reduction in tumor size or volume, a reduction in angiogenesis to the tumor, or a reduction in tumor metastasis. If a tumor has been removed, another treatment endpoint can be regeneration of the removed tissue or organ. The effectiveness of cancer treatment can be measured using methods in the art, such as imaging the tumor or detecting tumor markers or other indicators of the presence of cancer. Treatment endpoints for autoimmune disease treatment can include a reduction in the autoimmune response. The effectiveness of autoimmune disease treatment can be measured using methods in the art, such as measuring autoimmune antibodies, with a reduction in such antibodies in a treated subject indicating successful treatment. Treatment endpoints for neurodegenerative disorders can include a reduction in neurodegeneration-related deficits, such as a reduction in increased motor, memory, or behavioral deficits. Methods in the art can be used to measure the effectiveness of neurodegenerative disorder treatment, for example, by measuring cognitive impairment, with a reduction in such impairment in a treated subject indicating successful treatment. Therapeutic endpoints for nerve injury treatment can include a reduction in injury-related deficits, such as a reduction in increased motor, memory, or behavioral deficits. Methods in the art can be used to measure the effectiveness of nerve injury treatment, for example, by measuring mobility and flexibility, and increases in such in a treated subject indicate successful treatment. Treatment does not need to be 100% effective. For example, a reduction of at least about 10%, about 15%, about 25%, about 40%, about 50%, or more of the disease (or its symptoms) compared to the absence of treatment with the agent is considered effective.

[0123] Temperature-sensitive agents (e.g., temperature-sensitive therapeutic agents) of the present disclosure can also be used to treat atherosclerosis and / or coronary artery disease in a subject in need thereof, for example, by administering the temperature-sensitive agent (e.g., temperature-sensitive therapeutic agent) to the bloodstream of the subject so as to introduce / contact vascular endothelial cells and improve the properties of the vascular endothelial cells, thereby treating atherosclerosis and / or coronary artery disease in said subject.

[0124] The temperature-sensitive agents (e.g., temperature-sensitive therapeutic agents) of the present disclosure may also be used to provide resistance to one or more genotoxic agents in one or more human cells and / or subjects in need thereof. Treatment of Diseases and Disorders with Enhanced ZSCAN4 Expression

[0125] As disclosed herein, expression of ZSCAN4 increases telomere length, improves genomic stability, corrects genomic and / or chromosomal abnormalities, protects cells from DNA damage, and / or enhances DNA repair. DNA repair can refer to a group of processes by which cells identify and correct damage to DNA molecules in their genome. Thus, for example, methods are provided herein involving transiently enhancing ZSCAN4 expression in human cells to increase telomere length, improve genomic stability, correct genomic and / or chromosomal abnormalities, protect cells from DNA damage, and / or enhance DNA repair in such cells. In some embodiments, the present disclosure provides methods involving transiently enhancing ZSCAN4 expression in human cells to increase telomere length, for example, to treat diseases of the blood or hematopoietic organs. In some embodiments, the disease includes bone marrow failure.

[0126] Mammalian cells (e.g., human bone marrow cells) into which a ts agent that enhances ZSCAN4 expression has been introduced are referred to herein as "ZSCAN4* cells." "ZSCAN4* cells" include, but are not limited to, cells that transiently express ZSCAN4. That is, ZSCAN4* cells do not require continuous measurable ZSCAN4 or continuous expression of ZSCAN4 mRNA or protein. In some embodiments, the effect of ZSCAN4 is rapid and requires only transient expression of ZSCAN4 (e.g., on the order of hours to days). In the case of telomeres, once telomeres are lengthened by ZSCAN4 action, telomeres only gradually shorten, so further ZSCAN4 expression is not required for a long period of time. Thus, "ZSCAN4* cells" include both cells containing a ts agent that enhances ZSCAN4 expression and cells into which a ts agent has been introduced but is no longer present.

[0127] Methods and compositions are provided for treating subjects in need thereof, such as those suffering from telomere abnormalities.Telomere abnormalities refer to any alteration of telomeres that disrupts one or more telomere functions, such as telomere shortening, interruptions in telomere DNA repeats, or mutations in telomere DNA.Diseases or disorders associated with telomere abnormalities that may benefit from high ZSCAN4 expression include, but are not limited to, telomere shortening disorders, bone marrow failure syndromes, age-related telomere shortening disorders, and premature aging disorders.

[0128] Telomere shortening disorders (encompassed by the term "telomere biology disorders") that may benefit from temperature-sensitive agents that enhance ZSCAN4 expression in human cells include, but are not limited to, dyskeratosis congenita, Wheeler-Leiderson syndrome, Rewes syndrome, Coats-Plus syndrome, and idiopathic pulmonary fibrosis. In some embodiments, the telomere shortening disorder is dyskeratosis congenita.

[0129] Bone marrow failure syndromes that may benefit from temperature-sensitive agents that enhance ZSCAN4 expression in human cells include, but are not limited to, Fanconi anemia, amegakaryocytic thrombocytopenia, aplastic anemia, Diamond-Blackfan anemia, paroxysmal nocturnal hemoglobinuria, Pearson syndrome, Schwachman-Diamond syndrome, and myelodysplastic syndrome. In some embodiments, the bone marrow failure syndrome is Fanconi anemia. In some embodiments, the subject in need of treatment suffers from both a telomere biology disorder and a bone marrow failure disorder (e.g., dyskeratosis congenita).

[0130] Age-related telomere shortening or premature aging disorders that may benefit from temperature-sensitive agents that enhance ZSCAN4 expression in human cells include, but are not limited to, Werner syndrome, Bloom syndrome, Hutchison-Gilford progeria syndrome, Cockayne syndrome, xeroderma pigmentosum, ataxia-telangiectasia, Rothmond-Thomson syndrome, dyssulfuroides trichodystrophy, Joberg-Marsig syndrome, and Down syndrome.

[0131] Methods and compositions are provided for treating subjects in need thereof, such as those suffering from chromosomal abnormalities. Chromosomal abnormalities refer to any irregularity, change, or mutation in a chromosome that results in missing, extra, or irregular segments of chromosomal DNA. In certain embodiments, chromosomal abnormalities result in an abnormal number of chromosomes or structural abnormalities in one or more chromosomes. As used herein, aneuploidy can refer to an abnormal number of whole chromosomes or chromosomal segments. Aneuploidies that can benefit from temperature-sensitive agents that enhance ZSCAN4 expression in human cells include, but are not limited to, chromosomal nullisomy, chromosomal monosomy, chromosomal trisomy, chromosomal tetrasomy, and chromosomal pentasomy. Examples of human aneuploidies include, but are not limited to, trisomy 21, trisomy 16, trisomy 18 (Edwards syndrome), trisomy 13 (Patau syndrome), monosomy X (Turner syndrome), XXX aneuploidy, XXY aneuploidy, and XYY aneuploidy. Examples of human partial aneuploidies include, but are not limited to, 1p36 duplication, chromosome 17 (p11.2p11.2) duplication syndrome, Pelizaeus-Merzbacher disease, chromosome 22 (q11.2q11.2) duplication syndrome, and cat eye syndrome. In some embodiments, the aneuploidy comprises one or more deletions of a sex chromosome or an autosome, which deletions can result in conditions such as cricket-like syndrome, Wolf-Hirschhorn syndrome, Williams-Beuren syndrome, Charcot-Marie-Tooth disease, hereditary neuropathy with liability to pressure palsies, Smith-Magenis syndrome, neurofibromatosis, Alagille syndrome, palatocardiofacial syndrome, DiGeorge syndrome, steroid sulfatase deficiency, Kallmann syndrome, microphthalmia in linear cutis coloboma, adrenal hypoplasia, glycerol kinase deficiency, Pelizaeus-Merzbacher disease, testis-determining factor on the Y chromosome, azoospermia (factor a), azoospermia (factor b), azoospermia (factor c), or 1p36 deletions. ZSCAN4 Polynucleotide

[0132] In some embodiments, a therapeutic temperature-sensitive agent of the present disclosure that enhances ZSCAN4 expression is a nucleic acid molecule comprising a nucleic acid sequence (coding region) encoding a ZSCAN4 protein. Nucleic acid molecules include DNA, cDNA, and RNA (mRNA) molecules encoding a ZSCAN4 protein. It is understood that all polynucleotides encoding a ZSCAN4 protein are encompassed herein as long as they encode a ZSCAN4 protein, variant, or fragment thereof that has ZSCAN4 activity, such as the ability to regulate genomic stability or telomere length. Genomic stability refers to a cell's ability to faithfully replicate DNA and maintain the integrity of the DNA replication machinery. Long telomeres are thought to provide a buffer against cellular senescence and generally indicate genomic stability and overall cellular health. Chromosomal stability (e.g., few mutations, no chromosomal rearrangements, or no changes in chromosome number) is also associated with genomic stability. Loss of genomic stability is associated with cancer, neurological disorders, and premature aging. Signs of genomic instability include elevated mutation rates, large-scale chromosomal rearrangements, changes in chromosome number, and telomere shortening.

[0133] The sequence of ZSCAN4 nucleic acid molecules is known in the art. ZSCAN4 nucleic acid sequences include, but are not limited to, any one of mouse ZSCAN4 genes (including ZSCAN4a, ZSCAN4b, ZSCAN4c, ZSCAN4d, ZSCAN4e, and ZSCAN4f) that exhibit 2-cell embryo stage-specific or ES cell-specific expression, or their orthologs. In some embodiments, the ortholog is human ZSCAN4. Nucleic acid sequences encoding human ZSCAN4 and its orthologs are disclosed in the sequence listing of U.S. Patent No. 10,335,456 B1 to Ko, which is incorporated herein by reference.

[0134] Fragments and variants of ZSCAN4 polynucleotides can be prepared by those skilled in the art using standard molecular techniques. In some embodiments, ZSCAN4 polynucleotides encode truncated forms of ZSCAN4 proteins that lack one or more zinc finger domains of naturally occurring ZSCAN4 proteins. In some embodiments, ZSCAN4 polynucleotides encode variants of ZSCAN4 proteins. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single-stranded and double-stranded forms of DNA. Recombinant nucleic acids are those that have a sequence that is not naturally occurring or that is created by the artificial joining of two otherwise separate sequence fragments.

[0135] The ZSCAN4 coding region can be operably linked to a promoter to direct transcription of the coding region. A promoter refers to a nucleic acid control sequence that directs transcription of an operably linked coding region. A promoter includes necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements. A constitutive promoter is a promoter that is continuously active and is not subject to regulation by external signals or molecules. In contrast, the activity of an inducible promoter is regulated by external signals or molecules (e.g., transcription factors). A first nucleic acid sequence is operably linked to a second nucleic acid sequence when it is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Typically, operably linked nucleic acid sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. A heterologous polypeptide or heterologous polynucleotide refers to a polypeptide or polynucleotide derived from a different source or species. A promoter includes essential nucleic acid sequences near the transcription start site, such as a TATA sequence in the case of a polymerase II type promoter. A promoter may also include distal enhancer or repressor sequences, which may be located as many as several thousand base pairs from the transcription start site. In one example, the promoter is a constitutive promoter, such as a CAG promoter (Niwa et al., Gene 108(2):193-9, 1991). In some embodiments, the promoter is an inducible promoter, such as a tetracycline-inducible promoter (Masui et al., Nucleic Acids Res. 33:e43, 2005).Other exemplary promoters that can be used to drive expression of Zscan4 include, but are not limited to, the lac system, the trp system, the tac system, the trc system, the lambda phage major operator and promoter region, the fd coat protein regulatory region, the SV40 early and late promoters; promoters from polyoma virus, adenovirus, retrovirus, baculovirus, and simian virus; the 3-phosphoglycerate kinase promoter, the yeast acid phosphatase promoter, and the yeast alpha mating factor promoter. In some embodiments, the native ZSCAN4 promoter is used. ZSCAN4 Polypeptide

[0136] It is understood that all ZSCAN4 polypeptides are encompassed herein as long as they have ZSCAN4 activity, such as the ability to regulate genomic stability or telomere length. The terms "polypeptide" and "protein" are used interchangeably herein and include naturally occurring ZSCAN4 proteins, variants, or fragments thereof that have ZSCAN4 activity.

[0137] The amino acid sequence of the ZSCAN4 protein is known in the art. ZSCAN4 amino acid sequences include, but are not limited to, any one of mouse ZSCAN4 genes (including ZSCAN4a, ZSCAN4b, ZSCAN4c, ZSCAN4d, ZSCAN4e, and ZSCAN4f) that exhibit 2-cell embryo stage-specific or ES cell-specific expression, or their orthologs. In some embodiments, the ortholog is human ZSCAN4. The amino acid sequences encoding human ZSCAN4 and its orthologs are disclosed in the sequence listing of U.S. Patent No. 10,335,456 B1 to Ko, which is incorporated herein by reference.

[0138] Fragments and variants of ZSCAN4 proteins can be prepared by those skilled in the art using standard molecular techniques. In some embodiments, the ZSCAN4 protein is a truncated form of ZSCAN4 that lacks one or more zinc finger domains of the naturally occurring ZSCAN4 protein. In some embodiments, the ZSCAN4 protein is a variant of the naturally occurring ZSCAN4 protein.

[0139] In some preferred embodiments, the amino acid sequence of the human ZSCAN4 protein comprises SEQ ID NO: 38 or one of the group consisting of SEQ ID NOs: 39-42.

[0140] hZSCAN4, (aa1-433:): [ka]

[0141] hZSCAN4(aa1-311): [ka]

[0142] hZSCAN4(aa1-339): [ka]

[0143] hZSCAN4(aa1-367): [ka]

[0144] hZSCAN4(aa1-395): [ka]

[0145] In some embodiments, the amino acid sequence of the human ZSCAN4 protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to one of the group consisting of SEQ ID NOs: 38-42.

[0146] Identity / similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity, with the higher the percentage, the more identical the sequences. Sequence similarity can be measured in terms of percentage similarity (taking into account conservative amino acid substitutions), with the higher the percentage, the more similar the sequences. Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence identity / similarity when aligned using standard methods. This homology is more significant when the orthologous proteins or cDNAs are derived from more closely related species (e.g., human and monkey sequences) compared to more distantly related species (e.g., human and mouse sequences).

[0147] The term "identical" or percent "identity" in the context of two or more sequences (e.g., nucleic acid or amino acid sequences) can refer to two or more sequences or subsequences that are identical. Two sequences are substantially identical if they have a specified percentage of amino acid residues or nucleotides that are identical (i.e., 95%, 96%, 97%, 98%, 99%, or 100% identity over the specified region, or, if not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region, as assessed using one of the following sequence comparison algorithms or by manual alignment and visual inspection.

[0148] For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, and sequence algorithm program parameters are designated, if necessary. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. When comparing two sequences for identity, the sequences do not have to be contiguous, although a penalty is associated with any gaps that will reduce the overall percent identity. For blastp, the default parameters are a gap opening penalty of 11 and a gap extension penalty of 1. For blastn, the default parameters are a gap opening penalty of 5 and a gap extension penalty of 2.

[0149] The comparison window may include matching against any one segment of a number of contiguous positions, including, but not limited to, 20 to 600, typically about 50 to about 200, and more typically about 100 to about 150, and a sequence can be compared against the reference sequence of the same number of contiguous positions after optimally aligning the two sequences within the comparison window. Methods of sequence alignment for comparison are well known. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970) J Mol Biol 48(3):443-453, by the similarity search method of Pearson and Lipman (1988) Proc Natl Acad Sci USA 85(8):2444-2448, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Dr., Madison, WI)), or by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou Ed.)).

[0150] Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nucleic Acids Res 25(17):3389-3402 and Altschul et al. (1990) J. Mol Biol 215(3)-403-410, respectively. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short word lengths W in the query sequence that, when aligned with words of the same length in a database sequence, match or satisfy a certain positive threshold score T, where 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 these initial neighborhood word hits. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. The cumulative score is calculated using, for nucleotide sequences, the parameter M (reward score for a pair of matching residues; always above 0) and the parameter N (penalty score for mismatching residues; always below 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from the maximum achieved cumulative alignment score, when the cumulative score falls to 0 or below due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.For amino acid sequences, the BLASTP program uses as default a word length (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix [see Henikoff and Henikoff, (1992) Proc Natl Acad Sci USA 89(22):10915-10919] alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For nucleotide sequences, the BLASTN program uses as default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0151] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, (1993) Proc Natl Acad Sci USA 90(12):5873-5877). One measure of similarity provided by the BLAST algorithm is the minimum sum likelihood (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the minimum sum likelihood in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0152] In certain embodiments, the Zscan4 polynucleotide encoding the Zscan4 polypeptide is a human ZSCAN4 polynucleotide or a homolog thereof. In some embodiments, the Zscan4 polynucleotide encodes a human ZSCAN4 protein comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to one of the group consisting of SEQ ID NOs: 38-42.

[0153] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Thus, "comprising A or B" means including A or B, or including A and B. It should be further understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for illustrative purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All patent publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including explanations of terms, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting. Illustrative Embodiments 1. 1. A method for transiently inducing temperature-sensitive activity of a temperature-sensitive agent, comprising: i) incubating one or more cells containing a temperature-sensitive agent at a permissive temperature for inducing the temperature-sensitive activity for a period of time sufficient for the temperature-sensitive activity to produce an effect in the one or more cells; and ii) incubating one or more cells at a non-permissive temperature, wherein the non-permissive temperature reduces the temperature-sensitive activity of the temperature-sensitive agent; Including, wherein the temperature-sensitive agent comprises a therapeutic agent comprising human ZSCAN4 protein or a nucleic acid comprising a coding region of human ZSCAN4, and the effect comprises a therapeutic effect. 2. Before step i), contacting one or more cells with a temperature sensitive agent; 2. The method of embodiment 1, further comprising: 3. 3. The method of embodiment 2, wherein the one or more cells are at a permissive temperature when contacted with the temperature-sensitive agent. 4. 4. The method of any one of embodiments 1 to 3, further comprising administering the one or more cells to a subject in need of a therapeutic effect. 5. 4. The method of any one of embodiments 1-3, wherein incubating the one or more cells at a non-permissive temperature comprises administering the one or more cells to a subject in need of a therapeutic effect, wherein the subject's body temperature is the non-permissive temperature. 6. 6. The method of embodiment 4 or 5, wherein the one or more cells are further incubated at a non-permissive temperature prior to administering the one or more cells to a subject. 7. 7. The method of any one of embodiments 2-6, wherein the one or more cells are isolated from a subject prior to contacting the one or more cells with a temperature-sensitive agent. 8. The method of any one of embodiments 1 to 7, wherein the therapeutic effect comprises increasing telomere length in one or more cells. 9. 9. The method of any one of embodiments 1 to 8, wherein said one or more cells are mammalian cells. 10. The method of any one of embodiments 3 to 8, wherein the subject is a human subject. 11. 1. A method of transiently inducing a temperature-sensitive activity of a temperature-sensitive agent in a human subject, wherein one or more cells of the subject contain a temperature-sensitive agent, wherein the temperature-sensitive activity of the temperature-sensitive agent is induced at a permissive temperature, and wherein the permissive temperature is less than a body temperature of the subject, comprising: i) Lowering the subject's body temperature to a tolerable level; ii) maintaining the reduced body temperature for a period of time sufficient for the temperature-sensitive activity to elicit an effect in the subject; and iii) raising the subject's body temperature to normothermia; Including, wherein the temperature-sensitive agent comprises a therapeutic agent comprising human ZSCAN4 protein or a nucleic acid comprising a coding region of human ZSCAN4, and the effect is a therapeutic effect. 12. 1. A method of transiently inducing a temperature-sensitive activity of a temperature-sensitive agent in a human subject, wherein the temperature-sensitive activity of the temperature-sensitive agent is induced at a permissive temperature, and wherein the permissive temperature is less than the body temperature of the subject, comprising: i) Lowering the subject's body temperature to a tolerable level; ii) administering a temperature-sensitive agent to one or more cells of the subject; iii) maintaining the reduced body temperature for a period of time sufficient for the temperature-sensitive activity to elicit an effect in the subject; and iv) increasing the subject's body temperature and returning it to normothermia; Including, The method, wherein step (i) is performed before, after, or simultaneously with step (ii), wherein the temperature-sensitive agent comprises a therapeutic agent comprising human ZSCAN4 protein or a nucleic acid comprising the coding region of human ZSCAN4, and wherein the effect is a therapeutic effect. 13. 13. The method of embodiment 12, wherein the temperature-sensitive agent is administered systemically. 14. 14. The method of embodiment 13, wherein the temperature-sensitive agent is administered intravenously. 15. 13. The method of embodiment 12, wherein the temperature-sensitive agent is administered to a specific tissue or organ of the subject. 16. 16. The method of embodiment 15, wherein the temperature-sensitive agent is administered to the brain or spinal cord by epidural injection. 17. 16. The method of embodiment 15, wherein the temperature-sensitive agent is administered to the target organ by intradermal injection. 18. 16. The method of embodiment 15, wherein the temperature-sensitive agent is administered to the target organ endoscopically using an injection needle catheter. 19. 16. The method of embodiment 15, wherein the temperature-sensitive agent is administered to the target organ by a vascular catheter. 20. 20. The method of any one of embodiments 17-19, wherein the target organ is selected from the group consisting of liver, kidney, skeletal muscle, cardiac muscle, pancreas, spleen, heart, brain, spinal cord, skin, eye, lung, intestine, thymus, bone marrow, bone, and cartilage. twenty one. 13. The method of embodiment 12, wherein the temperature-sensitive agent is administered by inhalation. twenty two. 22. The method of any one of embodiments 11-21, wherein lowering the subject's body temperature comprises using a targeted temperature management (TTM) procedure, wherein the TTM procedure comprises applying one of the group consisting of a cooling catheter, a cooling blanket, and ice to the subject. twenty three. The method of any one of embodiments 11-22, wherein the subject is a mammalian subject, optionally wherein the subject is a human. twenty four. 24. The method of any one of embodiments 1-23, wherein the temperature-sensitive agent comprises a human ZSCAN4 protein. twenty five. 24. The method of any one of embodiments 1-23, wherein the temperature-sensitive agent comprises a nucleic acid comprising a coding region of human ZSCAN4. 26. 26. The method of embodiment 25, wherein the temperature-sensitive viral vector comprises a nucleic acid comprising a coding region of human ZSCAN4. 27. 27. The method of embodiment 26, wherein said temperature-sensitive viral vector is selected from the group consisting of Sendai virus, adenovirus, adeno-associated virus, retrovirus, and alphavirus. 28. 27. The method of embodiment 26, wherein the temperature-sensitive viral vector is an alphavirus. 29. 29. The method of embodiment 28, wherein said alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 30. 27. The method of embodiment 26, wherein the temperature-sensitive viral vector is Sendai virus. 31. The method of embodiment 30, wherein the Sendai virus is SeV18+ / TS15ΔF. 32. 32. The method of any one of embodiments 26 to 31, wherein the temperature-sensitive activity comprises replication and transcription of a temperature-sensitive viral vector. 33. 26. The method of embodiment 25, wherein the temperature-sensitive self-replicating RNA comprises a nucleic acid comprising the coding region of human ZSCAN4. 34. 34. The method of embodiment 33, wherein said self-replicating RNA comprises an alphavirus replicon lacking a viral structural protein coding region. 35. 35. The method of embodiment 34, wherein said alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 36. 36. The method of any one of embodiments 33 to 35, wherein the temperature-sensitive activity comprises one or both of replication and transcription of a temperature-sensitive self-replicating RNA. 37. 37. The method of any one of embodiments 25 to 36, wherein the coding region is operably linked to a promoter. 38. 11. The method of any one of embodiments 1-10, wherein the period of time sufficient for the temperature-sensitive activity to produce a therapeutic effect ranges from about 12 hours to about 12 weeks, optionally wherein the period of time is 1 to 7 days. 39. The method of any one of embodiments 11-37, wherein the period of time sufficient to induce a therapeutic effect in the subject is from about 12 hours to about 7 days, optionally wherein the period of time is from about 12 hours to about 72 hours. 40. 40. The method of any one of the preceding embodiments, wherein the permissible temperature is in the range of 30°C to 36°C or 38°C to 50°C. 41. 41. The method of embodiment 40, wherein the permissible temperature is 33°C ± 0.5°C. 42. 42. The method of embodiment 40 or embodiment 41, wherein the non-permissive temperature is 37°C ± 0.5°C. 43. 43. The method of any one of embodiments 1 to 42, wherein said one or more cells are human cells. 44. 44. The method of embodiment 43, wherein the one or more human cells are adult stem cells, tissue stem cells, progenitor cells, embryonic stem cells, or induced pluripotent stem cells. 45. 44. The method of embodiment 43, wherein said one or more human cells are selected from the group consisting of hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, adipose stem cells, neural stem cells, and germline stem cells. 46. 44. The method of embodiment 43, wherein the one or more human cells are somatic cells, mature cells, or differentiated cells. 47. 47. The method of embodiment 46, wherein the one or more human cells are selected from the group consisting of epidermal cells, fibroblasts, lymphocytes, hepatocytes, epithelial cells, myocytes, chondrocytes, osteocytes, adipocytes, cardiomyocytes, pancreatic cells, pancreatic beta cells, keratinocytes, erythrocytes, peripheral blood mononuclear cells (PBMCs), neurons, glial cells, neural cells, astrocytes, germ cells, sperm cells, and oocytes. 48. 44. The method of embodiment 43, wherein said one or more human cells are human bone marrow cells. 49. 49. The method of embodiment 48, wherein said human bone marrow cells are CD34+ hematopoietic stem cells. 50. The method of embodiment 48 or embodiment 49, wherein the human subject is afflicted with a telomere biology disorder, optionally wherein the subject is afflicted with a bone marrow failure disorder. 51. 1. A method for treating a disease of the blood or blood-forming organs, comprising: i) mobilizing hematopoietic stem cells from bone marrow into the peripheral blood of a human subject suffering from said disease; ii) isolating CD34+ cells from a sample of peripheral blood mononuclear cells obtained from the subject; iii) incubating the isolated CD34+ cells at a temperature of 33°C ± 0.5°C; iv) contacting the incubated CD34+ cells with a temperature-sensitive Sendai virus vector containing a heterologous nucleic acid comprising a coding region of human ZSCAN4; v) maintaining the contacted CD34+ cells at a permissive temperature of 33°C ± 0.5°C for a period of at least about 12 to 72 hours, wherein replication and transcription of the temperature-sensitive Sendai virus vector occurs at the permissive temperature, leading to increased expression of human ZSCAN4; and vi) injecting the contacted CD34+ cells into a subject under conditions suitable for cell engraftment to treat the disease; A method comprising: 52. 1. A method for treating a disease of the blood or blood-forming organs, comprising: i) mobilizing hematopoietic stem cells from bone marrow cells into the peripheral blood of a human subject suffering from the disease; ii) isolating CD34+ cells from a sample of peripheral blood mononuclear cells obtained from the subject; iii) contacting the isolated CD34+ cells with a temperature-sensitive Sendai virus vector containing a heterologous nucleic acid comprising a coding region of human ZSCAN4; iv) incubating the contacted CD34+ cells at a permissive temperature of 33°C ± 0.5°C for a period of at least about 12 to 72 hours, wherein replication and transcription of the temperature-sensitive Sendai virus vector occurs at the permissive temperature, leading to increased expression of ZSCAN4; and v) injecting the contacted CD34+ cells into a subject under conditions suitable for cell engraftment to treat the disease; A method comprising: 53. incubating the contacted CD34+ cells at a non-permissive temperature of 37°C ± 0.5°C prior to infusion of the contacted CD34+ cells into a subject, wherein replication and transcription of the temperature-sensitive Sendai virus vector and expression of human ZSCAN4 are stopped at the non-permissive temperature; 52. The method of embodiment 51, further comprising, after step v), 54. incubating the contacted CD34+ cells at a non-permissive temperature of 37°C ± 0.5°C prior to infusion of the contacted CD34+ cells into a subject, wherein replication and transcription of the temperature-sensitive Sendai virus vector and expression of human ZSCAN4 are stopped at the non-permissive temperature; 53. The method of embodiment 52, further comprising after step iv). 55. 55. The method of embodiment 53 or embodiment 54, wherein the contacted CD34+ cells are incubated at a non-permissive temperature of 37°C ± 0.5°C for about 30 minutes to about 10 days, optionally for about 30 to 180 minutes. 56. 56. The method of any one of embodiments 51-55, wherein the hematopoietic stem cells are mobilized by administering to the subject one or both of granulocyte colony-stimulating factor and plerixafor. 57. 57. The method of any one of embodiments 51-56, wherein the peripheral blood mononuclear cells are obtained from the subject by apheresis. 58. 58. The method of any one of embodiments 51 to 57, wherein the CD34+ cells are isolated from peripheral blood mononuclear cells by positive selection using anti-CD34 antibodies and magnetic beads. 59. 59. The method of any one of embodiments 51-58, wherein the contacted CD34+ cells are washed and resuspended in a sterile, isotonic aqueous solution prior to infusion. 60. The method described in embodiment 59, wherein the contacted CD34+ cells are injected intravenously at a dose of about 1.0 x 10^5 cells / kg to about 1.0 x 10^7 cells / kg, optionally about 2.0 to 8.0 x 10^6 cells / kg. 61. 1. A method for treating a disease of the blood or blood-forming organs, comprising: i) administering to a human subject suffering from the disease a temperature-sensitive Sendai virus vector comprising a heterologous nucleic acid comprising a coding region of human ZSCAN4; ii) reducing the subject's core body temperature to a tolerable temperature of 33°C ± 0.5°C; iii) maintaining the subject's core body temperature at a permissive temperature for a period of about 12 hours to about 7 days, or about 12 to 72 hours, wherein replication and transcription of the temperature-sensitive Sendai virus vector occurs at the permissive temperature, leading to increased expression of human ZSCAN4; and iv) restoring the subject's core body temperature to a normal, non-permissive temperature of 37°C ± 0.5°C, wherein replication and transcription of the temperature-sensitive Sendai virus vector and expression of human ZSCAN4 are stopped at the non-permissive temperature; A method comprising: 62. 1. A method for treating a disease of the blood or blood-forming organs, comprising: i) reducing the core body temperature of a subject suffering from the disease to a tolerable temperature of 33°C ± 0.5°C; ii) administering to the subject a temperature-sensitive Sendai virus vector containing a heterologous nucleic acid comprising a coding region of human ZSCAN4; iii) maintaining the subject's core body temperature at a permissive temperature for a period of about 12 hours to about 7 days, or about 12 to 72 hours, wherein replication and transcription of the temperature-sensitive Sendai virus vector occurs at the permissive temperature, leading to increased expression of human ZSCAN4; and iv) restoring the subject's core body temperature to a normal, non-permissive temperature of 37°C ± 0.5°C, wherein replication and transcription of the temperature-sensitive Sendai virus vector and expression of human ZSCAN4 are stopped at the non-permissive temperature; A method comprising: 63. The method of embodiment 61 or embodiment 62, wherein the subject's core body temperature is lowered using a targeted temperature management (TTM) procedure, wherein the TTM procedure comprises applying one of the group consisting of a cooling catheter, a cooling blanket, and ice to the subject. 64. The method of any one of embodiments 51-63, wherein the human subject is diagnosed with bone marrow failure prior to treatment, optionally wherein the bone marrow failure comprises one or more of neutropenia, thrombocytopenia, and anemia. 65. The method of any one of embodiments 51-64, wherein the subject does not have cancer. 66. 66. The method of any one of embodiments 51-65, wherein said disease is a telomere biology disorder. 67. 67. The method of embodiment 66, wherein said telomere biology disorder is selected from the group consisting of dyskeratosis congenita, Wheeler-Leiderson syndrome, Rewes syndrome, Coats-Plus syndrome, idiopathic pulmonary fibrosis, and liver cirrhosis. 68. The telomere biology disorder is one of: i) age-adjusted mean telomere length below the 1st percentile in one or more of peripheral blood lymphocytes, B cells, and naive T cells; and ii) a pathogenic mutation in a gene selected from the group consisting of DKC1, TERC, TERT, NOP10, NHP2, TINF2, CTC1, PARN, RTEL1, ACD, USB1, and WRAP53; 67. The method of embodiment 66, as defined by one or both of: 69. The method of any one of embodiments 51 to 63, wherein the disease is a bone marrow failure syndrome. 70. 70. The method of embodiment 69, wherein said bone marrow failure syndrome is selected from the group consisting of Fanconi anemia, amegakaryocytic thrombocytopenia, aplastic anemia, Diamond-Blackfan anemia, paroxysmal nocturnal hemoglobinuria, Pearson syndrome, Schwachman-Diamond syndrome, and myelodysplastic syndrome. 71. 65. The method of embodiment 64, wherein the disease is associated with a karyotypic abnormality. 72. 72. The method of any one of embodiments 1 to 71, wherein the amino acid sequence of human ZSCAN4 comprises SEQ ID NO:38 or is at least 95% identical to SEQ ID NO:38. 73. The method of any one of embodiments 1 to 71, wherein the amino acid sequence of human ZSCAN4 comprises one of the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, or is at least 95% identical to one of the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42. 74. 51. The method of any one of embodiments 26 to 50, wherein the temperature-sensitive viral vector or temperature-sensitive self-replicating RNA comprises a nonstructural protein coding region with an insertion of 12 to 18 nucleotides, wherein the insertion results in expression of nonstructural protein 2 (nsP2 = helicase proteinase) nsP2 comprising 4 to 6 additional amino acids between beta-sheet 5 and beta-sheet 6, optionally wherein the additional amino acids confer temperature sensitivity to the viral vector or self-replicating RNA. 75. 75. The method of embodiment 74, wherein the additional amino acids comprise one sequence selected from the group consisting of SEQ ID NO: 43 (GCGRT), SEQ ID NO: 44 (TGAAA), and SEQ ID NO: 45 (LRPHP). 76. 75. The method of embodiment 74, wherein the additional amino acids comprise the sequence of SEQ ID NO: 44 (TGAAA). 77. 77. The method of embodiment 76, wherein the amino acid sequence of NsP2 comprises one sequence selected from the group consisting of SEQ ID NOs: 29 to 36. 78. A temperature-sensitive agent, wherein the agent is a temperature-sensitive viral vector or a temperature-sensitive self-replicating RNA comprising a heterologous nucleic acid comprising a coding region for human ZSCAN4 and a nonstructural protein coding region with an insertion of 12 to 18 nucleotides, wherein the insertion results in expression of nonstructural protein 2 (nsP2 = helicase proteinase) comprising 4 to 6 additional amino acids between beta sheet 5 and beta sheet 6, and optionally wherein the additional amino acids confer temperature sensitivity to the viral vector or self-replicating RNA. 79. The temperature-sensitive agent of embodiment 78, wherein the additional amino acids comprise one sequence selected from the group consisting of SEQ ID NO: 43 (GCGRT), SEQ ID NO: 44 (TGAAA), and SEQ ID NO: 45 (LRPHP). 80. 79. The temperature-sensitive agent of embodiment 78, wherein the additional amino acids comprise the sequence of SEQ ID NO: 44 (TGAAA). 81. The temperature-sensitive agent of embodiment 81, wherein the amino acid sequence of NsP2 comprises one sequence selected from the group consisting of SEQ ID NOs: 29-36. 82. The temperature sensitive agent of any one of embodiments 78-81, wherein the agent is a temperature sensitive alphavirus vector. 83. 82. The temperature-sensitive agent of any one of embodiments 78-81, wherein the agent is a temperature-sensitive self-replicating RNA comprising an alphavirus replicon lacking viral structural protein coding regions. 84. The temperature sensitive agent of embodiment 82 or embodiment 83, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 85. The temperature sensitive agent of embodiment 82 or embodiment 83, wherein the alphavirus is Venezuelan equine encephalitis virus. 86. 1. A method for transiently inducing temperature-sensitive activity of a temperature-sensitive agent (ts agent) in a subject, wherein the ts agent is a temperature-sensitive viral vector or a temperature-sensitive self-replicating RNA comprising a heterologous nucleic acid comprising a coding region of human ZSCAN4, wherein one or more cells contain the ts agent at or near the surface of the body of the subject, wherein the temperature-sensitive activity of the ts agent comprises expression of human ZSCAN4 at a permissive temperature, and wherein the permissive temperature is the surface body temperature of the subject, comprising: i) maintaining the subject's surface body temperature at a permissive temperature for a period of time sufficient for the temperature-sensitive activity to induce an effect in the subject; and ii) raising the surface body temperature of the subject to a non-permissive temperature for a period of time sufficient to cause temperature-sensitive activity to cease in the subject; A method comprising: 87. 1. A method for transiently inducing a temperature-sensitive activity of a temperature-sensitive agent (ts agent) in a subject, wherein the ts agent is a temperature-sensitive viral vector or a temperature-sensitive self-replicating RNA comprising a heterologous nucleic acid comprising a coding region of human ZSCAN4, wherein the temperature-sensitive activity of the ts agent comprises expression of human ZSCAN4 at a permissive temperature, and wherein the permissive temperature is the surface body temperature of the subject, comprising: i) administering a ts agent to one or more cells at or near the surface of the body of a subject; and ii) maintaining the subject's surface body temperature at a permissive temperature for a period of time sufficient for the temperature-sensitive activity to elicit an effect in the subject; A method comprising: 88. iii) raising the surface body temperature of the subject to a non-permissive temperature for a period of time sufficient to cause temperature-sensitive activity to cease in the subject; 88. The method of embodiment 87, further comprising: 89. The method of embodiment 86 or embodiment 87, wherein the temperature-sensitive agent is administered i) intradermally or subcutaneously; or ii) intramuscularly. 90. The method of embodiment 86 or embodiment 87, wherein the temperature-sensitive agent is administered intranasally. 91. 91. The method of any one of embodiments 86-90, wherein the non-permissive temperature is greater than 36°C and the permissive temperature is less than 36°C, optionally wherein the permissive temperature is about 31°C to about 34°C, or about 33°C±0.5°C, and the non-permissive temperature is 37°C±0.5°C. 92. The method of any one of embodiments 86 to 91, wherein the effect of human ZSCAN4 expression is a prophylactic or therapeutic effect. 93. The method of any one of embodiments 86 to 92, wherein the ts agent is a temperature-sensitive viral vector and the temperature-sensitive activity further comprises replication and transcription of the temperature-sensitive viral vector. 94. 94. The method of embodiment 93, wherein said temperature-sensitive viral vector is selected from the group consisting of Sendai virus, adenovirus, adeno-associated virus, retrovirus, and alphavirus. 95. 94. The method of embodiment 93, wherein said temperature-sensitive viral vector is an alphavirus, optionally wherein said alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 96. 94. The method of embodiment 93, wherein the temperature-sensitive viral vector is Sendai virus. 97. 93. The method of any one of embodiments 86-92, wherein the ts agent is a temperature-sensitive self-replicating RNA and the temperature-sensitive activity further comprises one or both of replication and transcription of the temperature-sensitive self-replicating RNA. 98. 98. The method of embodiment 97, wherein said self-replicating RNA comprises an alphavirus replicon that lacks the viral structural protein coding region of the alphavirus. 99. 99. The method of embodiment 98, wherein said alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 100. 99. The method of embodiment 98, wherein said alphavirus is Venezuelan equine encephalitis virus. 101. The method of any one of embodiments 86-100, wherein the period of time sufficient for the temperature-sensitive activity to have an effect ranges from about 12 hours to about 12 weeks, optionally, the period of time is 1 to 7 days. 102. The method of any one of embodiments 86-100, wherein the period of time sufficient to induce an effect in the subject is from about 12 hours to about 7 days, optionally wherein the period of time is from about 12 hours to about 72 hours. 103. The method of any one of embodiments 86-102, wherein said subject is a mammalian subject, optionally wherein said subject is a human. [Example]

[0154] Abbreviations: Aura (Aura virus); BFV (Barmah Forest virus); GFP (Green fluorescent protein); GOI (Gene of interest); IRES (Internal ribosome entry site); LUC (Luciferase); ONNV (Onyong-Nyong virus); RRV (Ross River virus); SeV (Sendai virus); SeVt (Temperature-sensitive Sendai virus); SFV (Semliki Forest virus); shRNA (Short hairpin RNA); SINV (Sindbis virus); srRNA (Self-replicating RNA); ts (Temperature sensitive); ts agent (Temperature-sensitive agent); VEEV (Venezuelan equine encephalitis virus); and WEEV (Western equine encephalitis virus).

[0155] The following examples are provided to illustrate certain particular features and / or embodiments and are not intended to limit the claimed disclosure. Example 1: Temperature-sensitive agents

[0156] This example describes temperature-sensitive agents (ts agents) that function at temperatures below or above normal body temperature but are non-functional or exhibit reduced functionality at normal body temperature. ts agents are suitable for use in ex vivo, semi-in vivo, and in vivo therapies. Temperature-sensitive viral vectors and self-replicating RNAs are engineered to express genes of interest (GOIs), short hairpin RNAs (shRNAs), long non-coding RNAs, and / or other genetic elements. For example, proteins with temperature-sensitive mutations are functional at lower temperatures (e.g., at 30°C) but not functional at normal body temperature (e.g., at 37°C). Unless otherwise specified, normal body temperature is the normal human body temperature of 37°C ± 0.5°C.

[0157] A particular GOI is the ZSCAN4 gene, which is also referred to herein as the coding region of the ZSCAN4 gene or the nucleic acid encoding the ZSCAN4 protein. The amino acid sequence of the human ZSCAN4 protein is set forth as (SEQ ID NO: 38). Example 2: Temperature-sensitive Sendai virus vector (SeVt)

[0158] This example describes a temperature-sensitive Sendai virus vector (SeVt), which can be used for temperature-specific gene expression. Sendai virus vectors are based on Sendai virus, a single-stranded RNA virus of the paramyxovirus subfamily. SeV18 / TS15ΔF is a temperature-sensitive Sendai virus vector that is capable of viral replication and gene expression when maintained at 32–35°C. However, viral replication is halted at nonpermissive temperatures above 37°C (Ban et al., PNAS 2011). Example 3: Temperature-sensitive self-replicating RNAs (srRNAs)

[0159] This example describes the finding that mutations in the nsP2 protein encoded by a Venezuelan equine encephalitis virus (VEEV) vector exhibit temperature sensitivity. The temperature-sensitive system allows expression of a gene of interest (GOI) at 30°C to 33°C but disables expression above 37°C. The srRNA vector allows higher expression of the GOI than synthetic RNA encoding the GOI. GOI expression ceases when the temperature is shifted to 37°C (e.g., a nonpermissive temperature). The specific temperature-sensitive mutation (mutation 2) identified in this study lies within a well-conserved region within alphaviruses. Compared to Sendai virus vectors (SeVt), srRNAts may be more attractive for some applications because they can be utilized in nonviral RNA expression systems. Materials and Methods cell culture

[0160] Human adipose stem cell-derived iPS cell line (ADSC-iPSC) was purchased from System Biosciences (Palo Alto, CA). Cells were routinely maintained as undifferentiated human pluripotent cells (hPSC) according to standard hPSC culture methods. Briefly, cells were cultured in StemFit basic02 (Ajinomoto, Japan) supplemented with 100 ng / ml FGF2. Furthermore, cells were cultured on cell culture dishes coated with laminin-511 matrix (iMatrix-511, Nippi, Japan). VEEV vector

[0161] The VEEV vector plasmid was assembled using a synthetic DNA fragment based on publicly available sequence information (T7-VEE-IRES-Puro, hereafter referred to as "srRNA1wt"). According to Yoshioka et al., 2013, the VEEV vector backbone was originally derived as described by Petrakova et al., 2005. Seventy-four hundred candidate sequences identified by insertional mutagenesis and massively parallel sequencing (Beitzel et al., 2010) were used to derive potential temperature-sensitive mutants. The original large-scale screen was performed using a 15-bp transposon-mediated insertion into the VEEV genome (Figure 1A). Subsequently, many 15-bp insertion VEEV mutants capable of growth at 30°C or 40°C were isolated. These data provided initial mutants for further investigation; however, these sequences were not known to exhibit temperature sensitivity, such as permissiveness at 32°C or 33°C and nonpermissiveness at 37°C. Three mutant sequences—variant 1 (ts1, Figure 1B), variant 2 (ts2, Figure 1C), and variant 3 (ts3, Figure 1D)—were selected from a total of 7,480 candidate mutant sequences (Data Set S1 from Beitzel et al., 2010). These mutant DNA fragments (Figure 2) were synthesized and cloned into the VEEV vector and designated srRNA1ts1 (variant 1), srRNA1ts2 (variant 2), and srRNA1ts3 (variant 3). Variant 4 was designed and contains the 5'-region of the viral sequence (the 5'-UTR and part of the N-terminal protein sequence of the RNA-dependent RNA polymerase, which is known to contain a 51-nt conserved sequence element (CSE)). In this case, nucleotides were systematically changed to less thermostable variants (e.g., G → A) while maintaining the amino acid sequence (Figure 3). The sequence of this region within srRNA1ts2 was replaced to create srRNA1ts4 (i.e., containing both variant 4 and variant 2). Synthetic RNAs were produced from these vectors according to Yoshioka et al., 2013. result Evaluation of temperature sensitivity of srRNA1ts2-GFP and srRNA1ts3-GFP at 30°C, 32°C, and 37°C

[0162] ADSC-iPSC cells were plated on 24-well plates at a density of 80,000 cells / well. 24 hours later, cells were transfected with srRNA1wt-GFP, srRNA1ts2-GFP, or srRNA1ts3-GFP. For transfection, each well of the 24-well plate was treated with 0.5 μg of synthetic RNA (srRNA) mixed with 1 μl of JetMessenger (Polyplus) transfection reagent in a final volume of 50 μl. After adding the transfection complex to the cells, 450 μl of medium was added. The cells were incubated at either 30°C, 32°C, or 37°C. Six hours after transfection, the medium was replaced and the transfection complex was removed. Phase contrast and fluorescence images were taken at 20 and 48 hours. Figure 4A shows that the wild-type (srRNA1wt-GFP) strongly expressed GFP at 37°C but only weakly at both 30°C and 32°C. In contrast, mutant 2 (srRNA1ts2-GFP) expressed GFP at 30°C and 32°C but not at 37°C. Mutant 3 (srRNA1ts3-GFP) expressed GFP at 30°C and 32°C but also at 37°C. Based on these results, mutant 2 was selected for further development. As expected, srRNA induced much higher GFP expression compared to the GFP expression level achieved by transfection of synthetic mRNA encoding GFP alone (Figure 4B). Evaluation of the temperature sensitivity of srRNA1ts1-GFP and srRNA1ts2-GFP at 32°C

[0163] ADSC-iPSC cells were plated on 24-well plates at a density of 50,000 cells / well. 24 hours later, the cells were transfected with srRNA1wt-GFP, srRNA1ts2-GFP, or srRNA1ts3-GFP. For transfection, each well of the 24-well plate was treated with 0.5 μg of synthetic RNA (srRNA) mixed with 1 μl of JetMessenger (Polyplus) transfection reagent in a final volume of 50 μl. After adding the transfection complex to the cells, 450 μl of medium was added. The cells were incubated at 32°C. Six hours after transfection, the medium was replaced and the transfection complex was removed. Phase-contrast and fluorescence images were taken at 24, 48, 72, 96, 120, 144, 168, 192, 240, and 288 hours.

[0164] Figure 5 shows the results. GFP expression from the wild type (srRNA1wt-GFP) began at 24 hours and continued until the end of the observation period (at 288 hours), but was very weak throughout the time course. In contrast, GFP expression from mutant 2 (srRNA1ts2-GFP) was very strong throughout the time course. Mutant 1 (srRNA1ts1-GFP) did not express any GFP (based on observations at 24 hours and 168 hours). Based on these results, mutant 2 was selected for further development. Evaluation of temperature sensitivity of srRNA1ts2-GFP and srRNA1ts4-GFP at 32°C, 33°C, and 37°C

[0165] ADSC-iPSC cells were plated on 24-well plates at a density of 50,000 cells / well. 24 hours later, the cells were transfected with srRNA1ts2-GFP or srRNA1ts4-GFP. For transfection, each well of the 24-well plate was treated with 0.5 μg of synthetic RNA (srRNA) mixed with 1 μl of JetMessenger (Polyplus) transfection reagent in a final volume of 50 μl. After adding the transfection complex to the cells, 450 μl of medium was added. The cells were incubated at either 32°C, 33°C, or 37°C. Six hours after transfection, the medium was replaced and the transfection complex was removed. Phase contrast and fluorescence images were taken at 20, 48, and 96 hours.

[0166] Figure 6 shows the results. At 32°C and 33°C, GFP expression from mutant 2 (srRNA1ts2-GFP) began as early as 20 h but significantly increased at 48 h and further increased at 96 h. GFP expression was stronger at 33°C than at 32°C. Consistent with previous experiments, GFP was not expressed at all at 37°C. srRNA1ts4-GFP (including both mutant 2 and mutant 4) showed a similar temperature profile to srRNA1ts2-GFP, but GFP expression was much weaker overall. Based on these results, mutant 2 was selected for further development. Evaluation of temperature sensitivity of srRNA1ts2-GFP at 32°C

[0167] ADSC-iPSC cells were plated on 24-well plates at a density of 80,000 cells / well. 24 hours later, the cells were transfected with srRNA1ts2-GFP. For transfection, each well of the 24-well plate was treated with 0.5 μg of synthetic RNA (srRNA) mixed with 1 μl of JetMessenger (Polyplus) transfection reagent in a final volume of 50 μl. After adding the transfection complex to the cells, 450 μl of medium was added. The cells were incubated at 32°C. Six hours after transfection, the medium was changed to remove the transfection complex. The medium was changed daily. The srRNA1ts2-GFP vector contains a puromycin N-acetyltransferase (pac) selection gene inserted after the "IRES" sequence, allowing selection using puromycin. Experiments were performed in the absence (upper panel) or presence (lower panel) of 1 μg / ml puromycin. For cell selection with puromycin, puromycin was added at 48 and 72 hours. Phase contrast and fluorescence images were taken at 24, 48, 72, 96, 144, 168, and 192 hours.

[0168] Figure 7 shows the results. At 32°C, GFP expression from srRNA1ts2-GFP began as early as 24 h, increased significantly at 48 h, and peaked at 72 and 96 h. GFP expression continued throughout the entire observation period (192 h). The GFP expression pattern did not appear to be altered by the addition of puromycin. Evaluation of the temperature sensitivity of srRNA1ts2-GFP after switching from 32°C to 37°C after 24 hours

[0169] ADSC-iPSC cells were plated on 24-well plates at a density of 80,000 cells / well. 24 hours later, the cells were transfected with srRNA1ts2-GFP. For transfection, each well of the 24-well plate was treated with 0.5 μg of synthetic RNA (srRNA) mixed with 1 μl of JetMessenger (Polyplus) transfection reagent in a final volume of 50 μl. After adding the transfection complex to the cells, 450 μl of medium was added. The cells were incubated at 32°C. Six hours after transfection, the medium was changed to remove the transfection complex. The medium was changed daily. The srRNA1ts2-GFP vector contains a puromycin N-acetyltransferase (pac) selection gene inserted after the "IRES" sequence, allowing selection using puromycin. Experiments were performed in the absence (upper panel) or presence (lower panel) of 1 μg / ml puromycin. For cell selection with puromycin, puromycin was added at 48 and 72 h. To test the effect of temperature shift, cell cultures were transferred to a CO2 incubator maintained at 37 °C for 24 h (24 h after transfection). Phase contrast and fluorescence images were taken at 24, 48, 72, 96, 144, 168, and 192 h.

[0170] Figure 8 shows the results. At 32°C, GFP expression from srRNA1ts2-GFP began as early as 24 h and continued to increase even after the temperature was switched to 37°C at 24 h. GFP expression peaked at 48 h and then began to decline. By 96 h, GFP expression became very weak, and by 144 h, GFP expression was no longer detectable. Subsequently, GFP expression was absent until the end of the 192-h observation period. Thus, expression of the GOI (referred to here as GFP) rapidly ceased when the temperature was shifted from 33°C (the permissive temperature) to 37°C (the nonpermissive temperature). The GFP expression pattern did not appear to be altered by the addition of puromycin. Evaluation of the temperature sensitivity of srRNA1ts2-GFP after switching from 32°C to 37°C after 48 hours

[0171] ADSC-iPSC cells were plated on 24-well plates at a density of 80,000 cells / well. 24 hours later, the cells were transfected with srRNA1ts2-GFP. For transfection, each well of the 24-well plate was treated with 0.5 μg of synthetic RNA (srRNA) mixed with 1 μl of JetMessenger (Polyplus) transfection reagent in a final volume of 50 μl. After adding the transfection complex to the cells, 450 μl of medium was added. The cells were incubated at 32°C. Six hours after transfection, the medium was changed to remove the transfection complex. The medium was changed daily. The srRNA1ts2-GFP vector contains a puromycin N-acetyltransferase (pac) selection gene inserted after the "IRES" sequence, allowing selection using puromycin. Experiments were performed in the absence (upper panel) or presence (lower panel) of 1 μg / ml puromycin. For cell selection with puromycin, puromycin was added at 48 and 72 h. To test the effect of temperature shift, cell cultures were transferred to a CO2 incubator maintained at 37 °C for 48 h (48 h after transfection). Phase contrast and fluorescence images were taken at 24, 48, 72, 96, 144, 168, and 192 h.

[0172] Figure 9 shows the results. At 32°C, GFP expression from srRNA1ts2-GFP began as early as 24 h and further increased by 48 h. GFP expression continued for up to 96 h, even after a temperature shift to 37°C at 48 h. However, GFP expression began to decline from 72 h, and by 96 h, GFP expression was very weak. By 144 h, GFP expression was barely detectable and had completely ceased by 192 h. Thus, expression of the GOI (referred to here as GFP) rapidly ceased when the temperature was shifted from 33°C (the permissive temperature) to 37°C (the nonpermissive temperature). The GFP expression pattern did not appear to be altered by the addition of puromycin. Evaluation of the temperature sensitivity of srRNA1ts2-GFP after switching from 32°C to 37°C after 72 hours

[0173] ADSC-iPSC cells were plated on 24-well plates at a density of 80,000 cells / well. 24 hours later, the cells were transfected with srRNA1ts2-GFP. For transfection, each well of the 24-well plate was treated with 0.5 μg of synthetic RNA (srRNA) mixed with 1 μl of JetMessenger (Polyplus) transfection reagent in a final volume of 50 μl. After adding the transfection complex to the cells, 450 μl of medium was added. The cells were incubated at 32°C. Six hours after transfection, the medium was changed to remove the transfection complex. The medium was changed daily. The srRNA1ts2-GFP vector contains a puromycin N-acetyltransferase (pac) selection gene inserted after the "IRES" sequence, allowing selection using puromycin. Experiments were performed in the absence (upper panel) or presence (lower panel) of 1 μg / ml puromycin. For cell selection with puromycin, puromycin was added at 48 and 72 h. To test the effect of temperature shift, cell cultures were transferred to a CO2 incubator maintained at 37 °C at 72 h (72 h after transfection). Phase contrast and fluorescence images were taken at 24, 48, 72, 96, 144, 168, and 192 h.

[0174] Figure 10 shows the results. At 32°C, GFP expression from srRNA1ts2-GFP began as early as 24 h and further increased by 48 h. GFP expression continued for up to 96 h, even after the temperature was switched to 37°C at 48 h. However, GFP expression began to decline from 72 h, and by 144 h, GFP expression was very weak. By 168 h, GFP expression was barely detectable and had completely ceased by 192 h. Thus, expression of the GOI (referred to here as GFP) rapidly ceased when the temperature was shifted from 33°C (the permissive temperature) to 37°C (the nonpermissive temperature). The GFP expression pattern did not appear to be altered by the addition of puromycin. Evaluation of temperature sensitivity of srRNA1ts2-GFP in fibroblasts

[0175] Human neonatal dermal fibroblasts (HDFn, passage 20) were plated on 24-well plates at a density of 10,000 cells / well. 24 hours later, cells were transfected with srRNA1wt-GFP. Transfection of srRNA1wt-GFP (0.5 μg of synthetic RNA) was performed using either JetMessenger (Polyplus) transfection reagent or Lipofectamine MessengerMax (Thermo-Fisher). Cells were incubated at 37°C. To examine the effect of B18R, which is known to suppress interferon responses, transfection and cell culture were performed in the absence (upper panel) or presence (lower panel) of 200 ng / ml B18R. The medium was changed daily. Phase-contrast and fluorescence images were taken at 0, 24, 48, and 96 hours.

[0176] Figure 11 shows the results. In the absence of B18R, GFP expression was barely detectable. In contrast, in the presence of B18R, GFP expression from srRNA1wt-GFP began as early as 24 hours and continued for 48 and 72 hours. GFP expression was strong in GFP+ cells, but the frequency of GFP+ cells was not high. This was likely due to the low transfection efficiency of srRNA1wt-GFP for human primary fibroblasts. Alignment of amino acid sequences of the alphavirus family corresponding to variant 2 (ts2)

[0177] As shown in Figure 12, even at the amino acid level, the structure of alphavirus nsP2 proteins is well conserved among family members. Based on a 3D structural model (Russo et al., 2006), the protein region where the five amino acids SEQ ID NO:44 (TGAAA) are inserted in Mutant 2 is the division between two beta-sheet structures, which is also well conserved among alphavirus family members. Therefore, it is likely that the temperature sensitivity of Mutant 2 is transferable to other alphavirus family members, including Aura (Aura virus), WEEV (Western equine encephalitis virus), BFV (Barmah Forest virus), ONNV (Onyong-Nyong virus), RRV (Ross River virus), SFV (Semliki Forest virus), and SINV (Sindbis virus). Suitable positions for insertion into nsP2 of various alphaviruses to confer temperature sensitivity are listed in Table 3-1. [Table 1]

[0178] Example 4: Temperature-sensitive antibodies This example describes temperature-sensitive antibodies. Antibodies that function at permissive temperatures (e.g., 32°C) and exhibit no or reduced functionality at nonpermissive temperatures (e.g., 37°C) are engineered by inserting or substituting amino acid sequences. Temperature-sensitive antibodies can be generated by inserting a linker oligonucleotide encoding a temperature-sensitive helix-coil transition peptide (-Glu-Ala-Ala-Ala-Lys-, set forth as SEQ ID NO: 37) as described (Kamihara and Iijima, 2000; Merutka and Stellwagen, 1990). In this manner, engineered antibodies can be generated that function at permissive temperatures (e.g., 32°C) but not at nonpermissive temperatures (e.g., 37°C). Alternatively, antibody DNA sequences from animals that naturally live in low-temperature environments (e.g., Atlantic salmon or shrimp) can be used, as these antibodies function optimally at permissive temperatures (e.g., low temperatures) but exhibit reduced functionality at nonpermissive temperatures (e.g., 37°C). Example 5: Temperature-sensitive proteins

[0179] This example describes temperature-sensitive proteins that function at permissive temperatures (e.g., 32°C) but exhibit no or reduced functionality at non-permissive temperatures (e.g., 37°C). Temperature-sensitive proteins are engineered by substituting amino acid sequences. Alternatively, temperature-sensitive proteins from animals that naturally live in cold environments (e.g., Atlantic salmon or shrimp) can be used, as these proteins function optimally at permissive temperatures (low temperatures) but exhibit reduced functionality at non-permissive temperatures (e.g., 37°C). Example 6: Temperature-sensitive RNA

[0180] This example describes temperature-sensitive RNA molecules. RNA molecules include, but are not limited to, mRNA, mRNA precursors, non-coding RNA, siRNA, and shRNA. Temperature-sensitive RNAs function at permissive temperatures (e.g., 32°C) and exhibit no or reduced functionality at non-permissive temperatures (e.g., 37°C). Temperature-sensitive RNAs were engineered by systematically changing the nucleotides of the RNA molecule (e.g., G to A) to make the mutant less thermostable, while ensuring that the functional properties of the RNA are maintained. Furthermore, the difference in thermostability of nucleotide pairs induced by temperature shifts alters the secondary structure of the RNA. Example 7: Ex vivo treatment of cells with temperature-sensitive agents

[0181] This example demonstrates a method for transiently delivering RNA or protein to cells ex vivo (Figure 13). The temperature-sensitive therapeutic agent can be any of the temperature-sensitive therapeutic agents disclosed herein. ts agents, such as srRNAs or Sendai virus vectors, are functional at permissive temperatures (e.g., 33°C) but not at non-permissive temperatures (e.g., 37°C). Target cells treated with the ts agent are cultured ex vivo at the permissive temperature for a specific duration (e.g., 3 days), and then cultured at the non-permissive temperature for a specific duration (e.g., 10 days). The level of the GOI RNA (protein translated from the RNA) increases and reaches a high value at the permissive temperature. After switching to the non-permissive temperature, the expected level of RNA gradually decreases and then reaches a non-expression level (Figure 13). Example 8: Ex vivo therapeutic use of temperature-sensitive agents

[0182] This example demonstrates a method for transiently delivering RNA or protein to cells ex vivo (Figures 14 and 15). ts agents, such as srRNAs or Sendai virus vectors, are functional at permissive temperatures (e.g., 33°C) but not at non-permissive temperatures (e.g., 37°C; human body temperature). Typically, target cells are harvested from the patient (autologous cell transplant; Figure 14), but it is also possible to use target cells isolated from a donor (allogeneic cell transplant; Figure 15). For example, target cells may be isolated using antibody-conjugated magnetic beads. Target cells are incubated with the ts agent ex vivo at a permissive temperature, e.g., 33°C, for a specific duration, e.g., 24 hours. The level of the GOI RNA (or protein translated from the RNA) increases and reaches a high level at the permissive temperature. After a therapeutic effect is induced, the cells are transplanted back into the patient to treat the patient. The activity of a temperature-sensitive therapeutic agent is not induced at the subject's normal body temperature (i.e., normal body temperature is a non-permissive temperature). Degradation of the temperature-sensitive therapeutic agent begins after the therapeutic effect is induced, and eventually, the temperature-sensitive therapeutic agent is completely degraded. Body temperature is maintained above 37°C throughout the patient's life, which prevents the ts agent from being reactivated and prevents cells other than the target cells from being treated with the ts agent. Mobilization of human peripheral blood cells

[0183] Human blood cells isolated from a patient, or donor bone marrow or peripheral blood, are treated ex vivo with ts agents at a permissive temperature. After injection of G-CSF or other mobilizing agents, human leukocytes are collected from peripheral blood using an apheresis device (e.g., COBE Spectra). Leukocytes collected from bone marrow after mobilization include granulocytes, monocytes, lymphocytes, dendritic cells, mesenchymal stem cells (MSCs), vascular endothelial cells (VECs), and CD34+ hematopoietic / progenitor cells. Treatment of these cells with ts agents is carried out ex vivo at a functional temperature (e.g., 33°C) for a specific duration (hours to weeks), ideally using a functional closed system such as Miltenyi's CliniMacs Prodigy. The treated cells are then infused into the patient at a non-permissive temperature (37°C). The ts agent, cells containing the ts agent, or products of the ts agent are non-functional in the patient. Human CD34+ hematopoietic stem / progenitor cells

[0184] Human CD34+ hematopoietic stem / progenitor cells are isolated from mobilized human peripheral blood or bone marrow cells using antibody-conjugated magnetic beads (against CD34) and used as target cells for treatment with ts agents ex vivo at a permissive temperature. After treatment with the ts agents, the human CD34+ cells are infused into the patient's body and transplanted into the patient's bone marrow. These cells ultimately produce all blood cells in the patient's body, making them suitable targets for various diseases. Any human cell, including tissue stem cells

[0185] Any human cells isolated from a patient or donor and used as target cells are treated ex vivo with a ts agent at a permissive temperature. Such cells include, but are not limited to, skin fibroblasts, follicular cells, skeletal muscle cells, liver cells, and neural tissue. Such cells also include stem cells from various tissues, such as mesenchymal stem cells, neural stem cells, muscle stem cells, skin stem cells, and intestinal stem cells. Example 9: Semi-in vivo therapeutic use of temperature-sensitive agents

[0186] This example describes a semi-in vivo method for transient delivery of RNA or protein to cells (Figure 16). A temperature-sensitive therapeutic agent is any temperature-sensitive therapeutic agent disclosed herein. A ts agent is functional at a permissive temperature (e.g., 33°C) but not at a non-permissive temperature (e.g., 37°C).

[0187] The patient is subjected to therapeutic hypothermia: the patient's core body temperature is maintained below normal body temperature (e.g., 33° C.). Target cells (any cells—autologous or allogeneic) are treated ex vivo with a ts agent and immediately infused into the patient's circulation or injected into the patient's organs.

[0188] While the patient is maintained at the target temperature, e.g., 33°C, for a period of time, e.g., 24 hours, the ts agents exert their expected function. The level of the GOI's RNA (and the protein translated from that RNA) increases and reaches a high level at the permissive temperature. Subsequently, the patient's body temperature is returned to normothermia at 37°C. The ts agents no longer function at 37°C, a non-permissive condition within the patient's body. Body temperature is maintained above 37°C throughout the patient's life, thereby preventing the ts agents from being reactivated and preventing cells other than the target cells from being treated with the ts agents. Notably, this therapeutic approach is applicable to any cell type, including those described above. Example 10: In vivo therapeutic uses of temperature-sensitive agents

[0189] This example demonstrates how a temperature-sensitive viral vector is administered to a subject and transiently activated when mild hypothermia is induced in the subject (FIG. 17). The temperature-sensitive therapeutic agent can be any of the temperature-sensitive therapeutic agents disclosed herein. A temperature-sensitive therapeutic agent is functional at a permissive temperature (e.g., 33°C) but not at a non-permissive temperature (e.g., 37°C; human body temperature).

[0190] A subject's core body temperature was lowered using a targeted temperature management (TTM) procedure, which was used in outpatient clinics for patients suffering from cardiac and brain injuries. The TTM procedure is designed to achieve and maintain a specific subject's body temperature for a sustained period of time. Such procedures have previously been used therapeutically to reduce the negative effects resulting from various acute health problems, such as heart attack and stroke. Devices and general methods for using the TTM procedure are known in the art and can be used with the methods described herein. The TTM procedure can be performed using many methods, including cooling catheters, cooling blankets, and the application of ice around the body. Various devices have been used for this purpose. For example, the ArcticSun™ is a device that can be used to lower or raise a patient's body temperature between 32°C and 38.5°C (Pittl et al., 2013). The procedure is safe, and no major side effects have been reported due to the device.

[0191] A patient is placed under hypothermic conditions using the TTM procedure, and the target body temperature is sufficient to induce activity of a temperature-sensitive therapeutic agent, which is delivered directly to the patient via a systemic route (e.g., intravenous) or direct injection into an organ / tissue (e.g., catheter or percutaneous needle injection) (Figure 17).

[0192] The patient's temperature is maintained at a permissive temperature for a time sufficient to allow induction of the desired activity of the temperature-sensitive therapeutic agent, which leads to a therapeutic effect in cells containing or exposed to the temperature-sensitive therapeutic agent.

[0193] After the desired therapeutic effect is achieved, the patient's body temperature is then returned to normothermia (i.e., a non-permissive temperature) to terminate the activity of the temperature-sensitive therapeutic agent, followed by degradation of the temperature-sensitive therapeutic agent. Systemic delivery via the circulation

[0194] The patient is placed under hypothermic conditions (e.g., 33°C). Once the patient's core body temperature has been stabilized at the target temperature, a ts agent is delivered directly intravenously to the patient. The ts agent is delivered to many organs and tissues via this systemic route. The patient's core body temperature is maintained at a functional temperature for a desired period of time (e.g., 24 hours). While the patient's body temperature is maintained at a temperature tolerable to the agent (e.g., 33°C), the agent functions. When the patient's body temperature returns to normal at 37°C, a temperature not tolerable to the agent, the agent ceases to work.

[0195] The ts-agent can be naked RNA (i.e., synthetic RNA). Systemic delivery via the circulation delivers naked RNA to many organs, with or without target organ specificity. Alternatively, the ts agent can be RNA encapsulated in a nanoparticle (i.e., synthetic RNA) that is engineered to target specific cell types, tissues, organs, cancers, tumors, or diseased cells. Thus, systemic delivery via the circulation delivers nanoparticle-encapsulated RNA to specific cell types, tissues, organs, cancers, tumors, or diseased cells. Alternatively, the ts agent can be RNA packaged within a viral particle. Depending on the envelope type and other characteristics, the viral particle targets specific cell types, tissues, organs, cancers, tumors, or diseased cells. Thus, systemic delivery via the circulation delivers RNA packaged within a viral particle to specific cell types, tissues, organs, cancers, tumors, or diseased cells. Alternatively, the ts agent can be a temperature-sensitive viral vector. Depending on the envelope type and other characteristics, viral particles target specific cell types, tissues, organs, cancers, tumors, or diseased cells. Thus, systemic delivery via the circulation delivers temperature-sensitive viral vectors to specific cell types, tissues, organs, cancers, tumors, or diseased cells. Targeted delivery to the brain and spinal cord via the cerebrospinal fluid

[0196] The patient is placed under hypothermic conditions (e.g., 33°C). Once the patient's core body temperature has been stabilized at the target temperature, a ts agent is delivered directly to the patient's cerebrospinal fluid via epidural injection. The ts agent is delivered to the brain and spinal cord. The patient's core body temperature continues to be maintained at a tolerable temperature for a desired period of time (e.g., 24 hours). While the patient's body temperature is maintained at a temperature tolerable to the agent (e.g., 33°C), the agent functions. When the patient's body temperature returns to normal at 37°C, a temperature not tolerable to the agent, the agent ceases to work. Targeted delivery to the liver, kidney, skeletal muscle, cardiac muscle, pancreas, bone marrow, and other organs via intradermal injection

[0197] The patient is placed under hypothermic conditions (e.g., at 33°C). Once the patient's core body temperature has stabilized at the target temperature, the ts agent is injected through the skin (percutaneously) into an organ such as the liver, kidney, skeletal muscle, cardiac muscle, pancreas, or other organ using a fine needle with ultrasound or CT visual guidance. The patient's core body temperature is maintained at a tolerable temperature for the desired period of time (e.g., 24 hours). While the patient's body temperature is maintained at the agent's tolerable temperature (e.g., at 33°C), the agent functions. When the patient's body temperature returns to normal at 37°C, the agent's non-tolerable temperature, the agent ceases to work. Targeted delivery to the liver, kidney, skeletal muscle, cardiac muscle, pancreas, bone marrow, and other organs via an endoscope equipped with an injection needle catheter

[0198] The patient is placed under hypothermic conditions (e.g., at 33°C). Once the patient's core body temperature has been stabilized at the target temperature, the ts agent is then delivered directly to specific organs and tissues via an endoscopic injection needle catheter. The patient's core body temperature is maintained at a tolerable temperature for the desired period of time (e.g., 24 hours). While the patient's body temperature is maintained at the agent's tolerable temperature (e.g., at 33°C), the agent functions. When the patient's body temperature returns to normal at 37°C, the agent's non-tolerable temperature, the agent ceases to work. Targeted delivery to the liver, kidney, skeletal muscle, cardiac muscle, pancreas, and other organs via vascular catheters

[0199] The patient is placed under hypothermic conditions (e.g., at 33°C). Once the patient's core body temperature has been stabilized at the target temperature, the ts agent is then delivered directly to specific organs and tissues via a vascular catheter. The patient's core body temperature is maintained at a tolerable temperature for a desired period of time (e.g., 24 hours). While the patient's body temperature is maintained at the agent's tolerable temperature (e.g., at 33°C), the agent functions. When the patient's body temperature returns to normal at 37°C, the agent's non-functional temperature, the agent ceases to work. Targeted delivery to the lungs and other organs via inhalation

[0200] The patient is placed under hypothermic conditions (e.g., at 33°C). Once the patient's core body temperature has been stabilized at the target temperature, the ts agent is then delivered directly to the patient via inhalation. The ts agent is delivered to the lungs and other organs via pulmonary inhalation. The patient's core body temperature is maintained at a tolerable temperature for a desired period of time (e.g., 24 hours). While the patient's body temperature is maintained at a temperature tolerable to the agent (e.g., at 33°C), the agent functions. When the patient's body temperature returns to normal at 37°C, a temperature not tolerable to the agent, the agent ceases to work. Targeted delivery to spleen-mobilized bone marrow cells

[0201] The patient receives an injection of G-CSF, plerixafor, or other cytokines to mobilize bone marrow cells (including, but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) into the subject's spleen. The patient is placed under hypothermic conditions (e.g., at 33°C). Once the patient's core body temperature has been stabilized at the target temperature, a ts agent is then delivered to the spleen via the methods described above. The ts agent is subsequently delivered to the bone marrow cells mobilized to the spleen. The patient's core body temperature is maintained at a tolerable temperature for a desired period of time (e.g., 24 hours). While the patient's body temperature is maintained at a tolerable temperature for the agent (e.g., at 33°C), the agent functions. When the patient's body temperature returns to normal at 37°C, a non-tolerable temperature for the agent, the agent ceases to function. For example, the method can include administering a therapeutically effective amount of a temperature-sensitive agent (e.g., a temperature-sensitive therapeutic agent) to one or more bone marrow cells (including, but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells) in the spleen. Example 11: Optimal ex vivo contact conditions for SeVts-ZSCAN4 of human mobilized peripheral blood CD34+ cells

[0202] This example describes the finding that 16 hours of ex vivo incubation at 33°C is sufficient for a temperature-sensitive Sendai virus vector to be effective on human CD34+ cells. This example demonstrates that a multiplicity of infection (MOI) of 1 to 25 is sufficient for the vector to infect the majority of human CD34+ cells ex vivo. Materials and Methods cell culture

[0203] Frozen samples of human peripheral blood CD34+ cells purified by CD34+ magnetic beads were thawed and cultured in medium supplemented with StemMACS HSC Expansion Cocktail (containing a combination of recombinant human stem cell factor (SCF), Flt3-ligand, and thrombopoietin (TPO)). Under these culture conditions, most CD34+ cells did not divide within the first few days, as cells only underwent a maximum of two cell divisions even when cultured for 10 days. Sendai virus vector encoding the human ZSCAN4 gene

[0204] SeV18+TS15ΔF is a temperature-sensitive version of the Sendai virus vector with a TS15 backbone (Ban et al., PNAS 2011), custom-made by ID Pharma (Tsukuba, Japan). This vector backbone lacks the F(usion) gene (required for reproducing infectious progeny virus). Therefore, this vector does not transmit virus from infected to uninfected cells. This vector encodes two RNA polymerase genes (P and L) and three structural protein genes (NP, M, and HN). It contains point mutations in the M, HN, P, and L genes, which render the vector temperature-sensitive: it replicates at 33°C (below 35°C) but stops replicating at 37°C. SeV18+hZSCAN4 / TS15ΔF (also referred to herein as "SeVts-ZSCAN4") is a SeV18+TS15ΔF Sendai virus vector encoding the human ZSCAN4 gene, and it was custom-ordered by ID Pharma (Tsukuba, Japan). A diagram of the genome of SeV18+hZSCAN4 / TS15ΔF (i.e., SeVts-ZSCAN4) is shown in Figure 18. Multiplicity of infection (MOI)

[0205] The optimal multiplicity of infection (MOI) varies among different experimental conditions. For example, we found that not only the MOI but also the total volume of culture medium influences the infection efficiency of Sendai virus vectors. Our standard MOI of 25 was determined as follows. First, we previously showed that an MOI of 20 resulted in 100% infection efficiency in CD34+ cells, whereas an MOI of 2 resulted in 43% (Ban et al., 2011). For mouse embryonic stem cells, MOIs of 10, 30, and 100 were compared, and MOI of 30 showed the highest infection efficiency (Amano et al., 2015). For human fibroblasts, an MOI of 25 resulted in 55.6% infection efficiency, whereas an MOI of 5 resulted in 14% and an MOI of 10 resulted in 25.4% (Amano et al., 2015). Our CD34+ data showed that an MOI of 25 resulted in a 53% efficiency, while an MOI of 10 resulted in a 33% efficiency. Further studies showed that an MOI of 25 consistently resulted in an efficiency of 75.6 ± 14.2% (mean ± SD, n = 16) of CD34+ cells. Subsequent studies showed that an MOI of 1.1 resulted in an 89.8% efficiency in human CD34+ cells. Therefore, depending on the experimental conditions, an MOI of 1–25 is selected for SeVts-ZSCAN4 infection. result

[0206] To determine the optimal duration and conditions for ex vivo exposure of CD34+ cells to SeVts-ZSCAN4, a series of incubation times at functional temperature (33°C) was evaluated using the intended clinical CD34+ incubation protocol. CD34+ cells were plated (1 × 10) onto 12-well plates. 5 or 5 x 10 4Cells (1000 cells / well) were then incubated with SeVts-ZSCAN4 (MOI = 25) for 0, 3, 6, 16, 24, 48, or 72 hours at 33°C in 5% CO2. The cells were then incubated at 37°C in 5% CO2 for up to 10 days. Incubation at 33°C allowed for Sendai virus infection, replication, and transgene expression, whereas increasing the temperature to 37°C inactivated the virus and stopped transgene expression. After the designated 33°C incubation, cells were immunostained for ZSCAN4 protein expression using an anti-hZSCAN4 antibody. The number of human ZSCAN4-expressing cells was compared with the total number of cells identified by DAPI fluorescent staining.

[0207] Incubation for 3 and 6 hours was too short to express ZSCAN4 protein at detectable levels. However, incubation for 16 and 24 hours at 33°C resulted in ZSCAN4 protein expression in 82% and 95% of CD34+ cells, respectively (Figure 19). After incubation for 48 and 72 hours at 33°C, there was no further increase in transfection efficiency and protein expression. Example 12: ZSCAN4 protein dynamics in human CD34+ cells

[0208] This example describes the finding that a temperature shift from 33° C. to 37° C. stops the expression of ZSCAN4 protein, which disappears rapidly. Materials and Methods Sendai virus vector encoding the human ZSCAN4 gene

[0209] SeVts-ZSCAN4 (also called SeV18+hZSCAN4 / TS15ΔF) expresses human ZSCAN4 in a temperature-sensitive manner (FIG. 18). result

[0210] To determine the exposure time to ZSCAN4 protein, we investigated the dynamics of ZSCAN4 protein expression in CD34+ cells. To accurately mimic the proposed clinical trial conditions, CD34+ cells isolated by mobilization of peripheral HSCs were obtained from Hemacare, Inc.

[0211] CD34+ cells were either left untreated or contacted with SeVts-ZSCAN4 for 24 hours at 33° C. and further incubated for 9 days at 37° C. Cells were sampled on days 1, 3, 7, and 10 and immunostained with antibodies against CD34 and ZSCAN4.

[0212] During the 10-day incubation period, nearly 100% of the cells retained their CD34 marker, indicating that contact with SeVts-ZSCAN4 did not alter the CD34+ cell fraction or CD34 marker expression profile (Figures 20A and 20B). Based on immunostaining with ZSCAN4 on day 1, contact with SeVts-ZSCAN4 (MOI = 25) exposed 77% of the CD34+ cells to the ZSCAN4 protein (Figure 20A). As expected, upon temperature shift to 37°C, the number of cells bearing the ZSCAN4 protein decreased very rapidly: only 7% of ZSCAN4-positive cells were present on day 7 and only 2% on day 10. In contrast, control experiments showed that without SeVts-ZSCAN4 contact, no ZSCAN4-positive cells were present, but nearly 100% of the cells remained CD34+. The rapid decline in ZSCAN4 protein after the switch to the nonpermissive temperature of 37°C was not simply caused by cell division, because cell number increased only 3.5-fold (on average, less than two cell divisions) over 10 days, whereas the number of control cells (without SeVts-ZSCAN4 contact) increased 6.1-fold during the same time period. Example 13: Effect of SeVts-ZSCAN4 on telomere length of human CD34+ cells

[0213] This example describes the finding that transient expression of human ZSCAN4 using a temperature-sensitive viral vector enhanced telomere length in human CD34+ cells. Materials and Methods Sendai virus vector encoding the human ZSCAN4 gene

[0214] SeVts-ZSCAN4 (also called SeV18+hZSCAN4 / TS15ΔF) expresses human ZSCAN4 in a temperature-sensitive manner (FIG. 18). result

[0215] ZSCAN4 has been shown to localize to telomeres, upregulate meiosis-specific homologous recombination genes, and extend telomeres through telomere recombination in mouse embryonic stem (ES) cells (regardless of telomerase activity) (Zalzman et al., 2010; Amano et al., 2013). To evaluate this potential in human hematopoietic stem cells, human peripheral blood CD34+ cells were contacted with SeVts-ZSCAN4 ex vivo and incubated at 33°C. CD34+ cells were treated with SeVts-ZSCAN4 for 16, 24, 48, and 72 hours at 33°C and then cultured at 37°C for 10 days for telomere assays. Telomere length was measured by quantitative real-time PCR using a telomere-specific primer (T) and a single-copy gene-specific primer set (S) as described in (Cawthon 2002). Relative telomere length was calculated as the T / S ratio and further normalized by the T / S ratio of the control sample (untreated control).

[0216] Compared to untreated cells, incubation at 33°C for 24 hours extended telomeres by approximately 1.5-fold (Figure 21). Incubation for ≥24 hours did not further extend telomeres; thus, 24 hours of incubation at the permissive temperature (i.e., 33°C) was sufficient to extend telomeres in human CD34+ cells. Example 14: Effect of SeVts-ZSCAN4 on telomere length of human blood cells transplanted into immunodeficient mice

[0217] This example describes a procedure for evaluating the safety of administering CD34+ cells treated with a temperature-sensitive Sendai virus vector expressing the human ZSCAN4 gene to a subject and the efficacy of transplantation of the cells.

[0218] SeVts-ZSCAN4 (also referred to as SeV18+hZSCAN4 / TS15ΔF) expresses human ZSCAN4 in a temperature-sensitive manner (Figure 18). Human CD34+ cells were contacted with SeVts-ZSCAN4 (MOI = 25) in culture for 24 hours at the permissive temperature of 33°C, a manner suitable for intended clinical use. The cells were then washed to remove SeVts-ZSCAN4 and resuspended in physiological saline (test substance) (Figure 22). Aliquots of the test substance were cultured in vitro for 10 days and subjected to telomere length assay by qPCR (Figure 22). MNC was the mononuclear cell standard used for telomere length. The ratio of the telomere length of the sample to that of the MNC (T / S ratio) was expressed as relative telomere length. The telomeres of CD34+ cells treated with SeVts-ZSCAN4 for 24 hours were statistically significantly longer than those of untreated CD34+ cells (Figure 23). Therefore, SeVts-ZSCAN4 treatment for 24 hours at the permissive temperature (i.e., 33°C) was able to elongate the telomeres of human CD34+ cells in vitro.

[0219] To closely model intended clinical trials, severely immunodeficient NOG-EXL mice (Taconic) were treated with G-CSF and plerixafor (Figure 22). The study used NOG-EXL mice without irradiation (i.e., myeloablation). Similarly, unlike typical transplant studies that use more potent umbilical cord blood CD34+ cells, the study used G-CSF-mobilized peripheral blood CD34+ cells from healthy donors. NOG-EXL mice were treated with 2 x 10 7Either untreated CD34+ cells or CD34+ cells treated with SeVts-ZSCAN4 (test substance) were administered intravenously at a dose of 1000 cells / kg (Figure 22). The dose was approximately 10-fold higher than the dose intended for humans. No SeVts-ZSCAN4-related adverse events were observed in NOG-EXL mice receiving SeVts-ZSCAN4-treated CD34+ cells. Furthermore, 38 weeks after CD34+ cell injection, two mice (#492 and #493) receiving SeVts-ZSCAN4-treated CD34+ cells and one mouse (#496) receiving untreated (control) CD34+ cells were sacrificed to examine the engrafted cells derived from human CD34+ cells. Splenocytes isolated from these mice were FACS-sorted by the human CD45+ pan-hematopoietic marker and used for a qPCR-based telomere assay. As shown in Figure 24, the telomeres of human cells transplanted into mice receiving CD34+ cells treated with SeVts-ZSCAN4 were longer than those of mice receiving CD34+ cells alone (control). These data suggest that SeVts-ZSCAN4-treated human CD34+ cells could engraft into mouse bone marrow and participate in normal hematopoiesis. Furthermore, once telomeres were elongated by SeVts-ZSCAN4 treatment, the telomeres of these cells remained long even after transplantation and cell differentiation. This study also demonstrates the safety of SeVts-ZSCAN4 treatment. Example 15: Evaluation of SeVts-ZSCAN4 in human patients with telomere biology disorders and bone marrow failure

[0220] Telomere biology disorders associated with bone marrow failure, including dyskeratosis congenita, have a poor prognosis and high mortality rate. Currently, hematopoietic stem cell transplantation is the only curative treatment, which can alleviate the hematologic manifestations of the condition. However, its use can be challenging, fraught with difficulties in finding a well-matched donor and the toxicities associated with bone marrow ablation (chemotherapy and radiation) and immune complications. This example describes the evaluation of the safety and tolerability of administering CD34+ cells contacted ex vivo with a temperature-sensitive Sendai virus vector encoding human ZSCAN4 to human patients in need, as well as the efficacy of transplantation of the cells.

[0221] Therapeutic Temperature-Sensitive Agents. SeVts-ZSCAN4 (also referred to as SeV18+hZSCAN4 / TS15ΔF) expresses human ZSCAN4 in a temperature-sensitive manner ( FIG. 18 ). As used in this example, the investigational product is a pharmaceutical composition comprising a sterile, electrolyte-containing, isotonic aqueous solution in which autologous CD34+ cells that have been contacted ex vivo with SeVts-ZSCAN4 are suspended. PLASMA-LYTE Polyelectrolyte Injection, marketed by Baxter International Inc. (Deerfield, IL), is a suitable solution for resuspending virus-contacted CD34+ cells.

[0222] Rationale: Autologous CD34+ cells contacted ex vivo with SeVts-ZSCAN4 have been shown to elongate telomeres in in vitro and in vivo preclinical studies of human CD34+ cells. This treatment does not require a fully matched donor, as the patient's own cells can be used. Contact with SeVts-ZSCAN4 results in the transient production of human ZSCAN4 protein in the patient's own (autologous) CD34+ cells, which restores their function by elongating their abnormally short telomeres ex vivo. After administration, the contacted CD34+ cells are transplanted, subsequently proliferating in the patient's bone marrow and producing blood cells. In this way, the patient's bone marrow failure is effectively treated (Figure 25).

[0223] Patients. The study population will initially include adult men and women, but will be expanded to include pediatric patients. Inclusion criteria include a diagnosis of mild or moderate bone marrow failure and a telomere biology disorder. Mild or moderate bone marrow failure is defined by one or both of the following: 1) a peripheral blood absolute neutrophil count (ANC) of 0.5-1.5 x 10^9 / L; or platelets 20-100 x 10^9 / L; or hemoglobin <10 g / dL; and 2) low bone marrow cellularity for age. A diagnosis of telomere biology disorder was defined by one of the following: i) age-adjusted mean telomere length <1st percentile in one or more of peripheral blood lymphocytes (PBL), B cells, or naive T cells; and ii) pathogenic mutations in DKC1, TERC, TERT, NOP10, NHP2, TINF2, CTC1, PARN, RTEL1, ACD, USB1, or WRAP53. Exclusion criteria included one or more of the following: chemotherapy for cancer; myelodysplastic syndrome or clonal cytogenetic abnormalities associated with acute myeloid leukemia on bone marrow examination; uncontrolled bacterial, viral, or fungal infection; prior allogeneic marrow or stem cell transplant; subjects ineligible for G-CSF and plerixafor; subjects ineligible for apheresis; or subjects currently taking or who have taken danazol and androgens within 60 days prior to the start of the study.

[0224] Procedure. Briefly, the study involves: 1) mobilization of hematopoietic stem cells into the bloodstream and collection of mononuclear cells (MNC) by apheresis; 2) ex vivo cell processing; and 3) infusion of processed cells. The flow chart for the study is designed as shown in Figure 26, and a schematic diagram is shown in Figure 27.

[0225] Mobilization and apheresis Days 1-3: All eligible subjects received daily granulocyte colony-stimulating factor (G-CSF) injections (10 μg / kg SC). Day 4: After G-CSF injection (10 μg / kg SC), blood samples are collected and CD34+ cell counts are determined. Subjects with <5 cells / μL CD34+ cells are withdrawn from the study. Subjects with ≥5 cells / μL CD34+ cells are admitted and administered plerixafor (20 mg fixed dose or 0.24 mg / kg SC) approximately 11 hours before apheresis. Plerixafor 1,4-Bis((1,4,8,11 tetraazacyclotetradecan-1-yl)methyl)benzene, CAS No. 155148-31-5), such as MOZOBIL, marketed by Genzyme Corporation (Cambridge, MA), is a hematopoietic stem cell mobilizer. Day 5: G-CSF (10 μg / kg SC) is administered, and the first apheresis is initiated to collect MNCs. After apheresis, subjects are evaluated for their ability to tolerate a second apheresis. Plerixafor (20 mg fixed dose or 0.24 mg / kg SC) is administered to subjects deemed able to tolerate a second apheresis approximately 11 hours before the second apheresis. Subjects unable to tolerate a second apheresis or with <2.0 x 10^6 / kg CD34+ cells are withdrawn from the study, and all collected cells are infused back into the subject. Subjects unable to tolerate a second apheresis or with >2.0 x 10^6 / kg CD34+ cells continue on the study. Day 6: Subjects who can tolerate a second apheresis receive G-CSF (10 μg / kg SC) before the start of the second apheresis to collect additional MNCs. After apheresis, a complete blood count (CBC) is obtained, and if necessary, subjects receive a red blood cell or platelet transfusion to maintain a hemoglobin level >10.5 g / dL and platelets >100K. Subjects who underwent a second apheresis and subjects with <2.0 x 10^6 / kg CD34+ cells (first and second apheresis combined) are withdrawn from the study, and all collected cells are infused back into the subject. Subjects who underwent a second apheresis and subjects with ≥2.0 x 10^6 / kg CD34+ cells continue on the study.

[0226] Ex vivo cell processing. CD34+ cells were isolated from MNCs collected by apheresis using the CLINIMACS PRODIGY automated cell processing system, commercially available from Miltenyi Biotec (Germany), under good manufacturing practice. CD34+ cells were suspended in GMP-grade HSC Brew GMP Medium and cytokines (equivalent to StemMacs medium), contacted with SeVts-ZSCAN4 at an MOI of 1 to 25 (depending on the number of CD34+ cells collected), and cultured for 1 hour at the permissive temperature of 33°C. Additional HSC Brew GMP Medium was added to the virus-contacted CD34+ cells, which were then cultured for an additional 23 hours at the permissive temperature of 33°C. After incubation, virus-contacted CD34+ cells are washed three times with HSC Brew GMP Medium to remove SeVts-ZSCAN4 and resuspended in 100 mL of sterile PLASMA-LYTE to produce the investigational product.

[0227] Infusion. Subjects received a single intravenous infusion of the investigational product at a dose of 2.0-8.0 x 10^6 / kg CD34+ cells suspended in 100 mL of PLASMA-LYTE polyelectrolyte or other sterile, electrolyte-containing, isotonic aqueous solution for injection, marketed by Baxter Healthcare Corporation (Deerfield, IL). Cells were delivered over 30 minutes at an infusion rate of 3.3 mL / min. The investigational product infusion occurred approximately 32 hours after the first apheresis.

[0228] Safety assessments were performed up to 24, 36, or 48 hours post-infusion and included, but were not limited to, assessment of vital signs (temperature, pulse, respiratory rate, and blood pressure), body weight, electrocardiogram, clinical laboratory tests (hematology, blood chemistry, and urinalysis), adverse events, plasma cytokine levels, and immunogenicity of the investigational product. Plasma cytokine(s) measured included one or more of GM-CSF, IFN-gamma, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, and TNF-alpha. Immunogenicity of the investigational product was assessed by measuring Sendai virus vector-reactive antibodies and human SCAN4-reactive antibodies in blood samples obtained from the subjects.

[0229] Study endpoints: Increased telomere length in any of the following cells: lymphocytes, granulocytes, B cells, naive T cells, memory T cells, and NK cells in peripheral blood, as well as improved blood counts (neutrophils, platelets, or hemoglobin). Telomere length will be measured by Flow FISH. Example 16: Expression of human ZSCAN4 protein in vivo

[0230] Temperature-sensitive agents (ts agents), such as srRNAs or Sendai virus vectors, are functional at permissive temperatures (e.g., 31-34°C) but not at non-permissive temperatures (e.g., >37°C). The core body temperature of a human subject is approximately 37°C, whereas the surface body temperature of a human subject is approximately 31-34°C. Thus, a ts agent administered to cells at or near the body surface of a human patient (e.g., intradermally, subcutaneously, or intramuscularly) is functional without lowering the core body temperature of the human patient (Figure 28). No further action is required.

[0231] Similarly, the temperature of the nasal cavity and upper trachea of ​​a human subject is approximately 32°C, and the temperature of the subsegmental bronchi of a human subject is approximately 35°C (McFadden et al., 1985). Thus, ts agonists administered intranasally to cells of the upper respiratory tract (nasal cavity, pharynx, and / or larynx) and / or upper trachea of ​​a human patient are functional without lowering the core body temperature of the human patient (Figure 29). Intranasal administration may be by insufflation, inhalation, or instillation. No further action is required.

[0232] Alternatively, ts agents administered intranasally to cells at or near the surface of a human patient's body (e.g., intradermally, subcutaneously, or intramuscularly) can be rendered inoperative by subsequently elevating the human patient's surface temperature, for example, by applying a heat patch or heat pad to the treatment area of ​​the patient's skin, immersing in a hot bath, or sitting in a hot sauna. This therapeutic approach is highly safe in that the ts agent is functional only in the intended area and not in other areas of the patient's body. Similarly, ts agents administered intranasally to cells of a human patient's upper respiratory tract (nasal cavity, pharynx, and / or larynx) and / or upper trachea can be rendered inoperative by placing the human patient in an environment having a non-permissive temperature (e.g., ≥ 37°C).

[0233] For example, the coding region for human ZSCAN4 is introduced into srRNA1ts2 or SeV18 / TS15ΔF, as previously described for expression of human ZSCAN4 on or near the body surface of a human patient. The structure of srRNA1ts2 is described above in Example 3. Briefly, srRNA1ts2 contains a Venezuelan equine encephalitis virus (VEEV) replicon lacking the VEEV structural protein coding region. The VEEV replicon contains the VEEV nonstructural protein coding region with a 15-18 nucleotide insertion that results in expression of nonstructural protein 2 (nsP2, helicase proteinase) with five or six additional amino acids (SEQ ID NO: 44 = TGAAA) between beta-sheet 5 and beta-sheet 6 of nsP2. The additional amino acids confer temperature sensitivity to the srRNA.

[0234] The RNA of the srRNA1ts2 vector can be transcribed in vitro using T7 RNA polymerase without using materials of animal or human origin. Thus, ts agents using the srRNA1ts2 vector can be easily adapted for production using current pharmaceutical manufacturing and quality control standards. The RNA is transfected into cells in the dermal tissue of the subject. A suitable method for transfection is patch electroporation of naked RNA. Alternatively, microneedles can be used to intradermally transfect the RNA. For example, dissolvable microneedles made of hyaluronic acid or chitosan-hyaluronic acid complexes can be used to intradermally transfect the RNA.

Claims

1. A composition comprising a temperature-sensitive Sendai virus vector containing a heterogeneous nucleic acid including the coding region of human ZSCAN4 for use in a method of treating blood or hematopoietic organs, wherein the method is as follows: Human CD34+ hematopoietic stem cells are brought into contact with the temperature-sensitive Sendai virus vector; The contacted CD34+ cells were incubated at an acceptable temperature of 33°C ± 0.5°C for approximately 12 to 24 hours, during which replication and transcription of the temperature-sensitive Sendai virus vector occurred at the acceptable temperature, leading to increased expression of human ZSCAN4; and To treat the blood or hematopoietic organs by injecting contacted CD34+ cells into a human subject in need under conditions suitable for cell transplantation. A composition containing the following:

2. The composition according to claim 1, further comprising isolating the CD34+ cells from human peripheral blood mononuclear cells (PBMCs).

3. The composition according to claim 2, wherein the human PBMC is self-derived from the human subject.

4. The composition according to claim 3, further comprising recruiting hematopoietic stem cells from the bone marrow to the peripheral blood of the subject before isolating the CD34+ cells.

5. The composition according to claim 4, wherein the mobilization is completed on day 1 and the injection is carried out on day 3.

6. The composition according to claim 2, wherein the human PBMC is of the same origin as the human subject.

7. The composition according to claim 2, wherein the method further comprises collecting the human PBMC from the human subject or human donor by a first apheresis or by a first apheresis and a second apheresis.

8. The composition according to claim 7, wherein the injection is performed within about 24 to about 36 hours from the first apheresis or the second apheresis.

9. The method comprises incubating the contacted CD34+ cells at an unacceptable temperature of 37°C ± 0.5°C before injecting the contacted CD34+ cells into the human subject, wherein the replication and transcription of the temperature-sensitive Sendai virus vector and the expression of human ZSCAN4 are stopped at the unacceptable temperature. The composition according to claim 2, further comprising:

10. The composition according to claim 9, wherein the contacted CD34+ cells are incubated at a non-acceptable temperature of 37°C ± 0.5°C for a period of approximately 30 to 180 minutes.

11. The composition according to any one of claims 1 to 10, wherein the contacted CD34+ cells are washed and resuspended in a sterile isotonic aqueous solution before injection.

12. The composition according to claim 11, wherein the contacted CD34+ cells are injected intravenously at a dose of approximately 1.0 × 10^5 cells / kg to approximately 1.0 × 10^7 cells / kg.

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