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

Abstract

The present disclosure relates to methods of transiently activating a temperature sensitive agent in one or more cells, e.g., by contacting one or more cells with a temperature sensitive agent and transiently incubating the cells at a permissive temperature for inducing activity of the temperature sensitive agent in the cells.SOLUTION: In addition, the present disclosure relates to a method of contacting one or more cells of a subject with a temperature-sensitive agent and then lowering the core body temperature of the subject to a permissive temperature in order to trigger the activity of the temperature-sensitive agent in the cells. The present disclosure also relates to methods of contacting one or more cells of a subject with a temperature-sensitive agent and maintaining the surface body temperature of the subject at a permissive temperature to induce the activity of the temperature-sensitive agent in the cells. Further disclosed are methods of treating a subject with a temperature-sensitive therapeutic agent.SELECTED DRAWING: Figure 21

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 992,700, filed March 20, 2020, and U.S. Provisional Patent Application No. 62 / 955,801, 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: 699442001240SEQLIST.TXT, Recorded: December 27, 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 the ts agent is subsequently transiently activated by lowering the subject's core body temperature to a permissive temperature that induces activity of the therapeutic ts agent within the subject's cells. [Background technology]

[0004] background Delivery of therapeutic gene products to human cells, tissues, and organs presents significant challenges. For traditional gene therapy (which requires continuous expression of a gene to compensate for a patient's genetic deficiency), this has been achieved by using viral vectors such as retroviruses, 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 or foreign antigens (e.g., antigens of pathogens). 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 methods for transiently inducing 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 that lacks 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. In some embodiments, the gene product of interest is not 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 shift from 32°C to 37°C at 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 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.

[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 the differentiation of ES / iPS cells into human neurons. A schematic diagram of a typical experimental procedure is shown. Human ES / iPS cells were plated on cell culture dishes on day -1. On day 0, the cells were transfected with srRNA1ts2-NGN3. The cells were cultured at 33°C for 72 hours. On day 3, the cells were replated on new culture dishes, and the cell culture was then transferred to 37°C. On day 3, puromycin was added to the medium for 24 hours. Phase-contrast images were taken on days 0 (before transfection), 1, 2, 3 (before passage), 4 (before medium change), 5, and 6. A magnified image of the day 6 image is also shown. Cells were fixed on day 9 and used for immunostaining with anti-TUBB3 (β3-tubulin) (red signal), which is specific for mature neurons (10x and 20x objective lenses).

[0032] [Figure 19] Figure 19 shows the differentiation of human ES / iPS cells into vascular endothelial cells. A schematic diagram of a typical experimental procedure is shown. Human ES / iPS cells were plated on cell culture dishes on day -1. On day 0, the cells were transfected with srRNA1ts2-ETV2. The cells were cultured at 33°C for 72 hours. On day 3, the cell culture was transferred to 37°C. Puromycin was added to the medium for 48 hours on day 3. (Lower panel) Phase-contrast microscopy images were taken on days 1, 2, 3, 4, 5, 6, 7, and 8. The cells were fixed on day 8 and used for immunostaining with anti-CD31 (10x and 20x objective lenses).

[0033] [Figure 20]Figure 20 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.

[0034] [Figure 21] Figure 21 illustrates a schematic diagram showing an exemplary srRNA1ts2 vector encoding the spike protein (or a portion thereof) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, also known as 2019-nCoV). SARS-CoV-2 is the causative agent of coronavirus disease 2019 (COVID-2019). The nonstructural proteins (nsP1-nsP4) of the srRNA1ts2 vector are required for replication and transcription of the RNA genome, while the gene of interest (GOI) encodes the spike protein or a fragment thereof. "srRNA1ts2-2019-nCoV-spike" encodes the full-length spike protein (SEQ ID NO: 41) of 2019-nCoV. "srRNA1ts2-2019-nCoV-RBD1" encodes a fusion protein (SEQ ID NO: 42) containing the signal peptide of CD5 (residues 1-24) and the RBD of the spike protein of 2019-nCoV. "srRNA1ts2-2019-nCoV-RBD2" encodes a fusion protein (SEQ ID NO: 43) containing the signal peptide, RBD, transmembrane domain, and cytoplasmic tail of the spike protein of 2019-nCoV. The amino acid sequence of the RBD of 2019-nCoV is listed as SEQ ID NO: 44. Abbreviations: SP (signal peptide); RBD (receptor-binding domain); TM (transmembrane domain); and CT (cytoplasmic tail).

[0035] [Figure 22] Figure 22 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.

[0036] [Figure 23] FIG. 23 illustrates the expression of a gene of interest in vivo as a result of intradermal administration of RNA encoding the gene of interest (luciferase) into the hind leg of outbred mice.

[0037] [Figures 24A-24B]Figures 24A-24B show the frequency of cytokine-secreting cells in samples of splenocytes obtained from mice immunized by intradermal injection of temperature-sensitive srRNA1ts2 RNA encoding the receptor-binding domain (RBD) of SARS-CoV-2 (srRNA1ts2-2019-CoV-RBD1) or placebo (buffer only). Figure 24A shows the frequency of interferon-gamma (INF-γ) spot-forming cells (SFCs), and Figure 24B shows the frequency of interleukin-4 (IL-4) SFCs in splenocytes from immunized mice cultured in the presence and absence of SARS-CoV-2 antigen, as measured by ELISpot assay. Black bars (stimulated) represent splenocytes stimulated with a pool of 53 peptides (15-mers with 11 amino acid overlaps) covering the SARS-CoV-2 RBD for 24 hours. Gray bars (control) represent splenocytes without stimulation. The mean and standard deviation (error bars) of triplicate samples are shown.

[0038] [Figures 25A-25C]Figures 25A-25C show SARS-CoV-2 antigen-reactive serum immunoglobulin G (IgG) levels in mice immunized by intradermal injection with temperature-sensitive srRNA1ts2 RNA encoding the receptor-binding domain (RBD) of SARS-CoV-2 (srRNA1ts2-2019-CoV-RBD1) or placebo (buffer only). Briefly, on days 0 and 14, mice received placebo or srRNA1ts2-2019-CoV-RBD1 with and without an RNase inhibitor (filled triangles). On day 49, all mice received recombinant RBD protein (open triangles). An asterisk (*) indicates an IgG level greater than 3 (OD450). Figure 25A shows the results for mice receiving two doses of placebo (buffer only). Figure 25B shows the results for mice receiving two doses of srRNA1ts2-2019-CoV-RBD1 RNA. Figure 25C shows the results for mice receiving two doses of srRNA1ts2-2019-CoV-RBD1 RNA in combination with an RNase inhibitor. An asterisk (*) indicates an IgG level greater than 3 (OD450). N=10 in all groups. DETAILED DESCRIPTION OF THE INVENTION

[0039] 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 nucleic acids and proteins into cells, provided that the nucleic acids and proteins are not ZSCAN4 nucleic acids and proteins.

[0040] 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.

[0041] 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.

[0042] Another aspect of the present disclosure relates to treating a disease or condition by mobilizing bone marrow cells from a subject suffering from the disease or condition, the method comprising: isolating the mobilized bone marrow cells from the subject; culturing the isolated bone marrow 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; 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 transplanting the contacted cells into a subject to be treated for the disease or condition. Alternatively, after isolating the mobilized bone marrow cells from the subject, the isolated bone marrow cells are contacted with a temperature-sensitive viral vector, such as a Sendai virus vector, or a temperature-sensitive srRNA, and then culturing the cells at a temperature of about 33°C ± 0.5°C.

[0043] 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 in need thereof, wherein the viral vector or srRNA comprises a heterologous nucleic acid; 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 molecule; 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.

[0044] 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.

[0045] In some embodiments of the "Procedures and Methods" section, the heterologous nucleic acid comprises a gene of interest (GOI) or encodes a protein of interest. In preferred embodiments, the protein of interest is a therapeutic agent. In some embodiments, the GOI is a dominant-negative form of the GOI or an artificial gene encoding an artificial protein (e.g., a hybrid protein created by fusing different protein domains). In some embodiments, the heterologous nucleic acid comprises a non-coding RNA, an siRNA, or an shRNA. In other embodiments, the heterologous nucleic acid comprises an endonuclease editing system. In some embodiments, the endonuclease editing system is selected from, but is not limited to, a ZFN system, a TALENs system, and a CRISPR / CAS9 system. In some embodiments, the protein of interest is selected from, but is not limited to, human neurogenin-3 (NGN3), human ETS translocation variant 2 (ETV2), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF). In some embodiments, the protein of interest is erythropoietin (EPO) or granulocyte colony-stimulating factor (G-CSF). In other embodiments, the protein of interest is an enzyme such as adenosine deaminase (ADA) for enzyme replacement therapy. In some embodiments, the protein of interest is an antigen encoded by a pathogen, such as a virus, protozoan, or bacterium, for purposes of vaccination against infectious diseases. definition

[0046] 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.

[0047] 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.

[0048] 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).

[0049] 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).

[0050] 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.

[0051] 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).

[0052] 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.

[0053] 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).

[0054] "Stimulating" an immune response refers to an increase in the immune response, which may result from eliciting a de novo immune response (e.g., as a result of an initial vaccination regimen) or enhancing an existing immune response (e.g., as a result of a booster vaccination regimen). In some embodiments, stimulating an immune response includes, but is not limited to, one or more of the following: stimulating CD4+ helper T-cell proliferation; stimulating cytokine production; stimulating B-lymphocyte proliferation; stimulating stimulatory antibody production; stimulating CD8+ cytotoxic T-cell proliferation; and stimulating cytolysis of infected cells. In some preferred embodiments, stimulating an immune response comprises enhancing an antigen-specific antibody response in a subject. Preferably, enhancing an antigen-specific antibody response comprises enhancing the concentration of antigen-specific antibodies at least 2-, 3-, or 4-fold above pre-administration levels. In some embodiments, enhancing an antigen-specific antibody response comprises enhancing the concentration of antigen-specific antibodies above a minimal level, preferably above a seroprotective level. Temperature sensitive agents

[0055] 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.

[0056] 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.

[0057] 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

[0058] 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.

[0059] 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 a viral vector). The nucleic acid may comprise a genetic element. As used herein, the term "genetic element" refers to any nucleic acid encoding an RNA or polypeptide of interest. Exemplary genetic elements include, but are not limited to, artificial genes encoding a gene of interest (GOI), a dominant-negative form of the gene of interest, an artificial protein such as a hybrid protein created by fusing different protein domains, a non-coding RNA, an siRNA, an shRNA, and an endonuclease editing system. In some embodiments, the endonuclease editing system is selected from a ZFN system, a TALENs system, and a CRISPR / CAS9 system. In some embodiments, the GOI encodes a protein selected from, but not limited to, human neurogenin-3 (NGN3), human ETS translocation variant 2 (ETV2), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF). In some embodiments, the protein of interest is erythropoietin (EPO) or granulocyte colony-stimulating factor (G-CSF). In other embodiments, the protein of interest is an enzyme such as adenosine deaminase (ADA) for enzyme replacement therapy. In some embodiments, the protein of interest is an antigen encoded by a pathogen, such as a virus, protozoan, or bacterium, for purposes of vaccination against infectious diseases.

[0060] 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.

[0061] 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

[0062] 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.

[0063] In some embodiments, temperature-sensitive self-replicating RNAs are generated by engineering self-replicating RNAs, which are single-stranded RNAs typically 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) encoding a protein of interest and other genetic elements. 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, temperature-sensitive self-replicating RNAs may be generated by mutating genes encoding nsPs.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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

[0068] 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 antibody. In other embodiments, the temperature-sensitive polypeptide is selected from, but is not limited to, a transcription factor, a growth factor, a cell surface marker, a cell fusion protein, an epigenetic modifier, an enzyme, and a structural protein.

[0069] 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.

[0070] 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.

[0071] 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

[0072] 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 (PNA) molecules.

[0073] 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.

[0074] 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.

[0075] 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

[0076] 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.

[0077] 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).

[0078] 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 for nucleic acid introduction into human 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 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

[0079] 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.

[0080] 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.

[0081] 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

[0082] 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.

[0083] 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

[0084] 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.

[0085] 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

[0086] 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

[0087] 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

[0088] 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 normothermia. Certain aspects of the present disclosure relate to temperature-sensitive agents that do not function, e.g., replicate or express genes, at this normothermia (non-permissive 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

[0089] 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, human adult cells, hi certain embodiments, the one or more human cells are in need of treatment with the therapeutic agent in a subject.

[0090] 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).

[0091] 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.

[0092] 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.

[0093] 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

[0094] The temperature-sensitive agents of the present disclosure can 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 (IV) 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 composition is administered by subcutaneous injection and / or intravenous injection. In some embodiments, the composition is administered by injection into the subject's spleen.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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)).

[0099] Generally, the nature of the carrier will depend on the particular mode of administration being used. For example, parenteral formulations usually comprise injectable fluids that include a pharmaceutically and physiologically acceptable liquid, such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, or the like, as a vehicle. 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).

[0100] 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

[0101] 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.

[0102] 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 and peripheral blood 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 and peripheral blood 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 (IV) 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] Examples of disorders or diseases that can 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). Further examples of disorders or diseases that can benefit from the administration of a temperature-sensitive agent (e.g., a temperature-sensitive therapeutic agent) include cancer, autoimmune diseases, and neurological or neurodegenerative disorders, as well as diseases in which cellular regeneration is beneficial, such as blindness and hearing loss.

[0115] 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); 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);

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] Examples of disorders or diseases that can 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). Further examples of disorders or diseases that can benefit from the administration of a temperature-sensitive agent (e.g., a temperature-sensitive therapeutic agent) include cancer, autoimmune diseases, and diseases in which cell regeneration is beneficial, such as neurological or neurodegenerative disorders, as well as blindness and hearing loss.

[0123] 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 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 1 to 6, wherein said one or more cells are embryonic stem cells or induced pluripotent stem cells. 8. 8. The method of embodiment 7, wherein said therapeutic effect comprises differentiation of said cells into a desired cell type. 9. 9. The method of embodiment 8, wherein the desired cell type is selected from the group consisting of neurons, glial cells, and endothelial cells. 10. 10. The method of any one of embodiments 2-9, wherein the one or more cells are isolated from a subject prior to contacting the one or more cells with a temperature-sensitive agent. 11. 1. A method of transiently inducing a temperature-sensitive activity of a temperature-sensitive agent in a 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; A method comprising: 12. 1. A method of transiently inducing a temperature-sensitive activity of a temperature-sensitive agent in a 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 a 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 back to normothermia, wherein step (i) occurs before, after, or simultaneously with step (ii); A method comprising: 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 altering 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 11-23, wherein the temperature-sensitive agent comprises a therapeutic agent and the effect comprises a therapeutic effect. twenty five. 25. The method of any one of embodiments 1-10 or embodiment 24, wherein the therapeutic agent is encoded by a coding region of a heterologous nucleic acid of a temperature-sensitive viral vector. 26. 26. The method of embodiment 25, wherein said temperature-sensitive viral vector is selected from the group consisting of Sendai virus, adenovirus, adeno-associated virus, retrovirus, and alphavirus. 27. 27. The method of embodiment 26, wherein the temperature-sensitive viral vector is an alphavirus. 28. 28. The method of embodiment 27, wherein said alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 29. 27. The method of embodiment 26, wherein the temperature-sensitive viral vector is Sendai virus. 30. 30. The method of any one of embodiments 25-29, wherein the therapeutic agent is selected from the group consisting of non-coding RNA, siRNA, shRNA, and an endonuclease editing system. 31. 30. The method of any one of embodiments 25-29, wherein the therapeutic agent is a protein. 32. 32. The method of embodiment 31, wherein the protein is a transcription factor, optionally wherein the transcription factor is selected from the group consisting of human neurogenin-3 (NGN3), and human ETS translocation variant 2 (ETV2). 33. 32. The method of embodiment 31, wherein said protein is a growth factor, optionally wherein said growth factor is selected from the group consisting of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). 34. 34. The method of any one of embodiments 25 to 33, wherein the temperature-sensitive activity comprises replication and transcription of a temperature-sensitive viral vector. 35. The method of any one of embodiments 1 to 10 or embodiment 24, wherein the therapeutic agent is encoded by a coding region of a temperature-sensitive self-replicating RNA. 36. 36. The method of embodiment 35, wherein said self-replicating RNA comprises an alphavirus replicon lacking a viral structural protein coding region. 37. 37. The method of embodiment 36, wherein said alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 38. 38. The method of any one of embodiments 35-37, wherein the therapeutic agent is selected from the group consisting of non-coding RNA, siRNA, shRNA, and an endonuclease editing system. 39. 38. The method of any one of embodiments 35-37, wherein the therapeutic agent is a protein. 40. 40. The method of embodiment 39, wherein the protein is a transcription factor, optionally wherein the transcription factor is selected from the group consisting of human neurogenin-3 (NGN3), and human ETS translocation variant 2 (ETV2). 41. 40. The method of embodiment 39, wherein said protein is a growth factor, optionally wherein said growth factor is selected from the group consisting of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). 42. 42. The method of any one of embodiments 35 to 41, wherein the temperature-sensitive activity comprises one or both of replication and transcription of a temperature-sensitive self-replicating RNA. 43. 43. The method of any one of embodiments 25 to 42, wherein the coding region is operably linked to a promoter. 44. 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. 45. The method of any one of embodiments 11-43, 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. 46. 46. ​​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. 47. 47. The method of embodiment 46, wherein the permissible temperature is 33°C ± 0.5°C. 48. 48. The method of embodiment 46 or embodiment 47, wherein the non-permissive temperature is 37°C ± 0.5°C. 49. 49. The method of any one of embodiments 1 to 48, wherein said one or more cells are human cells. 50. 50. The method of embodiment 49, wherein the one or more human cells are adult stem cells, tissue stem cells, progenitor cells, embryonic stem cells, or induced pluripotent stem cells. 51. 51. The method of embodiment 50, wherein said one or more human cells are selected from the group consisting of hematopoietic stem cells, mesenchymal stem cells, adipose stem cells, neural stem cells, and germline stem cells. 52. 50. The method of embodiment 49, wherein said one or more human cells are somatic cells, mature cells, or differentiated cells. 53. 53. The method of embodiment 52, 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. 54. 50. The method of embodiment 49, wherein said one or more human cells are human bone marrow cells. 55. 55. The method of embodiment 54, wherein said human bone marrow cells are selected from the group consisting of hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells. 56. 56. The method of embodiment 55, wherein the hematopoietic stem cells are CD34+. 57. 57. The method of any one of embodiments 25 to 56, 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) comprising 4 to 6 additional amino acids between beta-sheet 5 and beta-sheet 6 of nsP2, optionally wherein the additional amino acids confer temperature sensitivity to the viral vector or self-replicating RNA. 58. 58. The method of embodiment 57, wherein the additional amino acids comprise one sequence selected from the group consisting of SEQ ID NO: 38 (GCGRT), SEQ ID NO: 39 (TGAAA), and SEQ ID NO: 40 (LRPHP). 59. 58. The method of embodiment 57, wherein the additional amino acids comprise the sequence of SEQ ID NO: 39 (TGAAA). 60. 60. The method of embodiment 59, wherein the amino acid sequence of NsP2 comprises one sequence selected from the group consisting of SEQ ID NOs: 29-36. 61. A temperature-sensitive agent, the agent being a temperature-sensitive viral vector or a temperature-sensitive self-replicating RNA comprising 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, optionally wherein the additional amino acids confer temperature sensitivity to the viral vector or self-replicating RNA. 62. The temperature-sensitive agent of embodiment 61, wherein the additional amino acids comprise one sequence selected from the group consisting of SEQ ID NO: 38 (GCGRT), SEQ ID NO: 39 (TGAAA), and SEQ ID NO: 40 (LRPHP). 63. 62. The temperature-sensitive agent of embodiment 61, wherein the additional amino acids comprise the sequence of SEQ ID NO: 39 (TGAAA). 64. The temperature-sensitive agent of embodiment 63, wherein the amino acid sequence of NsP2 comprises one sequence selected from the group consisting of SEQ ID NOs: 29 to 36. 65. The temperature sensitive agent of any one of embodiments 61-64, wherein the agent is a temperature sensitive alphavirus vector. 66. 65. The temperature-sensitive agent of any one of embodiments 61-64, wherein the agent is a temperature-sensitive self-replicating RNA comprising an alphavirus replicon lacking viral structural protein coding regions. 67. The temperature sensitive agent of embodiment 65 or embodiment 66, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 68. The temperature sensitive agent of embodiment 65 or embodiment 66, wherein the alphavirus is Venezuelan equine encephalitis virus. 69. 1. A method of transiently inducing temperature-sensitive activity of a temperature-sensitive agent (ts agent) in a subject, 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 is induced 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: 70. 1. A method of transiently inducing temperature-sensitive activity of a temperature-sensitive agent (ts agent) in a subject, wherein the temperature-sensitive activity of the ts agent is induced 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: 71. 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; 71. The method of embodiment 70, further comprising: 72. 72. The method of embodiment 70 or embodiment 71, wherein the temperature-sensitive agent is administered intradermally or subcutaneously. 73. 72. The method of embodiment 70 or embodiment 71, wherein the temperature-sensitive agent is administered intramuscularly. 74. 74. The method of any one of embodiments 69-73, 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. 75. The method of any one of embodiments 69-74, wherein the pharmaceutical agent is encoded by a coding region of a ts agent, or wherein the ts agent comprises a pharmaceutical agent, and the effect comprises a pharmaceutical effect, optionally wherein the pharmaceutical agent is a therapeutic agent and the pharmaceutical effect is a therapeutic effect, or wherein the pharmaceutical agent is a prophylactic agent and the pharmaceutical effect is a prophylactic effect. 76. 76. The method of any one of embodiments 69 to 75, wherein the ts agent is a temperature-sensitive viral vector and the temperature-sensitive activity comprises replication and transcription of the temperature-sensitive viral vector. 77. 77. The method of embodiment 76, wherein said temperature-sensitive viral vector is selected from the group consisting of Sendai virus, adenovirus, adeno-associated virus, retrovirus, and alphavirus. 78. 77. The method of embodiment 76, 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. 79. 77. The method of embodiment 76, wherein the temperature-sensitive viral vector is Sendai virus. 80. 76. The method of any one of embodiments 69 to 75, wherein the ts agent is a temperature-sensitive self-replicating RNA and the temperature-sensitive activity comprises one or both of replication and transcription of the temperature-sensitive self-replicating RNA. 81. 81. The method of embodiment 80, wherein said self-replicating RNA comprises an alphavirus replicon that lacks the viral structural protein coding region of the alphavirus. 82. 82. The method of embodiment 81, wherein said alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 83. 82. The method of embodiment 81, wherein said alphavirus is Venezuelan equine encephalitis virus. 84. 84. The method of any one of embodiments 69 to 83, wherein the pharmaceutical agent is selected from the group consisting of non-coding RNA, siRNA, shRNA, and an endonuclease editing system. 85. 84. The method of any one of embodiments 69-83, wherein the pharmaceutical agent comprises a protein. 86. 86. The method of embodiment 85, wherein the protein comprises an antigen of a pathogen. 87. 87. The method of embodiment 86, wherein the pathogen comprises one or more of a virus, a bacterium, a protozoan, and a fungus. 88. 88. The method of embodiment 86 or embodiment 87, wherein the antigen comprises a surface protein or fragment thereof of a pathogen. 89. The method of any one of embodiments 69 to 88, wherein the period of time sufficient for the temperature-sensitive activity to have an effect is in the range of about 12 hours to about 12 weeks, and optionally, the period is 1 to 7 days. 90. The method of any one of embodiments 69-88, 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. 91. An immunogenic composition for stimulating an immune response to a pathogen in a subject, comprising an excipient and a temperature-sensitive agent (ts agent), wherein the ts agent is a temperature-sensitive viral vector or a temperature-sensitive self-replicating RNA encoding an antigen of the pathogen, and wherein the ts agent is capable of expressing the antigen at a permissive temperature but not at a non-permissive temperature. 92. 92. The composition of embodiment 91, wherein the pathogen comprises one or more of a virus, a bacterium, a protozoan, and a fungus. 93. 93. The composition of embodiment 91 or embodiment 92, wherein the antigen comprises a surface protein or fragment thereof of a pathogen. 94. 94. The composition of embodiment 93, wherein the pathogen is a virus and the virus is different from the viral vector. 95. 95. The composition of embodiment 94, wherein the virus is a coronavirus and the antigen comprises a coronavirus spike protein or a fragment thereof. 96. 96. The composition of embodiment 95, wherein the coronavirus is 2019-nCoV and the antigen comprises the receptor binding domain (RBD) of 2019-nCoV. 97. 97. The composition of embodiment 96, wherein the amino acid sequence of the RBD comprises SEQ ID NO:44, or an amino acid sequence at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44. 98. 96. The composition of embodiment 95, wherein the coronavirus is 2019-nCoV and the antigen comprises an extracellular region of a spike protein comprising the amino acid sequence of residues 16 to 1213 of SEQ ID NO: 41, or an amino acid sequence at least 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41. 99. 99. The composition of any one of embodiments 91-98, 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. 100. The composition of any one of embodiments 91-99, wherein the ts agent is a temperature-sensitive self-replicating RNA. 101. 101. The composition of embodiment 100, wherein said self-replicating RNA comprises an alphavirus replicon lacking a viral structural protein coding region. 102. The composition of embodiment 101, wherein said alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 103. The composition of embodiment 101, wherein the alphavirus is Venezuelan equine encephalitis virus. 104. The composition of any one of embodiments 91-99, wherein the ts agent is a temperature-sensitive viral vector. 105. The composition of embodiment 104, wherein said viral vector is selected from the group consisting of Sendai virus, adenovirus, adeno-associated virus, retrovirus, and alphavirus. 106. The composition of embodiment 105, wherein said alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 107. The composition of embodiment 105, wherein the viral vector is Sendai virus. 108. The method of any one of embodiments 69-90 or the composition of any one of embodiments 91-107, wherein the subject is a mammalian subject, optionally wherein the subject is a human. 109. The composition of any one of embodiments 91-108, wherein said pathogen is a mammalian pathogen, optionally wherein said pathogen is a human pathogen. 110. 110. A method for stimulating an immune response to a pathogen in a mammalian subject, comprising administering to the mammalian subject the immunogenic composition of embodiment 109 to stimulate an immune response to an antigen in the mammalian subject, optionally wherein the mammalian subject is a human subject. 111. the immunogenic composition comprising: i) intradermal or subcutaneous; or ii) intramuscularly; 111. The method of embodiment 110, wherein the patient is administered 112. 111. The method of embodiment 110, wherein the immunogenic composition is administered intranasally. [Example]

[0124] Abbreviations: Aura (Aura virus); BFV (Barmah Forest virus); GFP (Green fluorescent protein); GOI (Gene of interest); IRES (Internal ribosome entry site); LUC (Luciferase); OD (Optical density); ONNV (Onyong-Nyong virus); RBD (Receptor binding domain); 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).

[0125] 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

[0126] 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 a gene of interest (GOI), short hairpin RNA (shRNA), long non-coding RNA, and / or other genetic element(s). 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. Example 2: Temperature-sensitive Sendai virus vector (SeVt)

[0127] 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)

[0128] This example describes the finding that mutations in the nsP2 protein encoded by a Venezuelan equine encephalitis virus (VEEV) vector are temperature-sensitive. 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 non-permissive temperature). The specific temperature-sensitive mutation (mutation 2) described below is located 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 non-viral RNA expression systems. Materials and Methods cell culture

[0129] 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 stem 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

[0130] 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

[0131] 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

[0132] 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.

[0133] 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

[0134] 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.

[0135] 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

[0136] 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.

[0137] 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

[0138] 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.

[0139] 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

[0140] 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.

[0141] 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

[0142] 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.

[0143] 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

[0144] 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.

[0145] 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)

[0146] 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: 39 (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]

[0147] 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

[0148] 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 because these proteins function optimally at permissive temperatures (low temperatures) but exhibit reduced functionality at non-permissive temperatures (e.g., 37°C) (e.g., shrimp alkaline phosphatase). Example 6: Temperature-sensitive RNA

[0149] 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

[0150] 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

[0151] 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

[0152] 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

[0153] 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

[0154] 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

[0155] 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).

[0156] 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.

[0157] 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

[0158] 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).

[0159] 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.

[0160] 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).

[0161] 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.

[0162] 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

[0163] 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.

[0164] 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

[0165] 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

[0166] 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

[0167] 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, bone marrow, and other organs via vascular catheters

[0168] 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

[0169] 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

[0170] 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: Differentiation of human ES and iPS cells into desired cell types with self-replicating RNA as a temperature-sensitive agent

[0171] This example demonstrates the discovery that temperature-sensitive, self-replicating RNA expressing human transcription factors can induce hPSCs to differentiate into various differentiated cells. The findings presented in this example can be applied to the ex vivo treatment of cells with ts agents. Differentiated cells generated in this way have many useful applications, such as in vitro modeling of human disease and drug screening using patient-derived iPS cells. The findings presented in this example can be applied to the ex vivo therapeutic use of ts agents. Differentiated cells generated in this way can be transplanted to repair defective organs or tissues in patients. For example, neurons (more specifically, dopaminergic neurons) generated ex vivo by this method can be transplanted into the substantia nigra (part of the brain) of a patient to treat Parkinson's disease. The findings presented in this example can be applied to the semi-in vivo therapeutic use of ts agents. Cells treated with a neuron-inducing ts agent are transplanted into a patient undergoing therapeutic hypothermia to allow ts-induced cell differentiation to occur in the patient's body. Once differentiated cells appear, the ts agonist is terminated by returning the patient's body temperature to a non-permissive temperature (i.e., 37°C). The findings presented in this example can be applied to the "in vivo therapeutic use of ts agonists." For example, a ts agonist expressing a set of transcription factors is injected directly into the pancreas of a diabetic patient undergoing therapeutic hypothermia (at a permissive temperature) so that the patient's pancreatic duct cells are converted in vivo to insulin-secreting beta cells. Once the desired cells appear, the ts agonist is terminated by returning the patient's body temperature to a non-permissive temperature (i.e., 37°C). Materials and Methods cell culture

[0172] Human adipose stem cell-derived iPS cell line (ADSC-iPSC) was purchased from System Biosciences (Palo Alto, CA). Cells were routinely maintained as undifferentiated pluripotent cells 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). Temperature-sensitive self-replicating RNA (srRNA1ts2)

[0173] The open reading frame of the gene of interest (GOI) was cloned into the srRNA1ts2 vector so that expression of the GOI was controlled in a temperature-sensitive manner. Synthetic RNA was produced by in vitro transcription from the vector according to Yoshioka et al., 2013, and used for transfection. The following GOIs were cloned into the srRNA1ts2 vector: srRNA1ts2-NGN3: human neurogenin 3 (NGN3) [NCBI GeneID: 50674]; and srRNA1ts2-ETV2: Human ETS variant 2 (ETV2) [NCBI GeneID: 2116]. result Neuron generation

[0174] ADSC-iPSC cells were plated on 24-well plates at a density of 1.2 x 10^5 cells / well. The cells were pretreated with a Rock inhibitor for 1 hour before plating. 24 hours after plating, the cells were transfected with srRNA1ts2-NGN3. 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 33°C for 72 hours. The cells were then passaged and cultured on ornithine / laminin-coated glass coverslips. The cells were then cultured at 37°C. The medium was changed daily. The srRNA1ts2-NGN3 vector contains a puromycin N-acetyltransferase (pac) selection gene inserted after the "IRES" sequence, allowing selection using puromycin. After passage, the cells were cultured for 24 hours in the presence of 1 μg / ml puromycin. Phase-contrast images were taken on days 0, 1, 2, 3, 4, 5, and 6 (Figure 18). A magnified image of day 6 is also shown to more clearly demonstrate the neurite formation. On day 9, the cells were fixed and stained with an antibody against tubulin βIII (TUBB3), a neural marker. Fluorescence microscopy images at two different magnifications (10x and 40x) are shown (Figure 18). The results demonstrate that srRNA1ts2-NGN3 can rapidly and efficiently differentiate human iPS cells into neurons. Generation of vascular endothelial cells

[0175] ADSC-iPSC cells were plated onto 24-well plates at a density of 1.2 x 10^5 cells / well. The cells were pretreated with a Rock inhibitor for 1 hour before plating. 24 hours after plating, the cells were transfected with srRNA1ts2-ETV2. 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 for 3 days and then cultured at 37°C for an additional 5 days (8 days in total). The medium was changed daily. The srRNA1ts2-ETV2 vector contains a puromycin N-acetyltransferase (pac) selection gene inserted after the "IRES" sequence, allowing selection using puromycin. At the time of temperature switching from 33°C to 37°C, 1 μg / ml puromycin was added to the culture. The next day, the medium was replaced with medium containing 1 μg / ml puromycin. Therefore, the cells were cultured in the presence of 1 μg / ml puromycin for 2 days. Phase-contrast images were taken on days 1, 2, 3, 4, 5, 6, 7, and 8 (Figure 19). On day 8, the cells were fixed and stained with an antibody against CD31 (a marker for vascular endothelial cells). Fluorescence microscopy images at two different magnifications (10x, 20x) are shown (Figure 19). The results indicate that srRNA1ts2-ETV2 can rapidly and efficiently differentiate human iPS cells into vascular endothelial cells. Example 12: Genome editing

[0176] Genome editing is a genetic engineering method that alters an organism's genome by substituting, deleting, or adding nucleotide sequences. It has been proposed to use genome editing to correct genomic mutations for gene therapy. As a first step, genomic DNA must be cleaved at specific locations by DNA nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), or the clustered regularly interspaced short palindromic repeats (CRISPR)-CAS9 system. The introduction of these nucleases must be carefully controlled because the double-stranded DNA breaks introduced by these enzymes are highly toxic to cells. Targeted expression of these nucleases requires precise control of timing and duration. It has been reported that both guide RNAs and CAS9 can be encoded in a single Sendai virus vector, enabling the delivery of these components for genome editing in human cells with high efficiency (Park et al., Molecular Therapy 2016). However, continuous expression of CAS9 can induce uncontrollable DNA breaks and the introduction of mutations in human cells. Therefore, for therapeutic use of gene editing systems, it is desirable to express CAS9 for short periods of time, on the order of hours rather than days. To this end, temperature-sensitive agents can be used as delivery vehicles for these components, particularly nucleases.

[0177] First, we replaced the Sendai virus vector used in the previously reported CRISPR / CAS9 system (Park et al., 2016) with a temperature-sensitive Sendai virus vector (SeVts-CAS9-guide RNA). In one embodiment, SeVt is SeV18 / TS15ΔF (Ban et al., PNAS 2011). Human primary fibroblasts were infected with the temperature-sensitive Sendai virus vector at an MOI of 25 at 33°C and maintained in a CO2 incubator for 24-48 hours. Subsequently, the cell culture temperature was shifted to 37°C for the remainder of the cell culture. Viral replication and increased CAS9 expression were observed only when the cell culture was maintained at 33°C, thereby limiting cellular exposure to CAS9 nuclease for this short period. During cell culture at 37°C, the temperature-sensitive Sendai virus vector was eventually lost from the cells, eliminating concerns about reactivation of CAS9 expression in vitro and in vivo. Similarly, a temperature-sensitive self-replicating RNA such as srRNA1ts2 (srRNA1ts2-CAS9-guideRNA) could be used instead of SeVts-CAS9-guideRNA. Example 13: CAR T cell therapy

[0178] CAR T cell therapy involves the transfer of a vector encoding a chimeric antigen receptor (CAR) into a patient's own T cells and the stable expression of the CAR in cytotoxic T cells (Maus and June 2016). CAR T cell therapy aims to reprogram a patient's own T cells to invade malignant cells. For example, CAR T cells targeting CD19 have been successfully used against B cell malignancies. However, CD19 is also expressed on normal B cells, making continuous expression of the CAR undesirable due to potential side effects. Therefore, a recent clinical trial involves the transfer of synthetic mRNA encoding the CAR into a patient's own T cells so that CAR expression is transient (ClinicalTrials.gov Identifier: NCT02624258). However, the short turnover (<12 hours) and relatively low protein expression levels of synthetic RNA present challenges in achieving sufficient CAR expression levels. To address this issue, ts agents are used as delivery vehicles for CARs into T cells. Ex vivo therapeutic use of temperature-sensitive agents to deliver CARs to T cells

[0179] T cells are collected from peripheral blood using an apheresis device (COBE Spectra) and a magnetic bead-based enrichment method (Miltenyi's CliniMacs Prodigy). Next, the T cells are transfected with srRNA1ts2-CAR and cultured ex vivo for 24 hours (or longer) at a permissive temperature (33°C). Ideally, this procedure is performed in a functionally closed system, such as Miltenyi's CliniMacs Prodigy. The treated cells are then infused back into the patient. In the patient's body, at a nonpermissive temperature (37°C), srRNA1ts2-CAR ceases to produce CAR, but sufficient CAR is already present on the surface of T cells to exert its expected function. Alternatively, a temperature-sensitive Sendai virus vector encoding a CAR (SeVts-CAR) is used instead of srRNA1ts2-CAR. For example, SeVt may be SeV18 / TS15ΔF (Ban et al., PNAS 2011). Semi-in vivo therapeutic use of temperature-sensitive agents to deliver CARs to T cells

[0180] T cells treated with srRNA1ts2-CAR are immediately infused into patients maintained at a permissive temperature (e.g., 33°C) by therapeutic hypothermia. While maintained at a permissive temperature, CAR-srRNA1ts2 remains functional. However, when the patient's temperature is switched to normothermia (37°C), srRNA1ts2-CAR ceases CAR production. Alternatively, a temperature-sensitive Sendai virus vector encoding a CARS (SeVts-CAR) can be used. For example, SeVt can be SeV18 / TS15ΔF (Ban et al., PNAS 2011). In vivo therapeutic use of temperature-sensitive agents to deliver CARs to T cells

[0181] The srRNA1ts2-CAR is directly targeted to T cells by in vivo delivery into the body of a patient maintained at a permissive temperature (33°C). While maintained at the permissive temperature, the CAR-srRNA1ts2 remains functional. However, when the patient's temperature is switched to normal body temperature (37°C), the srRNA1ts2-CAR ceases to produce CAR. Alternatively, a temperature-sensitive Sendai virus vector encoding a CARS (SeVts-CAR) can be used. For example, SeVt can be SeV18 / TS15ΔF (Ban et al., PNAS 2011). Example 14: Dominant-negative mutants of PD1 and CTLA4

[0182] Programmed death-1 (PD1) and cytotoxic T-lymphocyte antigen-4 (CTLA4) are known to function as immune checkpoints. Systemic delivery of antibodies against PD1 and CTLA4 (e.g., nivolumab and pembrolizumab) has been used in cancer therapy. However, up to 20% of patients who received these therapies experienced adverse events, such as autoimmunity (Roberts et al., 2017). Because immune checkpoints naturally function to limit immune activation to prevent autoimmunity, systemic disruption of these molecules by administering antibodies could potentially activate the immune system not only against cancer but also against the patient's own normal cells. To address this issue, dominant-negative mutants of PD1 and CTLA1 were specifically expressed in T cells, thereby blocking PD1 and CTLA4 function only in T cells (Shin et al., 2016). Regarding PD1, a mutant containing the extracellular and transmembrane domains but lacking the cytoplasmic domain has been shown to function as a dominant-negative mutant (PD1 decoy or PD1Δ) (Shin et al., 2016). Retroviral vectors were used to deliver PD1 decoy to isolated T cells in mouse studies, but integration of the retroviral vector into the host genome and persistent expression of the PD1 decoy are undesirable in humans, given the potential side effects of long-term inhibition of immune checkpoints. Ideally, expression of PD1 decoy in T cells (or other immune cells) should be permanently silenced after exerting its beneficial functions. To this end, ts agonists are used as delivery vehicles for dominant-negative mutants of PD1 and CTLA4. Ex vivo therapeutic use of temperature-sensitive agents to deliver dominant-negative PD1 mutants

[0183] Target cells (e.g., T cells) are collected from peripheral blood using an apheresis machine (COBE Spectra) and a magnetic bead-based enrichment method (Miltenyi CliniMacs Prodigy). The target cells are then transfected with srRNA1ts2-PD1Δ and cultured at a permissive temperature (e.g., 33°C) for the desired time (e.g., 24 hours or 1 week). The treated cells are then infused into the patient. At the nonpermissive temperature (37°C) in the patient's body, srRNA1ts2-PD1Δ silences PD1Δ production. However, PD1 function is prevented as long as the PD1Δ protein is present in the target cells. Alternatively, a temperature-sensitive Sendai virus vector encoding PD1Δ may be used instead of srRNA1ts2-PD1Δ. For example, SeVt may be SeV18 / TS15ΔF (Ban et al., PNAS 2011). Semi-in vivo therapeutic use of temperature-sensitive agents to deliver dominant-negative PD1 mutants

[0184] T cells treated with srRNA1ts2-PD1Δ are immediately infused into patients maintained at a permissive temperature (e.g., 33°C) by therapeutic hypothermia. PD1Δ-srRNA1ts2 is functional during permissive temperature maintenance. However, when the patient's temperature is switched to normothermia (37°C), srRNA1ts2-PD1Δ ceases production of PD1Δ. Alternatively, a temperature-sensitive Sendai virus vector encoding PD1Δ can be used instead of srRNA1ts2-PD1Δ. For example, SeVt can be SeV18 / TS15ΔF (Ban et al., PNAS 2011). In vivo therapeutic use of temperature-sensitive agents to deliver dominant-negative PD1 mutants

[0185] srRNA1ts2-PD1Δ is directly targeted to T cells by in vivo delivery into patients maintained at a permissive temperature (33°C). PD1Δ-srRNA1ts2 is functional during maintenance at the permissive temperature. However, when the patient's temperature is switched to normothermia (37°C), srRNA1ts2-PD1Δ ceases production of PD1Δ. Alternatively, a temperature-sensitive Sendai virus vector encoding PD1Δ can be used instead of srRNA1ts2-PD1Δ. For example, SeVt can be SeV18 / TS15ΔF (Ban et al., PNAS 2011). Example 15: Combination therapy of CAR T cells with dominant-negative mutants of PD1 and CTLA4

[0186] Systemic administration of PD1-blocking antibodies promotes tumor eradication by CAR T cells (John et al., 2013). Because srRNA1ts2 has a high payload capacity and can accommodate multiple genes in the same vector, ts agents (especially srRNA1ts2) provide a delivery vehicle for both PD1-blocking function and CAR to the same T cells. Ex vivo therapeutic use of temperature-sensitive agents to deliver CAR and dominant-negative PD1 to T cells

[0187] The coding regions of PD1Δ and CAR are fused with a P2A peptide (self-cleaving peptide) inserted between them. This fusion protein-encoding sequence is inserted into the srRNA1ts2 vector, which serves as the GOI. Synthetic RNA (srRNA1ts2-PD1ΔCAR) is produced from the srRNA1ts2-PD1Δ-CAR vector. T cells are collected from peripheral blood using an apheresis machine (COBE Spectra) and a magnetic bead-based enrichment method (Miltenyi CliniMacs Prodigy). Next, T cells are transfected with srRNA1ts2-PD1ΔCAR and cultured at the permissive temperature (33°C) for 24 hours (or longer). The treated cells are then infused back into the patient. At the nonpermissive temperature (37°C) in the patient's body, srRNA1ts2-PD1ΔCAR silences the production of PD1Δ and CAR. However, sufficient amounts of PD1Δ and CAR are already present on the surface of T cells. Alternatively, a temperature-sensitive Sendai virus vector encoding PD1Δ-CAR can be used instead of srRNA1ts2-PD1ΔCAR. For example, SeVt can be SeV18 / TS15ΔF (Ban et al., PNAS 2011). Semi-in vivo therapeutic use of temperature-sensitive agents to deliver CAR and dominant-negative PD1 to T cells

[0188] T cells treated with srRNA1ts2-PD1ΔCAR are immediately infused into patients maintained at a permissive temperature (e.g., 33°C) by therapeutic hypothermia. While maintained at the permissive temperature, srRNA1ts2-PD1ΔCAR remains functional. However, when the patient's temperature is switched to normothermia (37°C), srRNA1ts2-PD1ΔCAR ceases production of CAR and PD1Δ. Alternatively, a temperature-sensitive Sendai virus vector encoding PD1Δ-CAR can be used instead of srRNA1ts2-PD1ΔCAR. For example, SeVt can be SeV18 / TS15ΔF (Ban et al., PNAS 2011). In vivo therapeutic use of temperature-sensitive agents to deliver CAR and dominant-negative PD1 to T cells

[0189] srRNA1ts2-PD1ΔCAR is directly targeted to T cells by in vivo delivery into patients maintained at a permissive temperature (33°C). While maintained at the permissive temperature, srRNA1ts2-PD1ΔCAR remains functional. However, when the patient's temperature is switched to normothermia (37°C), srRNA1ts2-PD1ΔCAR ceases production of CAR and PD1Δ. Alternatively, a temperature-sensitive Sendai virus vector encoding PD1Δ-CAR can be used instead of srRNA1ts2-PD1ΔCAR. For example, SeVt can be SeV18 / TS15ΔF (Ban et al., PNAS 2011). Example 16: Ribonucleoproteins

[0190] Ribonucleoproteins function by forming complexes with RNA. The therapeutic application of ribonucleoproteins is challenging due to the difficulty of expressing both protein and RNA from the same vector. For example, the main components of telomerase are human telomerase reverse transcriptase (TERT) and telomerase RNA (TERC). Abnormal shortening of telomeres causes disease. Therefore, delivery of telomerase (TERT + TERC) to elongate telomeres is a desirable therapeutic intervention. However, persistent telomerase presence can induce adverse events such as tumor formation. Therefore, timed delivery of both TERT and TERC is desirable. To this end, ts agents can be used as delivery vehicles for TERT and TERC. Ex vivo therapeutic use of temperature-sensitive agents to deliver TERT and TERC

[0191] The srRNA1ts2 vector is constructed to express the protein TERT in a temperature-sensitive manner. The vector also contains an RNA component sandwiched between TERC, a self-cleaving ribozyme (e.g., Hammerhead ribozyme). The resulting vector is used to produce synthetic RNA (srRNA1ts2-TERT-TERC). Target cells (e.g., hematopoietic stem cells) are transfected with srRNA1ts2-TERT-TERC. At a permissive temperature (e.g., 33°C), srRNA1ts2-TERT-TERC is replicated and TERT (protein) is produced. Simultaneously, a portion of the RNA molecule (srRNA1ts2-TERT-TERC) is self-cleaved by the ribozyme to produce TERC (RNA). TERT and TERC then form a telomerase complex and elongate telomeres. The target cells (e.g., hematopoietic stem cells) are then infused into the patient's circulation. At the non-permissive temperature of 37°C, srRNA1ts2-TERT-TERC is deactivated. Alternatively, a temperature-sensitive Sendai virus vector encoding TERT-TERC can be used instead of srRNA1ts2-TERT-TERC. For example, SeVt can be SeV18 / TS15ΔF (Ban et al., PNAS 2011). Semi-in vivo therapeutic use of temperature-sensitive agents to deliver TERT and TERC

[0192] The srRNA1ts2-TERT-TERC-treated target cells are immediately transplanted into patients maintained at a permissive temperature (e.g., 33°C) by therapeutic hypothermia. While maintained at the permissive temperature, srRNA1ts2-TERT-TERC remains functional. However, when the patient's temperature is switched to normothermia (37°C), srRNA1ts2-TERT-TERC ceases production of TERT and TERC. Alternatively, a temperature-sensitive Sendai virus vector encoding TERT-TERC can be used instead of srRNA1ts2-TERT-TERC. For example, SeVt can be SeV18 / TS15ΔF (Ban et al., PNAS 2011). In vivo therapeutic use of temperature-sensitive agents to deliver TERT and TERC

[0193] srRNA1ts2-TERT-TERC is directly targeted to cells by in vivo delivery into a patient maintained at a permissive temperature (33°C). While maintained at the permissive temperature, srRNA1ts2-TERT-TERC is functional. However, when the patient's temperature is switched to normothermia (37°C), srRNA1ts2-TERT-TERC stops the production of TERT and TERC. Alternatively, a temperature-sensitive Sendai virus vector encoding TERT-TERC can be used instead of srRNA1ts2-TERT-TERC. For example, SeVt can be SeV18 / TS15ΔF (Ban et al., PNAS 2011). Example 17: Gene knockdown or silencing

[0194] RNAi, including microRNA, siRNA, and shRNA, has become an attractive option for knocking down the function of specific genes for therapeutic purposes (Kaczmarek et al., 2017). However, a drawback of RNAi technology is its very short duration of action, which requires repeated administration of large amounts of siRNAs. On the other hand, plasmids and viruses provide strong expression of shRNA from Pol III promoters, but it is difficult to turn off the expression when needed. Furthermore, DNA-based shRNA expression systems can integrate into the organism's genome and cause mutations. In this case, temperature-sensitive srRNA or Sendai virus vectors may offer an ideal solution because they provide controllable and long-term expression without leaving an RNAi footprint (no integration into the organism's genome).

[0195] shRNA. shRNA is incorporated into srRNAts or SeVt as a GOI flanked by self-cleaving ribozymes, similar to the guide RNA flanked by self-cleaving ribozymes described in Park et al., 2016. The srRNA1ts2-shRNA is delivered to target cells, where the target gene is silenced when the target cells are maintained at a permissive temperature. However, when the temperature is shifted to a nonpermissive temperature, shRNA production stops and the target gene is not silenced. Alternatively, a temperature-sensitive Sendai virus vector encoding the shRNA can be used instead of the srRNA1ts2-shRNA. For example, the SeVt can be SeV18 / TS15ΔF (Ban et al., PNAS 2011).

[0196] dsRNA. A long double-stranded RNA (dsRNA) expression unit is created by connecting a long sense strand of the target RNA (i.e., to be knocked down) and its antisense strand with a short linker RNA sequence. This dsRNA expression unit is inserted into the srRNA1ts2 vector as a GOI flanked by self-cleaving ribozymes, in a manner similar to a guide RNA flanked by self-cleaving ribozymes (Park et al., 2016 and Shinagawa T1, Ishii S. 2003). To facilitate the formation of siRNAs from dsRNA, the srRNA1ts2 vector also expresses human DICER1 (NCBI Reference Sequence: NG_016311.1). The srRNA1ts2-dsRNA or srRNA1ts2-DICER1-dsRNA is delivered to target cells, where the target gene is silenced when the target cells are maintained at a permissive temperature. However, when the temperature is shifted to the non-permissive temperature, production of dsRNA (and DICER1, if present) ceases, and the target gene is not silenced. Alternatively, a temperature-sensitive Sendai virus vector encoding dsRNA or dsRNA-DICER1 can be used instead of srRNA1ts2-dsRNA or srRNA1ts2-dsRNA-DICER1. For example, SeVt can be SeV18 / TS15ΔF (Ban et al., PNAS 2011).

[0197] asRNA. The target RNA (asRNA) (i.e., to be knocked down) is inserted into the srRNA1ts2 vector as a GOI flanked by self-cleaving ribozymes, in a manner similar to the guide RNA flanked by self-cleaving ribozymes described in Park et al., 2016 and Shinagawa T1, Ishii S. 2003. To facilitate the formation of siRNAs from dsRNA, the srRNA1ts2 vector also expresses human DICER1 (NCBI Reference Sequence: NG_016311.1). The srRNA1ts2-asRNA or srRNA1ts2-DICER1-asRNA is delivered to target cells, where the target gene is silenced when the target cells are maintained at the permissive temperature. However, when the temperature is shifted to the nonpermissive temperature, production of the asRNA (and DICER1, if present) stops, and the target gene is not silenced. Alternatively, a temperature-sensitive Sendai virus vector encoding asRNA or asRNA-DICER1 can be used instead of srRNA1ts2-asRNA or srRNA1ts2-asRNA-DICER1. For example, SeVt may be SeV18 / TS15ΔF (Ban et al., PNAS 2011).

[0198] The gene knockdown or gene silencing methods detailed above apply to either "ex vivo treatment of cells with ts agonists," "ex vivo therapeutic use with ts agonists," "semi-in vivo therapeutic use with ts agonists," or "in vivo therapeutic use with ts agonists." Example 18: Cell fusion therapy

[0199] One strategy in the field of regenerative medicine is to differentiate human pluripotent stem cells, such as embryonic stem (ES) cells or induced pluripotent stem (iPS) cells, into desired cell types, such as neurons or muscle, and then transplant these differentiated cells into patients. In many cases, it is desirable to use iPS cells generated from the patient's own cells, such as blood cells or fibroblasts. For example, to treat patients suffering from muscular dystrophy, skeletal muscle cells differentiated ex vivo from ES or iPS cells can be transplanted into the patient's skeletal muscle. One technical challenge is ensuring proper engraftment of the foreign muscle cells and replacing or supplementing the patient's defective muscle function. To address this issue, RNA encoding a fusogenic protein can be delivered into the foreign muscle cells, which facilitates cell-to-cell fusion between the foreign muscle cells and the patient's own muscle cells. Semi-in vivo therapeutic use of temperature-sensitive agents for delivery of fusion proteins

[0200] The srRNA1ts2 vector was constructed to express a fusogenic protein, such as the Sendai virus F and HN proteins. F and HN are fused into a single protein via the P2A self-cleaving peptide. The srRNA1ts2-F-HN vector was used to produce a synthetic RNA (srRNA1ts2-F-HN). It has been established that the presence of F and HN proteins on the cell surface can induce cell fusion (Rawling et al., 2008). Alternatively, the human respiratory syncytial virus F protein, which can induce cell-cell fusion by itself (Rawling et al., 2008), was cloned into the srRNA vector to produce a synthetic RNA (srRNA1ts2-RSVF). Another example of a fusogenic protein is Myomaker (Mymk) and Myomixer (Mymx), also known as Myomerger (Bi et al., 2017). Myomaker and myomixer are fused into a single protein via the P2A self-cleaving peptide. The srRNA1ts2-Mymk-Mymx vector is used to produce the synthetic RNA (srRNA1ts2-Mymk-Mymx). The presence of both Myomaker and Myomixer induces cell-cell fusion not only in muscle but also in fibroblasts (Bi et al., 2017). Human iPS cells are differentiated into skeletal muscle using a previously described method. Skeletal muscle is then transfected with srRNA1ts2-HN-F, srRNA1ts2-SRVF, or srRNA1ts2-Mymk-Mymx and immediately injected into the skeletal muscle of patients whose body temperature has previously been lowered to 33°C by therapeutic hypothermia (Figure 15). The fusion protein is expressed in the transplanted skeletal muscle cells (which then fuse with the patient's own defective skeletal muscle cells) while the patient is maintained at the target temperature (33° C.) for 24 hours. The patient's body temperature is then returned to normothermia at 37° C. The ts agent and fusion protein no longer function in this non-functional condition of 37° C. inside the patient's body.

[0201] Tissues that can be treated with this semi-in vivo use of ts agents are not limited to skeletal muscle. Cardiomyocytes and hepatocytes are usually polyploid, so cardiac tissue and the liver are suitable targets for this therapy. Cardiomyocytes and hepatocytes are tissues that can be easily generated from human ES / iPS cells. Furthermore, semi-in vivo use of ts agents can be used to treat neurodegenerative diseases such as spinal cord injury and neurological diseases such as Parkinson's disease. Alternatively, a temperature-sensitive Sendai virus vector can be used instead of srRNA1ts. For example, SeVt can be SeV18 / TS15ΔF (Ban et al., PNAS 2011).

[0202] The gene knockdown or gene silencing methods detailed above may be applied to either "ex vivo treatment of cells with ts agonists," "ex vivo therapeutic use with ts agonists," "semi-in vivo therapeutic use with ts agonists," or "in vivo therapeutic use with ts agonists." Example 19: Vaccine

[0203] 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 20). No further action is required.

[0204] 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 22). Intranasal administration may be by insufflation, inhalation, or instillation. No further action is required.

[0205] 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).

[0206] Immunogenic compositions and vaccines using srRNA1ts2 as a vector are suitable for eliciting immune responses against all types of pathogens. For example, recombinant srRNA1ts2 vectors can be constructed relatively quickly once the coding region for a pathogen's antigen is known. Furthermore, the RNA of srRNA1ts2 vectors is transcribed in vitro without the use of materials of animal or human origin. Thus, vaccines using srRNA1ts2 vectors are easily adapted for production using current manufacturing and quality control standards. Alternatively, temperature-sensitive Sendai virus vectors encoding pathogen antigens may be used (e.g., SeV18 / TS15ΔF).

[0207] 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:39 = TGAAA) between beta-sheet 5 and beta-sheet 6. The additional amino acids confer temperature sensitivity to the self-replicating RNA.

[0208] The genome of an exemplary srRNA1ts2 vector encoding the spike protein (or a portion thereof) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, also known as 2019-nCoV) is shown in Figure 21. The spike protein and receptor-binding domain (RBD) of the spike protein of related coronaviruses have previously been identified as targets for vaccine and drug development (Du et al., Nat Rev Microbiol, 7:226-236, 2009). The sequence of the RNA genome of 2019-nCoV is designated NC_045512 under NCBI accession number NC_045512. Three different temperature-sensitive srRNA1ts2 vectors were constructed. srRNA1ts2-2019-nCoV-Spike encodes the full-length spike protein. srRNA1ts2-2019-nCoV-RBD1 encodes the CD5 signal peptide fused to the RBD of the spike protein. srRNA1ts2-2019-nCoV-RBD2 encodes the RBD, transmembrane domain, and signal peptide of the spike protein fused to the cytoplasmic tail of the spike protein. Expression of the spike protein or its fragment is driven by the 26S promoter of the VEEV replicon.

[0209] Total RNA is transcribed in vitro using T7 RNA polymerase. The RNA is then transfected into cells of the subject's dermal tissue. A suitable method for transfection is by patch electroporation of naked RNA. Alternatively, microneedles can be used to intradermally transfect RNA. For example, dissolvable microneedles made of hyaluronic acid or chitosan-hyaluronic acid complexes can be used to intradermally transfect RNA. In some embodiments, the standard Mantoux procedure can be used. Alternatively, special injection devices designed to facilitate intradermal injection can be used.

[0210] The amino acid sequence of the spike protein of srRNA1ts2-2019-nCoV-spike is SEQ ID NO: 41: [ka] The signal peptide spans residues 1-15, the extracellular region spans residues 16-1213, the transmembrane domain spans residues 1214-1236, and the cytoplasmic region spans residues 1237-1273.

[0211] The amino acid sequence of the spike protein fragment of srRNA1ts2-2019-nCoV-RBD1 is SEQ ID NO: 42: [ka] The CD5 signal peptide spans residues 1-24, and the RBD spans residues 25-192.

[0212] The amino acid sequence of the spike protein fragment of srRNA1ts2-2019-nCoV-RBD2 is SEQ ID NO: 43: [ka] The signal peptide spans residues 1-15, the RBD spans residues 16-207, the transmembrane domain spans residues 208-230, and the cytoplasmic region spans residues 231-267.

[0213] The amino acid sequence of the RBD is SEQ ID NO:44: [ka] It is stipulated that: Example 20: In vivo expression of a GOI after insertion of a temperature-sensitive srRNA

[0214] As described in Example 19 and shown in Figure 20, temperature-sensitive agents (ts agents), such as srRNAs or Sendai virus vectors, which are functional at permissive temperatures (e.g., approximately 31-34°C) but not at non-permissive temperatures (e.g., 37°C), are delivered to the surface of the human body (e.g., skin) for controlled expression of a gene of interest (GOI). Thus, ts agents encoding a GOI have an inherent safety feature in that expression of the GOI is limited to the local (permissive temperature) to which the ts agent is delivered. That is, unintended expression of the GOI by the ts agent does not occur in areas of the subject's body that do not naturally have temperatures above or below the permissive temperature. This example demonstrates that this safety feature functions in vivo in a model mammalian subject, i.e., a mouse. Because mouse skin temperature is similar to that of humans (Mortola 2013), it is expected that intradermal delivery of ts agents to mice will mimic intradermal delivery of ts agents to humans.

[0215] RNA was formulated as naked RNA in lactated Ringer's solution without lipid nanoparticles or other transfection reagents. Luciferase (LUC)-encoding RNA (5 μg) was injected intradermally into a single site on the right hind leg of CD-1 outbred mice. Luciferase activity was visualized and quantified using a bioluminescence imaging system, AMI HTX (Spectral Instruments Imaging, Tucson, AZ).

[0216] Figure 23 shows the time course of in vivo luciferase activity from day 0 (the day of injection) to day 26 in recipients of either the control synRNA-LUC (TriLink, San Diego, CA) or the temperature-sensitive srRNA (srRNA1ts2-LUC). Luciferase imaging demonstrated that intradermal injection of naked RNA encoding luciferase resulted in luciferase expression in vivo. Remarkably, due to its self-replicating characteristics, in vivo expression of luciferase driven by srRNA1ts2-LUC continued for nearly a month. In contrast, in vivo expression of luciferase driven by synRNA-LUC lasted only a little over a week. Furthermore, due to its self-replicating characteristics, the luciferase expression levels in recipients of srRNA1ts2-LUC were 10- to 100-fold higher than those in recipients of synRNA-LUC. Importantly, no luciferase expression was observed in non-injected areas of the recipient skin or in the recipient's internal organs, suggesting that the temperature-sensitive srRNA1ts2-LUC did not replicate and express luciferase under non-permissive conditions. Example 21: Cellular immunity induced by temperature-sensitive srRNA vaccine

[0217] In this example, cytokine-secreting splenocytes induced by intradermal administration of temperature-sensitive srRNA expressing the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 were measured. The RNA was formulated as naked RNA in lactated Ringer's solution without any lipid nanoparticles or other transfection reagents. To assess cellular immunity, enzyme-linked immunospot (ELISpot) assays (which quantify the number of cytokine-secreting cells) were performed on splenocytes obtained from CD-1 outbred mice that received a single dose of placebo (buffer only) or 5 μg, 25 μg, or 100 μg of srRNA1ts2-2019-CoV-RBD1 RNA (described in Example 19). Splenocytes isolated 12 days after injection were stimulated for 24 hours with a pool of 53 peptides (15mers with 11 amino acid overlaps) covering the SARS-CoV-2 RBD (PepMix SARS-CoV-2 [S-RBD], Technologies GmbH, JPT Peptide Berlin Germany).

[0218] As shown in Figure 24A, srRNA1ts2-2019-nCoV-RBD1 administered by intradermal injection induced cellular immunity against SARS-CoV-2 RBD in a dose-dependent manner. IFN-γ-secreting cells (characteristic of type 1 CD4+ T helper cells (Th1 cells) and CD8+ cytotoxic T cells) (Figure 24A) were preferentially expanded by the temperature-sensitive SARS-CoV-2 RBD srRNA vaccine. In contrast, IL4-secreting cells (characteristic of type 2 CD4+ T helper cells (Th2 cells)) (Figure 24B) were expanded by the temperature-sensitive SARS-CoV-2 RBD srRNA vaccine. In conclusion, we show that intradermal administration of srRNA1ts2-2019-nCoV-RBD1 elicits a Th1-dominated (Th1>Th2) cellular immune response against the SARS-CoV-2 RBD, a desirable characteristic of vaccines directed against viral pathogens. Example 22: Humoral immunity induced by a temperature-sensitive srRNA vaccine

[0219] In this example, we measured antibodies elicited by intradermal administration of a temperature-sensitive srRNA expressing the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. The RNA was formulated as naked RNA in lactated Ringer's solution without any lipid nanoparticles or other transfection reagents. Groups of CD-1 outbred mice (N=10) received one of three formulations by intradermal injection on days 0 and 14 (black triangles): placebo (buffer only) (Figure 25A), 5 μg of temperature-sensitive srRNA1ts2-2019-CoV-RBD1 RNA (Figure 25B), or 5 μg of temperature-sensitive srRNA1ts2-2019-CoV-RBD1 RNA combined with an RNase inhibitor (3 units of RNasin Plus) (Promega, Madison, WI) (Figure 25C). All mice received recombinant RBD protein (Ala319-Phe541 with a C-terminal 6-His tag, accession number YP_009724390.1: R&D Systems, Minneapolis, MN) by intradermal injection on day 49 (open triangles). To assess humoral immunity, enzyme-linked immunosorbent assay (ELISA) (quantitating the amount of immunoglobulin G (IgG) specific for the recombinant RBD protein (expressed as OD450 measurements)) was performed on serum obtained from injected mice on days 3, 14, 28, 46, 56, and 63. The amount of IgG in serum is shown as OD450.

[0220] As shown in Figures 25A-25C, no increase in RBD-specific IgG was observed in either group after the prime and first booster alone. However, when mice were given a second booster containing the recombinant RBD protein, the group receiving the prime and first booster containing srRNA1ts2-2019-CoV-RBD1 showed a rapid response with an increase in RBD-specific IgG (Figures 25B-C), whereas the placebo group showed no clear increase in RBD-specific IgG (Figure 25A). The rapid increase in RBD-specific IgG following injection of the recombinant RBD protein indicates that mice receiving the prime and first booster containing srRNA1ts2-2019-CoV-RBD1 maintained immune memory against the recombinant RBD and displayed a secondary humoral response. Some aspects of the invention are described below. 1. An immunogenic composition for stimulating an immune response to a pathogen in a subject, comprising an excipient and a temperature-sensitive agent (ts agent), wherein the ts agent is a temperature-sensitive viral vector or a temperature-sensitive self-replicating RNA encoding an antigen of the pathogen, and wherein the ts agent is capable of expressing the antigen at a permissive temperature but not at a non-permissive temperature. 2. The composition of item 1, wherein the pathogen comprises one or more of a virus, a bacterium, a protozoan, and a fungus. 3. The composition according to item 2, wherein the antigen comprises a surface protein or a fragment thereof of a pathogen. 4. The composition according to item 3, wherein the pathogen is a virus and the virus is different from the viral vector. 5. The composition of item 4, wherein the virus is a coronavirus and the antigen comprises a coronavirus spike protein or a fragment thereof. 6. The composition of item 5, wherein the coronavirus is 2019-nCoV and the antigen comprises the receptor binding domain (RBD) of 2019-nCoV. 7. The composition of item 6, wherein the amino acid sequence of the RBD comprises SEQ ID NO: 44 or an amino acid sequence at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 44. 8. The composition of item 5, wherein the coronavirus is 2019-nCoV and the antigen comprises an extracellular domain of a spike protein comprising the amino acid sequence of residues 16 to 1213 of SEQ ID NO: 41, or an amino acid sequence at least 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41. 9. The composition according to any one of items 1 to 8, wherein the permissible temperature is 30°C to 36°C, 31°C to 35°C, 32°C to 34°C, or 33°C±0.5°C, and the non-permissible temperature is 37°C±0.5°C. 10. The composition of item 9, wherein the ts agent is a temperature-sensitive self-replicating RNA. 11. The composition of item 10, wherein the self-replicating RNA comprises an alphavirus replicon lacking a viral structural protein coding region. 12. The composition of claim 11, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 13. The composition of claim 11, wherein the alphavirus is Venezuelan equine encephalitis virus. 14. The composition of item 9, wherein the ts agent is a temperature-sensitive viral vector. 15. The composition of item 14, wherein the viral vector is selected from the group consisting of Sendai virus, adenovirus, adeno-associated virus, retrovirus, and alphavirus. 16. The composition of claim 15, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 17. The composition according to item 15, wherein the viral vector is Sendai virus. 18. The composition of item 9, wherein the subject is a human and the pathogen is a human pathogen. 19. A method for stimulating an immune response to a pathogen in a mammalian subject, comprising administering to the mammalian subject the immunogenic composition of item 18 to stimulate an immune response to an antigen in the mammalian subject, wherein the mammalian subject is a human subject. 20. The immunogenic composition comprises: i) intradermal or subcutaneous; or ii) intramuscularly; 20. The method of item 19, wherein the patient is administered 21. The method of item 19, wherein the immunogenic composition is administered intranasally. 22. A temperature-sensitive agent, wherein the agent is a temperature-sensitive viral vector or a temperature-sensitive self-replicating RNA comprising a nonstructural protein coding region with an insertion of 12-18 nucleotides, wherein the insertion results in expression of nonstructural protein 2 (nsP2 = helicase proteinase) with 4-6 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. 23. The temperature-sensitive agent according to item 22, wherein the additional amino acids comprise one sequence selected from the group consisting of SEQ ID NO: 38 (GCGRT), SEQ ID NO: 39 (TGAAA), and SEQ ID NO: 40 (LRPHP). 24. The temperature-sensitive agent according to item 22, wherein the additional amino acids comprise the sequence of SEQ ID NO: 39 (TGAAA). 25. The temperature-sensitive agent according to item 24, wherein the amino acid sequence of NsP2 comprises one sequence selected from the group consisting of SEQ ID NOs: 29 to 36. 26. The temperature-sensitive agent of item 22, wherein the agent is a temperature-sensitive alphavirus vector. 27. The temperature-sensitive agent of item 22, wherein the agent is a temperature-sensitive self-replicating RNA comprising an alphavirus replicon lacking a viral structural protein coding region. 28. The temperature sensitive agent of claim 26 or 27, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 29. The temperature sensitive agent of claim 26 or 27, wherein the alphavirus is Venezuelan equine encephalitis virus. 30. A method of transiently inducing temperature-sensitive activity of a temperature-sensitive agent (ts agent) in a subject, wherein one or more cells at or near the surface of the subject's body contain the ts agent, and wherein the temperature-sensitive activity of the ts agent is induced 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: 31. A method for transiently inducing temperature-sensitive activity of a temperature-sensitive agent (ts agent) in a subject, wherein the temperature-sensitive activity of the ts agent is induced 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: 32.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; 32. The method of claim 31, further comprising: 33. The method of claim 31, wherein the temperature-sensitive agent is administered intradermally or subcutaneously. 34. The method of claim 31, wherein the temperature-sensitive agent is administered intramuscularly. 35. The method according to any one of items 30 to 34, wherein the permissive temperature is 30°C to 36°C, 31°C to 35°C, 32°C to 34°C, or 33°C±0.5°C, and the non-permissive temperature is 37°C±0.5°C. 36. The method of item 35, wherein the pharmaceutical agent is encoded by a coding region of a ts agent, or wherein the ts agent comprises a pharmaceutical agent and the effect comprises a pharmaceutical effect, optionally wherein the pharmaceutical agent is a therapeutic agent and the pharmaceutical effect is a therapeutic effect, or wherein the pharmaceutical agent is a prophylactic agent and the pharmaceutical effect is a prophylactic effect. 37. The method of claim 35, wherein the ts agent is a temperature-sensitive viral vector and the temperature-sensitive activity comprises replication and transcription of the temperature-sensitive viral vector. 38. The method of item 37, wherein the temperature-sensitive viral vector is selected from the group consisting of Sendai virus, adenovirus, adeno-associated virus, retrovirus, and alphavirus. 39. The method of item 37, wherein the temperature-sensitive viral vector is an alphavirus, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 40. The method according to item 37, wherein the temperature-sensitive viral vector is Sendai virus. 41. The method of item 35, wherein the ts agent is a temperature-sensitive self-replicating RNA and the temperature-sensitive activity comprises one or both of replication and transcription of the temperature-sensitive self-replicating RNA. 42. The method of claim 41, wherein the self-replicating RNA comprises an alphavirus replicon lacking the viral structural protein coding region of the alphavirus. 43. The method of claim 42, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 44. The method of claim 42, wherein the alphavirus is Venezuelan equine encephalitis virus. 45. The method of claim 36, wherein the pharmaceutical agent is selected from the group consisting of non-coding RNA, siRNA, shRNA, and an endonuclease editing system. 46. ​​The method of claim 36, wherein the pharmaceutical agent comprises a protein. 47. The method of claim 46, wherein the protein comprises an antigen of a pathogen. 48. The method of item 47, wherein the pathogen comprises one or more of a virus, a bacterium, a protozoan, and a fungus. 49. The method of claim 47, wherein the antigen comprises a surface protein or fragment thereof of a pathogen. 50. The method of claim 36, wherein the period of time sufficient for the temperature-sensitive activity to have an effect is in the range of about 12 hours to about 12 weeks, and optionally, the period is 1 to 7 days. 51. The method of item 36, 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. 52. 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 and the effect comprises a therapeutic effect. 53. Before step i), contacting one or more cells with a temperature sensitive agent; 53. The method of claim 52, further comprising: 54. The method of item 53, wherein the one or more cells are at a permissive temperature when contacted with the temperature-sensitive agent. 55. The method of paragraph 52, further comprising administering one or more cells to a subject in need of a therapeutic effect. 56. The method of item 52, 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. 57. The method of item 56, wherein the one or more cells are further incubated at a non-permissive temperature before administering the one or more cells to a subject. 58. The method of item 52, wherein the one or more cells are embryonic stem cells or induced pluripotent stem cells. 59. The method of item 58, wherein the therapeutic effect comprises differentiation of the cells into a desired cell type. 60. The method of item 59, wherein the desired cell type is selected from the group consisting of neurons, glial cells, and endothelial cells. 61. The method of item 53, wherein the one or more cells are isolated from a subject prior to contacting the one or more cells with a temperature-sensitive agent. 62. A method for transiently inducing a temperature-sensitive activity of a temperature-sensitive agent in a 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 lower than the 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; A method comprising: 63. A method for transiently inducing temperature-sensitive activity of a temperature-sensitive agent in a subject, wherein the temperature-sensitive activity of the temperature-sensitive agent is induced at a permissive temperature, and wherein the permissive temperature is lower 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 back to normothermia, wherein step (i) occurs before, after, or simultaneously with step (ii); A method comprising: 64. The method of item 63, wherein the temperature-sensitive agent is administered systemically. 65. The method of claim 64, wherein the temperature-sensitive agent is administered intravenously. 66. The method of item 63, wherein the temperature-sensitive agent is administered to a specific tissue or organ of the subject. 67. The method of claim 66, wherein the temperature-sensitive agent is administered to the brain or spinal cord by epidural injection. 68. The method of item 66, wherein the temperature-sensitive agent is administered to the target organ by intradermal injection. 69. The method of claim 66, wherein the temperature-sensitive agent is administered to the target organ by endoscopy using an injection needle catheter. 70. The method of item 66, wherein the temperature-sensitive agent is administered to the target organ by a vascular catheter. 71. The method of item 68, 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. 72. The method of item 63, wherein the temperature-sensitive agent is administered by inhalation. 73. The method of item 63, wherein altering 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. 74. The method of item 63, wherein the subject is a human. 75. The method of claim 63, wherein the temperature-sensitive agent comprises a therapeutic agent and the effect comprises a therapeutic effect. 76. The method of item 75, wherein the therapeutic agent is encoded by a coding region of a heterologous nucleic acid of a temperature-sensitive viral vector. 77. The method of item 76, wherein the temperature-sensitive viral vector is selected from the group consisting of Sendai virus, adenovirus, adeno-associated virus, retrovirus, and alphavirus. 78. The method of item 77, wherein the temperature-sensitive viral vector is an alphavirus. 79. The method of claim 78, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 80. The method according to item 77, wherein the temperature-sensitive viral vector is Sendai virus. 81. The method of item 75, wherein the therapeutic agent is selected from the group consisting of non-coding RNA, siRNA, shRNA, and an endonuclease editing system. 82. The method of claim 75, wherein the therapeutic agent is a protein. 83. The method of item 82, wherein the protein is a transcription factor, optionally wherein the transcription factor is selected from the group consisting of human neurogenin-3 (NGN3) and human ETS translocation variant 2 (ETV2). 84. The method of item 82, wherein the protein is a growth factor, optionally wherein the growth factor is selected from the group consisting of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). 85. The method of item 76, wherein the temperature-sensitive activity comprises replication and transcription of a temperature-sensitive viral vector. 86. The method of item 75, wherein the therapeutic agent is encoded by a coding region of a temperature-sensitive self-replicating RNA. 87. The method of item 86, wherein the self-replicating RNA comprises an alphavirus replicon lacking a viral structural protein coding region. 88. The method of item 87, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus. 89. The method of item 86, wherein the therapeutic agent is selected from the group consisting of non-coding RNA, siRNA, shRNA, and an endonuclease editing system. 90. The method of claim 86, wherein the therapeutic agent is a protein. 91. The method of item 90, wherein the protein is a transcription factor, optionally wherein the transcription factor is selected from the group consisting of human neurogenin-3 (NGN3) and human ETS translocation variant 2 (ETV2). 92. The method of item 90, wherein the protein is a growth factor, optionally wherein the growth factor is selected from the group consisting of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). 93. The method of item 86, wherein the temperature-sensitive activity comprises one or both of replication and transcription of a temperature-sensitive self-replicating RNA. 94. The method of item 76, wherein the coding region is operably linked to a promoter. 95. The method of item 75, wherein the period of time sufficient for the temperature-sensitive activity to produce a therapeutic effect is in the range of about 12 hours to about 12 weeks, optionally wherein the period is 1 to 7 days. 96. The method of item 75, 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. 97. The method according to any one of items 52 to 96, wherein the permissible temperature is in the range of 30°C to 36°C, 31°C to 35°C, or 32°C to 34°C. 98. The method according to item 97, wherein the permissible temperature is 33°C ± 0.5°C. 99. The method of claim 98, wherein the non-permissive temperature is 37°C ± 0.5°C. 100. The method of item 52, wherein the one or more cells are human cells. 101. The method of item 100, wherein the one or more human cells are adult stem cells, tissue stem cells, progenitor cells, embryonic stem cells, or induced pluripotent stem cells. 102. The method according to item 101, wherein the one or more human cells are selected from the group consisting of hematopoietic stem cells, mesenchymal stem cells, adipose stem cells, neural stem cells, and germline stem cells. 103. The method of item 100, wherein the one or more human cells are somatic cells, mature cells, or differentiated cells. 104. The method of item 103, 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, cardiac myocytes, pancreatic cells, pancreatic beta cells, keratinocytes, erythrocytes, peripheral blood mononuclear cells (PBMCs), neurons, glial cells, neural cells, astrocytes, germ cells, sperm cells, and oocytes. 105. The method of item 100, wherein the one or more human cells are human bone marrow cells. 106. The method according to item 105, wherein the human bone marrow cells are selected from the group consisting of hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells. 107. The method of item 106, wherein the hematopoietic stem cells are CD34+. 108. The method of item 97, 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) containing 4 to 6 additional amino acids between beta-sheet 5 and beta-sheet 6 of nsP2, optionally wherein the additional amino acids confer temperature sensitivity to the viral vector or self-replicating RNA. 109. The method according to item 108, wherein the additional amino acids comprise one sequence selected from the group consisting of SEQ ID NO: 38 (GCGRT), SEQ ID NO: 39 (TGAAA), and SEQ ID NO: 40 (LRPHP). 110. The method according to item 108, wherein the additional amino acids comprise the sequence of SEQ ID NO: 39 (TGAAA). 111. The method according to item 110, wherein the amino acid sequence of NsP2 comprises one sequence selected from the group consisting of SEQ ID NOs: 29 to 36.

Claims

1. An immunogenic composition for stimulating an immune response to a pathogen in a subject, comprising an excipient and a temperature-sensitive agent (ts agent), wherein the ts agent is a temperature-sensitive viral vector or a temperature-sensitive self-replicating RNA encoding an antigen of the pathogen, and wherein the ts agent is capable of expressing the antigen at a permissive temperature but not at a non-permissive temperature.

2. 10. The composition of claim 1, wherein the pathogen comprises one or more of a virus, a bacterium, a protozoan, and a fungus.

3. The composition of claim 2 , wherein the antigen comprises a surface protein or fragment thereof of a pathogen.

4. The composition of claim 3, wherein the pathogen is a virus and the virus is different from the viral vector.

5. 5. The composition of claim 4, wherein the virus is a coronavirus and the antigen comprises a coronavirus spike protein or a fragment thereof.

6. 6. The composition of claim 5, wherein the coronavirus is 2019-nCoV and the antigen comprises the receptor binding domain (RBD) of 2019-nCoV.

7. 7. The composition of claim 6, wherein the amino acid sequence of the RBD comprises SEQ ID NO:44, or an amino acid sequence at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

44.

8. 6. The composition of claim 5, wherein the coronavirus is 2019-nCoV and the antigen comprises an extracellular region of a spike protein comprising the amino acid sequence of residues 16 to 1213 of SEQ ID NO:41, or an amino acid sequence that is at least 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

41.

9. 9. The composition of any one of claims 1 to 8, wherein the permissive temperature is 30°C to 36°C, 31°C to 35°C, 32°C to 34°C, or 33°C ± 0.5°C, and the non-permissive temperature is 37°C ± 0.5°C.

10. The composition of claim 9, wherein the ts agent is a temperature-sensitive self-replicating RNA.

11. The composition of claim 10, wherein the self-replicating RNA comprises an alphavirus replicon lacking a viral structural protein coding region.

12. 12. The composition of claim 11, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus.

13. 12. The composition of claim 11, wherein the alphavirus is Venezuelan equine encephalitis virus.

14. The composition of claim 9, wherein the ts agent is a temperature-sensitive viral vector.

15. 15. The composition of claim 14, wherein the viral vector is selected from the group consisting of Sendai virus, adenovirus, adeno-associated virus, retrovirus, and alphavirus.

16. 16. The composition of claim 15, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus.

17. The composition of claim 15, wherein the viral vector is a Sendai virus.

18. 10. The composition of claim 9, wherein the subject is a human and the pathogen is a human pathogen.

19. 20. A method for stimulating an immune response to a pathogen in a mammalian subject, comprising administering to the mammalian subject the immunogenic composition of claim 18 to stimulate an immune response to an antigen in the mammalian subject, wherein the mammalian subject is a human subject.

20. The immunogenic composition comprises: i) intradermal or subcutaneous; or ii) intramuscularly; 20. The method of claim 19, wherein the patient is administered

21. 20. The method of claim 19, wherein the immunogenic composition is administered intranasally.

22. A temperature-sensitive agent, wherein the agent is a temperature-sensitive viral vector or a temperature-sensitive self-replicating RNA comprising 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) with 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.

23. 23. The temperature-sensitive agent of claim 22, wherein the additional amino acids comprise one sequence selected from the group consisting of SEQ ID NO: 38 (GCGRT), SEQ ID NO: 39 (TGAAA), and SEQ ID NO: 40 (LRPHP).

24. 23. The temperature-sensitive agent of claim 22, wherein the additional amino acids comprise the sequence of SEQ ID NO: 39 (TGAAA).

25. 25. The temperature-sensitive agent of claim 24, wherein the amino acid sequence of NsP2 comprises one sequence selected from the group consisting of SEQ ID NOs: 29 to 36.

26. 23. The temperature sensitive agent of claim 22, wherein the agent is a temperature sensitive alphavirus vector.

27. 23. The temperature-sensitive agent of claim 22, wherein the agent is a temperature-sensitive self-replicating RNA comprising an alphavirus replicon lacking viral structural protein coding regions.

28. 28. The temperature sensitive agent of claim 26 or claim 27, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus.

29. 28. The temperature sensitive agent of claim 26 or claim 27, wherein the alphavirus is Venezuelan equine encephalitis virus.

30. 1. A method of transiently inducing temperature-sensitive activity of a temperature-sensitive agent (ts agent) in a subject, wherein one or more cells at or near the surface of the subject's body contain the ts agent, wherein the temperature-sensitive activity of the ts agent is induced 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 elicit 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:

31. 1. A method of transiently inducing temperature-sensitive activity of a temperature-sensitive agent (ts agent) in a subject, wherein the temperature-sensitive activity of the ts agent is induced 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 a subject's body; 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:

32. 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; 32. The method of claim 31, further comprising:

33. 32. The method of claim 31 , wherein the temperature-sensitive agent is administered intradermally or subcutaneously.

34. 32. The method of claim 31 , wherein the temperature-sensitive agent is administered intramuscularly.

35. 35. The method of any one of claims 30 to 34, wherein the permissive temperature is 30°C to 36°C, 31°C to 35°C, 32°C to 34°C, or 33°C±0.5°C, and the non-permissive temperature is 37°C±0.5°C.

36. 36. The method of claim 35, wherein the pharmaceutical agent is encoded by a coding region of a ts agent, or wherein the ts agent comprises a pharmaceutical agent and the effect comprises a pharmaceutical effect, optionally wherein the pharmaceutical agent is a therapeutic agent and the pharmaceutical effect is a therapeutic effect, or wherein the pharmaceutical agent is a prophylactic agent and the pharmaceutical effect is a prophylactic effect.

37. 36. The method of claim 35, wherein the ts agent is a temperature-sensitive viral vector and the temperature-sensitive activity comprises replication and transcription of the temperature-sensitive viral vector.

38. 38. The method of claim 37, wherein the temperature-sensitive viral vector is selected from the group consisting of Sendai virus, adenovirus, adeno-associated virus, retrovirus, and alphavirus.

39. 38. The method of claim 37, wherein the temperature-sensitive viral vector is an alphavirus, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus.

40. The method of claim 37, wherein the temperature-sensitive viral vector is Sendai virus.

41. 36. The method of claim 35, wherein the ts agent is a temperature-sensitive self-replicating RNA and the temperature-sensitive activity comprises one or both of replication and transcription of the temperature-sensitive self-replicating RNA.

42. 42. The method of claim 41, wherein the self-replicating RNA comprises an alphavirus replicon lacking the viral structural protein coding region of the alphavirus.

43. 43. The method of claim 42, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus.

44. 43. The method of claim 42, wherein the alphavirus is Venezuelan equine encephalitis virus.

45. 37. The method of claim 36, wherein the pharmaceutical agent is selected from the group consisting of non-coding RNA, siRNA, shRNA, and an endonuclease editing system.

46. 37. The method of claim 36, wherein the pharmaceutical agent comprises a protein.

47. 47. The method of claim 46, wherein the protein comprises an antigen of a pathogen.

48. 48. The method of claim 47, wherein the pathogen comprises one or more of a virus, a bacterium, a protozoan, and a fungus.

49. 48. The method of claim 47, wherein the antigen comprises a surface protein or fragment thereof of a pathogen.

50. 37. The method of claim 36, wherein the period of time sufficient for the temperature-sensitive activity to take effect ranges from about 12 hours to about 12 weeks, and optionally, the period of time is 1 to 7 days.

51. 37. The method of claim 36, 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.

52. 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 and the effect comprises a therapeutic effect.

53. Before step i), contacting one or more cells with a temperature sensitive agent; 53. The method of claim 52, further comprising:

54. 54. The method of claim 53, wherein the one or more cells are at a permissive temperature when contacted with the temperature-sensitive agent.

55. 53. The method of claim 52, further comprising administering the one or more cells to a subject in need of the therapeutic effect.

56. 53. The method of claim 52, 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.

57. 57. The method of Claim 56, 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.

58. 53. The method of claim 52, wherein the one or more cells are embryonic stem cells or induced pluripotent stem cells.

59. 59. The method of claim 58, wherein the therapeutic effect comprises differentiation of the cells into a desired cell type.

60. 60. The method of claim 59, wherein the desired cell type is selected from the group consisting of neurons, glial cells, and endothelial cells.

61. 54. The method of claim 53, wherein the one or more cells are isolated from a subject prior to contacting the one or more cells with a temperature-sensitive agent.

62. 1. A method of transiently inducing a temperature-sensitive activity of a temperature-sensitive agent in a 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) reducing 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; A method comprising:

63. 1. A method of transiently inducing a temperature-sensitive activity of a temperature-sensitive agent in a 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 a body temperature of the subject, comprising: i) reducing 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 back to normothermia, wherein step (i) occurs before, after, or simultaneously with step (ii); A method comprising:

64. 64. The method of claim 63, wherein the temperature-sensitive agent is administered systemically.

65. 65. The method of claim 64, wherein the temperature-sensitive agent is administered intravenously.

66. 64. The method of claim 63, wherein the temperature-sensitive agent is administered to a specific tissue or organ of the subject.

67. 67. The method of claim 66, wherein the temperature-sensitive agent is administered to the brain or spinal cord by epidural injection.

68. 67. The method of claim 66, wherein the temperature-sensitive agent is administered to the target organ by intradermal injection.

69. 67. The method of claim 66, wherein the temperature-sensitive agent is administered to the target organ endoscopically using a needle catheter.

70. 67. The method of claim 66, wherein the temperature-sensitive agent is administered to the target organ by a vascular catheter.

71. 69. The method of claim 68, 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.

72. 64. The method of claim 63, wherein the temperature-sensitive agent is administered by inhalation.

73. 64. The method of claim 63, wherein altering 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.

74. 64. The method of claim 63, wherein the subject is a human.

75. 64. The method of claim 63, wherein the temperature-sensitive agent comprises a therapeutic agent and the effect comprises a therapeutic effect.

76. 76. The method of claim 75, wherein the therapeutic agent is encoded by a coding region of a heterologous nucleic acid of a temperature-sensitive viral vector.

77. 77. The method of claim 76, wherein the temperature-sensitive viral vector is selected from the group consisting of Sendai virus, adenovirus, adeno-associated virus, retrovirus, and alphavirus.

78. 78. The method of claim 77, wherein the temperature-sensitive viral vector is an alphavirus.

79. 79. The method of claim 78, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus.

80. 78. The method of claim 77, wherein the temperature-sensitive viral vector is Sendai virus.

81. 76. The method of claim 75, wherein the therapeutic agent is selected from the group consisting of non-coding RNA, siRNA, shRNA, and an endonuclease editing system.

82. 76. The method of claim 75, wherein the therapeutic agent is a protein.

83. 83. The method of claim 82, wherein the protein is a transcription factor, optionally wherein the transcription factor is selected from the group consisting of human neurogenin-3 (NGN3) and human ETS translocation variant 2 (ETV2).

84. 83. The method of claim 82, wherein the protein is a growth factor, optionally wherein the growth factor is selected from the group consisting of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF).

85. 77. The method of claim 76, wherein the temperature-sensitive activity comprises replication and transcription of a temperature-sensitive viral vector.

86. 76. The method of claim 75, wherein the therapeutic agent is encoded by a coding region of a temperature-sensitive self-replicating RNA.

87. 87. The method of claim 86, wherein the self-replicating RNA comprises an alphavirus replicon lacking a viral structural protein coding region.

88. 88. The method of claim 87, wherein the alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus.

89. 87. The method of Claim 86, wherein the therapeutic agent is selected from the group consisting of non-coding RNA, siRNA, shRNA, and an endonuclease editing system.

90. 87. The method of claim 86, wherein the therapeutic agent is a protein.

91. 91. The method of claim 90, wherein the protein is a transcription factor, optionally wherein the transcription factor is selected from the group consisting of human neurogenin-3 (NGN3) and human ETS translocation variant 2 (ETV2).

92. 91. The method of claim 90, wherein the protein is a growth factor, optionally wherein the growth factor is selected from the group consisting of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF).

93. 87. The method of claim 86, wherein the temperature-sensitive activity comprises one or both of replication and transcription of a temperature-sensitive self-replicating RNA.

94. 77. The method of claim 76, wherein the coding region is operably linked to a promoter.

95. 76. The method of claim 75, 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.

96. 76. The method of claim 75, 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.

97. 97. The method of any one of claims 52 to 96, wherein the permissive temperature is in the range of 30°C to 36°C, 31°C to 35°C, or 32°C to 34°C.

98. 98. The method of claim 97, wherein the permissible temperature is 33°C ± 0.5°C.

99. 99. The method of claim 98, wherein the non-permissive temperature is 37°C ± 0.5°C.

100. 53. The method of claim 52, wherein the one or more cells are human cells.

101. 101. The method of claim 100, wherein the one or more human cells are adult stem cells, tissue stem cells, progenitor cells, embryonic stem cells, or induced pluripotent stem cells.

102. 102. The method of claim 101, wherein the one or more human cells are selected from the group consisting of hematopoietic stem cells, mesenchymal stem cells, adipose stem cells, neural stem cells, and germline stem cells.

103. 101. The method of claim 100, wherein the one or more human cells are somatic cells, mature cells, or differentiated cells.

104. 104. The method of claim 103, 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, cardiac myocytes, pancreatic cells, pancreatic beta cells, keratinocytes, erythrocytes, peripheral blood mononuclear cells (PBMCs), neurons, glial cells, neural cells, astrocytes, germ cells, sperm cells, and oocytes.

105. 101. The method of claim 100, wherein the one or more human cells are human bone marrow cells.

106. 106. The method of claim 105, wherein the human bone marrow cells are selected from the group consisting of hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells.

107. The method of claim 106, wherein the hematopoietic stem cells are CD34+.

108. 98. The method of claim 97, 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) 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.

109. 109. The method of claim 108, wherein the additional amino acids comprise one sequence selected from the group consisting of SEQ ID NO: 38 (GCGRT), SEQ ID NO: 39 (TGAAA), and SEQ ID NO: 40 (LRPHP).

110. 109. The method of claim 108, wherein the additional amino acids comprise the sequence of SEQ ID NO: 39 (TGAAA).

111. 111. The method of claim 110, wherein the amino acid sequence of NsP2 comprises one sequence selected from the group consisting of SEQ ID NOs: 29 to 36.

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