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

Abstract

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

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 992,745, filed Mar. 20, 2020, and U.S. Provisional Patent Application No. 62 / 955,820, filed Dec. 31, 2019, the entire disclosures of which are incorporated herein by reference. Submission of Sequence Listing in ASCII Text File

[0002] The entire contents of the following submission in ASCII text file are incorporated herein by reference: Sequence listing in computer - readable format (CRF) (filename: 699442001340SEQLIST.TXT, recording date: Dec. 23, 2020, size: 25 KB). Field

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

[0004] background The delivery of therapeutic gene products to human cells, tissues, and organs presents a significant challenge. In conventional gene therapy (which requires continuous gene expression to compensate for a patient's genetic defect), this has been achieved using viral vectors such as retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses. However, equally important strategic gene therapy involves transient, short-term gene expression. For such applications, sustained gene expression is unnecessary and can even be harmful to cells.

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

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

[0007] Given the examples mentioned above, conventional gene therapy using viral vectors to achieve continuous gene expression may become undesirable. For time-limited expression of gene products, intracellular synthesis or delivery of in vitro transcribed mRNA has begun to be used (Warren et al., 2010). However, several problems exist with these methodologies. For example, the amount of mRNA delivered to cells, tissues, and organs is limited, which means that the amount of protein product may not be sufficient for a biologically significant effect in vivo.

[0008] Furthermore, due to the rapid metabolic turnover of RNA, which typically only survives for 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 twice a day for several days is required (Akiyama et al., 2016; Goparaju et al. 2017). To produce iPS cells from human fibroblasts, daily transfection with a synthetic RNA cocktail must be continued for more than two weeks (Warren et al., 2010). This is not only cumbersome but also inefficient.

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

[0010] The advantage of self-replicating RNA vectors lies in their self-replicating properties, which result in increased GOI expression levels. However, one drawback of self-replicating RNA vectors for delivering RNA / protein to mammalian cells is their sustained expression. Typically, this leads to positive feedback production of RNA-dependent RNA polymerase and GOIs, which can result in the death of cells transfected with the naked RNA form of the self-replicating RNA or infected with the viral form of the self-replicating RNA.

[0011] Therefore, what is needed in this field of gene therapy is a tool for the transient expression of GOIs encoding the target protein, such as a therapeutic agent (e.g., human ZSCAN4). In particular, control of transcription and translation of RNA vectors and self-replicating RNA is desirable. [Overview of the Initiative]

[0012] overview Based on the need for time-limited expression of a gene of interest (GOI), a transient gene product delivery system is required 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 stopped after achieving a biologically significant effect. To meet these and other needs, this disclosure relates to a tool for transiently inducing the activity of a temperature-sensitive activator (ts-activator), such as a therapeutic ts-activator, in a subject (in vivo) or in cultured cells (ex vivo). In some embodiments, the therapeutic ts-activator is used in combination with mild therapeutic hypothermia therapy. In other embodiments, the therapeutic ts-activator is used in combination with the local application of mild therapeutic hyperthermia or heating. In some embodiments, the ts-activator is a ts-RNA molecule or a ts-protein molecule. In some embodiments, the ts-activator is encoded by heterologous nucleic acid or self-replicating RNA inserted into a temperature-sensitive viral vector. In some embodiments, the viral vector is selected from, but is not limited to, Sendai virus vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and alphavirus vectors. In some embodiments, the self-replicating RNA comprises an alphavirus replicon lacking a viral structural protein-coding region. In some embodiments, the alphavirus is selected from, but is not limited to, Venezuelan encephalitis virus, Sindbis virus, and Semryki Forest virus. A specific gene product of interest is ZSCAN4, in particular human ZSCAN4.

[0013] The above and other issues and features of this disclosure will become apparent from the following detailed description in relation to the attached drawings. [Brief explanation of the drawing]

[0014] [Figure 1A-1D]Figures 1A-1D show the structure and location of mutant regions of the Venezuelan encephalitis virus (VEEV) genome. Figure 1A shows a schematic diagram of the wild-type VEEV genome (TC-83 strain: complete genome 11,446 bp linear RNA: NCBI registration: L01443.1 GI: 323714). The genes for non-structural proteins (nsP1, nsP2, nsP3, nsP4) encode RNA-dependent RNA polymerase, and the genes for structural proteins encode viral envelope proteins (C, E1, E2), 5'-UTR (5'-untranslated region), and 3'-UTR (3'-untranslated region). The gene for the nsP2 protein, represented as a bolded box, was mutated to produce 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] [Figure 2A-2C] Figures 2A-2C show subsequences of VEEV nsP2 corresponding to the mutated regions of ts1, ts2, and ts3. Figure 2A shows the wild-type sequence compared to mutant 1 (ts1), which contains a 15-nucleotide insertion resulting in five amino acid insertions. Figure 2B shows the wild-type sequence compared to mutant 2 (ts2), which contains a 15-nucleotide insertion resulting in five amino acid insertions. Figure 2C shows the wild-type sequence compared to mutant 3 (ts3), which contains a 15-nucleotide insertion resulting in five amino acid insertions.

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

[0017] [Figure 4A-4B] Figures 4A and 4B show the temperature sensitivity tests 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. RNA produced by in vitro transcription was transfected into human induced pluripotent stem cells (ADSC-iPSC line). Cells were cultured in CO2 incubators maintained at 30°C, 32°C, and 37°C, respectively. Cell images were obtained at 20 hours and 48 hours, respectively. The upper panel shows phase-contrast images, and the lower panel shows fluorescence images detecting green fluorescent protein (GFP) expression. Figure 4A shows the results of cell transfection with srRNA1wt-GFP, srRNA1ts2-GFP, and srRNA1ts3-GFP RNA. Figure 4B shows the results of cell transfection using synthetic mRNA encoding GFP (synRNA-GFP).

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

[0019] [Figure 6]Figure 6 shows the temperature sensitivity tests of srRNA1ts2 and srRNA1ts4 at 32 °C, 33 °C, and 37 °C. Mutant (srRNA1ts2-GFP and srRNA1ts4-GFP) self-replicating RNA (srRNA) vectors were constructed. RNAs produced by in vitro transcription were transfected into human induced pluripotent stem cells (ADSC-iPSC line). The 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 the phase-contrast images, and the lower panel shows the fluorescence images detecting the expression of green fluorescent protein (GFP).

[0020] [Figure 7] Figure 7 shows the temperature sensitivity test of the 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). The 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, and thereby transfected cells can be selected using puromycin. The experiment was conducted in the absence (upper panel) or presence (lower panel) of 1 μg / ml puromycin. Images of the cells were obtained at 24, 48, 72, 96, 144, 168, and 192 hours, respectively. For srRNA1ts1-GFP, images were taken only at 24 and 168 hours. The upper panel shows the phase-contrast images, and the lower panel shows the fluorescence images detecting the expression of GFP.

[0021] [Figure 8]Figure 8 shows the temperature sensitivity test of the mutant srRNA1ts2-GFP using temperature switching 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). The cells were cultured in a CO2 incubator maintained at 32°C. At 24 hours, the cells were transferred into 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, and thereby transfected cells can be selected using puromycin. The experiment was conducted in the absence (upper panel) or presence (lower panel) of 1 μg / ml puromycin. Images of the cells were obtained at 24, 48, 72, 96, 144, 168, 192 hours, respectively. For srRNA1ts1-GFP, images were taken only at 24 hours and 168 hours. The upper panel shows the phase contrast images, and the lower panel shows the fluorescence images detecting GFP expression.

[0022] [Figure 9]Figure 9 shows the temperature sensitivity test of the mutant srRNA1ts2-GFP 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 strain). The cells were cultured in a CO2 incubator maintained at 32°C. After 48 hours, the 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, thereby 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. Cell images were obtained at 24, 48, 72, 96, 144, 168, and 192 hours, respectively. For srRNA1ts1-GFP, only 24-hour and 168-hour images were taken. The upper panel shows phase-contrast images, and the lower panel shows fluorescence images detecting GFP expression.

[0023] [Figure 10]Figure 10 shows the temperature sensitivity test of the mutant srRNA1ts2-GFP using a temperature switch from 32°C to 37°C over 72 hours. RNA produced by in vitro transcription of the mutant vector (srRNA1ts2-GFP) was transfected into human induced pluripotent stem cells (ADSC-iPSC strain). The cells were cultured in a CO2 incubator maintained at 32°C. After 72 hours, the 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, thereby allowing selection using puromycin. Experiments were performed in the absence (upper panel) or presence (lower panel) of 1 μg / ml puromycin. Cell images were obtained at 24, 48, 72, 96, 144, 168, and 192 hours, respectively. For srRNA1ts1-GFP, only 24-hour and 168-hour images were taken. The upper panel shows phase-contrast images, and the lower panel shows fluorescence images detecting GFP expression.

[0024] [Figure 11A-11D] Figures 11A–11D show the temperature sensitivity testing of the 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. Cell images were obtained at 24, 48, and 96 hours. The upper panel shows phase-contrast images, and the lower panel shows fluorescence images detecting GFP expression. Figures 11A and 11B show transfection 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 transfection performed using MessengerMax (ThermoFisher). Cells were cultured in standard medium alone (Figure 11C) or standard medium supplemented with 200 ng / ml of B18R (Figure 11D).

[0025] [Figure 12] Figure 12 shows amino acid sequence alignments corresponding to nsP2 variant 2 (ts2) of various alphavirus family members. The left panel shows the alignment of wild-type sequences, designated as SEQ ID NOs. 21-28 (partially replicated from Figure 1 of Russo et al., 2006), while the right panel shows the alignment of variants, designated as SEQ ID NOs. 29-36, which include the insertion of five amino acids between "β5" and "β6" (the 5th and 6th β chains) in the secondary structure of nsP2. These include VEEV (Venezuelan Encephalitis Virus), Aura (Aura Virus), WEEV (Western Equine Encephalitis Virus), BFV (Bammer Forest Virus), ONNV (Onyon-Nyon Virus), RRV (Ross River Virus), SFV (Semryki Forest Virus), and SINV (Sindobis Virus).

[0026] [Figure 13] Figure 13 illustrates a schematic diagram of a typical ex vivo treatment of cells with a temperature-sensitive activator (ts activator). ts activators, such as srRNAs or Sendai virus vectors, are functional at tolerable temperatures (e.g., 33°C) but not at non-tolerable temperatures (e.g., 37°C). Target cells treated with a ts activator are cultured at tolerable temperatures for a specific duration (e.g., 3 days), and then cultured at non-tolerable temperatures 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 tolerable temperatures and reaches high levels. After switching to non-tolerant temperatures, transcription and translation cease, and the expected level of RNA (or protein) gradually decreases.

[0027] [Figure 14]Figure 14 illustrates a schematic diagram of an exemplary ex vivo therapeutic method. A temperature-sensitive activator (ts activator), such as srRNAs or Sendai virus vectors, is functional at an acceptable temperature (e.g., 33°C) but not at an unacceptable temperature (e.g., 37°C). Target cells are harvested from the patient's body (spontaneous transplantation) and incubated with the ts activator ex vivo at an acceptable temperature, e.g., 33°C, for a specific duration, e.g., 24 hours. The target cells with the ts activator are then transplanted into the patient. At the unacceptable temperature of 37°C, the ts activator is not functional in the patient's body.

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

[0029] [Figure 16]Figure 16 illustrates a schematic diagram of an exemplary semi-in vivo therapeutic method. A temperature-sensitive activator (ts activator), such as srRNAs or Sendai virus vectors, is functional at an acceptable temperature (e.g., 33°C) but not at an unacceptable temperature (e.g., 37°C). The patient undergoes therapeutic hypothermia therapy, and the patient's core body temperature is maintained at a low temperature (e.g., 33°C), which is lower than normal body temperature (e.g., 37°C). Target cells (either autologous or allogeneic) are treated with the ts activator ex vivo and immediately injected into the patient's circulation or into the patient's organs. The ts activator remains functional while the patient is maintained at a low temperature (e.g., 33°C) for a period of time (e.g., 24 hours). Subsequently, when the patient's core body temperature returns to normal (37°C), the ts activator is no longer functional.

[0030] [Figure 17] Figure 17 illustrates a schematic diagram of an exemplary semi-in vivo therapeutic approach. A temperature-sensitive activator (ts activator), such as srRNAs or Sendai virus vectors, is functional at tolerable temperatures (e.g., 33°C) but not at non-tolerable temperatures (e.g., 37°C). The patient undergoes therapeutic hypothermia therapy, and the patient's core body temperature is maintained at a low temperature (e.g., 33°C), which is lower than normal body temperature (e.g., 37°C). The ts activator is delivered systemically or to specific organs, tissues, or cell types. The ts activator remains functional while the patient is maintained at a low temperature (e.g., 33°C) for a period of time (e.g., 24 hours). Subsequently, when the patient's core body temperature returns to normal (37°C), the ts activator is no longer functional.

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

[0032] [Figure 19] Figure 19 shows that the majority of human CD34+ cells, after being in contact with SeVts-ZSCAN4 and incubated at an acceptable temperature (e.g., 33°C) for 16 or 24 hours, express the ZSCAN4 protein.

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

[0034] [Figure 21] Figure 21 shows that telomeres are elongated in human CD34+ cells that have been in contact with SeVts-ZSCAN4 and incubated at an acceptable temperature (e.g., 33°C) for a minimum of 24 hours.

[0035] [Figure 22] Figure 22 illustrates a schematic diagram of the ex vivo procedure of Example 14, in which human CD34+ cells are contacted with the ZSCAN4 therapeutic ts activator at an acceptable temperature (e.g., 33°C) and subsequently injected into immunodeficient mice with an unacceptable normal body temperature (e.g., 37°C) to evaluate the safety and efficacy of CD34+ cell transplantation.

[0036] [Figure 23] Figure 23 shows that 24-hour SeVts-ZSCAN4 treatment at an acceptable temperature (e.g., 33°C) was effective in extending the telomeres of human CD34+ cells in vitro.

[0037] [Figure 24] Figure 24 shows that the telomeres of human cells transplanted into immunodeficient mice were longer when human CD34+ cells injected into those mice in vitro were first exposed to SeVts-ZSCAN4 and then incubated at an acceptable temperature (e.g., 33°C). Mice 492 and 493 were given SeVts-ZSCAN4-exposed CD34+ cells, while mouse 496 was given CD34+ cells that were not exposed to SeVts-ZSCAN4.

[0038] [Figure 25] Figure 25 illustrates a schematic diagram of the ex vivo treatment procedure of Example 15, in which autologous CD34+ cells are contacted with the ZSCAN4 therapeutic ts activator at an acceptable temperature (e.g., 33°C), followed by injection into a patient with an unacceptable normal body temperature (e.g., 37°C).

[0039] [Figure 26] Figure 26 shows a flowchart of the clinical diagnostic method of Example 15 for evaluating autologous CD34+ cells exposed to SeVts-ZSCAN4 in human patients suffering from telomere biological disorders or bone marrow failure.

[0040] [Figure 27] Figure 27 illustrates another schematic diagram showing the ex vivo treatment procedure of Example 15 for evaluating autologous CD34+ cells exposed to SeVts-ZSCAN4 in human patients suffering from telomere biological disorders or bone marrow failure.

[0041] [Figure 28] Figure 28 illustrates a schematic diagram of an exemplary in vivo therapeutic approach. Temperature-sensitive activators (ts activators), such as srRNAs or Sendai virus vectors, are functional at tolerable temperatures (e.g., 31–34°C) but not at non-tolerable temperatures (e.g., >37°C). The patient's surface temperature (surface body temperature), which is approximately 31–34°C, is lower than the patient's core body temperature (approximately 37°C). The ts activator, functional at the patient's surface temperature, is delivered directly to the patient by intradermal, subcutaneous, or intramuscular administration. Further activity is required. Alternatively, when the function of the ts activator is no longer needed, it is transiently rendered inactive by raising the patient's surface temperature.

[0042] [Figure 29] Figure 29 illustrates a schematic diagram of an exemplary in vivo therapeutic approach. Temperature-sensitive activators (ts activators), such as srRNAs or Sendai virus vectors, are functional at tolerable temperatures (e.g., 31–35°C) but not at non-tolerable temperatures (e.g., >37°C). The patient's body airway temperature (airway temperature), which is approximately 32°C for the nasal cavity 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). The ts activator, functional at the patient's airway temperature, is delivered directly to the patient by intranasal administration (e.g., inhalation or infusion). Further activity is required. When the function of the ts activator is no longer needed, it is transiently rendered inactive by raising the patient's airway temperature. [Modes for carrying out the invention]

[0043] Detailed explanation Review The applicant demonstrated that cells can be cultured at a tolerable 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 the temperature-sensitive therapeutic agent can be reduced or inhibited by subsequently incubating the cells at an untolerable temperature. The applicant also initially provided a method for using temperature-sensitive agents (ts agents) in vivo. The same type of ts agent used in vivo can 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 tolerable 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 tolerable temperature to induce the activity of the ts agent. The subject's surface body temperature may be maintained naturally or artificially. These methods provide a novel way to deliver and transiently activate therapeutic agents such as nucleic acids and polypeptides. In particular, this disclosure provides a tool for temperature-sensitive delivery of ZSCAN4 nucleic acid and protein to cells.

[0044] Accordingly, this disclosure generally relates to a method 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 tolerable temperature to induce the activity of the temperature-sensitive therapeutic agent. The cells are cultured at the tolerable temperature for a sufficient period of time for the temperature-sensitive therapeutic agent to induce a therapeutic effect in the cells. The cells are then returned to a non-tolerable temperature, where the non-tolerable temperature reduces or inhibits the activity of the temperature-sensitive therapeutic agent. In another embodiment, one or more cells do not contain a temperature-sensitive therapeutic agent beforehand and come into contact 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 that needs it. In some embodiments, one or more cells are isolated from a subject in need of treatment and treated with the temperature-sensitive therapeutic agent, and then the cells are returned to the subject.

[0045] In another embodiment, the disclosure relates to a method 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 body temperature of the subject is lowered to an acceptable temperature for a sufficient period of time for the temperature-sensitive therapeutic agent to induce a therapeutic effect in the cells, and then the body temperature of the subject is returned to normal. In another embodiment, the temperature-sensitive therapeutic agent is administered to the subject either before or after the body temperature of the subject is lowered to an acceptable temperature.

[0046] Other aspects of the present disclosure relate to treating a disease or condition by recruiting CD34+ cells from the bone marrow of a subject (a subject in need thereof) suffering from the disease or condition, comprising: isolating recruiting cells from the subject; incubating the isolated cells at a temperature of approximately 33°C ± 0.5°C; contacting the cells with a temperature-sensitive viral vector or temperature-sensitive self-replicating RNA (srRNA), such as a Sendai virus vector, where the viral vector or srRNA contains a heterogeneous nucleic acid molecule encoding a protein of interest; maintaining the contacted cells at approximately 33°C ± 0.5°C for a sufficient period of time, where the viral vector or srRNA can be replicated at 33°C ± 0.5°C, and the replication of the viral vector or srRNA leads to an increase in the expression of the heterogeneous nucleic acid molecule; and injecting the contacted cells into the subject, thereby transplanting the contacted cells and treating the disease or condition. Alternatively, after isolating recruited cells from the target, the isolated cells are brought into contact with a temperature-sensitive viral vector such as a Sendai virus vector or a temperature-sensitive srRNA, and then the cells are incubated at a temperature of approximately 33°C ± 0.5°C. In some embodiments, the disease or condition is a telomere biological disorder, and the protein of interest is ZSCAN4, such as human ZSCAN4.

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

[0048] The references and claims relating to methods of treating a disease or condition by administering ts-active substances, or cells containing ts-active substances, in their general and specific forms, are as follows: a) Use of ts activators or cells containing ts activators for the manufacture of therapeutic agents for diseases or conditions; and b) A pharmaceutical composition comprising a ts-active substance or cells containing a ts-active substance for the treatment of a disease or condition. Regarding.

[0049] In some embodiments of the procedure paragraph, the heterologous nucleic acid contains the gene of interest (GOI) or, otherwise, encodes the protein of interest. In another of the above methods, the heterologous nucleic acid contains the coding region of the protein of interest. In a preferred embodiment, the protein of interest is ZSCAN4, such as human ZSCAN4 or a variant thereof. definition

[0050] As used herein and in the accompanying claims, the singular forms “a,” “an,” and “the” include the plural form unless otherwise indicated. For example, “a polynucleotide” includes one or more polynucleotides.

[0051] As used herein, the phrase "including" is open-ended and indicates that such embodiments may include further elements. In contrast, the phrase "consisting of" is closed and indicates that such embodiments do not include further elements (except for trace amounts of impurities). The phrase "essentially consisting of" is partially closed and indicates that such embodiments may include further elements that do not substantially alter the fundamental features of such embodiments. It is understood that embodiments described herein as "including" include embodiments "consisting of" and embodiments "essentially consisting of."

[0052] With the exception of temperature, the term "approximately" as used herein in reference to values ​​encompasses 90% to 110% of that value unless otherwise indicated (for example, approximately 30 minutes refers to 27 to 33 minutes). When used in reference to temperature in Celsius units, approximately encompasses -1°C to +1°C of that value unless otherwise indicated (for example, approximately 37°C refers to 36 to 38°C). In contrast, the use of plus and minus signs without further indication demarcates the indicated range (for example, 33°C ± 0.5°C refers to 32.5°C to 33.5°C).

[0053] Where used herein, a numerical range includes the numbers that define the range (for example, 12–18 nucleotides includes 12, 13, 14, 15, 16, 17 and 18 nucleotides).

[0054] Where used herein, the terms “isolated” and “purified” refer to the object (e.g., cells) that has been removed (e.g., isolated) from its environment (e.g., cell culture, biological sample, etc.). The “isolated” object is free from at least 50%, preferably 75%, more preferably at least 90%, and most preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) of the other components to which they are related.

[0055] The terms "individual" and "subject" refer to mammals. "Mammals" are not limited to these, but include humans, non-human primates (e.g., monkeys), domesticated animals, athletic animals, rodents (e.g., mice and rats), and companion animals (e.g., dogs and cats).

[0056] Where used herein, the term “dosage” in reference to a pharmaceutical composition refers at any given time to the measured portion of the composition ingested by the subject (administered to or given to) the subject.

[0057] The term “treating” a disease or condition means implementing a protocol that may involve administering one or more pharmaceutical compositions to an individual (human or other animal) in an attempt to alleviate the signs or symptoms of a disease. Thus, “treating” or “treatment” specifically includes protocols that do not require complete relief of signs or symptoms, nor require a cure, but only have a mitigating effect on the individual. As used herein and as is well understood in the art, “treatment” means a method for obtaining beneficial or desired results, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, relief or improvement of one or more symptoms, reduction of the severity of the disease, stabilization (i.e., non-exacerbation) of the disease, prevention of disease spread, delay or slowing of disease progression, improvement or alleviation of the disease condition, and remission (whether partial or complete). Temperature sensitive agents

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

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

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

[0061] In certain embodiments, the temperature-sensitive therapeutic agents of the present disclosure may include temperature-sensitive viral vectors. In some embodiments, the activity of a temperature-sensitive viral vector induced at an acceptable temperature may include vector replication. As used herein, the term “temperature-sensitive viral vector” refers to any viral vector having different levels of functionality at different temperatures. Examples of temperature-sensitive viral vectors include, but are not limited to, Sendai virus vectors, adeno-associated virus vectors, retroviral vectors, or alphavirus vectors. Examples of temperature-sensitive alphavirus vectors include, but are not limited to, Venezuelan encephalitis virus vectors, Sindbis virus vectors, and Semryki Forest virus vectors.

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

[0063] The tolerable temperature of the temperature-sensitive viral vectors 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 tolerable 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 tolerable temperature is 33°C ± 0.5°C. As a result, in some embodiments, the non-tolerable temperature of the temperature-sensitive viral vectors of the present disclosure is greater than 36°C and less than 38°C. In some preferred embodiments, the non-tolerable temperature is 37°C ± 0.5°C.

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

[0065] In certain embodiments, the temperature-sensitive therapeutic agents of this disclosure may include temperature-sensitive self-replicating RNA. As used herein, the term “temperature-sensitive self-replicating RNA” refers to any self-replicating RNA having different levels of functionality at different temperatures.

[0066] In some embodiments, temperature-sensitive self-replicating RNA is typically produced by manipulating self-replicating RNA, which is single-stranded RNA made from alphaviruses such as Venezuelan encephalitis virus (VEEV), Sindbis virus (SINV), and Semryki forest virus (SFV), by removing DNA that encodes structural proteins necessary for viral particle formation (Petrakova et al., 2005). In some embodiments, the self-replicating RNA encodes non-structural proteins (nsPs), which function as an RNA-dependent RNA polymerase that replicates the self-replicating RNA itself and produces a transcript for translation. In some embodiments, the self-replicating RNA may also include a gene of interest (GOI) containing the coding region for the protein of interest. The gene of interest may include one or more additional genetic elements, such as a promoter operably combined with the coding region. In a preferred embodiment, the protein of interest is ZSCAN4, such as human ZSCAN4 or a variant thereof. While we do not wish to be bound by any theory, in some embodiments, self-replicating RNA can express GOIs at high levels due to positive feedback production of its RNA. In some embodiments, temperature-sensitive self-replicating RNA may be produced by mutating genes encoding nsPs.

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

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

[0069] In some embodiments, the tolerable temperature of the temperature-sensitive self-replicating RNA of the Disclosure is typically in the range of 30°C to 36°C. In some embodiments, the tolerable 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 tolerable temperature is 33°C ± 0.5°C. Consequently, in some embodiments, the non-tolerable temperature of the temperature-sensitive self-replicating RNA of the Disclosure is greater than 36°C. In some preferred embodiments, the non-tolerable temperature is 37°C ± 0.5°C.

[0070] In other embodiments, the tolerable temperature of the temperature-sensitive self-replicating RNA of the Disclosure is typically in the range of 38°C to 50°C. As a result, in some embodiments, the non-tolerable temperature of the temperature-sensitive self-replicating RNA of the Disclosure is greater than 36°C and less than 38°C. In some preferred embodiments, the non-tolerable temperature is 37°C ± 0.5°C. Temperature-sensitive polypeptide

[0071] In certain embodiments, the temperature-sensitive therapeutic agents of the present disclosure may include temperature-sensitive polypeptides. As used herein, the term “temperature-sensitive polypeptide” refers to any temperature-sensitive polypeptide having different levels of functionality at different temperatures. In some embodiments, the temperature-sensitive polypeptide may be temperature-sensitive ZSCAN4. In other embodiments, the temperature-sensitive polypeptide may be selected from transcription factors for the ZSCAN4 gene, but is not limited thereto.

[0072] When the temperature-sensitive substance is a temperature-sensitive polypeptide, the activity of the temperature-sensitive protein induced at the permissible temperature may include conformational changes of the protein (e.g., changes in structure or shape).

[0073] The tolerable temperature range for the temperature-sensitive polypeptides 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 tolerable temperature range is about 31°C to about 35°C, or 32°C to 34°C (33°C ± 1.0°C). In some preferred embodiments, the tolerable temperature range is 33°C ± 0.5°C. Consequently, in some embodiments, the non-tolerable temperature range for the temperature-sensitive self-replicating polypeptides of the present disclosure is greater than 36°C and less than 38°C. In some preferred embodiments, the non-tolerable temperature range is 37°C ± 0.5°C.

[0074] Various aspects of this disclosure relate to substantially purified polypeptides. A substantially purified polypeptide may mean a polypeptide that is substantially free of other polypeptides, lipids, carbohydrates, or other substances that are naturally related to it. In one embodiment, the polypeptide is free of at least 50%, for example, at least 80%, of other polypeptides, lipids, carbohydrates, or other substances that are naturally related to it. In another embodiment, the polypeptide is free of at least 90% of other polypeptides, lipids, carbohydrates, or other substances that are naturally related to it. In yet another embodiment, the polypeptide is free of at least 95% of other polypeptides, lipids, carbohydrates, or other substances that are naturally related to it. Nucleic acids and polypeptides

[0075] Certain aspects of this disclosure relate to transiently inducing the activity of a temperature-sensitive therapeutic agent in one or more cells, where such 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 group of linked nucleotides linked by a phosphodiester bond. Oligonucleotides are polynucleotides between 6 and 300 nucleotides in length. Oligonucleotide analogs refer to a portion that functions similarly to an oligonucleotide but has a non-natural portion. For example, an oligonucleotide analog may include a non-natural portion, an altered sugar portion, or an intersugar bond, such as a phosphorothioate oligodeoxynucleotide. Functional analogs of natural polynucleotides can bind to RNA or DNA and include peptide nucleic acid molecules.

[0076] In certain embodiments, nucleic acid molecules or polynucleotides encode genetic elements. These polynucleotides include DNA (encoding the gene of interest), cDNA, and RNA sequences such as mRNA sequences. A coding sequence can be operably ligated to a heterologous promoter to directly transcribe its genetic elements. A promoter can refer to a nucleic acid regulatory sequence that transcribes the nucleic acid. The promoter includes an essential nucleic acid sequence near the transcription start site. The promoter may also include a distal enhancer sequence or a distal repressor sequence. A constitutive promoter is constantly active and is not subject to regulation by external signals or molecules. In contrast, the activity of an inductive promoter is regulated by external signals or molecules (e.g., transcription factors). A first nucleic acid sequence is operably ligated to a second nucleic acid sequence when the first nucleic acid sequence is functionally related to the second nucleic acid sequence. For example, a promoter is operably ligated to a coding sequence if the promoter affects the transcription or expression of that coding sequence. Typically, functionally linked nucleic acid sequences are contiguous and, when it is necessary to ligate two protein-coding regions, they are on the same reading frame. Heterogeneous polypeptides or heterogeneous polynucleotides refer to polypeptides or polynucleotides derived from different origins or species. Promoters include essential nucleic acid sequences located near the transcription start site, for example, the TATA sequence in the case of a polymerase type II promoter. Promoters may also include distal enhancer sequences or distal repressor sequences that can be located thousands of 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 inductive promoter, such as a tetracycline-inducible promoter (Masui et al., Nucleic Acids Res. 33:e43, 2005).Other exemplary promoters that may be used to express genetic elements include, but are not limited to, the LAC system, TRP system, TAC system, TRC system, the major operator and promoter regions of lambda phage, the regulatory region of FD coat proteins, the early and late promoters of SV40; promoters derived from polyomavirus, adenovirus, retrovirus, baculovirus, and Simian virus; the promoter of 3-phosphoglycerate kinase, the promoter of yeast acid phosphatase, and the promoter of yeast α-mating factor. The genetic elements of this disclosure may be under the control of constitutive promoters, inductive promoters, or other suitable promoters described herein or other suitable promoters readily recognizable to those skilled in the art.

[0077] In some embodiments, inducing the activity of a temperature-sensitive active substance leads to an increase in the expression of nucleic acids or polypeptides, which, for example, is at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.1 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, and at least 2.6 times compared to the expression of polynucleotides or polypeptides in human cells not in contact with the active substance. 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 This may include an increase in expression of at least 175,000 times, at least 200,000 times, at least 225,000 times, at least 250,000 times, at least 275,000 times, at least 300,000 times, at least 325,000 times, at least 350,000 times, at least 375,000 times, at least 400,000 times, at least 425,000 times, at least 450,000 times, at least 475,000 times, at least 500,000 times, at least 750,000 times, or at least 1,000,000 times.

[0078] Various aspects of this disclosure relate to isolated entities such as isolated nucleic acids or synthetic mRNA molecules. Isolated nucleic acids are Other nucleic acid sequences and their nucleic acids, i.e., other chromosomal and extrachromosomal DNA and RNA, were substantially isolated or purified from the cells of the organism where they naturally occur. Thus, the term “isolated” includes nucleic acids purified by standard nucleic acid purification methods. The term also includes nucleic acids prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acids. Similarly, isolated polypeptides were substantially isolated or purified from other polypeptides in the cells of the organism where the proteins naturally occur, as well as polypeptides prepared by recombinant expression in host cells and chemically synthesized polypeptides. Similarly, isolated cells were substantially isolated from other cell types. A method for introducing temperature-sensitive substances into cells.

[0079] In some embodiments, one or more cells are brought into contact with a temperature-sensitive substance. Contact refers to a direct physical relationship, including both solid and liquid arrangements. "Contact" may be used interchangeably with "exposure." In some cases, "contact" includes transfection, such as the transfection of nucleic acid molecules into cells. In some cases, "contact" includes the introduction of a temperature-sensitive substance into one or more cells.

[0080] In some embodiments, the temperature-sensitive agent is a polynucleotide (e.g., self-replicating RNA), and the polynucleotide is introduced into the cell. The introduction of nucleic acid molecules or proteins into the cell encompasses any means of delivery of nucleic acid molecules or proteins into the cell. For example, nucleic acid molecules may be transfected, transduced, or subjected to electoporation 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 polypeptides into the cell can be achieved by fusing a protein with 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). The protein transduction domain may refer to a small cationic peptide that facilitates the entry of larger molecules (proteins, nucleic acid molecules, etc.) into the cell by a mechanism independent of classical endocytosis. A polyarginine peptide tag may also refer to a short peptide (generally 7-11 residues) consisting of arginine residues that facilitates the delivery of larger molecules (such as proteins and nucleic acid molecules) into cells (see, for example, Fuchs and Raines, Protein Science 14:1538-1544, 2005).

[0081] The introduction of nucleic acids into cells using temperature-sensitive agents may involve the use of temperature-sensitive viral vectors (such as integrated or unintegrated viral vectors) or temperature-sensitive plasmid vectors. Each of these methods is described in the art and is therefore within the scope of those skilled in the art. A brief outline of each method that can be used to deliver nucleic acid molecules to one or more host cells (preferred mammalian host cells, such as human host cells) is provided herein. When introduced into host cells, resulting in transformed host cells, the vector may refer to a nucleic acid molecule. The vector may contain nucleic acid sequences that enable its replication in the host cell, such as replication origins (DNA sequences involved in the initiation of DNA synthesis). For example, an expression vector contains regulatory sequences necessary to enable the transcription and translation of the inserted (one or more) genes. The vector may also contain one or more selective marker genes and other genetic elements known in the art. The vector may include, for example, viral vectors and plasmid vectors. Allowable temperature Incubation of one or more cells at an acceptable temperature

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

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

[0084] In some embodiments, after incubation at an acceptable temperature, one or more cells are cultured at an unacceptable temperature, where the activity of temperature-sensitive activators is reduced or inhibited. For example, replication of temperature-sensitive viral vectors may be inhibited, replication of temperature-sensitive self-replicating RNA may be inhibited, and conformational changes to temperature-sensitive polypeptides may be inhibited. This temperature shift allows for a transient induction and subsequent inhibition of the activity of temperature-sensitive activators. In other embodiments, one or more cells are cultured at an acceptable temperature before being administered to a subject. One or more cells may be administered directly to a subject from culture at an acceptable temperature, or they may be first transferred from an acceptable temperature to an unacceptable temperature during culture and then administered to a subject. In certain embodiments, temperature-sensitive activators are subsequently degraded. For example, unintegrated temperature-sensitive viral vectors, RNA, and polypeptides are degraded. Lower the target's core body temperature to an acceptable temperature.

[0085] Certain aspects of this disclosure relate to transient induction of the activity of a temperature-sensitive therapeutic agent in target cells by lowering the target's core body temperature to an acceptable temperature in order to induce the activity of the temperature-sensitive agent. In some embodiments, the target's core body temperature is lowered using a target temperature control (TTM) procedure. The TTM procedure is designed to achieve and maintain a specific body temperature of the target over a period of time. Such procedures have previously been used therapeutically to mitigate the negative effects resulting from various acute health problems such as heart attacks and strokes. The apparatus and general methods using them are known in the art and can be used in the manner described herein. The procedure can be carried out using many methods, including cooling catheters, cooling blankets, and the application of ice around the body.

[0086] After lowering the subject's core body temperature to an acceptable temperature, the subject's core body temperature is maintained at the acceptable temperature for a sufficient time to induce the activity of a temperature-sensitive substance. The subject's core body temperature is then returned to a normal core body temperature (which is an unacceptable temperature), where the activity of the temperature-sensitive substance is reduced or inhibited. In certain embodiments, the temperature-sensitive substance is subsequently degraded. For example, unintegrated temperature-sensitive viral vectors, RNA, and polypeptides are degraded at an unacceptable temperature. As used herein, the term "body temperature" refers to "core body temperature" unless otherwise explicitly stated. Maintain the target's surface body temperature at an acceptable level.

[0087] Certain aspects of this disclosure relate to utilizing the normal temperature difference in a region of the body of a subject. For example, the surface temperature of or near the surface of a human body (surface body temperature) is approximately 31-34°C, which is lower than the core body temperature of a human body (which is approximately 37°C). As used herein, “surface” of the body of a subject means one or more of the epidermis, dermis, subcutaneous tissue, or muscle. “Skin” of the body of a subject means either or both of the epidermis and dermis. Therefore, preferred routes of administration to the epidermis, dermis, or subcutaneous tissue of the body of a subject include intradermal and subcutaneous administration. A preferred route of administration to the muscle near the surface of the body of a subject is intramuscular administration.

[0088] For example, the ts-active agent is delivered directly to a specific area of ​​the target skin (in the case of vaccination) or to a broader area of ​​the target skin (in the case of treating a skin disease). Skin temperature (approximately 31-34°C) is the acceptable temperature for the ts-active agent and allows it to function. Further activity is not required for the prolonged expression of the GOI. Since the cessation of the function of the ts-active agent is required or desired, the temperature of the treated skin is raised to an unacceptable temperature (>37°C) by local application of heat (e.g., a heating patch or heating blanket) or by mild therapeutic thermotherapy (e.g., a warm bath or a hot sauna) and then transiently maintained. Since the core body temperature is the unacceptable temperature (approximately 37°C), this therapeutic method is safe, meaning that the ts-active agent functions only in the intended area of ​​the body. In some embodiments, if the surface body temperature of the target must be higher than normal, the surface body temperature is lowered to match the acceptable temperature for the ts-active agent. Maintain the target upper airway temperature at an acceptable level.

[0089] Similar to the surface body temperature of human subjects, the temperature of the upper respiratory tract and upper trachea of ​​human subjects is within the acceptable temperature range for ts activators and allows them to function. Specifically, the temperature of the nasal cavity and upper trachea of ​​human subjects is approximately 32°C, and the temperature of the subsegmental bronchi of human subjects is approximately 35°C (McFadden et al., 1985). Thus, ts activators administered intranasally to cells of the upper respiratory tract (nasal cavity, pharynx, and / or larynx) and / or upper trachea of ​​human patients are functional without lowering the core body temperature of human patients. Intranasal administration may be performed by inhalation, suction, or intravenous infusion. Non-permissible temperature Incubate one or more cells at an unacceptable temperature.

[0090] Typically, in vitro culture of cells is performed at the normal body temperature of the subject from which the cells were obtained. For example, mammalian cells such as human cells and mouse cells are usually cultured at approximately 37°C. Specific aspects of this disclosure relate to temperature-sensitive substances 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-acceptable temperature for the temperature-sensitive substance. In some preferred embodiments, the non-acceptable temperature is 37°C ± 0.5°C. The normal core body temperature of the subject

[0091] In some embodiments, a temperature-sensitive substance, cells in contact with the temperature-sensitive substance, or cells carrying the temperature-sensitive substance are introduced into the body of an organism maintained at a normal core body temperature. Specific aspects of this disclosure relate to temperature-sensitive substances that do not function, for example, replicate or express genes, at this normal body temperature (non-tolerant temperature) of the organism. This feature provides a safety mechanism to prevent undesirable effects or reactivation of the temperature-sensitive substance throughout the organism's lifespan. human cells

[0092] Specific aspects of this disclosure, though not limited thereto, relate to transiently inducing the activity of a temperature-sensitive therapeutic agent in one or more human cells, including adult human cells. In certain embodiments, one or more human cells require treatment with the therapeutic agent in a subject.

[0093] Various human cells are useful in the methods described herein. As disclosed herein, the term “human cell” refers to any cell found in the human body during and after embryonic development, e.g., human embryonic cells, stem cells, pluripotent cells, differentiated cells, adult cells, somatic cells, and mature cells. In some embodiments, the human cells of this 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., non-embryonic cells). The human cells of this 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 tissue. Originating cells can refer to pluripotent or unipotent cells that differentiate into a specific type of cell or cell lineage. Originating cells are similar to stem cells but are further differentiated and exhibit limited self-renewal. Exemplary adult stem cells, tissue stem cells, and / or originating cells may include, but are not limited to, hematopoietic stem cells, mesenchymal stem cells, adipose-derived stem cells, neural stem cells, intestinal stem cells, skin stem cells, and germ cells (e.g., spermatids and oocytes).

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

[0095] Human cells may also include, but are not limited to, cells derived 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, proliferative cells, dysplasia cells, and atypical cells. Human cells may also include, but are not limited to, diploid cells, haploid cells, tetraploid cells, polyploid cells, cells with karyotype abnormalities, cells with chromosomal abnormalities, cells with mutant genes, cells with abnormal telomere lengths, cells with short telomeres, and cells with long telomeres. Human cells that exhibit epigenetic abnormalities, such as cells with hypomethylated genomic regions, cells with hypermethylated genomic regions, and cells with abnormal histone modifications like acetylation and methylation, are also examples, although they are not limited to these.

[0096] In some embodiments, the subject matter of this disclosure is non-human animals. Non-human animals may refer to all animals other than humans. Examples of non-human animals include, but are not limited to, non-human primates, livestock such as pigs, cattle, and poultry, sporting 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 use of temperature-sensitive substances

[0097] The temperature-sensitive substances of the present disclosure may be administered by any suitable method known in the art, including, but not limited to, oral administration, sublingual administration, oral administration, topical administration, rectal administration, inhalation, transdermal administration, subcutaneous injection, intravenous injection, intra-arterial injection, intramuscular injection, intracardiac injection, intraosseous injection, intradermal injection, intraperitoneal injection, transmucosal administration, intravaginal administration, intravitreal administration, intra-articular administration, peri-articular administration, topical administration, surface administration of the skin, or any combination thereof. In some embodiments, the composition is administered by subcutaneous injection and / or intravenous injection.

[0098] In some embodiments, the methods of the present disclosure involve the use of a therapeutically effective amount of a temperature-sensitive active substance. A therapeutically effective amount of an active substance can refer to a sufficient amount of therapeutic agent to achieve the intended purpose. For example, a therapeutically effective amount of a temperature-sensitive active substance for treating a disease or condition is a sufficient amount to alleviate the disease or condition, or one or more symptoms of that disease or condition. In some cases, a therapeutically effective amount may not cure the disease or condition, or the symptoms of that disease or condition, by 100%. However, a reduction in any known feature 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%, may be therapeutic.

[0099] The therapeutically effective dose of a given therapeutic agent varies depending on factors such as the properties of the agent, the route of administration, the size and / or age of the recipient, and the purpose of administration. The therapeutically effective dose in each individual case can be experimentally determined by a person skilled in the art without unnecessary experimentation, according to methods established in the art.

[0100] The term "subject" may refer to a category including living multicellular vertebrate organisms, humans, and non-human mammals. In some embodiments, the subject is human. Subjects that can be treated using the methods provided herein may include mammalian subjects, e.g., veterinary subjects or human subjects. Subjects may include fertilized eggs, zygotes, preimplantation embryos, embryos, fetuses, neonates, infants, children, and / or adults. In some embodiments, subjects to be treated are selected, for example, by selecting subjects who would benefit from a treatment, particularly a treatment involving the administration of a temperature-sensitive agent of the Disclosure.

[0101] The pharmaceutical compositions of this disclosure comprise a ts-active agent, such as a therapeutic ts-active 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 the ts-active agent). Suitable pharmaceutically acceptable carriers for use in this disclosure are conventional products. In particular, suitable compositions and formulations for the pharmaceutically effective delivery of compositions containing temperature-sensitive agents are those described above (see, for example, Gennaro, AR (editor) Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th edition (1990); and Felton, LA (editor) Remington Essentials of Pharmaceutics, Pharmaceutical Press, London, United Kingdom, 1st edition, (2013)).

[0102] Generally, the properties of the carrier depend on the specific mode of administration used. For example, parenteral formulations typically contain an injectable liquid encapsulating a carrier, such as a pharmaceutically and physiologically acceptable liquid, such as water, saline, equilibrium salt solution, glucose aqueous solution, glycerol, or similar. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the administered pharmaceutical composition may contain small amounts of non-toxic adjuvants such as humectants or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitan monolaurate. In some embodiments, the pharmaceutical compositions of this disclosure include a tsactive agent, such as a therapeutic tsactive agent, and one or more additional compounds (to facilitate the uptake of the tsactive agent into cells). In the case of RNA-based tsactive agents, the tsactive agent is encapsulated within nanoparticles. In some cases, the nanoparticles are lipid-based (e.g., lipofectamines).

[0103] The most appropriate therapeutic dose and treatment plan for a patient varies depending on the disease or condition being treated, and according to the patient's weight and other parameters. Effective dosages and treatment protocols can be determined by conventional methods, which involve starting with low doses in experimental animals, gradually increasing the dose while monitoring the effect, and systematically modifying the administration plan. When determining the optimal dosage for a given subject, physicians can consider numerous factors. These factors include the patient's physique, age, general condition, the specific disease being treated, the severity of the disease, and the presence of other medications in the patient. The test dose is selected after considering the results of animal studies and clinical literature. Mobilization of bone marrow cells

[0104] In some embodiments, the method includes recruiting 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 of a target. In some embodiments, the method includes administering a therapeutically effective amount of a temperature-sensitive agent of the Disclosure (e.g., a temperature-sensitive therapeutic agent) in the spleen under conditions suitable for the delivery of nucleic acids to one or more bone marrow cells (including, but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells).

[0105] In some embodiments of the methods disclosed herein, the recruitment 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 includes targeting a therapeutically effective amount of cytokines and / or chemotherapeutic agents. In some embodiments, the recruitment of bone marrow cells (including, but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) to the spleen includes targeting a therapeutically effective amount of cytokines. In some embodiments, the recruitment of bone marrow cells (including, but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) to the spleen includes targeting a therapeutically effective amount of chemotherapeutic agents. In some embodiments, the recruitment of bone marrow cells (including, but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) to the spleen includes targeting a therapeutically effective amount of cytokines and chemotherapeutic agents. Cytokines and / or chemokines may be administered by any suitable method known in the art, including, but not limited to, oral administration, sublingual administration, oral administration, topical administration, rectal administration, inhalation, transdermal administration, subcutaneous injection, intravenous injection, intra-arterial injection, intramuscular injection, intracardiac injection, intraosseous injection, intradermal injection, intraperitoneal injection, transmucosal administration, vaginal administration, intravitreal administration, intra-articular administration, peri-articular administration, topical administration, surface administration of the skin, or any combination thereof. In some embodiments, cytokines and / or chemokines are administered by subcutaneous injection and / or intravenous injection.

[0106] In some embodiments, the bone marrow cells of the subject (including, but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) are recruited 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 bone marrow cells of the subject (including, but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) are recruited over a continuous period of 7 days, 5 days, 4 days, 3 days, 2 days, or 1 day prior to administration of the composition. In some embodiments, the target bone marrow cells (including, but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) are recruited in parallel with the administration of the composition.

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

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

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

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

[0111] In some embodiments, the recruitment 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 involves administering a therapeutically effective dose of G-CSF and a therapeutically effective dose of plerixafor. In some embodiments, G-CSF and plerixafor are administered co-administered to the subject. In some embodiments, G-CSF and plerixafor are administered co-administered to the subject over a period of one, two, three, four, or more days. In some embodiments, G-CSF is administered to the subject before plerixafor. In some embodiments, G-CSF is administered to the subject before plerixafor over a period of one, two, three, four, or more days. In some embodiments, G-CSF is administered to the subject one, two, three, four, or more days before plerixafor, and then G-CSF and plerixafor are administered co-administered to the subject over a period of one, two, three, four, or more days. In some embodiments, plerixafor is administered to the subject before G-CSF. In some embodiments, plerixafor is administered to the subject one, two, three, four or more days before G-CSF. In some embodiments, plerixafor is administered to the subject one, two, three, four or more days before G-CSF, and then G-CSF and plerixafor are administered co-administered to the subject over one, two, three, four or more days.

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

[0113] In some embodiments, the methods of the present disclosure involve the use of a therapeutic amount of a temperature-sensitive activator (e.g., a temperature-sensitive therapeutic activator) for delivering nucleic acids to target cells in vitro or in vivo. The therapeutically effective amount of an activator can refer to an amount sufficient to achieve the intended purpose as a therapeutic agent. For example, the therapeutically effective amount of a temperature-sensitive activator (e.g., a temperature-sensitive therapeutic activator) for delivering 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 that disease or condition. In some cases, the therapeutically effective amount does not have to cure the disease or condition, or the symptoms of that disease or condition, 100%. However, a reduction in any known feature 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%, may be therapeutic.

[0114] In another example, a therapeutically effective dose of cytokines and / or chemokines 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 sufficient to induce the mobilization of one or more bone marrow cells (including, but not limited to, CD34+ cells, hematopoietic stem cells, mesenchymal stem cells, and endothelial stem cells) from the bone marrow into the peripheral blood.

[0115] The therapeutically effective dose of a given temperature-sensitive active substance (e.g., a temperature-sensitive therapeutic active substance) varies depending on factors such as the properties of the active substance, the route of administration, the physique and / or age of the recipient of the therapeutic active substance, and the purpose of administration. The therapeutically effective dose in each individual case can be experimentally determined by a person skilled in the art without unnecessary experimentation, according to methods established in the art.

[0116] The term "subject" may refer to a category including living multicellular vertebrate organisms, humans, and non-human mammals. In some embodiments, the subject is human. Subjects that can 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, for example, by selecting subjects who would benefit from the treatment.

[0117] Examples of disorders or diseases that may benefit from the administration of temperature-sensitive agents (e.g., temperature-sensitive therapeutic agents) include disorders or diseases associated with (one or more) gene mutations, abnormal telomere length, or (one or more) abnormal epigenetic modifications. In some embodiments, the disease or disorder is a telomere biological disorder. Further examples of disorders or diseases that may benefit from the administration of temperature-sensitive agents (e.g., temperature-sensitive therapeutic agents), but are not limited to, include cancer, autoimmune diseases, and neurological injuries or neurodegenerative disorders, as well as diseases in which cell regeneration is beneficial, such as blindness or hearing impairment. In some embodiments, the disease or disorder is a disease of the blood or hematopoietic organs.

[0118] Cancer includes malignant tumors characterized by abnormal or uncontrolled cell proliferation. Cancer is often associated with gene mutations and abnormal telomere regulation. Exemplary cancers that may benefit from treatment with ts-activators include, but are not limited to, cardiac cancers (e.g., sarcomas (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyomas, fibromas, lipomas, and teratomas); lung cancers (e.g., bronchogenic carcinomas (scaly cell, anaplastic small cell, anaplastic large cell, adenocarcinoma), alveolar epithelial (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondrotoxic hamartoma, mesothelioma); and gastrointestinal cancers (e.g., esophageal (squamous cell) carcinoma). 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, chorioadenoma, hamartoma, leiomyoma); urogenital tract cancer (e.g., kidney (adenocarcinoma, Wilms' tumor, nephroblastoma, lymphoma, leukemia); Bladder cancer and urethral cancer (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma); prostate cancer (adenocarcinoma, sarcoma); testicular cancer (seminocarcinoma, teratoma, fetal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenoid tumor, lipoma); liver cancer (e.g., hepatocellular carcinoma, cholangioblastoma, angiosarcoma, hepatocellular adenoma, hemangioma); bone cancer (e.g., osteogenic sarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulosarcoma), multiple myeloma, malignant Giant cell tumor, chordoma, osteochondroma (osteochondrogenic exostosis), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumor; neurological cancers (e.g., skull (osteoma, hemangioma, granuloma, xanthomas, osteoosteitis), meninges (meningioma, meningiosarcoma, glioma), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor, pineal glandoma, glioblastoma multiforme, oligodendrocyte, Schwann cell tumor, retinoblastoma, congenital tumor), spinal cord (neurofibroma, meningioma, glioma, sarcoma));Gynecological cancers (e.g., uterus (endometrial cancer), cervix (cervical cancer, pre-neoplastic cervical dysplasia), ovaries (ovarian cancer, serous cystadenocarcinoma, mucinous cystadenocarcinoma, endometrioid tumor, Brenner tumor, clear cell carcinoma, unclassified carcinoma, granulosa / theca cell tumor, Sertoli-Leydig cell tumor, undifferentiated germ cell tumor, malignant teratoma), vulva (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, staphyloid sarcoma, fetal rhabdomyosarcoma), fallopian tubes (cancer) Examples include blood cancers (e.g., myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin lymphoma (malignant lymphoma)); skin cancers (e.g., malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, lentigo, dysplasia, lipoma, hemangioma, dermatofibroma, keloid, psoriasis); and adrenal cancers (e.g., neuroblastoma).

[0119] Autoimmune diseases result in an abnormal immune response, such as the production of antibodies or cytotoxic T cells that are specific to the body's own antigens or cells or tissues. In some cases, autoimmune diseases are limited to a specific organ (e.g., thyroiditis) or can affect specific tissues in various locations (e.g., Goodpasture's disease). Examples of autoimmune diseases that may benefit from treatment with ts agonists include, but are not limited to, rheumatoid arthritis, juvenile oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjögren'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 syndrome, Addison's disease, systemic sclerosis, polymyositis, dermatomyositis, autoimmune hemolytic anemia, and pernicious anemia.

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

[0121] Neurodegenerative diseases are conditions in which cells of the brain and / or spinal cord are lost. Neurodegenerative diseases result from the deterioration of nerve cells or the myelin sheath of nerve cells, which leads to dysfunction and impairment over time. The resulting conditions can cause motor problems (such as ataxia) and memory problems (such as dementia). Therefore, in some embodiments, temperature-sensitive agents (e.g., temperature-sensitive therapeutic agents) of the present disclosure can be used to treat the target neurodegenerative disease by modifying tissue stem cells in the nervous system of a patient suffering from the neurodegenerative disease, thereby producing nerve cells and glial cells through the modification of tissue stem cells in the nervous system, and consequently repairing defects in the nervous system. In some embodiments, the agent modifies the target nervous system and reverses the degenerative state of the disease. Examples of neurodegenerative diseases, though not limited to these, include adrenoleukodystrophy (ALD), alcoholism, Alexander disease, Alperz disease, Alzheimer's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), ataxia with capillary dilatation, Batten disease (also known as Spielmeyer-Vogt-Sjögren-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-related dementia, Kennedy disease, and Krabben disease. These include diseases such as Lewy body dementia, 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, pernicious anemia associated with subacute combined degeneration of the spinal cord, Spielmaier-Vogt-Sjögren-Batten disease (also known as Batten disease), spinocerebellar ataxia, spinal muscular atrophy, Steele-Richardson-Olsewski disease, tabes dorsalis, and toxic encephalopathy.

[0122] Therefore, temperature-sensitive agents (e.g., temperature-sensitive therapeutic agents) are administered to target patients to alleviate or improve symptoms associated with specific disorders. Therapeutic endpoints for cancer treatment may include a reduction in tumor size or volume, a reduction in angiogenesis around the tumor, or a reduction in tumor metastasis. If the tumor is removed, another therapeutic endpoint may be the regeneration of the removed tissue or organ. Methods in the art, such as imaging the tumor or detecting tumor markers or other indicators of the presence of cancer, can be used to measure the effectiveness of cancer treatment. Therapeutic endpoints for autoimmune disease treatment may include a reduction in the autoimmune response. Methods in the art, such as measuring autoimmune antibodies, can be used to measure the effectiveness of autoimmune disease treatment, and a reduction in such antibodies in the patient indicates that the treatment was successful. Therapeutic endpoints for neurodegenerative disorder treatment may include a reduction in neurodegenerative-related defects, such as a reduction in the increase of motor defects, memory defects, or behavioral defects. Methods in the art, such as measuring cognitive impairment, can be used to measure the effectiveness of neurodegenerative disorder treatment, and a reduction in such impairment in the patient indicates that the treatment was successful. The treatment endpoints for nerve injury treatment may include a reduction in injury-related defects, such as a decrease in the increase of motor, memory, or behavioral defects. Using methods in the art, the effectiveness of nerve injury treatment can be measured, for example, by measuring motor function and flexibility, and an increase in such in the treated subject indicates that the treatment was successful. 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 than the reduction of the disease (or its symptoms) compared to when there is no treatment with the active ingredient is considered effective.

[0123] Atherosclerosis and / or coronary artery disease can also be treated in subjects requiring treatment of atherosclerosis and / or coronary artery disease by using a temperature-sensitive substance of the Disclosed (e.g., a temperature-sensitive therapeutic substance) by introducing / contacting it with vascular endothelial cells to improve the properties of the vascular endothelial cells and thereby treating atherosclerosis and / or coronary artery disease in the subject, for example by administering the temperature-sensitive substance of the Disclosed (e.g., a temperature-sensitive therapeutic substance) into the bloodstream of the subject.

[0124] The temperature-sensitive agents of the present disclosure (e.g., temperature-sensitive therapeutic agents) may also be used to provide resistance to one or more human cells and / or subjects to one or more genotoxic substances in need of them. Treatment of diseases and disorders by enhanced ZSCAN4 expression

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

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

[0127] Methods and compositions are provided for treating subjects in need of such treatment, such as subjects suffering from telomere abnormalities. Telomere abnormalities refer to any change in telomeres that disrupts one or more telomere functions, such as telomere shortening, interruption of telomere DNA repeats, or mutation of telomere DNA. Diseases or disorders associated with telomere abnormalities in which high ZSCAN4 expression may be beneficial include, but are not limited to, telomere shortening disorders, myelosufficiency syndromes, age-related telomere shortening disorders, and premature aging disorders.

[0128] Telomere shortening disorders that can benefit from temperature-sensitive agents that enhance ZSCAN4 expression in human cells (included by the term "telomere biological disorders") include, but are not limited to, congenital dyskeratosis, Heuerard-Raiderson syndrome, Löwes syndrome, Coates-Plass syndrome, and idiopathic pulmonary fibrosis. In some embodiments, telomere shortening disorders are congenital dyskeratosis.

[0129] Myelodysplastic syndromes that may benefit from temperature-sensitive agents that enhance ZSCAN4 expression in human cells include, but are not limited to, Fanconi anemia, amegakaryocytic thrombocytopenia, aplastic anemia, Diamond-Blackfan anemia, paroxysmal nocturnal hemoglobinuria, Pearson syndrome, Schbachermann-Diamond syndrome, and myelodysplastic syndromes. In some embodiments, the myelodysplastic syndrome is Fanconi anemia. In some embodiments, the subject requiring treatment suffers from both telomere biological disorders and myelodysplastic syndromes (e.g., congenital dyskeratosis).

[0130] Age-related telomere shortening disorders or premature aging disorders that can benefit from temperature-sensitive substances that enhance ZSCAN4 expression in human cells include, but are not limited to, Werner syndrome, Bloom syndrome, Hutchison-Gilford progeria syndrome, Cockayne syndrome, xeroderma pigmentosum, ataxia capillary dilatatum, Rothmond-Thomson syndrome, sulfur-deficient hair growth disorder, Juberg-Marcisgi syndrome, and Down syndrome.

[0131] Methods and compositions are provided for treating subjects in need of such treatment, such as subjects suffering from chromosomal abnormalities. Chromosomal abnormalities refer to any anomalies, changes, or mutations in a chromosome that result in a lost, extra, or irregular portion of chromosomal DNA. In certain embodiments, chromosomal abnormalities result in an abnormal number of chromosomes or structural abnormalities in one or more chromosomes. As used herein, aneuploidy can refer to an abnormal number of chromosomes as a whole or a portion of a chromosome. Aneuploidy that can benefit from temperature-sensitive agents that enhance ZSCAN4 expression in human cells includes, but is not limited to, chromosomal nulithomy, chromosomal monosomy, chromosomal trisomy, chromosomal tetrasomy, and chromosomal pentasomy. Examples of human aneuploidy include, but is not limited to, trisomy 21, trisomy 16, trisomy 18 (Edwards syndrome), trisomy 13 (Patau syndrome), monosomy X (Turner syndrome), XXX aneuploidy, XXY aneuploidy, and XYY aneuploidy. Examples of human partial aneuploidy, though not limited to these, include 1p36 region duplication, chromosome 17 (p11.2p11.2) region duplication syndrome, Pelizaeus-Merzbacher disease, chromosome 22 (q11.2q11.2) region duplication syndrome, and cat-eye syndrome. In some embodiments, aneuploidy includes deletions of one or more sex chromosomes or autosomes, which may result in conditions such as cat cry syndrome, Wolf-Hirschhorn syndrome, Williams-Beuren syndrome, Charcot-Marie-Tooth disease, hereditary sexual pressure fragility neuropathy, Smith-Magenis syndrome, neurofibromatosis, Alagille syndrome, palatine-cardiafacial syndrome, DiGeorge syndrome, steroid sulfatase deficiency, Kallmann syndrome, microphthalmia of linear cutaneous defects, adrenal hypoplasia, glycerol kinase deficiency, Pelizaeus-Merzbacher disease, testicular determinants on the Y chromosome, azoospermia (factor a), azoospermia (factor b), azoospermia (factor c), or 1p36 region deletion. ZSCAN4 Polynucleotide

[0132] In some embodiments, the thermosensitive therapeutic agents of the Disclosure that enhance ZSCAN4 expression are nucleic acid molecules containing a nucleic acid sequence (coding region) encoding the ZSCAN4 protein. Examples of nucleic acid molecules include DNA, cDNA, and RNA (mRNA) molecules encoding the ZSCAN4 protein. All polynucleotides encoding the ZSCAN4 protein are understood to be included herein insofar as they encode the ZSCAN4 protein, variants, or fragments thereof, possessing ZSCAN4 activity such as the ability to regulate genomic stability or telomere length. Genomic stability refers to a cell's ability to faithfully replicate DNA and maintain the integrity of the DNA replication mechanism. Long telomeres provide a buffer against cellular senescence and are generally considered to indicate genomic stability and overall cellular health. Chromosomal stability (e.g., minimal mutations, absence of chromosome rearrangements, or absence of chromosome number changes) is also related to genomic stability. Loss of genomic stability is associated with cancer, neurological disorders, and premature aging. Signs of genomic instability include increased mutation rates, large-scale chromosome rearrangements, changes in chromosome number, and telomere shortening.

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

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

[0135] The ZSCAN4 coding region can be operably ligated to a promoter to direct the transcription of the coding region. A promoter is a nucleic acid regulatory sequence that directs the transcription of an operably ligated coding region. The promoter contains the necessary nucleic acid sequence near the transcription start site. The promoter also optionally contains distal enhancer or repressor factors. A constitutive promoter is a promoter that is continuously active and not subject to external signaling or molecular regulation. In contrast, the activity of an inductive promoter is regulated by external signals or external molecules (e.g., transcription factors). A first nucleic acid sequence is operably ligated to a second nucleic acid sequence when the first nucleic acid sequence is positioned in a functionally related state with the second nucleic acid sequence. For example, a promoter is operably ligated to a coding sequence if it affects the transcription or expression of that coding sequence. Typically, operably ligated nucleic acid sequences are contiguous and are on the same reading frame when it is necessary to ligate two protein coding regions. Heterogeneous polypeptides or heterogeneous polynucleotides refer to polypeptides or polynucleotides derived from different origins or species. The promoter includes an essential nucleic acid sequence located near the transcription start site, for example, the TATA sequence in the case of a polymerase type II promoter. The promoter may also include a distal enhancer sequence or distal repressor sequence that can be located several thousand base pairs away from the transcription start site. In one example, the promoter is a constitutive promoter, such as the CAG promoter (Niwa et al., Gene 108(2):193-9, 1991). In some embodiments, the promoter is an inductive promoter, such as the tetracycline inductive promoter (Masui et al., Nucleic Acids Res. 33:e43, 2005).Other exemplary promoters that may be used to express Zscan4 include, but are not limited to, the lac system, trp system, tac system, trc system, major operator and promoter regions of lambda phage, regulatory regions of fd coat proteins, early and late promoters of SV40; promoters derived from polyomavirus, adenovirus, retrovirus, baculovirus, and Simian virus; promoters for 3-phosphoglycerate kinase, promoters for yeast acid phosphatase, and promoters for yeast α-coating factor. In some embodiments, a natural ZSCAN4 promoter is used. ZSCAN4 polypeptide

[0136] All ZSCAN4 polypeptides are included herein insofar as they possess ZSCAN4 activity, such as the ability to regulate genomic stability or telomere length. The terms “polypeptide” and “protein” are used interchangeably herein and include naturally occurring ZSCAN4 proteins, variants, or fragments thereof possessing ZSCAN4 activity.

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

[0138] Fragments and mutants of the ZSCAN4 protein can be prepared by those skilled in the art using standard molecular techniques. In some embodiments, the ZSCAN4 protein is a cleaved form of ZSCAN4 lacking one or more zinc finger domains of the naturally occurring ZSCAN4 protein. In some embodiments, the ZSCAN4 protein is a mutant of the naturally occurring ZSCAN4 protein.

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

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

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

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

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

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

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

[0146] The identity / similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of identity or similarity between those sequences. Sequence identity can be measured in terms of percentage identity, with a higher percentage indicating that the sequences are identical. Sequence similarity can be measured in terms of percentage similarity (considering conserved amino acid substitutions), with a higher percentage indicating that the sequences are similar. Homologs or orthologues of nucleic acid sequences or amino acid sequences exhibit a relatively high degree of sequence identity / similarity when aligned using standard methods. This homology is more significant when their orthologue proteins or cDNAs originate from more closely related species (e.g., human or monkey sequences) compared to more distantly related species (e.g., human or mouse sequences).

[0147] In the context of two or more sequences (e.g., nucleic acid sequences or amino acid sequences), the terms “identical” or percentage “identity” can refer to two or more sequences or subsequences that are identical. When two sequences are compared and aligned to find the maximum correspondence across a comparison window or specified region, either using one of the subsequent sequence comparison algorithms or by manual alignment and visual inspection, the two sequences are substantially identical if they have a specified percentage of identical amino acid residues or nucleotides (i.e., 95%, 96%, 97%, 98%, 99%, or 100% identity across a specified region, or across the entire sequence if not specified).

[0148] Typically, one sequence acts as a reference sequence for sequence comparison, and the test sequence is compared against this reference sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into the computer, subsequence coordinates are specified, and sequence algorithm program parameters are specified if necessary. Initial program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence compared to the reference sequence based on the program parameters. When comparing two sequences for identity, they do not need to be contiguous, but any gaps are associated with a penalty that will reduce the overall percentage identity. For blastp, the initial parameters are gap-opening penalty = 11 and gap-extension penalty = 1. For blastn, the initial parameters are gap-opening penalty = 5 and gap-extension penalty = 2.

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

[0150] Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, which are described in Altschul et al. (1997) Nucleic Acids Res 25(17):3389-3402 and Altschul et al. (1990) J. Mol Biol 215(3)-403-410, respectively. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information. This algorithm involves initially identifying high-scoring sequence pairs (HSPs) by identifying short word lengths W in the query sequence that, when aligned with words of the same length in the database sequence, match a certain positive threshold score T, or word lengths W that satisfy T. T is called the adjacent word score threshold (Altschul et al., cited above). These initial adjacent word hits serve as seeds to initiate a search for longer HSPs that contain those initial adjacent word hits. These word hits are extended in both directions along their respective sequences as long as the cumulative alignment score can increase. The cumulative score is calculated for nucleotide sequences using parameter M (reward score for matching residue pairs; always greater than 0) and parameter N (penalty score for mismatched residues; always less than 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of word hits in each direction stops when the cumulative alignment score falls by amount X from the maximum achieved value of the cumulative alignment score, when the cumulative score becomes 0 or less due to the accumulation of one or more negative score-forming residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.For amino acid sequences, the BLASTP program uses a word length of 3, an expected value of 10 (E), and a 50-point BLOSUM62 scoring matrix alignment (B) [see Henikoff and Henikoff, (1992) Proc Natl Acad Sci USA 89(22):10915-10919], an expected value of 10 (E), M=5, N=-4, and a comparison of both strands as initial settings. For nucleotide sequences, the BLASTN program uses a word length of 11 (W), an expected value of 10 (E), M=5, N=-4, and a comparison of both strands as initial settings.

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

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

[0153] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural nouns unless the context explicitly indicates otherwise. Similarly, the word “or” includes “and” unless the context explicitly indicates otherwise. Thus, “containing A or B” means containing A or B, or containing 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 approximations and are provided for illustrative purposes only. Methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this disclosure, but suitable methods and materials are listed below. All patent application publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of any inconsistency, this specification, including explanations of terms, shall prevail. In addition, the materials, methods, and examples are illustrative and not intended to be limiting. Exemplary Embodiments 1. A method for transiently inducing the temperature-sensitive activity of a temperature-sensitive substance, the following: i) Incubate one or more cells containing a temperature-sensitive agent at an acceptable temperature for inducing temperature-sensitive activity for a period of time sufficient for temperature-sensitive activity to produce an effect in one or more cells; and ii) Incubating one or more cells at an unacceptable temperature, where the unacceptable temperature reduces the temperature-sensitive activity of the temperature-sensitive substance. Includes, The present invention relates to a method wherein the temperature-sensitive active substance comprises a therapeutic agent comprising human ZSCAN4 protein or nucleic acid containing the coding region of human ZSCAN4, and the effect comprises a therapeutic effect. 2. Before step i), the following: Contacting one or more cells with a temperature-sensitive substance, The method according to Embodiment 1, further comprising: 3. The method according to Embodiment 2, wherein the one or more cells are at an acceptable temperature when in contact with a temperature-sensitive substance. 4. The method according to any one of Embodiments 1 to 3, further comprising administering one or more cells to a subject requiring a therapeutic effect. 5. The method according to any one of Embodiments 1 to 3, comprising incubating one or more of the aforementioned cells at an unacceptable temperature, and administering one or more cells to a subject requiring a therapeutic effect, wherein the subject's body temperature is at an unacceptable temperature. 6. The method according to Embodiment 4 or 5, wherein the one or more cells are further incubated at an unacceptable temperature before being administered to the target. 7. The method according to any one of embodiments 2 to 6, wherein the one or more cells are isolated from the subject before contacting the temperature-sensitive substance with the one or more cells. 8. The method according to any one of Embodiments 1 to 7, wherein the therapeutic effect includes increasing the telomere length of one or more cells. 9. The method according to any one of Embodiments 1 to 8, wherein the one or more cells are mammalian cells. 10. The method according to any one of embodiments 3 to 8, wherein the subject is a human subject. 11. A method for transiently inducing the temperature-sensitive activity of a temperature-sensitive substance in a human subject, wherein one or more cells of the subject contain the temperature-sensitive substance, wherein the temperature-sensitive activity of the temperature-sensitive substance is induced at an acceptable temperature, and wherein the acceptable temperature is lower than the body temperature of the subject, and furthermore: i) Lower the subject's body temperature to an acceptable level; ii) Maintain the lowered body temperature for a period of time sufficient for the temperature-sensitive activity to induce an effect in the subject; and iii) Raise the subject's body temperature to a normal body temperature. Includes, The method wherein the temperature-sensitive active substance comprises a therapeutic agent containing human ZSCAN4 protein or nucleic acid containing the coding region of human ZSCAN4, and the effect is a therapeutic effect. 12. A method for transiently inducing the temperature-sensitive activity of a temperature-sensitive substance in a human subject, wherein the temperature-sensitive activity of the temperature-sensitive substance is induced at an acceptable temperature, and wherein the acceptable temperature is lower than the subject's body temperature, and furthermore: i) Lower the subject's body temperature to an acceptable level; ii) Administer a temperature-sensitive substance to one or more target cells; iii) Maintain the lowered body temperature for a period of time sufficient for the temperature-sensitive activity to induce an effect in the subject; and iv) Raise the subject's body temperature and return it to a normal temperature. Includes, A method wherein step (i) is performed before, after, or simultaneously with step (ii), wherein the temperature-sensitive active substance comprises a therapeutic active substance comprising human ZSCAN4 protein or nucleic acid containing the coding region of human ZSCAN4, and the effect is a therapeutic effect. 13. The method according to Embodiment 12, wherein the temperature-sensitive substance is administered systemically. 14. The method according to Embodiment 13, wherein the temperature-sensitive substance is administered intravenously. 15. The method according to Embodiment 12, wherein the temperature-sensitive substance is administered to a specific tissue or organ of interest. 16. The method according to Embodiment 15, wherein the temperature-sensitive substance is administered to the brain or spinal cord by epidural injection. 17. The method according to Embodiment 15, wherein the temperature-sensitive substance is administered to a target organ by intradermal injection. 18. The method according to Embodiment 15, wherein the temperature-sensitive substance is administered to a target organ by an endoscope using an injection needle catheter. 19. The method according to Embodiment 15, wherein the temperature-sensitive substance is administered to a target organ by a vascular catheter. 20. The method according to any one of embodiments 17 to 19, wherein the target organ is selected from the group consisting of the liver, kidney, skeletal muscle, cardiac muscle, pancreas, spleen, heart, brain, spinal cord, skin, eye, lung, intestine, thymus, bone marrow, bone, and cartilage. twenty one. The method according to Embodiment 12, wherein the temperature-sensitive substance is administered by inhalation. twenty two. The method according to any one of embodiments 11 to 21, wherein lowering the body temperature of the subject involves using a target temperature control (TTM) procedure, wherein the TTM procedure involves applying one of the group consisting of a cooling catheter, a cooling blanket, and ice to the subject. twenty three. The method according to any one of Embodiments 11 to 22, wherein the subject is a mammal, and optionally the subject is a human. twenty four. The method according to any one of Embodiments 1 to 23, wherein the temperature-sensitive substance comprises human ZSCAN4 protein. twenty five. The method according to any one of Embodiments 1 to 23, wherein the temperature-sensitive substance comprises a nucleic acid including the coding region of human ZSCAN4. 26. The method according to Embodiment 25, wherein the temperature-sensitive viral vector comprises a nucleic acid including the coding region of human ZSCAN4. 27. The method according to Embodiment 26, wherein the temperature-sensitive viral vector is selected from the group consisting of Sendai virus, adenovirus, adeno-associated virus, retrovirus, and alphavirus. 28. The method according to Embodiment 26, wherein the temperature-sensitive viral vector is an alphavirus. 29. The method according to Embodiment 28, wherein the alphavirus is selected from the group consisting of Venezuelan encephalitis virus, Sindbis virus, and Semryki Forest virus. 30. The method according to Embodiment 26, wherein the temperature-sensitive viral vector is Sendai virus. 31. The method according to Embodiment 30, wherein the Sendai virus is SeV18+ / TS15ΔF. 32. The method according to any one of embodiments 26 to 31, wherein the temperature-sensitive activity includes replication and transcription of a temperature-sensitive viral vector. 33. The method according to Embodiment 25, wherein the temperature-sensitive self-replicating RNA comprises a nucleic acid including the coding region of human ZSCAN4. 34. The method according to Embodiment 33, wherein the self-replicating RNA includes an alphavirus replicon lacking a viral structural protein-coding region. 35. The method according to Embodiment 34, wherein the alphavirus is selected from the group consisting of Venezuelan encephalitis virus, Sindbis virus, and Semryki Forest virus. 36. The method according to any one of embodiments 33 to 35, wherein the temperature-sensitive activity includes one or both of replication and transcription of temperature-sensitive self-replicating RNA. 37. The method according to any one of embodiments 25 to 36, wherein the code region is operably linked to a promoter. 38. The method according to any one of Embodiments 1 to 10, wherein the period for which the temperature-sensitive activity is sufficient to produce a therapeutic effect is in the range of about 12 hours to about 12 weeks, and optionally therein the period is 1 to 7 days. 39. The method according to any one of embodiments 11 to 37, wherein the period sufficient for the therapeutic effect to be induced in the subject is approximately 12 hours to approximately 7 days, and optionally, the period is approximately 12 hours to approximately 72 hours. 40. The method according to any one of Embodiments 1 to 39, wherein the allowable temperature is in the range of 30°C to 36°C or 38°C to 50°C. 41. The method according to Embodiment 40, wherein the allowable temperature is 33°C ± 0.5°C. 42. The method according to Embodiment 40 or Embodiment 41, wherein the non-acceptable temperature is 37°C ± 0.5°C. 43. The method according to any one of Embodiments 1 to 42, wherein the one or more cells are human cells. 44. The method according to Embodiment 43, wherein the one or more human cells are adult stem cells, tissue stem cells, progenitor cells, embryonic stem cells, or induced pluripotent stem cells. 45. The method according to Embodiment 43, wherein the one or more human cells are selected from the group consisting of hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, adipose-derived stem cells, neural stem cells, and germline stem cells. 46. The method according to Embodiment 43, wherein the one or more human cells are somatic cells, mature cells, or differentiated cells. 47. The method according to Embodiment 46, wherein the one or more human cells are selected from the group consisting of epidermal cells, fibroblasts, lymphocytes, hepatocytes, epithelial cells, muscle cells, chondrocytes, osteocytes, adipocytes, cardiomyocytes, pancreatic cells, pancreatic β-cells, keratinocytes, erythrocytes, peripheral blood mononuclear cells (PBMCs), neurons, glial cells, nerve cells, astrocytes, germ cells, spermatocytes, and oocytes. 48. The method according to Embodiment 43, wherein the one or more human cells are human bone marrow cells. 49. The method according to Embodiment 48, wherein the human bone marrow cells are CD34+ hematopoietic stem cells. 50. The method according to Embodiment 48 or Embodiment 49, wherein the human subject suffers from telomere biological disorder, and optionally, the subject suffers from bone marrow failure. 51. A method for treating diseases of the blood or hematopoietic organs, the following: i) Recruit hematopoietic stem cells from bone marrow into the peripheral blood of a human subject suffering from the disease; ii) Isolate CD34+ cells from the peripheral blood mononuclear cell sample obtained from the subject; iii) Incubate the isolated CD34+ cells at a temperature of 33°C ± 0.5°C; iv) Incubated CD34+ cells are brought into contact with a temperature-sensitive Sendai virus vector containing heterologous nucleic acid including the coding region of human ZSCAN4; v) The contacted CD34+ cells were maintained at an acceptable temperature of 33°C ± 0.5°C for at least approximately 12 to 72 hours, during which replication and transcription of the temperature-sensitive Sendai virus vector occurred at the acceptable temperature, leading to increased expression of human ZSCAN4; and vi) Treating a disease by injecting contacted CD34+ cells into a target under conditions suitable for cell transplantation. A method that includes this. 52. A method for treating diseases of the blood or hematopoietic organs, the following: i) Recruit hematopoietic stem cells from bone marrow cells into the peripheral blood of a human subject suffering from the disease; ii) Isolate CD34+ cells from the peripheral blood mononuclear cell sample obtained from the subject; iii) The isolated CD34+ cells are contacted with a temperature-sensitive Sendai virus vector containing heterologous nucleic acids including the coding region of human ZSCAN4; iv) The contacted CD34+ cells were incubated at an acceptable temperature of 33°C ± 0.5°C for at least approximately 12 to 72 hours, during which replication and transcription of the temperature-sensitive Sendai virus vector occurred at the acceptable temperature, leading to increased ZSCAN4 expression; and v) Treating a disease by injecting contacted CD34+ cells into a target under conditions suitable for cell transplantation. A method that includes this. 53. Before injecting the contacted CD34+ cells, the contacted CD34+ cells are incubated at a non-permissible temperature of 37°C ± 0.5°C, where replication and transcription of the temperature-sensitive Sendai virus vector and expression of human ZSCAN4 are stopped at the non-permissible temperature. The method according to Embodiment 51, further comprising step v) after step v). 54. Before injecting the contacted CD34+ cells, the contacted CD34+ cells are incubated at a non-permissible temperature of 37°C ± 0.5°C, where replication and transcription of the temperature-sensitive Sendai virus vector and expression of human ZSCAN4 are stopped at the non-permissible temperature. The method of Embodiment 52, further comprising step iv). 55. The method according to Embodiment 53 or Embodiment 54, wherein the contacted CD34+ cells are incubated at a non-permissible temperature of 37°C ± 0.5°C for approximately 30 minutes to approximately 10 days, or optionally for approximately 30 to 180 minutes. 56. The method according to any one of embodiments 51 to 55, wherein the hematopoietic stem cells are recruited by administration of granulocyte colony-stimulating factor and / or plerixafor to the subject. 57. The method according to any one of embodiments 51 to 56, wherein the peripheral blood mononuclear cells are obtained from a subject by apheresis. 58. The method according to any one of embodiments 51 to 57, wherein the CD34+ cells are isolated from peripheral blood mononuclear cells by positive selection using an anti-CD34 antibody and magnetic beads. 59. The method according to any one of embodiments 51 to 58, wherein the contacted CD34+ cells are washed and resuspended in a sterile isotonic aqueous solution before injection. 60. The method according to Embodiment 59, wherein the contacted CD34+ cells are injected intravenously at a dose of approximately 1.0 × 10^5 cells / kg to approximately 1.0 × 10^7 cells / kg, and optionally approximately 2.0 to 8.0 × 10^6 cells / kg. 61. A method for treating diseases of the blood or hematopoietic organs, the following: i) A temperature-sensitive Sendai virus vector containing heterologous nucleic acid including the coding region of human ZSCAN4 is administered to a human subject suffering from the disease; ii) Lower the subject's core body temperature to an acceptable temperature of 33°C ± 0.5°C; iii) Maintain the subject's core body temperature at an acceptable temperature for a period of approximately 12 hours to approximately 7 days, or approximately 12 to 72 hours, during which replication and transcription of the temperature-sensitive Sendai virus vector occur at the acceptable temperature, leading to increased expression of human ZSCAN4; and iv) Return the target's core body temperature to a normal, non-acceptable temperature of 37°C ± 0.5°C, at which point replication and transcription of the temperature-sensitive Sendai virus vector and expression of human ZSCAN4 will cease. A method that includes this. 62. A method for treating diseases of the blood or hematopoietic organs, the following: i) Lower the core body temperature of the subject suffering from the disease to an acceptable temperature of 33°C ± 0.5°C; ii) The subjects were administered a temperature-sensitive Sendai virus vector containing heterologous nucleic acid including the coding region of human ZSCAN4; iii) Maintain the subject's core body temperature at an acceptable temperature for a period of approximately 12 hours to approximately 7 days, or approximately 12 to 72 hours, during which replication and transcription of the temperature-sensitive Sendai virus vector occur at the acceptable temperature, leading to increased expression of human ZSCAN4; and iv) Return the target's core body temperature to a normal, non-acceptable temperature of 37°C ± 0.5°C, at which point replication and transcription of the temperature-sensitive Sendai virus vector and expression of human ZSCAN4 will cease. A method that includes this. 63. The method according to Embodiment 61 or Embodiment 62, wherein the core body temperature of the subject is lowered using a target temperature control (TTM) procedure, the TTM procedure comprising applying one of the group consisting of a cooling catheter, a cooling blanket, and ice to the subject. 64. The method according to any one of Embodiments 51 to 63, wherein the human subject is diagnosed with bone marrow failure prior to treatment, and optionally, the bone marrow failure includes one or more of neutropenia, thrombocytopenia, and anemia. 65. The method according to any one of embodiments 51 to 64, wherein the subject does not have cancer. 66. The method according to any one of embodiments 51 to 65, wherein the disease is a telomere biological disorder. 67. The method according to Embodiment 66, wherein the telomere biological disorder is selected from the group consisting of congenital dyskeratosis, Heuerard-Raiderson syndrome, Löwes syndrome, Coats-Plus syndrome, idiopathic pulmonary fibrosis, and liver cirrhosis. 68. The aforementioned telomere biological damage is as follows: i) Age-adjusted mean telomere length below the 1st percentile in one or more peripheral blood lymphocytes, B cells, and unsensitized T cells; and ii) Pathogenic mutations in genes selected from the group consisting of DKC1, TERC, TERT, NOP10, NHP2, TINF2, CTC1, PARN, RTEL1, ACD, USB1, and WRAP53, The method according to Embodiment 66, as defined by one or both of the following. 69. The method according to any one of embodiments 51 to 63, wherein the disease is bone marrow failure syndrome. 70. The method according to Embodiment 69, wherein the bone marrow failure syndrome is selected from the group consisting of Fanconi anemia, amegakaryocytic thrombocytopenia, aplastic anemia, Diamond-Blackfan anemia, paroxysmal nocturnal hemoglobinuria, Pearson syndrome, Schbachmann-Diamond syndrome, and myelodysplastic syndromes. 71. The method according to Embodiment 64, wherein the disease is related to a karyotype abnormality. 72. The method according to any one of Embodiments 1 to 71, wherein the amino acid sequence of the human ZSCAN4 described above includes SEQ ID NO: 38 or is at least 95% identical to SEQ ID NO: 38. 73. The method according to any one of Embodiments 1 to 71, wherein the amino acid sequence of the human ZSCAN4 described above includes one of the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, or is at least 95% identical to one of the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42. 74. The method according to any one of embodiments 26 to 50, wherein the temperature-sensitive viral vector or temperature-sensitive self-replicating RNA comprises a non-structural protein-coding region with an insertion of 12 to 18 nucleotides, wherein the insertion results in the expression of nsP2 containing 4 to 6 additional amino acids between β-sheet 5 and β-sheet 6 of non-structural protein 2 (nsP2 = helicase proteinase), and optionally, wherein the additional amino acids result in the temperature sensitivity of the viral vector or self-replicating RNA. 75. The method according to Embodiment 74, wherein the additional amino acid comprises one sequence selected from the group consisting of SEQ ID NO: 43 (GCGRT), SEQ ID NO: 44 (TGAAA), and SEQ ID NO: 45 (LRPHP). 76. The method according to Embodiment 74, wherein the additional amino acid includes the sequence of Sequence ID No. 44 (TGAAA). 77. The method according to Embodiment 76, wherein the amino acid sequence of NsP2 includes one sequence selected from the group consisting of SEQ ID NOs: 29 to 36. 78. A temperature-sensitive active substance, wherein the active substance is a temperature-sensitive viral vector or temperature-sensitive self-replicating RNA comprising a heterogeneous nucleic acid containing the coding region of human ZSCAN4 and a non-structural protein coding region with an insertion of 12 to 18 nucleotides, wherein the insertion results in the expression of nsP2 containing 4 to 6 additional amino acids between β-sheet 5 and β-sheet 6 of non-structural protein 2 (nsP2 = helicase proteinase), and optionally, wherein the additional amino acids result in temperature sensitivity of the viral vector or self-replicating RNA. 79. The temperature-sensitive substance according to Embodiment 78, wherein the additional amino acid comprises one sequence selected from the group consisting of SEQ ID NO: 43 (GCGRT), SEQ ID NO: 44 (TGAAA), and SEQ ID NO: 45 (LRPHP). 80. The temperature-sensitive substance according to Embodiment 78, wherein the additional amino acid includes the sequence of Sequence ID No. 44 (TGAAA). 81. The temperature-sensitive substance according to Embodiment 81, wherein the amino acid sequence of NsP2 includes one sequence selected from the group consisting of SEQ ID NOs: 29 to 36. 82. The temperature-sensitive active substance according to any one of embodiments 78 to 81, wherein the active substance is a temperature-sensitive alphavirus vector. 83. The temperature-sensitive active substance according to any one of embodiments 78 to 81, wherein the active substance is a temperature-sensitive self-replicating RNA containing an alphavirus replicon lacking a viral structural protein-coding region. 84. The temperature-sensitive substance according to Embodiment 82 or Embodiment 83, wherein the alphavirus is selected from the group consisting of Venezuelan encephalitis virus, Sindbis virus, and Semliki Forest virus. 85. The temperature-sensitive substance according to Embodiment 82 or Embodiment 83, wherein the alphavirus is Venezuelan encephalitis virus. 86. A method for transiently inducing the temperature-sensitive activity of a temperature-sensitive activator (ts activator) in a subject, wherein the ts activator is a temperature-sensitive viral vector or temperature-sensitive self-replicating RNA containing a heterogeneous nucleic acid including the coding region of human ZSCAN4, wherein one or more cells contain the ts activator on or near the surface of the subject's body, wherein the temperature-sensitive activity of the ts activator includes the expression of human ZSCAN4 at an acceptable temperature, and wherein the acceptable temperature is the surface body temperature of the subject, and further: i) Maintain the surface body temperature of the subject at an acceptable temperature for a period of time sufficient for the temperature-sensitive activity to induce an effect in the subject; and ii) Raise the surface temperature of the subject to an unacceptable temperature for a period of time sufficient for temperature-sensitive activity to cease in the subject. A method that includes this. 87. A method for transiently inducing the temperature-sensitive activity of a temperature-sensitive activator (ts activator) in a subject, wherein the ts activator is a temperature-sensitive viral vector or temperature-sensitive self-replicating RNA containing a heterogeneous nucleic acid including the coding region of human ZSCAN4, wherein the temperature-sensitive activity of the ts activator includes the expression of human ZSCAN4 at an acceptable temperature, and wherein the acceptable temperature is the surface body temperature of the subject, and further: i) administering a ts-active substance to one or more cells on or near the surface of the target body; and ii) Maintain the surface body temperature of the subject at an acceptable temperature for a period of time sufficient for the temperature-sensitive activity to induce an effect in the subject. A method that includes this. 88. iii) Raise the surface temperature of the subject to an unacceptable temperature for a period of time sufficient for temperature-sensitive activity to cease in the subject. The method of embodiment 87, further including the following. 89. The method according to Embodiment 86 or Embodiment 87, wherein the temperature-sensitive substance is administered i) intradermally or subcutaneously; or ii) intramuscularly. 90. The method according to Embodiment 86 or Embodiment 87, wherein the temperature-sensitive substance is administered intranasally. 91. The method according to any one of embodiments 86 to 90, wherein the non-acceptable temperature is greater than 36°C, and the acceptable temperature is less than 36°C, wherein optionally the acceptable temperature is about 31°C to about 34°C, or about 33°C ± 0.5°C, and the non-acceptable temperature is 37°C ± 0.5°C. 92. The method according to any one of Embodiments 86 to 91, wherein the effect of human ZSCAN4 expression is a preventive effect or a therapeutic effect. 93. The method according to any one of Embodiments 86 to 92, wherein the ts-active substance is a temperature-sensitive viral vector, and the temperature-sensitive activity further comprises replication and transcription of the temperature-sensitive viral vector. 94. The method according to Embodiment 93, wherein the temperature-sensitive viral vector is selected from the group consisting of Sendai virus, adenovirus, adeno-associated virus, retrovirus, and alphavirus. 95. The method of Embodiment 93, wherein the temperature-sensitive viral vector is an alphavirus, and optionally the alphavirus is selected from the group consisting of Venezuelan encephalitis virus, Sindbis virus, and Semryki Forest virus. 96. The method according to Embodiment 93, wherein the temperature-sensitive viral vector is Sendai virus. 97. The method according to any one of Embodiments 86 to 92, wherein the tsactive agent is a temperature-sensitive self-replicating RNA, and the temperature-sensitive activity further comprises one or both of replication and transcription of the temperature-sensitive self-replicating RNA. 98. The method according to Embodiment 97, wherein the self-replicating RNA includes an alphavirus replicon lacking the viral structure protein-coding region of the alphavirus. 99. The method according to Embodiment 98, wherein the alphavirus is selected from the group consisting of Venezuelan encephalitis virus, Sindbis virus, and Semryki Forest virus. 100. The method according to Embodiment 98, wherein the alphavirus is Venezuelan encephalitis virus. 101. The method according to any one of embodiments 86 to 100, wherein the period for which the temperature-sensitive activity is sufficient to produce an effect is in the range of about 12 hours to about 12 weeks, optionally, the period is 1 to 7 days. 102. The method according to any one of embodiments 86 to 100, wherein the period sufficient to induce the effect in the subject is about 12 hours to about 7 days, and optionally, the period is about 12 hours to about 72 hours. 103. The method according to any one of embodiments 86 to 102, wherein the subject is a mammal, and optionally the subject is a human. [Examples]

[0154] Abbreviations: Aura (Aura virus); BFV (Bammer Forest virus); GFP (Green fluorescent protein); GOI (Gene of Interest); IRES (Internal ribosome entry site); LUC (Luciferase); ONNV (Onyon-Nyon virus); RRV (Ross River virus); SeV (Sendai virus); SeVt (Temperature-sensitive Sendai virus); SFV (Semryki Forest virus); shRNA (Short hairpin RNA); SINV (Sindobis virus); srRNA (Self-replicating RNA); ts (Temperature-sensitive); ts activator (Temperature-sensitive activator); VEEV (Venezuelan horse encephalitis virus); and WEEV (Western equine encephalitis virus).

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

[0156] This example describes temperature-sensitive activators (ts activators) that function at temperatures lower or higher than normal body temperature but do not function or exhibit reduced function at normal body temperature. ts activators 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 elements. For example, a protein with a temperature-sensitive mutation may function at lower temperatures (e.g., 30°C) but not at normal body temperature (e.g., 37°C). Unless otherwise specified, normal body temperature is the normal human body temperature of 37°C ± 0.5°C.

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

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

[0159] This example illustrates the finding that mutations in the nsP2 protein encoded by the Venezuelan encephalitis virus (VEEV) vector exhibit temperature sensitivity. The temperature-sensitive system allows expression of the gene of interest (GOI) at 30°C–33°C but inhibits expression above 37°C. The srRNA vector enables higher GOI expression than the synthetic RNA encoding the GOI. GOI expression ceases when the temperature rises to 37°C (e.g., the non-tolerant temperature). The specific temperature-sensitive mutation (mutation 2) identified in this study is located within a well-conserved region within the alphavirus. Compared to Sendai virus vectors (SeVt), srRNAts may be more attractive for some applications because they can be used in non-viral RNA expression systems. Materials and methods cell culture

[0160] Human adipose-derived stem cell (ADSC-iPS) cells were purchased from System Biosciences (Palo Alto, CA). The cells were maintained as undifferentiated human pluripotent cells (hPSCs) according to standard hPSC culture procedures. In short, the cells were cultured in StemFit basic02 (Ajinomoto, Japan) supplemented with 100 ng / ml of FGF2. Furthermore, the cells were cultured on cell culture dishes coated with laminin-511 substrate (iMatrix-511, Nippi, Japan). veEV vector

[0161] The VEEV vector plasmid was assembled using a synthetic DNA fragment based on publicly available sequence information (T7-VEE-IRES-Puro, hereafter referred to as "srRNA1wt"). According to Yoshioka et al., 2013, the VEEV vector backbone was originally induced as described by Petrakova et al., 2005. 7480 candidate sequences identified by insertion mutagenesis and large-scale parallel sequencing (Beitzel et al., 2010) were used to induce potentially temperature-sensitive mutants. The original large-scale screening was performed by 15 bp transposon-mediated insertions into the VEEV genome (Figure 1A). Subsequently, many 15 bp insertion VEEV mutants capable of growing at 30°C or 40°C were isolated. While these data provided initial mutants for further investigation, it was not known that these sequences exhibited temperature sensitivity, such as tolerance at 32°C or 33°C and non-tolerance at 37°C. Three mutant sequences—mutant 1 (ts1, Figure 1B), mutant 2 (ts2, Figure 1C), and mutant 3 (ts3, Figure 1D)—were selected from a total of 7480 candidate mutant sequences (Data Set S1 from Beitzel et al., 2010). These mutant DNA fragments (Figure 2) were synthesized and cloned into VEEV vectors and named srRNA1ts1 (mutant 1), srRNA1ts2 (mutant 2), and srRNA1ts3 (mutant 3). Mutant 4 was designed, and it contains the 5'-region of the viral sequence (part of the 5'-UTR and N-terminal protein sequence of RNA-dependent RNA polymerase, known to contain the 51-nt conserved sequence element (CSE)). In this case, the nucleotides were systematically changed to fewer heat-stable mutants (e.g., G->A) while simultaneously maintaining the amino acid sequence (Figure 3). The sequence in this region within srRNA1ts2 was substituted to create srRNA1ts4 (i.e., containing both mutant 4 and mutant 2). Synthetic RNA was prepared from these vectors according to Yoshioka et al., 2013. result Evaluation of the temperature sensitivity of srRNA1ts2-GFP and srRNA1ts3-GFP at 30°C, 32°C, and 37°C.

[0162] ADSC-iPSC cells were cultured on 24-well plates at a density of 80,000 cells / well. After 24 hours, 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 culture medium was added. The cells were incubated at 30°C, 32°C, or 37°C. Six hours after transfection, the medium was changed 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 mutant (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 showed much higher GFP expression compared to the GFP expression levels achieved by single transfection of synthetic mRNA encoding GFP (Figure 4B). Evaluation of the temperature sensitivity of srRNA1ts1-GFP and srRNA1ts2-GFP at 32°C.

[0163] ADSC-iPSC cells were cultured on 24-well plates at a density of 50,000 cells / well. After 24 hours, 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 culture medium was added. The cells were incubated at 32°C. Six hours after transfection, the medium was changed and the transfection complex was removed. Phase contrast and fluorescence images were taken at 24, 48, 72, 96, 120, 144, 168, 192, 240, and 288 hours.

[0164] Figure 5 shows the results. GFP expression from the wild type (srRNA1wt-GFP) began at 24 hours and continued until the end of the observation period (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 and 168 hours). Based on these results, mutant 2 is selected for further development. Evaluation of the temperature sensitivity of srRNA1ts2-GFP and srRNA1ts4-GFP at 32°C, 33°C, and 37°C.

[0165] ADSC-iPSC cells were plated on a 24-well plate at a density of 50,000 cells / well. After 24 hours, 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 at 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 changed to remove the transfection complex. Phase contrast and fluorescence images were taken at 20, 48, and 96 hours.

[0166] Figure 6 shows the results. At 32 °C and 33 °C, GFP expression from mutant 2 (srRNA1ts2-GFP) started as early as 20 hours, but was significantly enhanced at 48 hours and further enhanced at 96 hours. GFP expression was stronger at 33 °C than at 32 °C. Consistent with the previous experiment, GFP was not expressed at 37 °C at all. SrRNA1ts4-GFP (including both mutant 2 and mutant 4) showed a temperature profile similar to that of srRNA1ts2-GFP, but GFP expression was overall much weaker. Based on these results, mutant 2 was selected for further development. Evaluation of the temperature sensitivity of srRNA1ts2-GFP at 32 °C

[0167] ADSC-iPSC cells were plated on a 24-well plate at a density of 80,000 cells / well. After 24 hours, 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 at 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 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. The experiment was conducted in the absence (upper panel) or presence (lower panel) of 1 μg / ml puromycin. Puromycin was added at 48 and 72 hours for cell selection with puromycin. Phase contrast and fluorescence images were taken at 24, 48, 72, 96, 144, 168, and 192 hours.

[0168] Figure 7 shows the results. At 32°C, GFP expression from srRNA1ts2-GFP started as early as 24 hours, but was significantly enhanced at 48 hours and reached a peak at 72 and 96 hours. GFP expression continued until the end of the observation period (192 hours). The GFP expression pattern did not appear to change with the addition of puromycin. Evaluation of the temperature sensitivity of srRNA1ts2-GFP switched from 32°C to 37°C after 24 hours

[0169] ADSC-iPSC cells were cultured on 24-well plates at a density of 80,000 cells / well. After 24 hours, 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 using puromycin, puromycin was added at 48 and 72 hours. To test the effect of temperature shift, the cell cultures were transferred to a CO2 incubator maintained at 37°C for 24 hours (24 hours after transfection). Phase contrast and fluorescence imaging were taken at 24, 48, 72, 96, 144, 168, and 192 hours.

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

[0171] ADSC-iPSC cells were cultured on 24-well plates at a density of 80,000 cells / well. After 24 hours, 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 using puromycin, puromycin was added at 48 and 72 hours. To test the effect of temperature shift, the cell cultures were transferred to a CO2 incubator maintained at 37°C at 48 hours (48 hours after transfection). Phase contrast and fluorescence imaging were taken at 24, 48, 72, 96, 144, 168, and 192 hours.

[0172] Figure 9 shows the results. At 32°C, GFP expression from srRNA1ts2-GFP began as early as 24 hours and increased further at 48 hours. GFP expression continued until 96 hours, even after the temperature was changed to 37°C at 48 hours. However, GFP expression began to decrease from 72 hours, and by 96 hours, GFP expression was very weak. By 144 hours, GFP expression was barely detectable, and by 192 hours, function had completely ceased. Thus, GOI (represented here as GFP) expression rapidly ceased when the temperature was changed from 33°C (acceptable temperature) to 37°C (unacceptable temperature). The mode of GFP expression did not appear to change with the addition of puromycin. Evaluation of the temperature sensitivity of srRNA1ts2-GFP after switching from 32°C to 37°C after 72 hours.

[0173] ADSC-iPSC cells were cultured on 24-well plates at a density of 80,000 cells / well. After 24 hours, 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 using puromycin, puromycin was added at 48 and 72 hours. To test the effect of temperature shift, the cell cultures were transferred to a CO2 incubator maintained at 37°C at 72 hours (72 hours after transfection). Phase contrast and fluorescence imaging were taken at 24, 48, 72, 96, 144, 168, and 192 hours.

[0174] Figure 10 shows the results. At 32°C, GFP expression from srRNA1ts2-GFP began as early as 24 hours and increased further at 48 hours. GFP expression continued until 96 hours, even after the temperature was changed to 37°C at 48 hours. However, GFP expression began to decrease from 72 hours, and by 144 hours, GFP expression was very weak. By 168 hours, GFP expression was barely detectable, and by 192 hours, function had completely ceased. Thus, GOI (represented here as GFP) expression rapidly ceased when the temperature was changed from 33°C (acceptable temperature) to 37°C (unacceptable temperature). The mode of GFP expression did not appear to change with the addition of puromycin. Evaluation of temperature sensitivity of srRNA1ts2-GFP in fibroblasts

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

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

[0177] As shown in Figure 12, even at the amino acid level, the structure of the alphavirus nsP2 protein is well conserved within the family members. Based on the 3D structural model (Russo et al., 2006), the protein region in mutant 2 where the five amino acid sequence number 44 (TGAAA) is inserted is the boundary between two β-sheet structures, and this is also well conserved within the alphavirus family members. Therefore, it is highly probable that the temperature sensitivity of mutant 2 is transferable to other alphavirus family members, including Aura virus, WEEV (Western Equine Encephalitis Virus), BFV (Bammer Forest Virus), ONNV (Onyon-Nyon Virus), RRV (Roth River Virus), SFV (Semryki Forest Virus), and SINV (Sindobis Virus). Table 3-1 lists suitable sites for insertion into nsP2 of various alphaviruses to confer temperature sensitivity. [Table 1]

[0178] Example 4: Temperature-sensitive antibody This example describes a temperature-sensitive antibody. Antibodies that function at an acceptable temperature (e.g., 32°C) and do not function or have low function at an unacceptable temperature (e.g., 37°C) are designed by inserting or substituting amino acid sequences. Temperature-sensitive antibodies could be produced by inserting a linker oligonucleotide encoding a temperature-sensitive helix-coil transfer peptide (-Glu-Ala-Ala-Ala-Lys-, described as SEQ ID NO: 37) as described (Kamihara and Iijima, 2000; Merutka and Stellwagen, 1990). In this way, manipulated antibodies that function at an acceptable temperature (e.g., 32°C) but do not function at an unacceptable temperature (e.g., 37°C) can be produced. Alternatively, antibody DNA sequences from animals naturally living in low-temperature environments (e.g., Atlantic salmon or shrimp) can be used because these antibodies function optimally at an acceptable temperature (low temperature) but exhibit low functionality at an unacceptable temperature (e.g., 37°C). Example 5: Temperature-sensitive protein

[0179] This example describes a temperature-sensitive protein. Such a protein functions at an acceptable temperature (e.g., 32°C) but does not function or exhibits low function at an unacceptable temperature (e.g., 37°C). Temperature-sensitive proteins are manipulated by substituting amino acid sequences. Alternatively, temperature-sensitive proteins derived from animals that naturally live in low-temperature environments (e.g., Atlantic salmon or shrimp) can be used because these proteins function optimally at acceptable temperatures (low temperatures) but exhibit low functionality at unacceptable temperatures (e.g., 37°C). Example 6: Temperature-sensitive RNA

[0180] This example describes a temperature-sensitive RNA molecule. RNA molecules include, but are not limited to, mRNA, mRNA precursors, non-coding RNA, siRNA, and shRNA. The temperature-sensitive RNA functions at tolerable temperatures (e.g., 32°C) and is non-functional or exhibits low functionality at untolerable temperatures (e.g., 37°C). The temperature-sensitive RNA was manipulated by systematically altering the nucleotides of the RNA molecule to lower the thermal stability of the mutant (e.g., G->A), while simultaneously ensuring the maintenance of the RNA's functional properties. Furthermore, the difference in nucleotide pair thermal stability induced by temperature shifts alters the RNA's secondary structure. Example 7: Ex vivo treatment of cells using a temperature-sensitive substance

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

[0182] This example demonstrates a method for transiently delivering RNA or protein to cells ex vivo (Figures 14 and 15). ts-active agents, such as srRNAs or Sendai virus vectors, are functional at acceptable temperatures (e.g., 33°C) but not at unacceptable temperatures (e.g., 37°C: body temperature). Typically, target cells are harvested from the patient (autologous cell transplantation; Figure 14), but it is also possible to use target cells isolated from a donor (allogeneic cell transplantation; Figure 15). For example, target cells may be isolated by using antibody-conjugated magnetic beads. The target cells are incubated with the ts-active agent ex vivo at an acceptable temperature, e.g., 33°C, for a specific duration, e.g., 24 hours. The level of GOI RNA (or RNA-translated protein) increases at the acceptable temperature and reaches high levels. After the therapeutic effect is induced, the cells are transplanted back into the patient to treat the patient. The activity of the temperature-sensitive therapeutic agent is not induced at the target's normal body temperature (i.e., normal body temperature is an unacceptable 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, thereby preventing the reactivation of the ts activator and preventing the treatment of cells other than target cells with the ts activator. Mobilization of human peripheral blood cells

[0183] Human blood cells isolated from a patient, or the bone marrow or peripheral blood of a donor, are treated ex vivo with a ts agent at an acceptable temperature. After injection of G-CSF or other mobilizing agents, human white blood cells are recovered from peripheral blood by an apheresis device (e.g., COBE Spectra). White blood cells recovered from the bone marrow after mobilization include granulocytes, monocytes, lymphocytes, dendritic cells, mesenchymal stem cells (MSC), vascular endothelial cells (VEC), and CD34+ hematopoietic / progenitor cells. Treatment of these cells with a ts agent is carried out ex vivo at a functional temperature (e.g., 33°C) for a specific duration (hours to weeks), preferably using a functionally closed system such as the CliniMacs Prodigy from Miltenyi. Subsequently, the treated cells are injected into the patient at a non-acceptable temperature (37°C). The ts agent, the cells containing the ts agent, or the products of the ts agent do not function in the patient's body. Human CD34+ hematopoietic stem / progenitor cells

[0184] Human CD34+ hematopoietic stem / progenitor cells are isolated from mobilized human peripheral blood cells or bone marrow cells using antibody-conjugated magnetic beads (against CD34) and used as target cells to be treated ex vivo with a ts agent at an acceptable temperature. After treatment with the ts agent, the human CD34+ cells are injected into the patient's body and transplanted into the patient's bone marrow. Since these cells ultimately produce all blood cells in the patient's body, they are thus suitable targets for various diseases. All human cells including tissue stem cells

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

[0186] This example illustrates a semi-in vivo method for transiently delivering 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 an acceptable temperature (e.g., 33°C) but not at an unacceptable temperature (e.g., 37°C).

[0187] The patient undergoes therapeutic hypothermia: the patient's core body temperature is maintained at a temperature lower than normal (e.g., 33°C). Target cells (any cells - autologous or allogeneic) are treated with a ts activator ex vivo and immediately injected into the patient's circulation or into the patient's organs.

[0188] While the patient is maintained at a target temperature, e.g., 33°C, for a period of time, e.g., 24 hours, the ts-active substances exert their expected functions. The levels of GOI RNA (and the proteins translated from it) increase at the tolerable temperature and reach high levels. Subsequently, the patient's body temperature is returned to a normal temperature of 37°C. The ts-active substances no longer function at 37°C, which is an untolerable condition in the patient's body. Body temperature is maintained above 37°C throughout the patient's life, thereby preventing the ts-active substances from being reactivated and ensuring that cells other than target cells are not treated with the ts-active substances. In particular, this therapeutic method can be applied to all cell types, including those described above. Example 10: In vivo therapeutic use of temperature-sensitive substances

[0189] This embodiment demonstrates how a temperature-sensitive viral vector is administered to a subject and transiently activated when mild hypothermia is induced in the subject (Figure 17). The temperature-sensitive therapeutic agent may be any of the temperature-sensitive therapeutic agents disclosed herein. The temperature-sensitive therapeutic agent is functional at an acceptable temperature (e.g., 33°C) but not at an unacceptable temperature (e.g., 37°C: human body temperature).

[0190] The core body temperature of subjects was lowered using a target temperature management (TTM) procedure, which was used in an outpatient setting for patients with cardiac and brain injuries. The TTM procedure is designed to achieve and maintain a specific body temperature over a sustained period. Such procedures have previously been used therapeutically to mitigate the negative effects arising from various acute health problems, such as heart attacks and strokes. The devices and general methods for using the TTM procedure are known in the art and can be used in conjunction 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, 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 can be performed safely, and no major side effects caused by this device have been reported.

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

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

[0193] After the desired therapeutic effect is achieved, the patient's body temperature is then returned to normal (i.e., to an unacceptable temperature) to deactivate the temperature-sensitive therapeutic agent. This is followed by the decomposition of the temperature-sensitive therapeutic agent. Circulatory delivery to the whole body

[0194] The patient is placed under hypothermic conditions (e.g., 33°C). Once the patient's core body temperature is stably maintained at the target temperature, the ts activator is delivered directly into the patient's vein. The ts activator is then delivered to many organs and tissues via this systemic pathway. The patient's core body temperature is maintained at a functional temperature for a desired period (e.g., 24 hours). The activator functions while the patient's body temperature is maintained at the activator's acceptable temperature (e.g., 33°C). When the patient's body temperature returns to the normal temperature of 37°C, which is the activator's unacceptable temperature, the activator ceases to function.

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

[0196] The patient is placed under hypothermic conditions (e.g., 33°C). Once the patient's core body temperature is stably maintained at the target temperature, the ts activator is directly delivered to the patient's cerebrospinal fluid by epidural injection. The ts activator is delivered to the brain and spinal cord. The patient's core body temperature is maintained at an acceptable temperature for a desired period (e.g., 24 hours). The activator functions while the patient's body temperature is maintained at the activator's acceptable temperature (e.g., 33°C). When the patient's body temperature returns to the normal temperature of 37°C, which is the activator's non-acceptable temperature, the activator ceases to function. Targeted delivery to the liver, kidneys, skeletal muscle, cardiac muscle, pancreas, bone marrow, and other organs via intradermal injection.

[0197] The patient is placed under hypothermic conditions (e.g., 33°C). Once the patient's core body temperature is stably maintained at the target temperature, the ts active agent is injected percutaneously into organs such as the liver, kidneys, skeletal muscle, cardiac muscle, pancreas, or other organs using a very fine needle guided by ultrasound or CT. The patient's core body temperature is maintained at an acceptable temperature for a desired period (e.g., 24 hours). The active agent functions while the patient's body temperature is maintained at the acceptable temperature for the active agent (e.g., 33°C). When the patient's body temperature returns to the normal temperature of 37°C, which is the unacceptable temperature for the active agent, the active agent ceases to function. Targeted delivery to the liver, kidneys, skeletal muscle, cardiac muscle, pancreas, bone marrow, and other organs using an endoscope equipped with a needle catheter.

[0198] The patient is placed under hypothermic conditions (e.g., 33°C). Once the patient's core body temperature is stably maintained at the target temperature, the ts active 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 an acceptable temperature for a desired period (e.g., 24 hours). The active agent functions while the patient's body temperature is maintained at the acceptable temperature for the active agent (e.g., 33°C). When the patient's body temperature returns to the normal temperature of 37°C, which is the unacceptable temperature for the active agent, the active agent ceases to function. Targeted delivery to the liver, kidneys, skeletal muscle, cardiac muscle, pancreas, and other organs via vascular catheters.

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

[0200] The patient is placed under hypothermic conditions (e.g., 33°C). Once the patient's core body temperature is stably maintained at the target temperature, the ts active agent is then delivered directly to the patient by inhalation. The ts active agent is delivered to the lungs and other organs via lung inhalation. The patient's core body temperature is maintained at an acceptable temperature for the desired period (e.g., 24 hours). The active agent functions while the patient's body temperature is maintained at the acceptable temperature for the active agent (e.g., 33°C). When the patient's body temperature returns to the normal temperature of 37°C, which is the unacceptable temperature for the active agent, the active agent ceases to function. Targeted delivery to bone marrow cells mobilized to the spleen

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

[0202] This example illustrates the finding that a 16-hour incubation at 33°C ex vivo is sufficient for a temperature-sensitive Sendai virus vector to be effective against human CD34+ cells. This example demonstrates that an infection multiplicity (MOI) of 1–25 is sufficient for the vector to infect a large portion of human CD34+ cells ex vivo. Materials and methods cell culture

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

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

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

[0206] To determine the optimal duration and conditions for ex vivo contact of CD34+ cells with SeVts-ZSCAN4, a series of incubation times at functional temperature (33°C) were used to evaluate CD34+ cells using the intended clinical CD34+ incubation protocol. CD34+ cells were cultured on 12-well plates (1 × 10⁶). 5 or 5 x 10 4Cells were incubated with SeVts-ZSCAN4 (MOI=25) in 5% CO2 at 33°C for 0, 3, 6, 16, 24, 48, or 72 hours. The cells were then incubated in 5% CO2 at 37°C for up to 10 days. Incubation at 33°C allowed for Sendai virus infection, replication, and transgene expression, while the increase to 37°C inactivated the virus and stopped transgene expression. After the specified 33°C incubation, the cells were immunostained for ZSCAN4 protein expression using an anti-hZSCAN4 antibody. The number of human ZSCAN4-expressing cells was compared to the total number of cells identified by DAPI fluorescence staining.

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

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

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

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

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

[0212] During the 10-day incubation period, nearly 100% of the cells retained their CD34 marker, indicating that contact with SeVts-ZSCAN4 did not alter the properties of the CD34+ cells with respect to the CD34+ cell fraction and CD34 marker expression (Figure 20A, B). Based on immunostaining with ZSCAN4 on day 1, contact with SeVts-ZSCAN4 (MOI=25) exposed 77% of CD34+ cells to the ZSCAN4 protein (Figure 20A). As expected, when the temperature was increased to 37°C, the number of cells with the ZSCAN4 protein decreased very rapidly: only 7% of ZSCAN4-positive cells remained on day 7, and only 2% on day 10. In contrast, the control experiment showed that without SeVts-ZSCAN4 contact, while no ZSCAN4-positive cells were present, nearly 100% of the cells maintained CD34+. The rapid decrease in ZSCAN4 protein after switching to the non-acceptable temperature of 37°C was not simply caused by cell division, because the cell number increased only 3.5 times over 10 days (less than 2 cell divisions on average). During the same period, the number of control cells (without SeVts-ZSCAN4 contact) increased 6.1 times. Example 13: Effect of SeVts-ZSCAN4 on telomere length in human CD34+ cells

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

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

[0215] ZSCAN4 has been shown to localize to telomeres, upregulate meiotic-specific homologous recombination genes, and elongate telomeres by telomere recombination in mouse germinal stem (ES) cells (regardless of telomerase activity) (Zalzman et al., 2010; Amano et al., 2013). To evaluate this potential in human hematopoietic stem cells, human peripheral blood CD34+ cells were contacted with SeVts-ZSCAN4 ex vivo and incubated at 33°C. CD34+ cells were treated with SeVts-ZSCAN4 at 33°C for 16, 24, 48, and 72 hours, and then cultured at 37°C for 10 days for telomere assays. Telomere length was measured by quantitative real-time PCR using telomere-specific primers (T) and a single-copy gene-specific primer set (S) as described in (Cawthon2002). The relative telomere length was calculated as the T / S ratio and then normalized by the T / S ratio of the control sample (untreated control).

[0216] Compared to untreated cells, 24-hour incubation at 33°C resulted in approximately 1.5 times telomere elongation (Figure 21). Incubation ≥ 24 hours did not further elongate telomeres; therefore, 24-hour incubation at an acceptable temperature (i.e., 33°C) was sufficient to elongate telomeres in human CD34+ cells. Example 14: Effect of SeVts-ZSCAN4 on telomere length of human hematopoietic cells transplanted into immunodeficient mice

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

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

[0219] To rigorously model the intended clinical trial, severely immunodeficient NOG-EXL mice (Taconic) were treated with G-CSF and plerixafor (Figure 22). The study used NOG-EXL mice without irradiation (i.e., bone marrow resection). Similarly, unlike typical transplantation studies that use more potent umbilical cord blood CD34+ cells, the study used G-CSF-mobilized peripheral blood CD34+ cells from healthy donors. NOG-EXL mice received 2 × 10⁶ cells on day 1. 7Either CD34+ untreated cells or CD34+ treated with SeVts-ZSCAN4 (test substance) were administered intravenously at a dose of cells / kg (Figure 22). The dose was approximately 10 times higher than the dose intended for humans. No SeVts-ZSCAN4-related adverse events were observed in NOG-EXL mice that received CD34+ cells treated with SeVts-ZSCAN4. Furthermore, 38 weeks after CD34+ cell injection, two mice (#492 and #493) that received CD34+ treated with SeVts-ZSCAN4, and one mouse (#496) that received untreated (control) CD34+ cells were sacrificed, and transplanted cells derived from human CD34+ cells were examined. Splenocytes isolated from these mice were FACS sorted using the human CD45+ panhematopoietic marker and used for qPCR-based telomere assays. As shown in Figure 24, human cells transplanted into mice treated with SeVts-ZSCAN4-treated CD34+ cells had longer telomeres than mice treated with CD34+ cells alone (control). These data suggest that SeVts-ZSCAN4-treated human CD34+ cells could be transplanted into mouse bone marrow and participated in normal hematopoiesis. Furthermore, once telomere elongation occurred due to SeVts-ZSCAN4 treatment, these cells retained their long telomeres even after transplantation and cell differentiation. The study also demonstrates the safety of SeVts-ZSCAN4 treatment. Example 15: Evaluation of SeVts-ZSCAN4 in human patients with telomere biological damage and bone marrow failure.

[0220] Telomere biological disorders, including bone marrow failure such as congenital dyskeratosis, have a poor prognosis and high mortality rate. Currently, hematopoietic stem cell transplantation is the only therapeutic option, and it can alleviate the manifestation of the hematological state of the disease. However, its use can be challenging, involving difficulties in finding a suitable donor and in toxicity associated with bone marrow removal (chemotherapy and radiation) and immunological complications. This example describes the evaluation of the safety and tolerability of administering CD34+ cells ex vivo contacted with a temperature-sensitive Sendai virus vector encoding human ZSCAN4 to human patients in need, and the efficacy of transplanting these cells.

[0221] A thermosensitive therapeutic agent. SeVts-ZSCAN4 (also known as SeV18+hZSCAN4 / TS15ΔF) expresses human ZSCAN4 in a thermosensitive manner (Figure 18). As used in this example, the investigational drug product is a pharmaceutical composition comprising a sterile, electrolyte-containing isotonic aqueous solution in which autologous CD34+ cells contacted ex vivo with SeVts-ZSCAN4 are suspended. PLASMA-LYTE polyelectrolyte injection, commercially available from Baxter International Inc. (Deerfield, IL), is a suitable solution for resuspending virus-contacted CD34+ cells.

[0222] Theoretical basis: Autologous CD34+ cells exposed to SeVts-ZSCAN4 ex vivo have been shown to elongate telomeres in in vitro and in vivo non-clinical studies of human CD34+ cells. This treatment does not require a well-matched donor when the patient's own cells can be used. Exposure to SeVts-ZSCAN4 results in transient production of human ZSCAN4 protein in the patient's own (autologous) CD34+ cells, which restores their function by elongating their abnormally short telomeres ex vivo. After administration, the exposed CD34+ cells are transplanted, subsequently proliferating in the patient's bone marrow and resulting in the production of hematopoietic cells. In this way, the patient's bone marrow failure is effectively treated (Figure 25).

[0223] Patients. The study population initially includes adult men and women, but will be expanded to include pediatric patients. Inclusion criteria include a diagnosis of mild or moderate myelodeficiency and telomere biological disorder. Mild or moderate myelodeficiency is defined by one or both of the following: 1) absolute neutrophil count (ANC) in peripheral blood of 0.5–1.5 × 10⁹ / L; or platelet count of 20–100 × 10⁹ / L; or hemoglobin < 10 g / dL; and 2) low myelocyte count for age. The diagnosis of telomere biological impairment is defined by: i) age-adjusted mean telomere length at the <1st percentile in one or more peripheral blood lymphocytes (PBLs), B cells, or unsensitized T cells; and ii) one of the following pathogenic mutations in DKC1, TERC, TERT, NOP10, NHP2, TINF2, CTC1, PARN, RTEL1, ACD, USB1, or WRAP53. Exclusion criteria include: receiving chemotherapy for cancer; clonal cytogenetic abnormalities associated with myelodysplastic syndrome or acute myeloid leukemia in bone marrow examination; uncontrolled bacterial, viral, or fungal infection; pre-allogeneic mesenchymal or stem cell transplantation; ineligible for G-CSF and plerixafor; ineligible for apheresis; and one or more subjects currently taking danazol and androgens or who have taken them within 60 days prior to the start of this study.

[0224] Procedure. In short, this study involves: 1) recruitment of hematopoietic stem cells into the bloodstream and recovery of mononuclear cells (MNCs) by apheresis; 2) ex vivo cell treatment; and 3) injection of treated cells. The flowchart for this study was designed as shown in Figure 26, and illustrated in Figure 27.

[0225] Mobilization and Apheresis Days 1-3: All eligible subjects received daily injections of granulocyte colony-stimulating factor (G-CSF) (10 μg / kg SC). Day 4: After G-CSF injection (10 μg / kg SC), blood samples are collected and the CD34+ cell count is determined. Subjects with <5 cells / μL CD34+ cells were withdrawn from the study. Subjects with ≥5 cells / μL CD34+ cells were admitted to the hospital and administered plerixafor (fixed dose of 20 mg or 0.24 mg / kg SC) approximately 11 hours before apheresis. Plerixafor 1,4-Bis((1,4,8,11-tetraazacyclotetradecan-1-yl)methyl)benzene, such as MOZOBIL, marketed by Genzyme Corporation (Cambridge, MA), is a hematopoietic stem cell mobilizer. Day 5: Administer G-CSF (10 μg / kg SC), initiate the first apheresis, and collect MNCs. After apheresis, evaluate the subjects' ability to tolerate a second apheresis. Administer plerixafor (fixed dose of 20 mg or 0.24 mg / kg SC) to subjects deemed able to tolerate a second apheresis approximately 11 hours before the second apheresis. Subjects unable to tolerate a second apheresis and those with <2.0 × 10^6 / kg CD34+ cells are withdrawn from the study, and all collected cells are infused back into the subjects. Subjects unable to tolerate a second apheresis and those with >2.0 × 10^6 / kg CD34+ cells continue the study. Day 6: For subjects who can tolerate a second apheresis, administer G-CSF (10 μg / kg SC) before the start of the second apheresis to collect additional MNCs. After apheresis, obtain a complete blood count (CBC), and if necessary, administer red blood cell or platelet transfusions to maintain hemoglobin levels >10.5 g / dl and platelets >100 K. Remove subjects who have undergone a second apheresis and those with <2.0 × 10^6 / kg CD34+ cells (total from the first and second apheresis) from the study, and inject all recovered cells back into the subjects. Continue the study for subjects who have undergone a second apheresis and those with ≥2.0 × 10^6 / kg CD34+ cells.

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

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

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

[0229] The endpoint of the test. The following are required: an increase in telomere length in any of the following: lymphocytes, granulocytes, B cells, unsensitized T cells, memory T cells, and peripheral blood NK cells, as well as an improvement in blood cell count (neutrophils, platelets, or hemoglobin). Telomere length is measured by Flow FISH. Example 16: Expression of human ZSCAN4 protein in vivo

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

[0231] Similarly, the temperature of the nasal cavity and upper trachea in human subjects is approximately 32°C, and the temperature of the subsegmental bronchi in human subjects is approximately 35°C (McFadden et al., 1985). Thus, ts activators administered intranasally to cells of the upper respiratory tract (nasal cavity, pharynx, and / or larynx) and / or upper trachea in human patients are functional without lowering the core body temperature of human patients (Figure 29). Intranasal administration may be performed by inhalation, suction, or intravenous infusion. No further action is required.

[0232] Alternatively, a ts-active agent administered to cells on or near the body surface of a human patient (e.g., intradermally, subcutaneously, or intramuscularly) can be rendered ineffective by subsequently raising the surface body temperature of the human patient, for example, by applying a heat patch or heat pad to the treatment area of ​​the patient's skin, immersing in a warm bath, or sitting in a hot sauna. This therapeutic method is very safe in that the ts-active agent functions only in the intended area and does not function in other areas of the patient's body. Similarly, a ts-active agent administered intranasally to cells of the upper respiratory tract (nasal cavity, pharynx, and / or larynx) and / or upper trachea of ​​a human patient can be rendered ineffective by placing the human patient in an environment with an unacceptable temperature (e.g., ≥37°C).

[0233] For example, the coding region of human ZSCAN4 is introduced into srRNA1ts2 or SeV18 / TS15ΔF, as previously described for the expression of human ZSCAN4 on or near the surface of a human patient's body. The structure of srRNA1ts2 was previously described in Example 3. In short, srRNA1ts2 contains a Venezuelan encephalitis virus (VEEV) replicon lacking the VEEV structural protein-coding region. This VEEV replicon contains a VEEV non-structural protein-coding region with a 15-18 nucleotide insertion that results in the expression of nsP2 containing 5 or 6 additional amino acids (SEQ ID NO: 44 = TGAAA) between β-sheets 5 and β-sheet 6 of non-structural protein 2 (nsP2 = helicase proteinase). The additional amino acids result in temperature sensitivity of the srRNA.

[0234] The RNA in the srRNA1ts2 vector can be transcribed in vitro using T7 RNA polymerase without the use of animal or human-derived material. Thus, tsactive agents using the srRNA1ts2 vector can be easily adapted to current pharmaceutical manufacturing and quality control standards. The RNA is transfected into cells of the target dermal tissue. A suitable method for transfection is patch electroporation of naked RNA. Alternatively, microneedles can be used for intradermal RNA transfection. For example, soluble microneedles made of hyaluronic acid or a chitosan-hyaluronic acid complex can be used for intradermal RNA transfection.

Claims

1. A composition comprising a temperature-sensitive Sendai virus vector containing a heterogeneous nucleic acid including the coding region of human ZSCAN4, for use in a method for treating diseases of the blood or hematopoietic organs, wherein the method is as follows: i) Recruiting hematopoietic stem cells from bone marrow into the peripheral blood of a human subject suffering from the disease; ii) Isolate CD34+ cells from a sample of peripheral blood mononuclear cells obtained from the subject; iii) The isolated CD34+ cells are brought into contact with the temperature-sensitive Sendai virus vector; iv) The contacted CD34+ cells were incubated at an acceptable temperature of 33°C ± 0.5°C for approximately 12 to 24 hours, during which replication and transcription of the temperature-sensitive Sendai virus vector occurred at the acceptable temperature, leading to increased expression of human ZSCAN4; and v) Injecting the contacted CD34+ cells into the subject under conditions suitable for cell transplantation to treat the disease. A composition containing the following:

2. Before injecting the contacted CD34+ cells, the contacted CD34+ cells are incubated at a non-permissible temperature of 37°C ± 0.5°C, where replication and transcription of the temperature-sensitive Sendai virus vector and expression of human ZSCAN4 are stopped at the non-permissible temperature. The composition according to claim 1, further comprising after step iv).

3. The composition according to claim 1 or claim 2, wherein the contacted CD34+ cells are incubated at a non-permissible temperature of 37°C ± 0.5°C for approximately 30 minutes to approximately 10 days.

4. The composition according to any one of claims 1 to 3, wherein the hematopoietic stem cells are mobilized by the administration of granulocyte colony-stimulating factor and / or plerixafor to a subject.

5. The composition according to claim 4, wherein the peripheral blood mononuclear cells are obtained from the subject by apheresis.

6. The composition according to claim 5, wherein the CD34+ cells are isolated from peripheral blood mononuclear cells by positive selection using an anti-CD34 antibody and magnetic beads.

7. The composition according to claim 6, wherein the contacted CD34+ cells are washed and resuspended in a sterile isotonic aqueous solution before injection.

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

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