Peptide expression construct and its use

Abstract

This disclosure provides a gene transfer construct comprising a destabilization domain (DD) sequence, a translation separator sequence, and a sequence encoding one or more copies of one or more peptides of interest; a nucleic acid encoding the gene transfer construct; the use of a nucleic acid encoding the gene transfer construct to prevent mitochondrial hyperfission and fragmentation; the use of a nucleic acid encoding the gene transfer construct to induce apoptosis in cells, such as cancer cells; and therapeutic applications of a nucleic acid encoding the gene transfer construct, for example, in the treatment of mitochondrial dysfunction and mitochondrial diseases and disorders associated with various types of cancer.

Description

[Technical Field]

[0001] 1. Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 490,324, filed on 15 March 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] 2. Sequence Listing This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML sequence listing, created on February 29, 2024, is named RMG-002WO_SL.XML and has a size of 82,183 bytes. [Background technology]

[0003] 3.Background Many physiological processes are regulated through protein complexes, which typically involve protein-protein interactions (PPIs). Therefore, numerous diseases, including metabolic disorders, cancers, infectious diseases, and neurodegenerative diseases, may originate from abnormal PPIs (Lu et al., 2020, Sig Transduct and Target Ther 5:213). Many abnormal PPIs involve weakening or loss of essential interactions, while numerous others involve PPIs at inappropriate times, places, or between unlikely partners.

[0004] Targeting abnormal PPIs is a promising intervention strategy for various diseases, but challenges remain due to the large, flat, and relatively hydrophobic PPI interface region. One strategy is to design PPI modulators that act on PPI interface hotspots, which are amino acid residues that significantly contribute to binding. For example, small peptides generated using the amino acid sequence of a PPI hotspot can act as competitive inhibitors. In fact, several small peptides have been developed and shown to be effective as PPI inhibitors in cultured cells (Lu et al., 2020. Sig Transduct and Target Ther 5:213, Chang et al., 2013. PNAS 110:E3445-E3454).

[0005] Peptides that mimic PPI interface hotspots possess high target specificity and affinity, but their use as PPI modulators faces challenges such as limited biological availability when administered orally, low solubility, and poor membrane permeability.

[0006] Therefore, a novel approach is needed to provide short peptide modulators for PPIs. [Overview of the project]

[0007] 4. Overview Intracellular expression of short peptides, particularly those shorter than 50 amino acids, is challenging because ribosome binding to mRNA decreases as the coding sequence length decreases. This disclosure is partly based on the finding that intracellular expression of short peptides may be facilitated by using nucleic acid molecules encoding a nucleotide sequence encoding a short peptide, ligated to a nucleotide sequence encoding an analytic domain (DD), e.g., E. coli DHFR, via a translation separator, e.g., the self-cleaving peptide P2A. While not theoretically bound, it is thought that the DD-encoding sequence can act as a ribosome-binding fragment, resulting in increased expression of the target peptide compared to nucleic acids without the DD sequence. In the absence of a DD-stabilizing molecule, the DD is degraded post-translation.

[0008] Accordingly, in one embodiment, the present disclosure provides (a) a destabilization domain (DD), (b) a translation separator, and (c) an mRNA molecule encoding the target peptide.

[0009] Exemplary characteristics of the mRNA molecules of this disclosure are described in sections 6.2 and 6.3 below, as well as in specific embodiments 1 to 101.

[0010] In another embodiment, the Disclosure provides a DNA molecule encoding the mRNA of the Disclosure. For example, the DNA may be an expression vector, such as a plasmid, or a viral genome, such as an AAV genome.

[0011] Exemplary characteristics of the DNA molecules of this disclosure are described below in Section 6.3, for example in Section 6.3.1.3, and in Specific Embodiments 102-110.

[0012] In another embodiment, the disclosure provides particles comprising nucleic acids. For example, the nucleic acid-containing particles may be viral particles (e.g., retroviral particles or AAV particles) or lipid particles (e.g., lipid nanoparticles).

[0013] Exemplary characteristics of the particles of the present disclosure are described in Section 6.3 below, for example, Sections 6.3.1.3 and 6.3.3, as well as in Specific Embodiment 111.

[0014] In a further aspect, the present disclosure provides a host cell comprising the nucleic acid of the present disclosure. The host cell may be a prokaryote or a eukaryote, and can be used, for example, to amplify the nucleic acid of the present disclosure, or to amplify and package the particles of the present disclosure, or to express a target peptide.

[0015] Exemplary characteristics of the host cells of the present disclosure are described in Section 6.3.2 below and in Specific Embodiment 113.

[0016] In another aspect, the present disclosure provides a pharmaceutical composition comprising the nucleic acid of the present disclosure or the cell of the present disclosure and a pharmaceutically acceptable excipient.

[0017] Exemplary characteristics of the pharmaceutical composition of the present disclosure are further described in Section 6.5 below and in Specific Embodiment 112.

[0018] In another aspect, the present disclosure provides a method for introducing a nucleic acid into a host cell. The present disclosure also provides a method for expressing a target peptide in a host cell. In a further aspect, the present disclosure provides a method for (a) preventing mitochondrial hyperdivision and / or (b) inducing apoptosis in cancer cells. In yet another aspect, the present disclosure provides a method for treating a subject with the nucleic acid or pharmaceutical composition of the present disclosure.

[0019] Further exemplary characteristics of the methods of the present disclosure are described in Sections 6.3.3 and 6.4 below, as well as in Specific Embodiments 114 to 133. 5. Brief Description of the Drawings

Brief Description of the Drawings

[0020] [Figure 1]Figure 1 illustrates the regulation of the stability of the dihydrofolate reductase (DHFR) fusion protein. In the absence of a stabilizing drug, such as trimethoprim (TMP), the destabilizing domain (DD) of DHFR targets the fusion protein to the ubiquitin-proteasome system (UPS) for degradation. Upon addition of TMP, it binds to the destabilizing domain of DHFR, inhibiting UPS-mediated degradation of the fusion protein. [Figure 2-1] Figure 2A, shown in Figure 2-1, illustrates an exemplary peptide expression unit of the present disclosure. Figure 2A is a vector diagram illustrating the general configuration of the individual components of the exemplary peptide expression unit of the present disclosure, which include an optional marker, DHFR, a translation separator (e.g., P2A), and a coding sequence for the target peptide, embedded between the promoter and the poly(A) sequence. [Figure 2-2] Figures 2B to 2E in Figure 2-2 show exemplary peptide expression units of this disclosure. Figures 2B and 2C show diagrams of two peptide expression units that were evaluated. Figures 2D and 2E show a control construct. [Figure 2-3] Figure 2F, shown in Figure 2-3, shows an exemplary peptide expression unit of this disclosure. Figure 2F shows a pCAGGS expression plasmid, which includes the peptide expression unit shown in Figure 2B. [Figure 3] Figure 3 shows the pLenti-GFPβ1-10-puro plasmid used to generate cells expressing GFPβ1-10. [Figure 4] Figure 4 shows the split green fluorescent protein (GFP) system. Functional GFP has 11 β-chains. The split GFP system relies on the interaction of two non-fluorescent polypeptides, GFP β-chains 1-10 (GFPβ1-10) and GFP β-chain 11 (GFPβ11), to form a fluorescent GFP molecule. [Figure 5]Figure 5 shows fluorescence microscopy images of GFP+ cells and A549 cells transfected with the GFPβ1-10 plasmid shown in Figure 3. A549 cells transfected with the GFPβ1-10 plasmid possessed high levels of GFPβ1-10 mRNA, but lacked both GFP signaling and GFP mRNA, indicating that these cells do not express full-length GFP. [Figure 6-1] Figure 6A, shown in Figure 6-1, shows the results of transfection of A549 cells expressing GFPβ-1-10 with the plasmids shown in Figures 2B, 2D, and 2E, compared to untransfected cells. Figure 6A shows fluorescence microscopy images of each cell group. Figure 6B shows the results of FACS analysis. [Figure 6-2] Figure 6B, shown in Figure 6-2, shows the results of transfection of A549 cells expressing GFPβ-1-10 with the plasmids shown in Figures 2B, 2D, and 2E, compared to untransfected cells. Figure 6B shows the results of FACS analysis. [Figure 6-3] Figures 6C–6F in Figure 6-3 show the results of transfection of A549 cells expressing GFPβ-1-10 with the plasmids shown in Figures 2B, 2D, and 2E, compared to untransfected cells. Figures 6C, 6D, and 6E show the percentage of cells in each group expressing mCherry, GFP, or both mCherry and GFP, respectively. Figure 6F shows the GFP intensity in cells within each group. [Figure 6-4] Figures 6G-6I in Figure 6-4 show the results of transfection of A549 cells expressing GFPβ-1-10 with the plasmids shown in Figures 2B, 2D, and 2E, compared to untransfected cells. Figures 6G-6I show the mRNA levels for GAPDH expression in cells within each group. [Figure 7-1]Figure 7A, shown in Figure 7-1, illustrates the effect of TMP on peptide expression in cells transfected with a plasmid containing the mCherry-DHFR-P2A-GFPβ11 peptide expression unit. Figure 7A shows fluorescence microscopy images of GFP and mCherry in cells transfected with a plasmid containing mCherry-DHFR-P2A-GFPβ11, both in the absence and in the presence of TMP. [Figure 7-2] Figures 7B-7E in Figure 7-2 show the effect of TMP on peptide expression in cells transfected with a plasmid containing the mCherry-DHFR-P2A-GFPβ11 peptide expression unit. Figure 7B shows the results of FACS analysis of cells transfected with the plasmid containing mCherry-DHFR-P2A-GFPβ11. Figures 7C, 7D, and 7E show the percentage of cells expressing mCherry, GFP, or both mCherry and GFP, respectively, in the presence or absence of TMP. [Figure 7-3] Figure 7F, shown in Figure 7-3, illustrates the effect of TMP on peptide expression in cells transfected with a plasmid containing the mCherry-DHFR-P2A-GFPβ11 peptide expression unit. Figure 7F shows the results of FACS analysis of cells transfected with plasmids containing mCherry, GFP, GFP-G7-mCherry, and GFP-P2A-mCherry. [Figure 8-1] Figure 8A, shown in Figure 8-1, demonstrates that P110 integration protects cells from LPS-induced mitochondrial fragmentation. Figure 8A shows GFP and mCherry fluorescence microscopy images of untreated control H9c2 cells and H9c2 cells lipofected with mRNA from constructs containing GFPβ11 or P110. [Figure 8-2] Figure 8B, shown in Figure 8-2, demonstrates that the incorporation of P110 protects cells from LPS-induced mitochondrial fragmentation. Figure 8B also shows the results of FAC analysis and the average fluorescence intensity of GFP in the same cell population. [Figure 8-3]Figures 8C-8D in Figure 8-3 show that P110 integration protects cells from LPS-induced mitochondrial fragmentation. Figure 8C shows the results of FACs analysis and the average fluorescence intensity of GFP in the same cell population. Figure 8D shows fluorescence microscopy images of cells in each treatment group stained with mitotracker green. [Figure 8-4] Figures 8E-8G in Figure 8-4 show that the incorporation of P110 protects cells from LPS-induced mitochondrial fragmentation. Figures 8E-8G show the results of mitochondrial network analysis (MiNA). [Figure 8-5] Figures 8H to 8I in Figure 8-5 show that the incorporation of P110 protects cells from LPS-induced mitochondrial fragmentation. Figures 8H to 8I show the results of mitochondrial network analysis (MiNA). [Figure 8-6] Figures 8J-8L in Figure 8-6 show that the incorporation of P110 protects cells from LPS-induced mitochondrial fragmentation. Figures 8J-8L show the results of reactive oxygen species (ROS) and mitochondrial membrane potential evaluations. [Figure 8-7] Figures 8M to 8N in Figure 8-7 show that the incorporation of P110 protects cells from LPS-induced mitochondrial fragmentation. Figures 8M to 8N show the results of reactive oxygen species (ROS) and mitochondrial membrane potential evaluations. [Figure 9-1] Figures 9A-9D in Figure 9-1 show that the incorporation of P110 protects cells from doxorubicin-induced mitochondrial fragmentation and apoptosis. Figure 9A shows low-magnification (left panel) and high-magnification (right panel) fluorescence microscopy images of cells in each treatment group stained with mitotracker green. The frame in the left panel indicates the area magnified to the right. Figures 9B-9D show the MiNA results for each treatment group. [Figure 9-2]Figures 9E-9F in Figure 9-2 show that the incorporation of P110 protects cells from doxorubicin-induced mitochondrial fragmentation and apoptosis. Figures 9E-9F show the MiNA results for each treatment group. [Figure 9-3] Figures 9G-9H in Figure 9-3 show that the incorporation of P110 protects cells from doxorubicin-induced mitochondrial fragmentation and apoptosis. Figure 9G shows the results of FACS analysis, where the upper left, upper right, and lower right quartiles in each evaluation represent necrotic cells, late-stage apoptotic cells, and early-stage apoptotic cells, respectively. Figure 9H is a graph showing the percentage of late-stage apoptotic cells in each treatment group. [Figure 9-4] Figure 9I, shown in Figure 9-4, demonstrates that integration of P110 protects cells from doxorubicin-induced mitochondrial fragmentation and apoptosis. Figure 9I also shows the results of mitochondrial respiration evaluation by flux analysis. [Figure 9-5] Figures 9J-9L in Figure 9-5 show that integration of P110 protects cells from doxorubicin-induced mitochondrial fragmentation and apoptosis. Figures 9J-9L show the results of mitochondrial respiration evaluation by flux analysis. [Figure 9-6] Figures 9M to 9O in Figure 9-6 show that integration of P110 protects cells from doxorubicin-induced mitochondrial fragmentation and apoptosis. Figures 9M to 9O show the results of mitochondrial respiration evaluation by flux analysis. [Figure 10-1] Figure 10A, shown in Figure 10-1, demonstrates that P110 incorporation protects against mitochondrial dysfunction and associated cell death. Figure 10A shows the results of FACS analysis of apoptosis using DAPI and annexin V. [Figure 10-2]Figures 10B-10C in Figure 10-2 show that P110 integration protects against mitochondrial dysfunction-related cell death. Figure 10B shows the results of FACS analysis of apoptosis using DAPI and annexin V. Figure 10C shows the results of Western blot analysis of apoptosis-related proteins. [Figure 10-3] Figures 10D-10E in Figure 10-3 show that integration of P110 protects against cell death associated with mitochondrial dysfunction. Figures 10D-10E show the results of Western blot analysis of apoptosis-related proteins. [Figure 10-4] Figures 10F-10G in Figure 10-4 show that integration of P110 protects against cell death associated with mitochondrial dysfunction. Figures 10F-10G show the results of Western blot analysis of apoptosis-related proteins. [Figure 10-5] Figures 10H to 10I in Figure 10-5 show that P110 integration protects against cell death associated with mitochondrial dysfunction. Figure 10H shows the results of Western blot analysis of apoptosis-related proteins. Figure 10I shows the results of immunoprecipitation mediated by Fis1 antibody, analyzed by Western blot. [Figure 10-6] Figures 10J-10K in Figure 10-6 show that P110 integration protects against mitochondrial dysfunction and associated cell death. Figures 10J-10K show the results of immunoprecipitation mediated by Fis1 antibody, analyzed by Western blotting. [Modes for carrying out the invention]

[0021] 6. Detailed explanation 6.1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those widely understood by those skilled in the art to which this disclosure pertains. The following definitions are provided for a complete understanding of the terms used herein.

[0022] As used herein, the following terms are intended to have the following meanings:

[0023] One (a), one (an), the: Where used herein, the terms one (a), one (an), the, and similar terms as used in the context of this disclosure (in particular in the context of the claims) shall be construed to include both singular and plural forms unless otherwise indicated herein or the context clearly contradicts this. Thus, the terms "one (a)" (or "one (an)"), "one or more," and "at least one" may be used interchangeably herein.

[0024] and / or: The term "and / or" means that one, both, or all of the components or characteristics of the list, in particular, two or more of them, are possible variations in an alternative or cumulative manner.

[0025] Apoptosis: As used herein, “apoptosis” refers to a form of cell death in which a programmed series of events leads to cell death. Characteristics of apoptosis include morphological changes, cell shrinkage, caspase activation, nuclear and cytoplasmic condensation, and alterations in plasma membrane topology. Biochemically, apoptotic cells are characterized by increased intracellular calcium concentration, chromosomal DNA fragmentation, and the expression of novel cell surface components. In some embodiments, cells undergoing apoptosis may undergo mitochondrial outer membrane permeabilization (MOMP).

[0026] Cancer: As used herein, “cancer” is a condition characterized by abnormal and / or uncontrolled cell growth. The term cancer encompasses both benign and malignant cancers. Exemplary cancers include pancreatic cancer (e.g., ductal adenocarcinoma), lung cancer, small cell lung cancer or non-small cell lung cancer (e.g., lung adenocarcinoma), colorectal cancer, melanoma (e.g., with BRAF mutation), leukemia (e.g., acute myeloid leukemia or acute lymphoblastic leukemia), lymphoma (e.g., non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL)), myeloma (e.g., multiple myeloma, leiomyosarcoma), breast cancer, liver cancer, osteosarcoma, and head and neck cancer.

[0027] Destabilization domain (DD): The term "destabilization domain" or "destabilizing domain" refers to a polypeptide domain that, in the absence of stabilizing molecules, is unstable, readily ubiquitinated, and degraded by the proteasome. Degradation of peptides or fusion polypeptides containing DDs can be prevented by suitable stabilizers. Exemplary destabilization domains include the dihydrofolate reductase (DHFR) destabilization domain (which can be stabilized by the exemplary stabilizer trimethoprim), the FK506-binding protein (FKBP) destabilization domain (which can be stabilized by the exemplary stabilizers Shield-1 (Shld1), rapamycin, and FK506), the PDE5 destabilization domain (which can be stabilized by the exemplary stabilizers sildenafil, vardenafil, tadalafil, avanafil, rodenafil, mirodenafil, udenafil, benzamidenafil, dasantafil, and beminafil), and the CA2 destabilization domain (which can be stabilized by the exemplary stabilizers celecoxib (Celebrex), valdecoxib, rofecoxib (Vioxx), acetazolamide, and metazolamide). Examples include the dorsolamide, brinzolamide, diclofenamide, ethoxyzolamide, zonisamide, dansylamide, and dichlorphenamide, the PPAR gamma destabilization domain (which can be stabilized by exemplary stabilizers pioglitazone and posiglitazone), the NQO2 destabilization domain (which can be stabilized by exemplary stabilizers imatinib and melatonin), the ERLBD destabilization domain (which can be stabilized by exemplary stabilizers CMP8, 4-hydroxytamoxifene, tamoxifen, fulvestrant, and raloxifene), and the UnaG destabilization domain (which can be stabilized by exemplary stabilizer bilirubin).Exemplary DHFR destabilization domains are described in Iwamoto et al., 2010, Chem Biol. 17(9):981-8, Liu et al., 2014 Int. J. Parasitol. 44(10):729-735, and U.S. Patent No. 9,487,787, while an exemplary FKBP destabilization domain is described in Banaszynski et al., 2006, Cell. The exemplary PDE5 destabilization domain is described in 126(5):995-1004 and U.S. Patent No. 9,487,787, the exemplary CA2 destabilization domain is described in International Publication No. 2018 / 237323, the exemplary PPAR gamma destabilization domain and NQO2 destabilization domain are described in International Publication No. 2018 / 160993 and U.S. Patent Application Publication No. 2022 / 0213449, the exemplary ERLBD destabilization domain is described in Miyazaki et al., 2012, J Am Chem Soc. 134(9):3942-3945 and U.S. Patent Application Publication No. 2014 / 0255361, and the exemplary UnaG destabilization domain is described in Navarro et al., 2016, ACS Chem Biol. These are described in 11(8):2101-4, and the contents of each of these are incorporated herein by reference in their entirety.

[0028] Effective Dose: The term “effective dose” or “therapeutic effective dose” means the amount or quantity of a drug or composition that is sufficient to induce a requested or desired response, or in other words, sufficient to induce a recognizable biological response when administered to a subject (e.g., relief of one or more signs or symptoms of a disease or disorder, or improvement of biomarkers associated with a disease or disorder). The amount preferably relates to an amount that is therapeutically, or more broadly, preventively, effective against the progression of the disease or disorder disclosed herein. It is understood that the “effective dose” or “therapeutic effective dose” may vary from subject to subject due to variations in drug metabolism, the subject’s age, weight, overall condition, the condition being treated, the severity of the condition being treated, and the prescribing physician’s judgment.

[0029] mRNA: As used herein, the term "mRNA" refers to messenger ribonucleic acid. Unless otherwise required by context, the term "mRNA" encompasses modified and unmodified mRNA. Modified mRNA may include, for example, one or more modified and / or non-naturally occurring components, such as one or more non-naturally occurring nucleic acid bases, nucleosides, nucleotides, or internucleoside ligatures. mRNA may include a cap structure, a chain termination nucleoside, a stem-loop, a poly(A) sequence, and / or a polyadenylation signal. Conventionally, the basic components of an mRNA molecule include at least a coding region, a 5'-untranslated region (5'-UTR), a 3'UTR, a 5' cap, and a polyadenylation (poly(A)) sequence.

[0030] mmRNA: As used herein, “modified mRNA” or “mmRNA” refers to an mRNA molecule having at least one modified sugar group, nucleic acid base, and / or nucleoside linkage. In one embodiment, the mRNA molecules of this disclosure are modified, for example, with respect to the natural ribonucleotides A, U, G, and C, by the introduction of a non-natural nucleoside and / or nucleotide. Non-canonical nucleotides, such as cap structures, differ in chemical structure from the A, C, G, and U ribonucleotides but are not considered “modified.”

[0031] Or: Unless otherwise indicated, the conjunction "or" is intended to be used in the correct sense as a Boolean logical operator, encompassing both the selection of an alternative feature (A or B, the selection of A is mutually exclusive with B) and the selection of a feature in combination (A or B, both A and B are selected). In some parts of the document, the terms "and / or" are used for the same purpose, and this should not be interpreted as meaning that "or" is used to refer to a mutually exclusive alternative usage.

[0032] Peptides: The term "peptide" refers to a molecule containing two or more amino acids linked by the carboxyl group of one amino acid to the alpha-amino group of another. The peptides of this disclosure are typically 2 to 150 amino acids long, e.g., 3 to 100, 4 to 50, 5 to 20, or 6 to 10 amino acids long.

[0033] Identity Percentage: The identity percentage between two amino acid sequences or nucleotide sequences is calculated by multiplying the number of matches between the aligned sequence pair by 100 and dividing by the length of the aligned region. Identity scoring counts only exact matches; substitutions or deletions are not considered matches. For the calculation of sequence identity percentage, the two sequences are aligned using the EMBOSS Needle Pairwise Sequence Alignment software tool with the following parameters based on the Needleman and Wunsch algorithm (available at www.ebi.ac.uk / Tools / psa / emboss_needle): Matrix: BLOSUM62 (for protein sequences) or DNAfull (for DNA sequences), Gap Start: 10, Gap Extension: 0.5, End Gap Penalty: None, End Gap Start: 10, and End Gap Extension: 0.5.

[0034] Subject: As used herein, the term "subject" means any organism to which the compositions according to this disclosure may be administered, for example, for diagnostic, prophylactic, and / or therapeutic purposes. In some embodiments, the subject is a human.

[0035] Transfection: As used herein, the term “transfection” refers to a method for introducing a species (e.g., polynucleotides, e.g., mRNA) into a cell.

[0036] Translation Separator: As used herein, the term “translation separator” refers to a sequence that enables the generation of distinct peptide products from a single mRNA molecule containing multiple coding sequences. A translation separator may be a self-cleaving peptide, which enables the expression of distinct peptide or protein products from a single mRNA molecule through ribosome skipping. While not bound by theory, self-cleaving peptides are thought to function by inducing ribosomes to skip the synthesis of a peptide bond at their C-terminus. This peptide bond skipping causes separation between the end of the self-cleaving peptide sequence and the next peptide downstream, for example, without preventing downstream translation.

[0037] To treat, to treat, treatment: As used herein, the terms “to treat,” “to treat,” and “treatment,” as well as their grammatical variations, include reducing or mitigating a disease or disorder and / or associated signs or symptoms, or delaying or halting its progression. It will be understood, though not excluded, that treating a disease or disorder does not require the complete elimination of the disease, disorder, or associated symptoms. Treatments provided herein may be applied prophylactically (e.g., to subjects at risk of developing a disease or disorder), temporarily mitigating, or amelioratively. Prophylactic treatment may be administered to a subject before the onset of signs or symptoms, during the initial onset of signs or symptoms (e.g., at the first signs and symptoms), or after the onset of signs or symptoms has been established. Prophylactic administration may occur several days to several years prior to the manifestation of symptoms.

[0038] 6.2. Gene Transfer Construct This disclosure provides a gene transfer construct, such as mRNA, comprising a destabilization domain (DD) sequence, a translation separator sequence, and a sequence encoding one or more copies of one or more target peptides. Exemplary characteristics of the destabilization domain, translation separator, and target peptide are described in sections 6.2.1, 6.2.2, and 6.2.3, respectively.

[0039] The DD sequence and the sequence of the target peptide may be positioned on either side of the translation separator sequence. In some embodiments, the gene transfer construct is an mRNA molecule comprising the sequence of the target peptide, the translation separator sequence, and the DD sequence in 5' to 3' order. In preferred embodiments, the gene transfer construct is an mRNA molecule comprising the DD sequence, the translation separator sequence, and the sequence of the target peptide in 5' to 3' order.

[0040] 6.2.1. Destabilized Domains The gene transfer constructs of this disclosure include sequences encoding destabilization domains (DDs), which function as ribosome-binding fragments (though not theoretically bound) to enable translation of the gene transfer construct mRNA. Exemplary DDs include those derived from dihydrofolate reductase (DHFR), FK506-binding protein (FKBP), phosphodiesterase 5 (PDE5), carbonic anhydrase 2 (CA2), peroxisome proliferator-activated receptor gamma (PPAR gamma), NRH:quinone oxidoreductase 2 (NQO2), human estrogen receptor ligand-binding domain (ERLBD), and UnaG.

[0041] An example DHFR DD is described in U.S. Patent No. 9,487,787, which is incorporated herein by reference in its entirety. The amino acid sequence of wild-type Escherichia coli (E coli) DHFR is as follows:

[0042] [ka]

[0043] DHFR DD may contain the wild-type DHFR sequence or may contain one or more amino acid substitutions and / or shortenings at the N and / or C-terminus. For example, the DHFR DD sequence may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1. Examples of amino acid substitutions and combinations that can be included in DHFR DD include Y100I, G121V, N18T / A19V, F103L, H12Y / Y100I, H12L / Y100I, R98H / F103S, M42T / H114R, I61F / T68S, and W74R / T113S / E120D / Q146L (Nakahara et al., 2022, ACS Chem Biol 17:2877-2889). The aforementioned substitution combinations can also be used. In some embodiments, DHFR contains an amino acid sequence identical to SEQ ID NO: 1, except for substitutions of Y100I, G121V, N18T / A19V, F103L, H12Y / Y100I, H12L / Y100I, R98H / F103S, M42T / H114R, I61F / T68S, or W74R / T113S / E120D / Q146L, or combinations thereof. In some embodiments, DHFR DD lacks the N-terminal methionine. For example, in some embodiments, DHFR contains an amino acid sequence identical to SEQ ID NO: 1, except for the substitutions Y100I, G121V, N18T / A19V, F103L, H12Y / Y100I, H12L / Y100I, R98H / F103S, M42T / H114R, I61F / T68S, or W74R / T113S / E120D / Q146L, or combinations thereof, and lacks an N-terminal methionine.

[0044] In some embodiments, DHFR DD has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the following sequences.

[0045] [ka]

[0046] An example nucleotide sequence encoding Sequence ID No. 2 is as follows:

[0047] [ka]

[0048] An exemplary stabilizer for DHFR DD is trimethoprim (TMP). Another exemplary stabilizer for DHFR DD is methotrexate (MTX).

[0049] An exemplary FKBP DD is described in U.S. Patent No. 9,487,787, which is incorporated herein by reference in its entirety. The amino acid sequence of the exemplary FKBP DD (with the F36V substitution compared to the wild-type sequence) is as follows:

[0050] [ka]

[0051] FKBP DD may contain the wild-type FKBP sequence or may contain one or more amino acid substitutions. For example, the FKBP DD sequence may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4. Exemplary amino acid substitutions that may be included in FKBP DD include F15S, V24A, H25R, E60G, L106P, D100G, M66T, R71G, D100N, E102G, and K105I. Combinations of the aforementioned substitutions may also be used. In some embodiments, DD includes an amino acid sequence that is identical to SEQ ID NO: 4 except for substitutions of F15S, V24A, H25R, E60G, L106P, D100G, M66T, R71G, D100N, E102G, or K105I, or combinations thereof.

[0052] Examples of FKBP DD stabilizers include Shield-1 (Shld1), rapamycin, and FK506.

[0053] An exemplary PDE5 DD is described in International Publication No. 2018 / 237323, the contents of which are incorporated herein by reference in their entirety. PDE5 DD may be derived from PDE5A isoform 1 (SEQ ID NO: 5), PDE5A isoform 2 (SEQ ID NO: 6), and / or PDE5A isoform 3 (SEQ ID NO: 7). These isoforms differ in their N-terminal regions and share a common sequence of a unique first exon followed by 823 amino acids.

[0054] All PDE5A isoforms contain a catalytic domain located near the C-terminus of the protein, which is relatively selective to cGMP as a substrate at physiological levels. The substrate binding site is also the binding site for several known PDE5 inhibitors, such as sildenafil, which is used to treat cardiovascular disease and erectile dysfunction. Towards the N-terminus, two homologous GAF domains are located. One of the GAF domains, GAF-A, contains a high-affinity binding site to cGMP. Occupation of this domain by cGMP is known to cause activation of the catalytic domain. Furthermore, the affinity of this site for cGMP is increased by phosphorylation of serine 92 mediated by cGMP-dependent protein kinases. In another embodiment, PDE5A DD may contain the catalytic domain of PDE5A, extending from amino acid positions 535 to 860 of UniProt identifier: O76074 (SEQ ID NO: 5), as represented in SEQ ID NO: 8. In addition to the catalytic domain, PDE5A DD may also contain one or more GAF domains and / or a C-terminal portion extending beyond the catalytic domain. In one embodiment, the DD derived from PDE5A contains amino acids from positions 535 to 875 of SEQ ID NO: 5. In another embodiment, the PDE5 DD contains amino acids from positions 466 to 875 or 420 to 875 of SEQ ID NO: 5. Exemplary PDE5 DD sequences are shown in Table 1.

[0055] [Table 1-1]

[0056] [Table 1-2]

[0057] Exemplary amino acid substitutions that can be included in PDE5 DD include E535D, E536G, Q541R, K555R, F559L, S560G, F561L, F564L, F564S, V585A, N587S, K591E, I599V, K604E, K608E, N609H, K630R, K633E, N636S, I648V, N661S, S663P, L675P, Y676D, Y676N, C677R, H678R, D687A, T711A, T712S, D724N, L73 Examples of amino acid substitutions include one or more selected from 8H, N742S, F744L, L746S, F755L, A762S, D764V, D764N, D764G, S766F, K795E, L797F, I799T, L804P, T802P, S815C, M816A, M816T, I824T, C839S, F840S, and K852E. PDE5 DD may also include additional substitutions, such as Q589R. In some embodiments, the PDE5 DD sequence includes a sequence selected from the group of amino acid sequences identified by SEQ ID NOs. 19-35 and SEQ ID NOs. 66-69 of International Publication No. 2018 / 237323.

[0058] Examples of stabilizers for PDE5 DD include sildenafil, vardenafil, tadalafil, avanafil, rodenafil, mirodenafil, udenafil, benzamidenafil, dasantafil, and beminafil.

[0059] An exemplary CA2 DD is described in International Publication No. 2020 / 185632, the entirety of which is incorporated herein by reference. The amino acid sequence of wild-type human CA2 corresponding to GenBank accession number P00918 is as follows:

[0060] [ka]

[0061] CA2-derived DDs may contain the wild-type CA2 sequence or may contain one or more amino acid substitutions and / or shortenings at the N and / or C-terminus. For example, a CA2-derived DD sequence may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9. Exemplary amino acid substitutions and combinations that may be included in a CA2 DD are A115L, A116Q, A116V, A133L, A133T, A141P, A152D, A152L, A152R, A173C, A173G, A173L, A173T, A23P, A247L, A247S, A257L, A 257S, A38P, A38V, A54Q, A54V, A54X, A65L, A65N, A65V, A77I, A77P, A77Q, C205M, C2 05R, C205V, C205W, C205Y, D101G, D101M, D110I, D129I, D138G, D138M, D138N, D161 * , D161M, D161V, D164G, D164I, D174 * , D174T, D179E, D179I, D179R, D189G, D189I, D19T, D19V, D242G, D242T, D32T, D34T, D41T, D52I, D52L, D 71F, D71G, D71K, D71M, D71S, D71Y, D72I, D72S, D72T, D72X, D75T, D75V, D85M, E106D, E106G, E106S, E117 * E117N, E14N, E186 * , E186N, E204A, E204D, E204G, E204N, E213 * , E213G, E213N, E220K, E220R, E220S, E233D, E233G, E233R, E235 * , E235G, E235N, E237K, E237R, E238 *、E238N、E238R、E26S、E69D、E69K、E69S、F130L、F146V、F175I、F175L、F175S、F178L、F178S、F20L、F20S、F225I、F225L、F225S、F225Y、F230I、F230L、F230S、F259L、F259S、F66S、F70I、F70L、F95Y、G102D、G104R、G104V、G128R、G12D、G12E、G131E、G131R、G131W、G139D、G144D、G144V、G150A、G150S、G150W、G155A、G155C、G155D、G155S、G170A、G170D、G182A、G182W、G195A、G195R、G232R、G232W、G234L、G234V、G25E、G63D、G63V、G81E、G81V、G82D、G86A、G86D、G98V、H107I、H107Q、H119T、H119Y、H122T、H122Y、H15L、H15T、H15Y、H17D、H17I、H36I、H36Q、H64M、H94T、H96T、I145F、I145M、I166H、I166L、I209D、I209L、I215H、I215S、I22L、I255N、I255S、I33S、I59F、I59N、I59S、I91F、K111E、K111N、K112R、K113I、K113N、K126N、K132E、K132R、K148E、K148R、K153 * 、K153N、K158E、K158N、K167 * 、K169N、K169R、K171Q、K171R、K18R、K212N、K212Q、K212R、K212W、K224E、K224N、K227 * 、K227N、K24R、K251E、K251R、K256Q、K260F、K260L、K260Q、K39S、K45N、K45S、K80M、K80R、L118F、L120W、L140V、L140W、L143 * 、L147 * 、L147F、L156F、L156H、L156P、L156Q、L163A、L163W、L183P、L183S、L184F、L184P、L188P、L188W、L197 *、L197M、L197P、L197R、L197T、L202F、L202H、L202I、L202P、L202R、L202S、L203P、L203S、L203W、L211 * 、L211A、L211S、L223 * 、L223I、L223V、L228F、L228H、L228T、L239 * 、L239F、L239T、L250 * 、L250P、L250T、L44 * 、L44M、L47C、L47V、L57 * 、L57X、L60S、L79F、L79S、L84W、L90 * 、L90V、M240D、M240L、M240R、M240W、N11D、N11K、N124T、N177 * 、N177T、N229 *、N229T、N231D、N231F、N231K、N231L、N231M、N231Q、N231T、N243Q、N243T、N252E、N252T、N61R、N61T、N61Y、N62K、N62M、N67D、N67T、P137L、PDA、P13H、P13L、P13S、P154L、P154R、P154T、P180L、P180S、P185L、P185S、P185V、P194Q、P200A、P200L、P200S、P200T、P201A、P201L、P201R、P201S、P214T、P236L、P236T、P246L、P246Q、P249A、P249F、P249H、P249I、P249X、P30L、P30S、P42L、P83A、Q103K、Q135S、Q136N、Q157R、Q157S、Q221A、Q221R、Q248F、Q248L、Q248S、Q254A、Q254K、Q28S、Q53H、Q53K、Q53N、Q74R、Q92H、Q92S、R181H、RMS、R181V、R226H、R226P、R226V、R245A、R253G、R253Q、R27A、R58G、R89D、R89F、R89I、R89X、R89Y、S105L、S105Q、S151A、S151I、S151Q、S165F、S165P、S172E、S172V、S187I、S187P、S196H、S196L、S216A、S216Q、S218A、S218Q、S219A、S219Q、S258F、S258P、S29C、S29P、S43P、S43T、S48L、S50P、S56F、S56N、S56P、S56X、S73L、S73N、S73X、S99H、T108L、T125I、T125P、T168K、T168N、T168Q、T176H、T176L、T192D、T192F、T192I、T192N、T192P、T192X、T198D、T198I、T198P、T199A、T199H、T199P、T207D、T207I、T207P、T207S、T35I、T35L、T37Q、T55L、T87L、V109M、V109W、V121F、V134C、V134F、V142F、V149G、V149L、V159L、V159S、V160C、V160L、V162A、V162C、V206 *, V206C, V206M, V210C, V217L, V217R, V217S, V222A, V222C, V222G, V241 G, V241W, V241X, V31L, V49F, V68L, V68W, V78C, W123G, W123R, W16G, W191 * , W191G, WML, W208G, W208L, W208S, W244 * , W244G, W244L, W97C, W97G, Y114H, Y114M, Y127M, Y190 * Examples include Y190L, Y190T, Y193C, Y193F, Y193I, Y193L, Y193T, Y193V, Y193X, Y40M, Y51F, Y51M, Y51T, Y51X, Y88T, K9N, and S29A.

[0062] Exemplary stabilizers of CA2-derived DD include celecoxib (Celebrex), valdecoxib, rofecoxib (Vioxx), acetazolamide, metazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxyzolamide, zonisamide, dansylamide, and dichlorfenamide.

[0063] Exemplary PPAR-gamma-derived DDs are described in International Publication No. 2018 / 160993 and U.S. Patent Application Publication No. 2022 / 0213449, the contents of which are incorporated herein by reference in their entirety. The amino acid sequence of full-length wild-type human PPAR-gamma corresponding to GenBank accession number P37231 is as follows:

[0064] [ka]

[0065] The sequence of the truncated PPAR gamma corresponding to the ligand-binding domain of PPAR gamma, which includes amino acids 317-505 of SEQ ID NO: 10, is as follows:

[0066] [ka]

[0067] PPAR gamma-derived DDs may include the full-length wild-type PPAR gamma sequence or may include one or more amino acid substitutions and / or shortenings at the N and / or C-terminus. In some embodiments, PPAR gamma-derived DDs include shortened wild-type PPAR gamma sequences. For example, a PPAR gamma-derived DD sequence may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10 or SEQ ID NO: 11.

[0068] Exemplary stabilizers for PPAR-gamma-derived DD include pioglitazone and posaglitazone.

[0069] Exemplary NQO2-derived DDs are described in International Publication No. 2018 / 160993 and U.S. Patent Application Publication No. 2022 / 0213449, the contents of which are incorporated herein by reference in their entirety. The amino acid sequence of wild-type human NQO2 corresponding to GenBank accession number P16083 is as follows:

[0070] [ka]

[0071] The DD derived from NQO2 may contain the wild-type NQO2 sequence or may contain one or more amino acid substitutions and / or shortenings at the N and / or C-terminus. For example, the DD sequence derived from NQO2 may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12.

[0072] Exemplary stabilizers for NQO2-derived DD include imatinib and melatonin.

[0073] Exemplary estrogen receptor ligand-binding domain (ERLBD)-derived DDs are described by Miyazaki et al. (Miyazaki et al., 2012, J Am Chem Soc. 134(9): 3942-3945) and in U.S. Patent Application Publication No. 2014 / 0255361, the contents of which are incorporated herein by reference in their entirety. The amino acid sequence of wild-type human ERLBD corresponding to residues 305-549 of human estrogen receptor 1 (ERS1) with GenBank accession number AAI28574.1 is as follows:

[0074] [ka]

[0075] ERLBD-derived DDs may contain the wild-type ERLBD sequence or may contain one or more amino acid substitutions and / or shortenings at the N and / or C-terminus. For example, an ERLBD-derived DD sequence may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13. Exemplary amino acid substitutions and combinations that may be included in an ERLBD DD include T371A, L384M, M421G, N519S, G521R, and Y537S. Combinations of the aforementioned substitutions may also be used. In some embodiments, the DD contains an amino acid sequence that is identical to SEQ ID NO: 13 except for the T371A, L384M, M421G, N519S, G521R, or Y537S substitution, or combinations thereof.

[0076] Exemplary stabilizers for ERLBD-derived DD include CMP8, 4-hydroxytamoxifene (afimoxifene), tamoxifen, fulvestrant, and raloxifene.

[0077] Exemplary DDs derived from the fluorescent protein UnaG have been described by Navarro et al. (Navarro et al., 2016, ACS Chem Biol. 11(8):2101-4). The amino acid sequence of wild-type UnaG, corresponding to GenBank accession number AB763906, is as follows:

[0078] [ka]

[0079] UnaG-derived DDs may contain the wild-type UnaG sequence or may contain one or more amino acid substitutions and / or shortenings at the N and / or C-terminus. For example, an UnaG-derived DD sequence may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14. Exemplary amino acid substitutions and combinations that may be included in an UnaG DD include A36V and R136G. Combinations of the aforementioned substitutions may also be used. In some embodiments, the DD contains an amino acid sequence that is identical to SEQ ID NO: 14 except for the A36V or R136G substitution, or a combination thereof.

[0080] An example of a stabilizer for DD derived from UnaG is bilirubin.

[0081] 6.2.2. Translation Separator The gene transfer constructs of this disclosure include a translation separator located between the sequence encoding the DD and the sequence encoding the peptide of interest. In some embodiments, if the gene transfer construct includes a sequence of the peptide of interest encoding more than one copy of one or more of the peptide of interest, each copy is separated by the translation separator. The translation separator is a sequence encoding a self-cleaving peptide, which (though not theoretically constrained) may enable the efficient, stoichiometric, coordinated expression of distinct peptide products from a single mRNA molecule via ribosome skipping.

[0082] The term "self-cleaving," when used in this technical context, is not entirely accurate and is not intended to imply proteolytic cleavage. While not theoretically bound, self-cleaving peptides are thought to function by inducing ribosomes to skip the synthesis of peptide bonds at their C-terminus. This peptide bond skipping causes separation between the end of the self-cleaving peptide sequence and the next peptide downstream, for example, without preventing downstream translation.

[0083] The first self-cleaving peptides discovered in picornaviruses are typically short peptides, usually 18–22 amino acids long. By using self-cleaving peptides, picornaviruses and many other viruses can produce multiple genes at equimolar levels from the same mRNA molecule.

[0084] An exemplary self-cleaving peptide suitable for use as a translation separator in the gene transfer construct of this disclosure is the 2A peptide, which has the consensus motif DVEXNPGP (SEQ ID NO: 15), where X is any amino acid. Translational separation via the 2A peptide typically occurs between a glycine residue and a proline residue found at the C-terminus of the resulting peptide, meaning that the upstream cistron has several additional residues attached to its terminus, while the downstream cistron is initiated at the proline residue. Any 2A peptide, e.g., P2A peptide, E2A peptide, F2A peptide, T2A peptide, or any other 2A peptide described in Szymczak-Workman et al., 2012, Cold Spring Harb Protoc doi:10.1101 / pdb.ip067876 or Wang & Marchisio, 2021, Synth Syst Biotechnol 6:254-261, may be selected for use as a translation separator. The polynucleotide sequence encoding the 2A peptide may be codon-optimized or otherwise modified by methods known in the art. For example, an optional N-terminal GSG sequence may be included to increase the efficiency of translational separation.

[0085] In some embodiments, the translation separator encodes a P2A peptide, or a fragment or variant thereof. An exemplary amino acid sequence of the P2A peptide is ATNFSLLKQAGDVEENPGP (SEQ ID NO: 16). An exemplary amino acid sequence of the P2A peptide having an N-terminal GSG is GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 17).

[0086] In some embodiments, the translation separator encodes an E2A peptide, or a fragment or variant thereof. An exemplary amino acid sequence of the E2A peptide is QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18). An exemplary amino acid sequence of the E2A peptide having an N-terminal GSG is GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 19).

[0087] In some embodiments, the translation separator encodes an F2A peptide, or a fragment or variant thereof. An exemplary amino acid sequence of the F2A peptide is VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 20). The amino acid sequence of the F2A peptide having an N-terminal GSG is GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 21).

[0088] In some other embodiments, the translation separator encodes a T2A peptide, or a fragment or variant thereof. An exemplary amino acid sequence of a T2A peptide is EGRGSLLTCGDVEENPGP (SEQ ID NO: 22). An exemplary amino acid sequence of a T2A peptide having an N-terminal GSG is GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 23).

[0089] 6.2.3. Target Peptide The gene transfer constructs of this disclosure include sequences encoding one or more copies of one or more target peptides (POIs). A gene transfer construct comprising two or more POI coding sequences may include (1) two or more identical sequences encoding the same POI linked to each other via a translation separator, (2) two or more different sequences encoding the same POI linked to each other via a translation separator, (3) two or more different sequences encoding two or more different POIs linked to each other via a translation separator, or (4) any combination of the sequences described in (1) to (3), wherein the POI coding sequences are linked to each other via a translation separator.

[0090] In some embodiments, the gene transfer construct includes one POI coding sequence. In some embodiments, the gene transfer construct includes two or more POI coding sequences.

[0091] In some embodiments, the gene transfer construct includes two identical sequences encoding POIs, linked via a translation separator. In some other embodiments, the gene transfer construct includes two different sequences encoding the same POI, linked via a translation separator. In further embodiments, the gene transfer construct includes two different sequences encoding two different POIs, linked via a translation separator.

[0092] In some embodiments, the gene transfer construct includes three or more POI sequences, where each POI is linked to the others via a translation separator. In some embodiments, the gene transfer construct includes three or more identical POI sequences. In some embodiments, the gene transfer construct includes three or more different POI sequences, which encode the same POI. In some embodiments, the gene transfer construct includes three or more different POI sequences, which encode the same POI. In some embodiments, the gene transfer construct includes three or more different POI sequences, which encode different POIs. In some other embodiments, the gene transfer construct includes three or more POI sequences, some of which encode the same POI.

[0093] POI is typically the length of fewer than 150 amino acids, for example, not more than 130, not more than 100, not more than 80, not more than 60, not more than 40, not more than 20, or not more than 10 amino acids. In some embodiments, POI is the length of at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 7 amino acids, at least 10 amino acids, or at least 20 amino acids, but not more than 150 amino acids. In some embodiments, POI is the length of not more than 130, not more than 100, not more than 80, not more than 60, not more than 50, not more than 40, not more than 20, or not more than 10 amino acids. In some embodiments, the POI has a length within a range determined by any two values ​​described in this paragraph, for example, a length of 3 to 100 amino acids, 4 to 50 amino acids, 5 to 20 amino acids, or 6 to 10 amino acids.

[0094] In one embodiment, the POI of the present disclosure is a peptide that can be used as a PPI inhibitor. Non-limiting examples of PPI inhibitor peptides that can be included in the gene transfer construct of the present disclosure include dynamin-1 related protein (Drp1), T-lymphokine-activated killer cell-derived protein kinase (TOPK), Sal-like protein 4 (SALL4), Ras, p53, protein phosphatase 2A (PP2A), signaling and transcriptional activator 3 (STAT3), Yes-related protein (YAP), Bcl-2 family proteins (e.g., anti-apoptotic Bcl-2 family proteins), NOTCH, estrogen receptor, microtubule-associated protein light chain 3 (LC3), or peptides that inhibit E3 ligase MDM2 / MDMX.

[0095] Drp1 is a cytosolic protein that, in response to various stimuli, translocates to the surface of mitochondria and mediates mitochondrial fission (Chan, 2006, Cell 125:1241-1252; Chang and Blackstone, 2010; Ann NY Acad Sci, 1201:34-39). Drp1 is thought to wrap around mitochondria and induce fission using its GTPase activity as a driving force (Smirnova et al., 2001, Mol Biol Cell 12:2245-2256). Cell culture studies have shown that excessive mitochondrial fission and fragmentation induced by Drp1 plays an active role in apoptosis (Frank et al., 2001, Dev Cell, 1:515-525, Estaquier and Arnoult, 2007, Cell Death Differ, 14:1086-1094), autophagy cell death (Twig et al., 2008, EMBO J 27:433-446, Barsoum et al., 2006, EMBO J 25:3900-3911), and necrosis (Wang et al., 2012, Cell 148:228-243). Inhibition of Drp1 leads to a decrease in mitochondrial fragmentation.

[0096] In some embodiments, the gene transfer construct of this disclosure comprises one or more POI sequences encoding Drp1 inhibitor peptides. Exemplary Drp1 inhibitor peptides are described in Qi et al., 2013. J Cell Sci 126(3):789-802 and U.S. Patent No. 10,912,815, the contents of which are incorporated herein in their entirety. Exemplary amino acid sequences of Dp1 inhibitor peptides are listed in Table 2.

[0097] In a particular embodiment, the gene transfer construct of the present disclosure comprises one or more POI sequences encoding the Drp1 inhibitor peptide P110 having the amino acid sequence DLLPRGT (SEQ ID NO: 24) and the exemplary nucleotide sequence GATCTGCTGCCACGCGGGACG (SEQ ID NO: 25), or a variant of P110 having the amino acid sequence DLLPRGS (SEQ ID NO: 26), for example, P110 variant a.

[0098] In other embodiments, the gene transfer construct of the present disclosure comprises one or more POI sequences encoding a Drp1 inhibitor peptide selected from the group consisting of P108, P109, P111, P112, and P113. The amino acid sequences of P108, P109, P111, P112, and P113 are listed in Table 2 as SEQ ID NOs. 27-31, respectively.

[0099] TOPK, also known as PDZ-binding kinase or PBK, is an upregulated protein in various rapidly proliferating cells and is associated with mitotic progression and tumor cell proliferation (Han et al., 2021 Cells 10(2):371, Matsuo et al., 2014. Sci Transl Med, 6(259):259ra145). Inhibition of TOPK has been shown to reduce tumor growth (Kim et al., 2012. Cancer Res 72:3060-8).

[0100] In some embodiments, the gene transfer construct of this disclosure encodes one or more sequences encoding a peptide inhibitor of TOPK. An exemplary amino acid sequence of a TOPK peptide inhibitor is MEGISNFKTPSKLSEKKK (SEQ ID NO: 32), which is shown in Table 2.

[0101] SALL4 encodes a zinc finger transcription factor expressed early in development, and its expression is undetectable in most adult tissues. However, SALL4 is expressed in a variety of cancers, including liver, lung, ovarian, endometrial, and breast cancers, as well as acute myeloid leukemia (Jones, 2013. Nat Rev Clin Oncol 10(426)).

[0102] In some embodiments, the gene transfer construct of the present disclosure comprises one or more sequences encoding a peptide inhibitor of SALL4. An exemplary amino acid sequence of a SALL4 peptide inhibitor is MSRRKQAKPQHI (SEQ ID NO: 33), which is shown in Table 2.

[0103] The Ras family represents some of the earliest described oncogenes. Unregulated activity of Ras gene products is associated with a wide range of cancers. In fact, Ras mutations have been found to contribute to 20–30% of all human cancers (Gimple & Wang 2019. Front Oncol 9:965 doi:10.3389 / fonc.2019.00965), and Ras mutations are found in nearly 100% of pancreatic cancer tumors (O'Bryan, 2019. Pharmacol Res 139:503-511).

[0104] In some embodiments, the gene transfer construct of the present disclosure comprises one or more sequences encoding a Ras peptide inhibitor. An exemplary amino acid sequence of a Ras peptide inhibitor is HYPWFKARLYPL (SEQ ID NO: 34), which is shown in Table 2.

[0105] p53 is a transcription factor that functions as a tumor suppressor (Surget S et al., OncoTargets and Therapy 7: 57-68, 2013). The gene encoding p53 has been found to be mutated in approximately half of all human cancers (Marei et al., 2021 Cancer Cell Int 703 doi.org / 10.1186 / s12935-021-02396-8).

[0106] In some embodiments, the gene transfer constructs of the present disclosure include one or more sequences encoding p53 peptide inhibitors. Exemplary amino acid sequences of p53 peptide inhibitors are presented in Table 2, which include SEQ ID NOs. 35–40.

[0107] PP2A is a serine / threonine phosphatase that modulates protein activity in several oncogenic signaling cascades (Kurimchak & Grana, 2015. Cell Cycle 14:18-30).

[0108] In some embodiments, the gene transfer constructs of this disclosure include one or more sequences encoding PP2A peptide inhibitors. Exemplary amino acid sequences of PP2A peptide inhibitors are presented in Table 2, which include SEQ ID NOs. 41-44.

[0109] STAT3 is a transcription factor, and alterations of its activity, such as loss of function, gain of function, or constitutive activation, have been associated with recurrent infections, bone and dental developmental disorders, autoimmune diseases, and various cancers (e.g., Levy et al., 2007. New Eng J Medi 357:1655-1658; Milner et al., 2015. Blood 125: 591-9; Klampfer, 2006. Curr Cancer Drug Targ 6:107-121; Alvarez et al., 2006. Cancer Research 66: 3162-3168; Yin et al., 2006. Mol Cancer 5:15. Doi:10.1186 / 1476-4598-5-15; Kusaba et al., 2006. Onc Rep 15:1445-51). See Doi:10.3892 / or.15.6.1445.

[0110] In some embodiments, the gene transfer construct of this disclosure comprises one or more sequences encoding a STAT3 peptide inhibitor. An exemplary amino acid sequence of a STAT3 peptide inhibitor, having the sequence PLTAVFWLIYVLAKALVTVC (SEQ ID NO: 45), is shown in Table 2.

[0111] YAP is a transcription factor regulated by the Hippo pathway, and alterations in its activity, such as gain-of-function, have been associated with various cancers (Yu, et al., 2015. Cell. 163(4):811-28, Yimlamai et al., 2015. J Hepatol. 63(6):1491-501). The Hippo pathway controls several cellular functions central to oncogenicity, such as cell proliferation and apoptosis, and its regulation is impaired in several human cancers. When the Hippo pathway is functioning, YAP is degraded, and VGLL family members (including VGLL1-4) bind to TEAD1-TEAD4, downregulating downstream genes. When the Hippo pathway is arrested, YAP binds to TEAD1-TEAD4 and induces transcription of downstream genes.

[0112] In some embodiments, the gene transfer construct of this disclosure comprises one or more sequences encoding a YAP peptide inhibitor. In some embodiments, the YAP peptide inhibitor comprises sequence fragments derived from both VGLL4 and YAP. In some embodiments, the YAP peptide inhibitor comprises sequence fragments of TEAD-binding regions derived from VGLL4 and YAP. In other embodiments, the YAP peptide inhibitor comprises Super-TDUs described in Jiao et al., 2015. Cancer Cell, 25: 66-180 and International Publication No. 2017 / 127750, the contents of which are incorporated herein in their entirety. Exemplary amino acid sequences of YAP peptide inhibitors are presented in Table 2, which include SEQ ID NOs. 46-56.

[0113] Bcl-2 family members are important regulators of apoptosis. While we do not wish to be constrained by theory, it is well known in the art that the balance between pro-apoptotic and anti-apoptotic Bcl-2 family proteins in cells is crucial for regulating apoptosis. Anti-apoptotic Bcl-2 family proteins, such as Bxl-2, Bcl-xL, and Mcl-1, are expressed in a wide range of tumors, and their inhibition in cancer cells, including those resistant to conventional chemotherapy, induces apoptosis. The four Bcl-2 homology domains (BH1-BH4) share a common folding motif that creates a hydrophobic groove, which mediates binding to α-helix stretch proteins possessing a BH3 domain, referred to as BH3-only proteins (Pelay-Gimeno et al., 2015, Angew Chem Int Ed Engl 54, 8896-8927). Several BH3 domain mimes have been developed to target anti-apoptotic Bcl-2 family proteins (D'Aguanno and Del Bufalo, 2020. Cells 9(5):1287). Structural studies have shown that the BH3 domain of BH3-only proteins can bind as an amphiphilic helix in hydrophobic grooves exposed on the surface of anti-apoptotic Bcl-2 family members (Day et al., 2008. J. Mol. Biol. 380:958-971). An exemplary BH3 domain is described in International Publication No. 2017 / 127750, the entire contents of which are incorporated herein by reference.

[0114] In some embodiments, the BH3 peptide is a human BH3 domain. In other embodiments, the BH3 peptide may be derived from a non-human species, such as Caenorhabditis elegans, a rodent, a non-human primate, or any other species.

[0115] Typically, BH3 peptides are derived from pro-apoptotic Bcl-2 family members, including those derived from effector pro-apoptotic Bcl-2 family members (e.g., BAK or BAX) or from BH3-only family members (e.g., BID, BIM, BAD, BIK, BMF, bNIP3, HRK, Noxa, and PUMA). In some embodiments, the BH3 peptide is derived from a BH3-only family member.

[0116] In some embodiments, the BH3 peptide may directly bind to Bcl-2 family proteins. For example, in some embodiments, the BH3 peptide may directly bind to pro-apoptotic Bcl-2 family proteins. In some embodiments, the pro-apoptotic Bcl-2 family proteins are Bax and / or Bak. In some embodiments, the BH3 domain may directly interact with anti-apoptotic Bcl-2 family proteins. In some embodiments, the anti-apoptotic Bcl-2 family proteins may be BCL-2, BCL-XL, BCL-w, MCL-1, or BCL2-related protein A1 (BCL2A1).

[0117] Exemplary amino acid sequences of the BH3 peptide are shown in Table 2, including SEQ ID NOs. 57–82.

[0118] NOTCH signaling is an evolutionarily conserved pathway that regulates cell fate determination at the individual cell level, and abnormal NOTCH signaling has been linked to the development and progression of various types of cancer (Pelay-Gimeno et al., 2015, Angew Chem Int Ed Engl 54, 8896-8927, Moellering et al., 2010, Nature, 462(7270): 182-188).

[0119] In some embodiments, the gene transfer construct of the present disclosure includes one or more sequences encoding NOTCH peptide inhibitors. Exemplary amino acid sequences of NOTCH peptide inhibitors are presented in Table 2, which include SEQ ID NO: 83.

[0120] Estrogen receptors are hormone-activating transcription factors regulated by co-activator proteins. Overactivation of estrogen receptors (e.g., ERα) has been linked to several diseases, including several types of cancer (Pelay-Gimeno et al., 2015, Angew Chem Int Ed Engl 54, 8896-8927, Darnell, 2002, Nat. Rev. Cancer 2:740-749).

[0121] In some embodiments, the gene transfer constructs of this disclosure include one or more sequences encoding ERα peptide inhibitors. Exemplary amino acid sequences of ERα peptide inhibitors are presented in Table 2, which include SEQ ID NO: 84.

[0122] Microtubule-associated protein light chain 3 (LC3) is an essential protein for autophagosome maturation and shares structural homology with ubiquitin. Preclinical studies have shown that inhibiting LC3-mediated autophagy may improve the outcomes of cancer treatment (Yang et al., 2022, Front Oncol. (12) doi.org / 10.3389 / fonc.2022.992171, Gray et al., 2021, Chem Sci, 12(10):3526-3543).

[0123] In some embodiments, the gene transfer construct of this disclosure comprises one or more sequences encoding LC3 peptide inhibitors. An exemplary amino acid sequence of an LC3 peptide inhibitor, having the sequence MFPHRVTAZK (SEQ ID NO: 85), is shown in Table 2.

[0124] The E3 ligase MDM2 and its homolog MDMX regulate the tumor suppressor p53 by binding to its N-terminal transactivation domain, which leads to ubiquitination and degradation of p53. Upregulation of MDM2 / MDMX has been detected in various cancers, and strategies to inhibit MDM2 / MDMX may restore p53 function (Yang et al., 2022, Front Oncol. (12) doi.org / 10.3389 / fonc.2022.992171, Pelay-Gimeno et al., 2015, Angew Chem Int Ed Engl 54, 8896-8927; Phan et al., 2010, J Biol Chem 285(3): 2174-2183, Pazgier et al., 2009, PNAS, 106(12):4665-70).

[0125] In some embodiments, the gene transfer constructs of this disclosure include one or more sequences encoding MDM2 / MDMX peptide inhibitors. Exemplary amino acid sequences of MDM2 / MDMX peptide inhibitors are presented in Table 2, and include SEQ ID NOs. 86 and 87.

[0126] [Table 2-1]

[0127] [Table 2-2]

[0128] [Table 2-3]

[0129] [Table 2-4]

[0130] 6.3 Nucleic acids and host cells 6.3.1. Nucleic acids This disclosure provides, for example, a nucleic acid gene transfer construct as described in Section 6.2. The nucleic acid may be, for example, an mRNA molecule as described in Section 6.3.1.1, a modified mRNA molecule as described in Section 6.3.1.2, or a DNA molecule as described in Section 6.3.1.3, such as an expression plasmid or a vector.

[0131] 6.3.1.1.mRNA In one embodiment, the present disclosure provides a gene transfer construct in mRNA form.

[0132] The mRNA molecules of this disclosure may include naturally occurring nucleic acid bases, nucleosides, or nucleotides, as well as nucleic acid bases, nucleosides, or nucleotides that are not naturally occurring.

[0133] mRNA may contain a 5' untranslated region (5'-UTR), a 3' untranslated region (3'-UTR), and / or a coding region (e.g., an open reading frame). mRNA may contain any suitable number of bases, including tens (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100), hundreds (e.g., 200, 300, 400, 500, 600, 700, 800, or 900), or thousands (e.g., 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000) bases.

[0134] In some embodiments, the mRNA molecules described herein may include a 5' cap structure, a chain termination nucleotide, optionally a Kozak sequence (also known as a Kozak consensus sequence), a stem-loop, a poly-A sequence, and / or a polyadenylation signal.

[0135] In some embodiments, mRNA sequences are codon-optimized. Codon optimization methods are known in the art and may be useful for a variety of purposes, including matching codon usage in a host organism to ensure proper folding; biasing GC content to increase mRNA stability or reduce secondary structures; minimizing tandem repeat codons or nucleotide sequences that could damage gene construction or expression; customizing transcriptional and translational regulatory regions; inserting or removing protein transport sequences; removing / adding post-translational modification sites (e.g., glycosylation sites) in the encoded protein; inserting or deleting restriction sites to add, remove, or shuffle protein domains; modifying ribosome binding sites and mRNA degradation sites; regulating translation rates to allow various domains of a protein to fold properly; or reducing or eliminating problematic secondary structures within polynucleotides.

[0136] In some embodiments, the polynucleotide (e.g., mRNA) of the Disclosure comprises a sequence-optimized nucleotide sequence (e.g., ORF) encoding a peptide (e.g., P110 peptide), its functional fragment, or variant, wherein the peptide (e.g., P110 peptide), its functional fragment, or variant encoded by the sequence-optimized nucleotide sequence has improved properties (e.g., compared to a P110 peptide, its functional fragment, or variant encoded by a non-sequence-optimized reference nucleotide sequence), such as improved properties relating to expression efficacy after in vivo administration. Such properties include, but are not limited to, improved nucleic acid stability (e.g., mRNA stability), increased translational efficacy in target tissue, reduced number of truncated products, improved folding or prevention of misfolding of the expressed peptide product, reduced toxicity of the expressed peptide product, reduced immunogenic and / or inflammatory responses to the expressed peptide product, reduced cell death caused by the expressed peptide product, and increased and / or decreased peptide aggregation.

[0137] In some embodiments, sequence-optimized nucleotide sequences are codon-optimized for expression in human subjects and possess properties useful for avoiding one or more of the problems in the art, such as optimizing the formulation and delivery of nucleic acid-based therapeutics while maintaining structural and functional integrity, overcoming expression thresholds, improving expression rates, half-lives and / or protein concentrations, optimizing protein localization, and avoiding harmful biological responses and / or degradation pathways, such as immune responses.

[0138] In some embodiments, a desired characteristic of the gene transfer construct of this disclosure is the level of expression of the target peptide (e.g., P110) encoded by the optimized sequence disclosed herein. The peptide expression level can be measured using one or more expression systems. In some embodiments, expression can be measured in a cell culture system, e.g., CHO cells or HEK293 cells. In some embodiments, expression can be measured using an in vitro expression system prepared from a live cell extract, e.g., rabbit reticulocyte lysate, or an in vitro expression system prepared by an assembly of purified individual components. In other embodiments, peptide expression is measured in an in vivo system, e.g., mouse, rabbit, or monkey.

[0139] In some embodiments, peptide expression in solution (e.g., the soluble form of the peptide of interest) may be desirable. Therefore, in some embodiments, the reference sequence may be sequence-optimized to obtain a sequence-optimized nucleic acid sequence having an optimized peptide expression level in the soluble form. The level of peptide expression and other properties, such as solubility, aggregation level, and the presence of truncated products (i.e., fragments resulting from proteolysis, hydrolysis, or missing translation), can be measured according to methods known in the art, for example, using electrophoresis (e.g., natural or SDS-PAGE) or chromatographic methods (e.g., HPLC, size exclusion chromatography, etc.).

[0140] 6.3.1.2. Modified mRNA In one embodiment, the disclosure provides a gene transfer construct in an mRNA molecule having a modified mRNA form, such as a modified nucleic acid base, nucleoside, or nucleotide.

[0141] In some embodiments, modified mRNA may possess useful properties compared to unmodified reference mRNA, including enhanced stability, intracellular retention, enhanced translation, and / or virtually no induction of the innate immune response in the cell into which the mRNA is introduced. Thus, the use of modified mRNA may enhance the efficiency of protein production, intracellular retention of nucleic acids, and similarly, reduce immunogenicity.

[0142] Any number (e.g., all, some, or none) nucleic acid bases, nucleosides, or nucleotides may be analogs of canonical species, may be substituted, may be modified, or may not otherwise exist naturally. In some embodiments, all of a particular nucleic acid base type may be modified. In some embodiments, mRNA contains one or more (e.g., one, two, three, or four) different modified nucleic acid bases, nucleosides, or nucleotides. In some embodiments, mRNA contains one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, one hundred, or more) different modified nucleic acid bases, nucleosides, or nucleotides. In some embodiments, modified mRNA may be less degraded in the cell into which the mRNA is introduced compared to the corresponding unmodified mRNA.

[0143] The mMRNA molecules of this disclosure may include combinations of modifications to sugar groups, nucleic acid bases, and / or nucleoside linkages. These combinations may include any one or more modifications described herein.

[0144] In some embodiments, the mRNA molecule of this disclosure may be modified in a coding region (e.g., an open reading frame encoding one of the domains of a gene transfer construct). In other embodiments, the mRNA may be modified in a region other than the coding region. For example, in some embodiments, a 5'-UTR and / or 3'-UTR are provided, where either or both independently contain one or more different nucleoside modifications. In some other embodiments, the mRNA molecule may be modified in both the coding region and the non-coding region.

[0145] Examples of nucleoside modifications and combinations that may be included in the mmRNAs of this disclosure are, but are not limited to, those described in PCT Patent Application Publications: International Publication Nos. 2012 / 045075, 2014 / 081507, 2014 / 093924, 2014 / 164253, 2014 / 159813, and 2017 / 127750, the contents of each of these publications being incorporated herein by reference in their entirety.

[0146] 6.3.1.3. Expression Vectors and Plasmids In another embodiment, the Disclosure relates to an expression vector or expression plasmid that includes or encodes a gene transfer construct of the Disclosure, for example, as described in Section 6.2.

[0147] Recombinant expression vectors may contain nucleic acid sequences encoding components of a gene transfer construct in a form suitable for expression in host cells. Thus, recombinant expression vectors may include one or more regulatory elements, such as TATA boxes, CAAT boxes, GC boxes, and promoters. Exemplary promoters include SV40, CMV, PGK1, EF1a, Ubs, TRE, UAS, CaMKIIa, and CAG promoters. The promoter may be selected based on the host cell intended for expression, and it is operably ligated to the nucleic acid sequence to be expressed, where "operably ligated" means that the target nucleotide sequence is ligated to the regulatory element in a manner that enables the expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in the host cell if the vector is introduced into the host cell).

[0148] Mammalian expression vectors can be used to introduce specific nucleic acid fragments into mammalian systems for mRNA or protein expression. Non-exclusive examples of mammalian expression vectors include adenovirus vectors, vaccinia vectors, retrovirus vectors, pSV and pCMV lineage plasmid vectors, and baculoviruses.

[0149] An exemplary mammalian expression vector that can be used to deliver a gene transfer construct to a host cell and express it there is pCAGGS (Niwa, 1991 Gene, 108 (2):193-200). In some embodiments, the expression vector is pCAGGS.

[0150] The gene transfer construct of this disclosure can be expressed in host cells using other vectors, for example, any of the vectors described by Okayama and Berg, 1983 Mol. Cell. Biol. 3:280. Another high-expression vector, PMLSV N1 / N4, is described by Cosman et al., 1984 Nature 312:768.Additional exemplary mammalian expression vectors are described in International Publication No. 2009 / 102569, the contents of which are incorporated herein in their entirety, and include pDC406, pFN11 A(BIND)Flexi(registered trademark), pGL4.31, pFC14A(HaloTag(registered trademark)7)CMV Flexi(registered trademark), pFC14K(HaloTag(registered trademark)7)CMV Flexi(registered trademark), pFN24A(HaloTag(registered trademark)7)CMVd3 Flexi(registered trademark), and pFN24K(HaloTag(registered trademark)7)CMVd3 Flexi®, HaloTag® pHT2, pACT, pAdVAntage®, pALTER®-MAX, pBIND, pCAT®3-Basic, pCAT®3-Control, pCAT®3-Enhancer, pCAT®3-Promoter, pCI, pCMVTNT®, pGδluc, pSI, pTARGET®, pTNT®, pF12A RM Flexi®, pF12K RM Flexi®, pReg neo, pYES2 / GS, pAckCMW5-DEST Examples include Gateway® vectors, pAckPL-DEST® Gateway® vectors, Gateway® pDEST® 27 vectors, Gateway® pEF-DEST51 ​​vectors, Gateway® pcDNA®-DEST47 vectors, pCMV / Bsd vectors, pEF6 / His A, B, and c, pcDNA® 6.2-DEST, pLenti6 / TR, pLP-AcGFP1-C, pLPS-AcGFP1-N, pLP-IRESneo, pLP-TRE2, pLP-RevTRE, pLP-LNCX, pLP-CMV-HA, pLP-CMV-Myc, pLP-RetroQ, and pLP-CMVneo.

[0151] In some embodiments, the mammalian expression vectors of this disclosure are vaccinia virus, poliovirus, adenovirus (e.g., Li et al., 1994, Invest Opthalmol Vis Sci 35:2543-2549, Borras et al, 1999, Gene Ther 6:515-524, Li and Davidson, 1995, PNAS 92:7700-7704, Sakamoto et al., 1999, H Gene Ther 5:1088-1097). See International Publications 94 / 12649, 93 / 03769, 93 / 19191, 94 / 28938, 95 / 11984, and 95 / 00655, 1999, Adeno-associated viruses (AAVs) (see, for example, Ali et al., 1998, Hum Gene Ther 9:81-86, Flannery et al., 1997, PNAS 94:6916-6921, Bennett et al., 1997, Invest Opthalmol Vis Sci 38:2857-2863, Jomary et al., 1997, Gene Ther 4:683-690, Rolling et al., 1999, Hum Gene Ther These include vectors based on SV40, herpes simplex virus, human immunodeficiency virus (see, e.g., Miyoshi et al., 1997, PNAS 94:10319-23, Takahashi et al., 1999, J Virol 73:7812-7816), retroviral vectors (e.g., mouse leukemia virus, splenic necrosis virus, and retroviral vectors such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus). In some embodiments, the recombinant expression vectors of this disclosure are recombinant lentiviral vectors.In some embodiments, the recombinant expression vector of the Disclosure is a recombinant retroviral vector. In some embodiments, the recombinant expression vector of the Disclosure is an AAV vector.

[0152] In some embodiments, the vector includes a retroviral genome. In some embodiments, the vector includes an AAV genome. Nucleic acids, such as retroviral genomes and AAV genomes, may be provided in the form of particles, such as viral particles (e.g., retroviral particles or AAV particles).

[0153] 6.3.2.Host cells In another embodiment, the Disclosure provides a host cell comprising the nucleic acid of the Disclosure. The host cell may be a prokaryote (e.g., a bacterium, e.g., Escherichia coli) or a eukaryote (e.g., a human cell line, e.g., HEK293 or 293T). The host cell may be used, for example, to expand nucleic acids, e.g., retroviral genomes or plasmids, or to expand and package particles, e.g., retroviral particles, or to express peptides of interest.

[0154] In some embodiments, the host cells are animal cells. In some embodiments, the host cells are mammalian cells. Non-limiting examples of suitable animal or mammalian host cells for expressing and producing inhibitory peptides include Chinese hamster ovary cells (CHO), e.g., CHO-K1 (ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec. Genet., 12:555-556 and Kolkekar et al., 1997, Biochemistry, 36:10901-10909), CHO-K1 Tet-On cell line (Clontech), CHO registry number ECACC 85050302 (CAMR, Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B (GEIMG, Genova, IT), and CHO-K1 / SF registry number ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK), RR-CHOK1 registration number ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK), dihydrofolate reductase-negative CHO cells (CHO / -DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA, 77:4216), and dp12.CHO cells (U.S. Patent No. 5,721,121), SV40-transformed monkey kidney CV1 cells (COS cells, COS-7, ATCC CRL-1651), human embryonic kidney cells (e.g., 293 cells, or 293T cells, or 293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen. Virol., 36:59), baby hamster kidney cells (BHK, ATCC CCL-10), monkey kidney cells (CV1, ATCC CCL-70), African green monkey kidney cells (VERO-76, ATCC CRL-1587, VERO, ATCC CCL-81), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod.)Examples include human cervical cancer cells (HELA, ATCC CCL-2), canine kidney cells (MDCK, ATCC CCL-34), human lung cells (W138, ATCC CCL-75), human hepatoma cells (HEP-G2, HB 8065), mouse mammary tumor cells (MMT 060562, ATCC CCL-51), buffalo rat liver cells (BRL 3A, ATCC CRL-1442), TRI cells (Mather, 1982, Annals NY Acad. Sci., 383:44-68), MCR 5 cells, and FS4 cells.

[0155] In some embodiments, the host cells are human cells, e.g., human cell lines, e.g., HEK293 cells and HeLa cells. Exemplary cells that can be used in this method include bone marrow cells, stem cells, e.g., hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs), immune cells, e.g., T cells, phagocytes, small glial cells, and macrophages. In some embodiments, the cells are T cells, e.g., CD4+ and / or CD8+ T cells. Primary cells obtained from the subject, as well as their offspring, can be used.

[0156] The host cells obtained from the subject may be normal cells (e.g., derived from a healthy donor) or may contain dysfunctional mitochondria (e.g., derived from a subject with disease or disorder). For example, the cells may be derived from a subject with age-related disease or disorder, such as autoimmune disease, metabolic disease, genetic disorder, cancer, neurodegenerative disease, or immunosenescence.

[0157] In some embodiments, the host cells are healthy human cells. In some embodiments, the host cells are human cells having dysfunctional mitochondria. In some other embodiments, the host cells are human cancer cells.

[0158] In some embodiments, the cancer cells are liver cancer cells. In some embodiments, the cancer cells are colorectal cancer cells. In some embodiments, the cancer cells are hematopoietic cells. In some embodiments, the cancer cells are bone marrow cells. In some embodiments, the cancer cells are hematopoietic stem cells (e.g., bone marrow-derived hematopoietic stem cells, erythroid stem cells, myeloid stem cells, platelet stem cells).

[0159] In some embodiments, the host cell is a transgenic cell that has been genetically modified prior to the introduction of the nucleic acid of the Disclosure, for example, mRNA, mMRNA, or expression plasmid containing a sequence encoding a gene transfer construct.

[0160] In some embodiments, the host cell is an in vitro host cell. In some embodiments, the host cell is an in vivo host cell. In some embodiments, the host cell is a multicellular organism, such as a mammal, such as a human.

[0161] 6.3.3. Method for introducing nucleic acids into host cells In one embodiment, the present disclosure provides a method for introducing nucleic acids into host cells in vitro and in vivo.

[0162] Methods for introducing nucleic acids into host cells are known in the art. For example, nucleic acids (e.g., mRNA, mMRNA, or expression vectors) can be introduced into host cells by transfection, electroporation, lipofection, injection, via carriers or delivery agents, or by any other means known in the art for delivering nucleic acids to cells.

[0163] Transfection is the process of introducing nucleic acids into eukaryotic cells by non-viral methods, for example, by using various chemical or physical methods. In some embodiments, the nucleic acids of this disclosure are introduced into host cells by transfection.

[0164] Electroporation involves applying a voltage to create transient pores in the cell membrane, thereby allowing nucleic acids and other macromolecules to enter the cell. In some embodiments, the nucleic acids of this disclosure are introduced into host cells by electroporation.

[0165] Lipofection is a liposome-based method that uses lipid complexes to introduce nucleic acids into host cells. Liposomes have the same lipid composition as the cell membrane, and contact between the liposome and the cell membrane causes fusion of the liposome and the cell membrane, releasing the contents encapsulated in the liposome into the cell's cytoplasm. In some embodiments, the host cell comes into contact with mRNA or mMRNA containing the sequence of the gene transfer construct of this disclosure by lipofection.

[0166] Nucleic acids can be introduced into host cells in vivo, and methods for introducing nucleic acids into host cells in vivo are known in the art. For example, nucleic acids can be introduced into host cells in vivo via viral and nonviral vectors. Nucleic acids can also be introduced into host cells in vivo by other methods, for example, lipid-based methods, for example, via lipid nanoparticles.

[0167] 6.4. Methods for expressing short peptides in host cells and their therapeutic use. The peptides for which this disclosure is intended can be introduced into host cells and expressed within them using any of the methods described in Section 6.3.3.

[0168] In some embodiments, host cells are cultured in a medium without a stabilizer. The omission of the stabilizer allows for the rapid degradation of the DD peptide translated from the gene transfer construct mRNA of this disclosure. Therefore, when the medium lacks a stabilizer, the expression product of the gene transfer construct of this disclosure in successfully transfected host cells is solely the target peptide.

[0169] Alternatively, host cells may be exposed to the stabilizer, for example, by culturing the cells in a medium containing the stabilizer. The cells may be cultured in a medium containing the stabilizer for a period of time. Although not bound by theory, it is thought that including the stabilizer may enhance the translation of the polynucleotide sequence encoding the gene transfer construct, thereby increasing the yield of the target peptide. Including the stabilizer can also be used to evaluate the effectiveness of transfection. For example, a sequence encoding a reporter or marker protein can be fused to the DD sequence to produce a marker-DD fusion polypeptide. The presence of the stabilizer ensures that the marker-DD fusion polypeptide remains intact until transfection is confirmed by detection of the marker. Methods for detecting marker and reporter proteins are well known in the art. After transfection is confirmed, the stabilizer can be removed from the medium, resulting in the degradation of the marker-DD fusion polypeptide.

[0170] In some embodiments, host cells are cultured in a medium containing a stabilizer for at least 8 hours (e.g., at least 12 hours, at least 1 day, at least 2 days, or more) and / or up to 5 days (e.g., up to 4 days, up to 3 days, or up to 2 days). The stabilizer can then be removed, for example, by culturing the host cells in a culture medium without the stabilizer. Once the stabilizer is removed, DD is destabilized and degradation of DD occurs, while the target peptide remains intact.

[0171] In other embodiments, the host cells are in vivo cells, for example, cells in a subject that have not been treated with a stabilizer. While not theoretically bound, it is thought that the absence of a stabilizer allows for the production of the target peptide without significant accumulation of DD protein.

[0172] In some other embodiments, the subject is treated with a stabilizer for a period of time, which, although not theoretically bound, is thought to increase the yield of the target peptide or evaluate the effectiveness of transfection, as described above. Although not theoretically bound, the removal of the stabilizer is thought to allow for the destabilization of DD, thereby promoting its degradation while leaving the target peptide intact.

[0173] The intracellular expression of short peptide inhibitors (PPIs) can be used to treat a variety of disease conditions. Some non-limiting examples of diseases that can be targeted using the intracellular expression of the peptide of interest include mitochondrial disorders, proliferative disorders (e.g., cancer), metabolic disorders, infectious diseases, and neurodegenerative diseases.

[0174] Under physiological conditions, the balance between mitochondrial fission and fusion in a cell is essential for maintaining healthy and functional mitochondria. In contrast, mitochondrial hyperfission leads to mitochondrial fragmentation and damage, decreased mitochondrial membrane potential, increased permeability, and reduced ATP production, as well as, in some cases, apoptosis in otherwise healthy cells. Therefore, methods to reduce mitochondrial hyperfission can prevent the changes associated with it, such as decreased ATP production and apoptosis.

[0175] Cell culture studies have shown that excessive mitochondrial fission and fragmentation induced by Drp1 plays an active role in apoptosis (Frank et al., 2001, Dev Cell, 1:515-525, Estaquier and Arnoult, 2007, Cell Death Differ, 14:1086-1094), autophagy cell death (Twig et al., 2008, EMBO J 27:433-446, Barsoum et al., 2006, EMBO J 25:3900-3911), and necrosis (Wang et al., 2012, Cell 148:228-243).

[0176] Drp1 peptide inhibitors, such as P110, can prevent mitochondrial hyperfission and fragmentation. Therefore, Drp1 inhibitors, such as P110, can be used to treat mitochondrial disorders, neurodegenerative and age-related disorders associated with mitochondrial dysfunction (e.g., eye or hearing disorders, Huntington's disease, etc.), heart failure, muscle weakness and atrophy, or proliferative disorders, including but not limited to these.

[0177] In some embodiments, the gene transfer constructs of the present disclosure, comprising one or more copies of one or more small peptide inhibitors of Drp1, are used to prevent mitochondrial hyperfibriation. In some embodiments, the gene transfer constructs of the present disclosure, comprising one or more copies of one or more small peptide inhibitors of Drp1, are used to treat mitochondrial disorders, as well as other diseases and disorders, as described above.

[0178] In some embodiments, the gene transfer construct of the present disclosure includes one or more copies of P110. In some embodiments, the gene transfer construct includes one copy of P110. In some embodiments, the gene transfer construct includes two or more copies of P110, for example, two, three, four, or five copies of P110.

[0179] In some embodiments, a gene transfer construct containing P110 is used to treat mitochondrial disorders, neurodegenerative and age-related disorders associated with mitochondrial dysfunction, heart failure, muscle weakness and atrophy, or proliferative disorders, or any other disease or disorder characterized by mitochondrial hyperfibrillation, such as those described in Section 6.4.1.

[0180] Small peptide inhibitors can also be used to target cancer cell proliferation. For example, inhibiting anti-apoptotic Bcl-2 family proteins in cancer cells can induce apoptosis in such cells, including cancer cells resistant to conventional chemotherapy. Similarly, peptides that inhibit TOPK, SALL4, Ras, p53, PP2A, STAT3, and YAP can also induce apoptosis in cancer cells.

[0181] In some embodiments, the gene transfer constructs of the present disclosure, comprising one or more copies of one or more small peptide inhibitors of anti-apoptotic Bcl-2 family proteins, are used to induce apoptosis in cancer cells. In some embodiments, the gene transfer constructs of the present disclosure, comprising one or more copies of one or more small peptide inhibitors of anti-apoptotic Bcl-2 family proteins, are used to treat various cancers, such as liver cancer and colorectal cancer.

[0182] In some other embodiments, a gene transfer construct of the present disclosure comprising one or more copies of one or more small peptide inhibitors of one or more non-Bcl-2 family proteins associated with uncontrolled cell division (e.g., TOPK, SALL4, Ras, p53, PP2A, STAT3, YAP) is used to induce apoptosis in cancer cells. In some embodiments, a gene transfer construct of the present disclosure comprising one or more copies of one or more such small peptide inhibitors is used to treat various cancers, such as liver cancer and colorectal cancer.

[0183] Accordingly, in another embodiment, the present disclosure provides a method for treating subjects having age-related diseases, mitochondrial diseases or disorders, neurodegenerative diseases, retinal diseases, diabetes, hearing impairment, genetic disorders, heart failure, immunodeficiency, cancer, or infectious diseases by in vivo expression of a gene transfer construct or by administering a therapeutically effective number of cells obtained or obtainable by the methods described herein. For example, subjects may have any of the diseases or disorders described in this section.

[0184] 6.4.1. Methods to prevent mitochondrial hyperfibrillation In another embodiment, the present disclosure provides a method for preventing mitochondrial hyperfibriation in a cell, comprising the step of contacting the cell with the nucleic acid of the present disclosure (e.g., mRNA, mMRNA, or expression vector). The contact step may occur in vitro or in vivo.

[0185] The short peptides for which this disclosure is intended (e.g., P110) can be introduced into and expressed within host cells using any of the methods described in Section 6.3.3 to prevent mitochondrial hyperfibriation.

[0186] In some embodiments, the methods of the present disclosure result in the prevention of mitochondrial hyperfibrillation. In some embodiments, inhibition of mitochondrial hyperfibrillation is associated with reduced mitochondrial fragmentation, a reduction in the number of damaged mitochondria, an increase in mitochondrial membrane potential, restoration of mitochondrial membrane permeability to healthy levels, restoration of ATP production, and reduction of cell death.

[0187] Diseases associated with mitochondrial hyperfibrillation have been described by Serasinghe and Chipuk (Serasinghe and Chipuk, 2017. Handb Exp Pharmacol. 240:159-188.), and include various atrophies (e.g., muscular atrophy and autosomal dominant optic atrophy), developmental defects and disorders (e.g., abnormal brain development, microcephaly, hypoplasia, and persistent lactic acidemia), neuropathy, neurodegenerative disorders (e.g., Parkinson's disease (PD), Alzheimer's disease (AD), and Huntington's disease (HD)), cardiovascular diseases and cardiomyopathy, metabolic disorders (e.g., type 1 and type 2 diabetes mellitus), and obesity.

[0188] The methods described herein can be used to generate therapeutic cells to be administered to a subject in order to treat a disease, for example, any of the aforementioned diseases associated with mitochondrial hyperfibrillation.

[0189] In another embodiment, the Disclosure also provides a method for targeting or administering nucleic acids encoding an effective amount of gene transfer construct to treat a disease, for example, any of the aforementioned diseases associated with mitochondrial hyperfibrillation.

[0190] 6.4.2. Methods for inducing apoptosis in cancer cells In another embodiment, the present disclosure provides a method for inducing apoptosis in cells, comprising the step of contacting the cells with the nucleic acid of the present disclosure (e.g., mRNA, mMRNA, or expression vector). The contact step may occur in vitro or in vivo.

[0191] Short peptides of interest, such as those inhibiting anti-apoptotic Bcl-2 family proteins TOPK, SALL4, Ras, p53, PP2A, STAT3, and YAP, can be introduced into host cells and expressed within them using one of the methods described in Section 6.3.3 to induce apoptosis in cells, such as cancer cells.

[0192] The methods described herein can be used to generate therapeutic cells to be administered to a target in order to treat various forms of cancer.

[0193] In another aspect, the Disclosure provides a method for treating a subject having cancer, comprising the step of providing or administering to the subject a nucleic acid encoding an effective amount of gene transfer construct for treating various forms of cancer.

[0194] In some embodiments, cancer is liver cancer or colorectal cancer. In some embodiments, liver cancer is hepatocellular carcinoma. In some embodiments, colorectal cancer is a primary tumor or metastasis. In some embodiments, cancer is a hematopoietic cancer. In some embodiments, cancer is acute myeloid leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, muscular dystrophy (including refractory anemia and refractory cytopenia), or myeloproliferative neoplasm or disease (including polycythemia vera, essential thrombocytosis, and primary myelofibrosis). In other embodiments, cancer is a blood-based cancer or a hematopoietic cancer.

[0195] In some embodiments, the Disclosure provides methods for which the nucleic acids of the Disclosure are used in combination therapy. Suitable therapeutic agents for use in combination therapy include small molecule chemotherapeutic agents, including protein tyrosine kinase inhibitors, and biological anticancer agents, such as anticancer antibodies.

[0196] 6.5. Pharmaceutical compositions and kits In another embodiment, the Disclosure provides a pharmaceutical composition comprising nucleic acids of the Disclosure (e.g., as described in Section 6.2 or Section 6.3) or cells of the Disclosure (e.g., cells described in Section 6.3 or cells obtained by the method described in Section 6.4) and pharmaceutically acceptable excipients. For example, the pharmaceutical composition may be prepared, for example, in the form of an aqueous solution or suspension by mixing the nucleic acid or cells with one or more physiologically acceptable carriers, excipients, or stabilizers (see, for example, Hardman et al., 2001, Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro, 2000, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; and Weiner and Kotkoskie, 2000, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0197] In another embodiment, the Disclosure provides a pharmaceutical composition comprising (e.g., mRNA or mMRNA as described in sections 6.3.1.1 and 6.3.1.2, respectively) a nucleic acid of the Disclosure containing a sequence encoding a gene transfer construct of the Disclosure (e.g., as described in section 6.2), and (a) a lipid compound (e.g., lipid nanoparticles) and (b) additional components (e.g., phospholipids, structural lipids, polyethylene glycol lipids, quaternary amine compounds, ionizable aminolipids, other lipid composition components, and nanoparticle compositions).

[0198] Pharmaceutical compositions comprising nucleic acids of the present disclosure (e.g., as described in Section 6.3) may be administered to a subject by any preferred route. In some embodiments, the compositions of the present disclosure are administered by one or more of a variety of routes, including parenteral (e.g., subcutaneous, intradermal, intravenous, intraperitoneal, intramuscular, intra-articular, intra-arterial, intra-sacral, intrasternal, intrathecal, intralesional, or intracranial injection, and any preferred injection technique), oral, percutaneous or intradermal, interdermal, intrarectal, intravaginal, topical (e.g., by powder, ointment, cream, gel, lotion, and / or drops), mucous membrane, nose, cheek, intestine, intravitreal, intratumoral, sublingual, intranasal, intratracheal, intrabronchial, and / or inhalation, as oral sprays and / or powders, nasal sprays and / or aerosols, and / or through a portal vein catheter. In some embodiments, the compositions may be administered by intravenous, intramuscular, intradermal, intra-arterial, intratumoral, subcutaneous, or inhalation. However, this disclosure encompasses the delivery of the compositions of this disclosure by any suitable route, taking into account the advances expected in the science of drug delivery. Generally, the most suitable route of administration will depend on a variety of factors, including the properties of the pharmaceutical composition containing one or more mRNAs (e.g., its stability in various bodily environments, e.g., bloodstream and gastrointestinal tract) and the condition of the subject (e.g., whether the subject can tolerate a particular route of administration).

[0199] In another embodiment, the Disclosure provides a kit comprising nucleic acids of the Disclosure (e.g., as described in Section 6.3) and stabilizers. For example, the kit may include trimethoprim (TMP) or methotrexate (MTX) if the DD sequence of the gene transfer construct is a DHFR DD sequence; Shield-1, rapamycin, or FK506 if the DD sequence of the gene transfer construct is an FKBP DD sequence; sildenafil, vardenafil, tadalafil, avanafil, rodenafil, mirodenafil, udenafil, benzamidenafil, dasantafil, or beminafil if the DD sequence of the gene transfer construct is a PDE5 DD sequence; and CA2 If the DD sequence is celecoxib (Celebrex), valdecoxib, rofecoxib (Vioxx), acetazolamide, metazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxyzolamide, zonisamide, dansylamide, and dichlorfenamide; if the DD sequence of the gene transfer construct is PPAR gammaDD, it may include pioglitazone and posiglitazone; or if the DD sequence of the gene transfer construct is NQO2 DD, it may include imatinib and melatonin.

[0200] 7. Specific Embodiments This disclosure is illustrated by the following specific embodiments. 1. (a) Destabilization domain (DD), (b) Translation separator, and (c) Target peptide An mRNA molecule that codes for something. 2. The mRNA according to Embodiment 1, wherein DD is a protein, peptide, or peptide fragment that is rapidly degraded during translation of mRNA in the absence of a stabilizing molecule. 3. The mRNA according to Embodiment 1 or Embodiment 2, wherein the sequence encoding the DD enhances ribosome targeting of the mRNA compared to mRNA that encodes a translation separator and the target peptide but does not encode a destabilizing domain. 4. mRNA according to any one of Embodiments 1 to 3, wherein DD is derived from dihydrofolate reductase (DHFR), FK506-binding protein (FKBP), carbonic anhydrase 2 (CA2), phosphodiesterase 5 (PDE5), peroxisome proliferator-activated receptor gamma (PPAR gamma), NRH:quinone oxidoreductase 2 (NQO2), human estrogen receptor ligand-binding domain (ERLBD), or UnaG. 5. The mRNA according to any one of Embodiments 1 to 4, wherein the nucleotide sequence encoding DD is codon-optimized for expression in human cells. 6. mRNA according to any one of Embodiments 1 to 5, wherein DD is DHFR (e.g., Escherichia coli DHFR (ecDHFR)). 7. The mRNA according to Embodiment 6, wherein the nucleotide sequence encoding DD comprises the amino acid sequence of SEQ ID NO: 1. 8. The mRNA according to Embodiment 6, wherein the nucleotide sequence encoding DD comprises the amino acid sequence of SEQ ID NO: 2. 9. The nucleic acid sequence encoding ecDHFR,

[0201] [ka] The mRNA according to Embodiment 6, including the mRNA described in Embodiment 6. 10. mRNA according to any one of Embodiments 1 to 5, wherein DD is FK506-binding protein (FKBP). 11. The mRNA according to Embodiment 10, wherein the nucleotide sequence encoding DD comprises the amino acid sequence of SEQ ID NO: 4. 12. mRNA according to any one of Embodiments 1 to 5, wherein DD is derived from carbonic anhydrase 2 (CA2). 13. The mRNA according to Embodiment 12, wherein the nucleotide sequence encoding DD comprises the amino acid sequence of SEQ ID NO: 9. 14. mRNA according to any one of Embodiments 1 to 5, wherein the DD is derived from phosphodiesterase 5 (PDE5). 15. The mRNA according to Embodiment 14, wherein the nucleotide sequence encoding DD comprises the amino acid sequence of SEQ ID NO: 5. 16. The mRNA according to Embodiment 14, wherein the nucleotide sequence encoding DD comprises the amino acid sequence of SEQ ID NO: 6. 17. The mRNA according to Embodiment 14, wherein the nucleotide sequence encoding DD comprises the amino acid sequence of SEQ ID NO: 7. 18. The mRNA according to Embodiment 14, wherein the nucleotide sequence encoding DD comprises the amino acid sequence of SEQ ID NO: 8. 19. mRNA according to any one of Embodiments 1 to 5, wherein DD is derived from peroxisome proliferator-activated receptor gamma (PPAR gamma). 20. The mRNA according to Embodiment 19, wherein the nucleotide sequence encoding DD comprises the amino acid sequence of SEQ ID NO: 10. 21. The mRNA according to Embodiment 19, wherein the nucleotide sequence encoding DD comprises the amino acid sequence of SEQ ID NO: 11. 22. mRNA according to any one of Embodiments 1 to 5, wherein the DD is derived from NRH:quinone oxidoreductase 2 (NQO2). 23. The mRNA according to Embodiment 22, wherein the nucleotide sequence encoding DD comprises the amino acid sequence of Sequence ID No. 12. 24. mRNA according to any one of Embodiments 1 to 5, wherein the DD is derived from the human estrogen receptor ligand-binding domain (ERLBD). 25. The mRNA according to Embodiment 24, wherein the nucleotide sequence encoding DD comprises the amino acid sequence of SEQ ID NO: 13. 26. mRNA according to any one of Embodiments 1 to 5, wherein DD is derived from UnaG. 27. The mRNA according to Embodiment 26, wherein the nucleotide sequence encoding DD comprises the amino acid sequence of SEQ ID NO: 14. 28. mRNA according to any one of Embodiments 1 to 27, wherein the nucleotide sequence encoding the translation separator is located at 3' relative to the nucleotide sequence encoding DD and at 5' relative to the nucleotide sequence encoding the target peptide. 29. mRNA according to any one of Embodiments 1 to 28, wherein the translation separator is a self-cleaving peptide. 30. The mRNA according to Embodiment 29, wherein the self-cleaving peptide is a P2A, E2A, F2A, or T2A self-cleaving peptide. 31. The mRNA according to Embodiment 30, wherein the self-cleaving peptide is a P2A self-cleaving peptide. 32. The mRNA according to Embodiment 31, wherein the amino acid sequence of the P2A self-cleaving peptide includes DVEXNPGP (SEQ ID NO: 15). 33. The mRNA according to Embodiment 31, wherein the amino acid sequence of the P2A self-cleaving peptide includes ATNFSLLKQAGDVEENPGP (SEQ ID NO: 16). 34. The mRNA according to Embodiment 31, wherein the amino acid sequence of the P2A self-cleaving peptide includes GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 17). 35. The mRNA according to Embodiment 30, wherein the self-cleaving peptide is an E2A self-cleaving peptide. 36. The mRNA according to Embodiment 35, wherein the amino acid sequence of the E2A self-cleaving peptide includes QCTNYALLKLAGDVESNPGP (SEQ ID NO: 18). 37. The mRNA according to Embodiment 35, wherein the amino acid sequence of the E2A self-cleaving peptide includes GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 19). 38. The mRNA according to Embodiment 30, wherein the self-cleaving peptide is an F2A self-cleaving peptide. 39. The mRNA according to Embodiment 38, wherein the amino acid sequence of the F2A self-cleaving peptide includes VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 20). 40. The mRNA according to Embodiment 38, wherein the amino acid sequence of the F2A self-cleaving peptide includes GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 21). 41. The mRNA according to Embodiment 30, wherein the self-cleaving peptide is a T2A self-cleaving peptide. 42. The mRNA according to Embodiment 41, wherein the amino acid sequence of the T2A self-cleaving peptide is EGRGSLLTCGDVEENPGP (SEQ ID NO: 22). 43. The mRNA according to Embodiment 41, wherein the amino acid sequence of the T2A self-cleaving peptide is GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 23). 44. The mRNA according to any one of Embodiments 1 to 43, wherein the nucleotide sequence encoding a self-cleaving peptide is codon-optimized for expression in human cells. 45. The mRNA according to any one of Embodiments 1 to 44, wherein the nucleotide sequence encoding the target peptide is codon-optimized for expression in human cells. 46. ​​The mRNA according to any one of Embodiments 1 to 45, wherein the target peptide is a peptide that cannot be effectively delivered to target cells by conventional drug delivery methods. 47. mRNA according to any one of Embodiments 1 to 46, wherein the target peptide has a length of 2 to 150 amino acids. 48. The mRNA according to Embodiment 47, wherein the target peptide has a length of at least three amino acids. 49. The mRNA according to Embodiment 47, wherein the target peptide has a length of at least four amino acids. 50. The mRNA according to Embodiment 47, wherein the target peptide has a length of at least 5 amino acids. 51. The mRNA according to Embodiment 47, wherein the target peptide has a length of at least 7 amino acids. 52. The mRNA according to Embodiment 47, wherein the target peptide has a length of at least 10 amino acids. 53. The mRNA according to Embodiment 47, wherein the target peptide has a length of at least 20 amino acids. 54. The mRNA according to any one of Embodiments 47 to 53, wherein the target peptide has a length of not more than 130 amino acids. 55. The mRNA according to any one of Embodiments 47 to 53, wherein the target peptide has a length of not more than 100 amino acids. 56. The mRNA according to any one of Embodiments 47 to 53, wherein the target peptide has a length of not more than 80 amino acids. 57. The mRNA according to any one of embodiments 47 to 53, wherein the target peptide has a length of not more than 60 amino acids. 58. The mRNA according to any one of Embodiments 47 to 53, wherein the target peptide has a length of not more than 40 amino acids. 59. The mRNA according to any one of Embodiments 47 to 53, wherein the target peptide has a length of not more than 20 amino acids. 60. mRNA according to any one of embodiments 47 to 52, wherein the target peptide has a length of not more than 10 amino acids. 61. mRNA according to any one of Embodiments 1 to 46, wherein the target peptide has a length of 3 to 100 amino acids. 62. The mRNA according to any one of Embodiments 1 to 46, wherein the target peptide has a length of 4 to 50 amino acids. 63. mRNA according to any one of Embodiments 1 to 46, wherein the target peptide has a length of 5 to 20 amino acids. 64. mRNA according to any one of Embodiments 1 to 46, wherein the target peptide has a length of 6 to 10 amino acids. 65. mRNA according to any one of Embodiments 1 to 64, wherein the target peptide is a therapeutic peptide. 66. The mRNA according to any one of Embodiments 1 to 65, wherein the target peptide can inhibit protein-protein interactions. 67. mRNA according to any one of Embodiments 1 to 66, wherein the target peptide is dynamin-1 related protein (Drp1), T-lymphokine-activated killer cell-derived protein kinase (TOPK), Sal-like protein 4 (SALL4), Ras, p53, protein phosphatase 2A (PP2A), signaling and transcriptional activator 3 (STAT3), Yes-related protein (YAP), Bcl-2 family proteins, NOTCH, estrogen receptor, microtubule-associated protein light chain 3 (LC3), or an inhibitor of MDM2 / MDMX. 68. mRNA according to any one of Embodiments 1 to 67, wherein the target peptide is one of the peptides listed in Table 2. 69. The mRNA according to any one of Embodiments 1 to 68, wherein the target peptide is a Drp1 inhibitor. 70. mRNA according to any one of Embodiments 1 to 69, wherein the target peptide is P110. 71. The mRNA according to Embodiment 70, wherein the nucleotide sequence encoding P110 is codon-optimized for expression in human cells. 72. mRNA according to Embodiment 70 or Embodiment 71, wherein the nucleotide sequence encoding P110 includes GATCTGCTGCCACGCGGGACG (SEQ ID NO: 25). 73. mRNA according to any one of Embodiments 1 to 68, wherein the target peptide is an inhibitor of T-lymphokine-activated killer cell-derived protein kinase (TOPK). 74. mRNA according to any one of Embodiments 1 to 68, wherein the target peptide is an inhibitor of Sal-like protein 4 (SALL4). 75. The mRNA according to any one of Embodiments 1 to 68, wherein the target peptide is an inhibitor of Ras. 76. The mRNA according to any one of Embodiments 1 to 68, wherein the target peptide is a p53 inhibitor. 77. mRNA according to any one of Embodiments 1 to 68, wherein the target peptide is an inhibitor of protein phosphatase 2A (PP2A). 78. The mRNA according to any one of Embodiments 1 to 68, wherein the target peptide is an inhibitor of signal transduction and transcription activator 3 (STAT3). 79. mRNA according to any one of Embodiments 1 to 68, wherein the target peptide is an inhibitor of Yes-related protein (YAP). 80. The mRNA according to any one of Embodiments 1 to 68, wherein the target peptide is an inhibitor of a Bcl-2 family protein. 81. The mRNA according to any one of Embodiments 1 to 68, wherein the target peptide is an inhibitor of NOTCH. 82. The mRNA according to any one of Embodiments 1 to 68, wherein the target peptide is an estrogen receptor inhibitor. 83. mRNA according to any one of Embodiments 1 to 68, wherein the target peptide is an inhibitor of microtubule-associated protein light chain 3 (LC3). 84. mRNA according to any one of Embodiments 1 to 68, wherein the target peptide is an inhibitor of MDM2 / MDMX. 85. mRNA according to any one of Embodiments 1 to 84, wherein the mRNA molecule encodes a single copy of the peptide of interest. 86. mRNA according to any one of Embodiments 1 to 84, wherein the mRNA molecule encodes two or more copies of the target peptide. 87. The mRNA according to Embodiment 86, wherein the mRNA molecule encodes two copies of the target peptide. 88. The mRNA according to Embodiment 86, wherein the mRNA molecule encodes three copies of the target peptide. 89. The mRNA according to Embodiment 86, wherein the mRNA molecule encodes four copies of the target peptide. 90. The mRNA molecule according to Embodiment 86, wherein the mRNA molecule encodes five copies of the target peptide. 91. The mRNA according to any one of embodiments 86 to 90, wherein the nucleotide sequences encoding each copy of the target peptide are identical. 92. The mRNA according to any one of embodiments 86 to 90, wherein the nucleotide sequences encoding two or more copies of the target peptide are not identical. 93. The mRNA according to any one of embodiments 1 to 92, which further encodes a second target peptide different from the first target peptide. 94. The mRNA according to embodiment 93, which further encodes a third target peptide different from the first and second target peptides. 95. The mRNA according to any one of embodiments 1 to 94, which encodes four or more different target peptides. 96. The mRNA according to any one of embodiments 1 to 95, which encodes four different target peptides. 97. The mRNA according to any one of embodiments 1 to 95, which encodes five different target peptides. 98. The mRNA according to any one of embodiments 86 to 97, wherein each sequence encoding the target peptide is separated by a translation separator, and optionally, each translation separator is independently selected from the translation separators described in embodiments 30 to 44. 99. The mRNA according to any one of embodiments 1 to 98, which further encodes a marker protein. 100. (a) A target peptide, (b) means for destabilizing the amino acid chain, and (c) means for separating the target peptide and means for destabilizing the amino acid chain An mRNA molecule encoding. 101. The mRNA molecule according to embodiment 100, wherein the peptide of interest is the peptide of interest described in any one of embodiments 1 to 99, and optionally, the means for destabilizing the amino acid chain is the destabilizing domain described in any one of embodiments 1 to 99, and optionally, the means for separating the peptide of interest and the means for destabilizing the amino acid chain are the translation separator described in any one of embodiments 1 to 99. 102. A DNA encoding the mRNA according to any one of embodiments 1 to 101. 103. The DNA according to embodiment 102, further comprising one or more regulatory elements, and optionally, the one or more regulatory elements include a TATA box, a CAAT box, a GC box, a promoter, or a combination thereof. 104. The DNA according to embodiment 102, further comprising a promoter operably linked to the sequence encoding the mRNA. 105. The DNA according to embodiment 104, wherein the promoter is a CMV immediate enhancer / chicken beta-actin (CAG) promoter. 106. The DNA according to any one of embodiments 102 to 105, further comprising a polyadenylation signal sequence located 3' to the sequence encoding the peptide of interest. 107. The DNA according to any one of embodiments 102 to 106, which is an expression plasmid. 108. The DNA according to embodiment 107, wherein the expression plasmid is a pCAGGS plasmid. 109. The DNA according to any one of embodiments 102 to 106, which is a viral genome. 110. The DNA according to embodiment 109, wherein the viral genome is an AAV genome. 111. A particle comprising the mRNA according to any one of embodiments 1 to 101 or the DNA according to any one of embodiments 102 to 110, optionally being a viral particle or a lipid particle. 112. A pharmaceutical composition comprising mRNA according to any one of Embodiments 1 to 101, DNA according to any one of Embodiments 102 to 110, or particles according to Embodiment 111, and one or more excipients. 113. A host cell containing mRNA as described in any one of Embodiments 1 to 101, DNA as described in any one of Embodiments 102 to 110, or a particle as described in Embodiment 111. 114. A method for introducing mRNA into cells, comprising the step of contacting the cells with mRNA according to any one of Embodiments 1 to 101, DNA according to any one of Embodiments 102 to 110, particles according to Embodiment 111, or a pharmaceutical composition according to Embodiment 112. 115. The method according to Embodiment 114, comprising the step of bringing cells into contact with mRNA by lipofection. 116. A method for introducing DNA into cells, comprising transfection of an expression plasmid described in Embodiment 107 or Embodiment 108 into cells. 117. A method for producing mRNA, comprising the step of transcribing mRNA from DNA as described in any of Embodiments 102 to 110. 118. A method for expressing a target peptide in cells, comprising the step of contacting the cells with mRNA according to any one of Embodiments 1 to 101, DNA according to any one of Embodiments 102 to 110, particles according to Embodiment 111, or a pharmaceutical composition according to Embodiment 112. 119. A method for inhibiting Drp1 / Fis1 binding in cells, comprising the step of contacting cells with mRNA encoding a Drp1 inhibitor, e.g., P110, as described in any one of Embodiments 1 to 101, DNA encoding a Drp1 inhibitor, e.g., P110, as described in any one of Embodiments 102 to 110, or a pharmaceutical composition according to Embodiment 112 comprising mRNA or DNA encoding a Drp1 inhibitor, e.g., P110. 120. A method for inhibiting mitochondrial hyperfiring in cells, comprising the step of contacting cells with mRNA according to any one of embodiments 1 to 101 encoding a Drp1 inhibitor, e.g., P110, DNA according to any one of embodiments 102 to 110 encoding a Drp1 inhibitor, e.g., P110, or a pharmaceutical composition according to embodiment 112 comprising mRNA or DNA encoding a Drp1 inhibitor, e.g., P110. 121. A method for inhibiting apoptosis of the mitochondrial pathway in cells, comprising the step of contacting cells with mRNA according to any one of embodiments 1 to 101 encoding a Drp1 inhibitor, e.g., P110, DNA according to any one of embodiments 102 to 110 encoding a Drp1 inhibitor, e.g., P110, or a pharmaceutical composition according to embodiment 112 comprising mRNA or DNA encoding a Drp1 inhibitor, e.g., P110. 122. A method for inducing apoptosis in cancer cells, comprising the step of contacting the cells with mRNA according to any one of Embodiments 1 to 101 encoding a peptide that inhibits Bcl-2 family proteins, TOPK, SALL4, Ras, p53, PP2A, STAT3, or YAP, DNA according to any one of Embodiments 102 to 110 encoding a peptide that inhibits Bcl-2 family proteins, TOPK, SALL4, Ras, p53, PP2A, STAT3, or YAP, or a pharmaceutical composition according to Embodiment 112 comprising mRNA or DNA encoding a peptide that inhibits Bcl-2 family proteins, TOPK, SALL4, Ras, p53, PP2A, STAT3, or YAP. 123. The method according to any one of embodiments 114 to 122, wherein the contact step is exovivo. 124. The method according to any one of embodiments 114 to 122, wherein the contact step is in vivo. 125. The method according to any one of Embodiments 114 to 124, comprising the step of bringing cells into contact with a DD stabilizing molecule for an initial period. 126. The method according to embodiment 125, further comprising the step of removing DD stabilizing molecules. 127. The method according to Embodiment 125 or Embodiment 126, wherein DD is DHFR and the stabilizing molecule is trimethoprim (TMP). 128. The method according to any one of embodiments 114 to 124, wherein the cells do not come into contact with the DD stabilizing molecule. 129. A method for treating a neurodegenerative disease, cardiovascular disease, or diabetes, comprising the step of administering to a subject in need thereof a therapeutically effective amount of mRNA encoding P110 as described in any one of Embodiments 1 to 101, DNA encoding P110 as described in any one of Embodiments 102 to 110, or a pharmaceutical composition according to Embodiment 112 comprising mRNA or DNA encoding P110. 130. A method for treating a neurodegenerative disease, cardiovascular disease, or diabetes, comprising the step of administering to a subject in need thereof a therapeutically effective amount of mRNA according to any one of embodiments 1 to 101 encoding a Drp1 inhibitor, e.g., P110, DNA according to any one of embodiments 102 to 110 encoding a Drp1 inhibitor, e.g., P110, or a pharmaceutical composition according to embodiment 112 comprising mRNA or DNA encoding a Drp1 inhibitor, e.g., P110. 131. The method according to Embodiment 129 or Embodiment 130, wherein the subject has a neurodegenerative disease. 132. The method according to Embodiment 129 or Embodiment 130, wherein the subject has a cardiovascular disease. 133. The method according to Embodiment 129 or Embodiment 130, wherein the subject has diabetes. [Examples]

[0202] 8. Examples 8.1. [Example 1] Design of a peptide expression unit By utilizing the rapid degradation of dihydrofolate reductase (DHFR) with an destabilizing domain (DD) in the absence of a stabilizer (e.g., trimethoprim (TMP)) (Figure 1), we devised a gene transfer system using DHFR as a ribosomal binding fragment. To generate individual peptide products rather than a fusion polypeptide, a translation separator (e.g., P2A) sequence was incorporated between the DHFR-coding sequence and the sequence encoding the target peptide. Given that DHFR is rapidly targeted for degradation in the absence of TMP, this design would result in the expression of only the target peptide in target cells. Therefore, the peptide expression unit was designed to include, from 5' to 3', an optional marker protein (e.g., mCherry reporter) coding sequence, a dihydrofolate reductase (DHFR)-coding sequence, a translation separator (e.g., P2A peptide) sequence, and a sequence encoding the target peptide (Figure 2). The peptide expression unit was then incorporated between the promoter and poly(A) sequence of a suitable expression vector, e.g., pCAGGS (Figure 2A).

[0203] 8.2. [Example 2] Gene transfer using peptide expression units The split GFP system relies on the association of two non-fluorescent polypeptides, GFP β chains 1-10 (GFPβ1-10) and GFP β chain 11 (GFPβ11), for the formation of a fluorescent molecule (Figure 4). Therefore, the split GFP system was used to evaluate gene transfer using the peptide expression unit described in Example 1, in which cells expressing GFPβ1-10 were transfected with an expression plasmid generated using the peptide expression unit containing GFPβ11.

[0204] In short, a pCAGGS expression plasmid was generated by incorporating the coding sequences of mCherry, Escherichia coli (E. coli) DHFR (ecDHFR), P2A, and GFPβ11 between the chicken β-actin promoter and the β-globin poly(A) signaling sequence (Figures 2B and 2F). Because the pCAGGS expression plasmid used in this example had limited gene transfer efficiency, only cells in which gene transfer was successful in the presence of TMP using mCherry fused to ecDHFR were evaluated. Next, A549 cells were transfected with the pLenti-GFPβ1-10-puro plasmid (Figure 3) to generate cells expressing GFPβ1-10. After confirming that A549 cells express GFPβ1-10 but not full-length GFP (Figure 5), they were transfected with an expression plasmid containing the mCherry-DHFR-P2A-GFPβ11 peptide expression unit (Figure 2B) or a control plasmid, such as a plasmid containing only mCherry (Figure 2D) or a plasmid containing only GFPβ11 (Figure 2E), or left untreated.

[0205] No GFP fluorescence was observed in A549 cells transfected with either a control plasmid containing only mCherry or a control plasmid containing only GFPβ11 (Figure 6A). In contrast, most mCherry-positive cells transfected with an expression plasmid containing the peptide expression unit were GFP-positive (Figure 6A). FACS analysis revealed that a very small fraction of transgenic cells transfected with the GFPβ11-only plasmid expressed GFP at low intensity (Figure 6B), however, most mCherry-expressing cells transfected with a plasmid containing the peptide expression unit were GFP-positive (Figures 6B-6F).

[0206] Next, mRNA expression levels of GFPβ1-10, GFPβ11, and mCherry were evaluated in GFPβ1-10-positive A549 cells transfected with one of three expression vectors (m-Cherry only, GFPβ11 only, or the mCherry-DHFR-P2A-GFPβ11 peptide expression unit) or left untreated (Figures 6G-6I). GFPβ11 expression was significantly higher in cells transfected with the GFPβ11-only expression vector than in cells transfected with the expression vector containing the peptide expression unit (Figure 6I), indicating that shorter expression units are associated with higher transcriptional efficiency. In any case, almost all cells transfected with the expression vector containing the mCherry-DHFR-P2A-GFPβ11 peptide expression unit not only expressed GFP but also showed the highest GFP intensity (Figure 6F). In summary, these results suggest that mRNA from short expression units is translated inefficiently regardless of their abundance, while mRNA from recombinant peptide expression units is translated much more efficiently.

[0207] 8.3. [Example 3] DHFR is rapidly broken down after translation. The mCherry-DHFR-P2A-GFPβ11 peptide expression unit was introduced into A549 cells expressing GFPβ1-10, and the expression levels of mCherry and GFP were tested in the presence and absence of TMP. In the presence of TMP, most mCherry-positive cells were also positive for GFP, but in the absence of TMP, GFP expression was very low in these cells (Figure 7A).

[0208] By FACS analysis, it was found that mCherry expression increased rapidly after the addition of TMP (Figure 7B), suggesting stabilization of the mCherry-DHFR fusion protein by TMP. Indeed, the percentage of cells expressing mCherry after TMP treatment was similar in cells transfected with a plasmid expressing only m-Cherry and in cells transfected with a plasmid containing the mCherry-DHFR-P2A-GFPβ11 peptide expression unit (Figures 7C and 7E). Furthermore, the percentage of cells with GFP signal was somewhat lower in the absence of TMP compared to its expression in the presence of TMP (Figure 7D), suggesting that TMP exposure enables more cells to express GFPβ11. Cells transfected with plasmids containing GFP and mCherry connected via P2A (GFP-P2A-mCherry) or GFP and mCherry connected via a control separator sequence (GFP-T7-mCherry) showed similar diagonal signal profiles, so the expression levels of GFP and mCherry were not significantly affected by the translation separator (Figure 7F).

[0209] [[ID=​​​​​​​​​​​​​​A peptide expression unit containing codon-optimized P110, mCherry-DHFR-P2A-P110 (Figure 2C), was transcribed using an in vitro transcription system, and the resulting mRNA was introduced into H9c2 cardiomyocytes by lipofection. The efficiency of mRNA gene transfer into naive H9c2 cells was observed to be approximately 40% by fluorescence microscopy (Figure 8A).

[0212] Next, H9c2 cells expressing GFPβ1-10 were generated by transfection with the pLenti-GFPβ1-10-puro plasmid (Figure 3). mRNA of a peptide expression unit containing GFPβ11 (mCherry-DHFR-P2A-GFPβ11) was introduced into GFPβ1-10 expressing H9c2 cells by lipofection. Approximately half of the resulting cells were mCherry-positive, and approximately 40% of the resulting mCherry-positive cells were also GFP-positive (Figure 8B). In contrast, GFPβ1-10 expressing H9c2 cells lipofected with mRNA of a construct containing P110 did not express GFP (Figures 8B and 8C).

[0213] Next, lipopolysaccharide (LPS) was used as an external stressor to trigger hypermitosis. Briefly, H92c cells were seeded on day 0, lipofection with mRNA of a peptide expression unit containing the P110 coding sequence was performed on day 1, and the cells were treated with TMP. After 12 hours, the medium was changed, and the cells were treated with LPS for 12 hours. Mitochondrial morphology of the cells was evaluated using mitotracker green staining and mitochondrial network analysis (MiNA). LPS-induced changes in mitochondrial reactive oxygen species (ROS) production were evaluated using MitoSOX staining. LPS-induced changes in mitochondrial membrane potential were evaluated using TMRM staining. For the purpose of this evaluation, mCherry-positive cells were assumed to express transgenic P110, while mCherry-negative cells were assumed to lack transgenic P110, and were evaluated separately (Figure 8D). The results showed that mCherry-positive cells reversed indicators of hypermitosis with respect to branch length, branching region, and branching number (Figures 8D-8I), suggesting that P110 expression in these cells suppressed LPS-triggered hypermitosis. LPS treatment was associated with increased mitochondrial ROS (Figures 8J-8K) and increased mitochondrial membrane potential per unit volume (Figures 8L-8N), which were inhibited in P110-expressing cells. In summary, P110 expression in cells suppressed LPS-induced mitophagy-related changes.

[0214] 8.5. [Example 5] Genetic transfer of the P110 peptide using peptide expression unit mRNA suppresses doxorubicin-induced apoptosis. The anti-apoptotic activity of peptide expression unit mRNA containing the P110 coding sequence was evaluated in H9c2 cells after doxorubicin exposure, a treatment that induces mitochondrial stress, hyperdivision, and cell death. Briefly, H92c cells were seeded on day 0, lipofection with peptide expression unit mRNA containing the P110 coding sequence was performed on day 1, and the cells were treated with TMP. After 12 hours, the medium was changed, and the cells were treated with doxorubicin for 3–6 hours. Similar to the LPS exposure evaluation in Example 4, the effect of P110 on hyperdivision was evaluated by mitotracker green staining, followed by MiNA.

[0215] The doxorubicin-induced increase in cell division was accompanied by an increase in mitochondrial footprint, a measure of mitochondrial volume, as well as increases in branch length, number, and region, attributable to P110 expression from mRNA expression units (Figures 9A-9F). Cell death was assessed using FACS by quantifying DAPI / annexin 5 double-positive cells as a cell death marker (Figure 9G), where doxorubicin addition resulted in cell death in approximately 15% of cells, whereas cell death in cells transfected with P110 mRNA expression units was reduced to approximately 7% of all cells (Figure 9H). These results indicate that peptide expression of P110 in cells lipofected with peptide expression unit mRNA containing the P110 coding sequence possesses effective anti-apoptotic activity.

[0216] Next, the effect of P110 expression on mitochondrial hyperfission was evaluated in the context of mitochondrial respiratory function. Briefly, oligomycin (2 μM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 2 μM), and rotenone / antimycin A (0.5 μM), prepared using reagents from the Seahorse XF Cell Mito Stress Test Kit (103015-100, Agilent Technologies), were measured at 1 × 10⁶ after baseline measurement using an XFe96 extracellular flux analyzer (Agilent Technologies, Santa Clara, CA, USA). 5 The doxorubicin solution was sequentially added to each well containing individual cells, and the data were expressed as oxygen consumption rate (pmol / min). Doxorubicin treatment reduced mitochondrial respiration, and this reduction was attenuated in cells expressing P110 (Figure 9I). Doxorubicin treatment significantly reduced basal respiration, maximal respiration, respiratory reserve, and ATP production in control cells (Figures 9J-9M), and also significantly reduced non-mitochondrial oxygen consumption (Figure 9O). Cells expressing P110 showed a significant attenuation of the doxorubicin-induced reductions in basal respiration, maximal respiration, and ATP production (Figures 9J-9L). Similarly, cells expressing P110 showed a significant attenuation of the doxorubicin-induced reduction in non-mitochondrial oxygen consumption (Figure 9O). P110 expression restored respiratory reserve to levels comparable to those observed in untreated control cells (Figure 9M). The reduction in doxorubicin-induced proton leakage was slightly improved by P110 expression (Figure 9N).

[0217] Doxorubicin-induced apoptosis was evaluated by FACS analysis of cells stained with the nuclear marker DAPI and the apoptosis marker annexin V. Control cells and GFPβ11-expressing cells treated with doxorubicin showed similar levels of apoptosis induction, and evidence of toxicity existed at the gene transfer level (Figure 10A-10B). Cells expressing P110 resisted doxorubicin-induced apoptosis as well as gene transfer-related cell death induction (Figure 10A-10B). Next, doxorubicin-induced caspase-3 activation was evaluated, which is a cysteine-aspartate protease that plays a central role in the execution phase of cellular apoptosis. mRNA cell transfection strongly induced cleavage caspase-3 expression in response to doxorubicin exposure, but transfection of cells with P110 reduced the effects associated with both gene transfer and doxorubicin (Figure 10C-10D).

[0218] The release of cytochrome c from mitochondria associated with hyperfiring triggers apoptosis, and therefore, doxorubicin-responsive cytochrome c release was evaluated. While mRNA gene transfer did not elicit cytochrome c release as potently as cleavage caspase-3 induction, it further enhanced doxorubicin-induced cytochrome c release, whereas transfection of cells with P110 completely suppressed cytochrome c release (Figures 10E-10F). This observation indicates that apoptosis associated with mitochondrial hyperfiring was completely suppressed by P110 expression, while non-mitochondrial apoptotic pathways were unaffected by P110. Given that P110 inhibits the recruitment of Drp1 to the mitochondrial membrane during fission, Drp1 in the mitochondrial fraction was evaluated to determine whether P110 mRNA introduction affects this mechanism. Doxorubicin exposure caused a strong accumulation of Drp1 in mitochondria, but P110 treatment significantly prevented Drp1 aggregation and maintained it at nearly the same level as the control group, as shown in the results (Figures 10G-10H).

[0219] The association of Drp1 and Fis1 is an intracellular PPI associated with mitochondrial dysfunction. To determine whether the interaction between Fis1 and Drp1 was associated with the accumulation of Drp1 in the mitochondrial membrane, immunoprecipitation with Fis1 was followed by immunoblotting with Drp1. Drp1 expression was significantly enhanced by doxorubicin treatment in both untransfected and GFPβ11-transfected cells. On the other hand, Drp1 showed little aggregation and recovered to almost control levels in P110-transfected cells (Figures 10I-10K). In conclusion, P110 mRNA gene transfer inhibited the association of Drp1-Fis1, an intracellular PPI, preventing excessive cell division that contributes to the pathogenesis and thus reducing cell death.

[0220] 9. Citation of References All publications, patents, patent applications, and other documents cited herein are incorporated herein by reference in whole for any purpose to the same extent that each individual publication, patent, patent application, or other document is individually indicated as being incorporated by reference for any purpose. In the event of any conflict between one or more teachings of the references incorporated herein and the present disclosure, the teachings herein shall prevail.

Claims

1. (a) Destabilization domain (DD), (b) Translation separator, and (c) Target peptide An mRNA molecule that codes for something.

2. The mRNA according to claim 1, wherein the DD is a protein, peptide, or peptide fragment that is rapidly degraded during translation of the mRNA in the absence of a stabilizing molecule.

3. The mRNA according to claim 1 or claim 2, wherein the sequence encoding the DD enhances ribosome targeting of the mRNA compared to mRNA that encodes the translation separator and the target peptide but does not encode the destabilization domain.

4. The mRNA according to any one of claims 1 to 3, wherein the DD is derived from dihydrofolate reductase (DHFR), FK506-binding protein (FKBP), carbonic anhydrase 2 (CA2), phosphodiesterase 5 (PDE5), peroxisome proliferator-activated receptor gamma (PPAR gamma), NRH:quinone oxidoreductase 2 (NQO2), human estrogen receptor ligand-binding domain (ERLBD), or UnaG.

5. The mRNA according to any one of claims 1 to 4, wherein the nucleotide sequence encoding DD is codon-optimized for expression in human cells.

6. The mRNA according to any one of claims 1 to 5, wherein the DD is a DHFR (for example, E. coli DHFR (ecDHFR)), and optionally the nucleotide sequence encoding the DD comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

7. The mRNA according to any one of claims 1 to 6, wherein the nucleotide sequence encoding the translation separator is located 3' with respect to the nucleotide sequence encoding DD and 5' with respect to the nucleotide sequence encoding the target peptide.

8. The mRNA according to any one of claims 1 to 7, wherein the translation separator is a self-cleaving peptide.

9. The mRNA according to claim 8, wherein the self-cleaving peptide is a P2A, E2A, F2A, or T2A self-cleaving peptide.

10. The mRNA according to claim 9, wherein the self-cleaving peptide is a P2A self-cleaving peptide, and optionally the amino acid sequence of the P2A self-cleaving peptide includes the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:

17.

11. The mRNA according to any one of claims 1 to 10, wherein the nucleotide sequence encoding the self-cleaving peptide is codon-optimized for expression in human cells.

12. The mRNA according to any one of claims 1 to 11, wherein the nucleotide sequence encoding the target peptide is codon-optimized for expression in human cells.

13. The mRNA according to any one of claims 1 to 12, wherein the target peptide is a peptide that cannot be effectively delivered to target cells by conventional drug delivery methods.

14. The mRNA according to any one of claims 1 to 13, wherein the target peptide has a length of 2 to 150 amino acids.

15. The mRNA according to any one of claims 1 to 14, wherein the target peptide is a therapeutic peptide.

16. The mRNA according to any one of claims 1 to 15, wherein the target peptide can inhibit protein-protein interactions.

17. The mRNA according to any one of claims 1 to 16, wherein the target peptide is dynamin-1 related protein (Drp1), T-lymphokine-activated killer cell-derived protein kinase (TOPK), Sal-like protein 4 (SALL4), Ras, p53, protein phosphatase 2A (PP2A), signaling and transcriptional activator 3 (STAT3), Yes-related protein (YAP), Bcl-2 family proteins, NOTCH, estrogen receptor, microtubule-associated protein light chain 3 (LC3), or an inhibitor of MDM2 / MDMX.

18. The mRNA according to any one of claims 1 to 17, wherein the target peptide is one of the peptides listed in Table 2.

19. The mRNA according to any one of claims 1 to 18, wherein the target peptide is a Drp1 inhibitor.

20. The mRNA according to any one of claims 1 to 19, wherein the target peptide is P110.

21. The mRNA according to claim 20, wherein the nucleotide sequence encoding P110 is codon-optimized for expression in human cells.

22. The mRNA according to claim 20 or 21, wherein the nucleotide sequence encoding P110 includes the nucleotide sequence of SEQ ID NO:

25.

23. The mRNA according to any one of claims 1 to 22, wherein the mRNA molecule encodes a single copy of the target peptide.

24. The mRNA according to any one of claims 1 to 22, wherein the mRNA molecule encodes two or more copies of the target peptide.

25. The mRNA according to any one of claims 1 to 24, wherein the mRNA further encodes a second target peptide that is different from the first target peptide.

26. The mRNA according to claim 25, wherein each sequence encoding the target peptide is separated by a translation separator, and optionally, each translation separator is independently selected from the translation separators described in claims 9 to 10.

27. The mRNA according to any one of claims 1 to 26, further encoding a marker protein.

28. DNA encoding mRNA according to any one of claims 1 to 27.

29. The DNA according to claim 28, further comprising one or more regulatory elements, wherein optionally, one or more regulatory elements include a TATA box, a CAAT box, a GC box, a promoter, or a combination thereof.

30. The DNA according to claim 28, further comprising a promoter operably ligated to the sequence encoding the mRNA.

31. The DNA according to any one of claims 28 to 30, further comprising a polyadenylation signal sequence located at 3' relative to the sequence encoding the target peptide.

32. The DNA according to any one of claims 28 to 31, wherein the DNA is an expression plasmid or a viral genome, and optionally the viral genome is an AAV genome.

33. A particle comprising mRNA according to any one of claims 1 to 27 or DNA according to any one of claims 28 to 32, which is optionally a viral particle or a lipid particle.

34. A pharmaceutical composition comprising mRNA according to any one of claims 1 to 27, DNA according to any one of claims 28 to 32, or particles according to claim 33, and one or more excipients.

35. A host cell comprising mRNA according to any one of claims 1 to 27, DNA according to any one of claims 28 to 32, or a particle according to claim 33.

36. A method for introducing mRNA into cells, comprising the step of contacting the cells with mRNA according to any one of claims 1 to 27, DNA according to any one of claims 28 to 32, particles according to claim 33, or a pharmaceutical composition according to claim 34.

37. A method for expressing a target peptide in cells, comprising the step of contacting the cells with mRNA according to any one of claims 1 to 27, DNA according to any one of claims 28 to 32, particles according to claim 33, or a pharmaceutical composition according to claim 34.

38. A method for inhibiting Drp1 / Fis1 binding in cells, comprising the step of contacting the cells with mRNA according to any one of claims 1 to 27 encoding a Drp1 inhibitor, for example P110, DNA according to any one of claims 28 to 32 encoding a Drp1 inhibitor, for example P110, or a pharmaceutical composition according to claim 34 comprising mRNA or DNA encoding a Drp1 inhibitor, for example P110.

39. A method for inhibiting mitochondrial hyperfiring in cells, comprising the step of contacting the cells with mRNA according to any one of claims 1 to 27 encoding a Drp1 inhibitor, for example P110, DNA according to any one of claims 28 to 32 encoding a Drp1 inhibitor, for example P110, or a pharmaceutical composition according to claim 34 comprising mRNA or DNA encoding a Drp1 inhibitor, for example P110.

40. A method for inhibiting apoptosis of the mitochondrial pathway in cells, comprising the step of contacting the cells with mRNA according to any one of claims 1 to 27 encoding a Drp1 inhibitor, for example P110, DNA according to any one of claims 28 to 32 encoding a Drp1 inhibitor, for example P110, or a pharmaceutical composition according to claim 34 comprising mRNA or DNA encoding a Drp1 inhibitor, for example P110.

41. A method for inducing apoptosis in cancer cells, comprising the step of contacting the cells with mRNA according to any one of claims 1 to 27 encoding a peptide that inhibits Bcl-2 family proteins, TOPK, SALL4, Ras, p53, PP2A, STAT3, or YAP, DNA according to any one of claims 28 to 32 encoding a peptide that inhibits Bcl-2 family proteins, TOPK, SALL4, Ras, p53, PP2A, STAT3, or YAP, or a pharmaceutical composition according to claim 34 comprising mRNA or DNA encoding a peptide that inhibits Bcl-2 family proteins, TOPK, SALL4, Ras, p53, PP2A, STAT3, or YAP.

42. A method for treating a neurodegenerative disease, a cardiovascular disease, or diabetes, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the mRNA encoding P110 as described in any one of claims 1 to 27, the DNA encoding P110 as described in any one of claims 28 to 32, or the pharmaceutical composition according to claim 34 comprising the mRNA or DNA encoding P110.

43. A method for treating a neurodegenerative disease, a cardiovascular disease, or diabetes, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the mRNA according to any one of claims 1 to 27 encoding a Drp1 inhibitor, for example P110, the DNA according to any one of claims 28 to 32 encoding a Drp1 inhibitor, for example P110, or the pharmaceutical composition according to claim 34 comprising mRNA or DNA encoding a Drp1 inhibitor, for example P110.

Images