Compositions and methods for rejuvenating DNA and preventing DNA damage

Abstract

The present disclosure relates to a method for rejuvenating DNA in a cell nucleus and / or preventing DNA damage. The method generally comprises: administering a reagent having a vector containing a polynucleotide sequence encoding a peptide as shown in SEQ ID No. 1 or SEQ ID No. 2 into a cell, wherein the vector is used to express the encoded peptide in the cell; and overexpressing the encoded peptide, wherein the overexpressed peptide forms one or more complexes in the cell that are capable of rejuvenating DNA in the cell nucleus and preventing DNA damage.

Description

Technical Field

[0001] The present disclosure relates to compositions and methods for rejuvenating DNA in cells and preventing DNA damage. More specifically, the disclosed compositions and methods contribute to the formation of one or more biocomplexes within a cell, preferably within the nucleus, to produce significant DNA stability, thereby leading to resistance to DNA damaging agents or limiting DNA damage in the nucleus. Due to aging, the above-mentioned biocomplexes are generally depleted in the elderly. The present disclosure also encompasses promoting the production of Box A of HMGB1 peptides in the nucleus or transporting Box A of HMGB1 into the nucleus to produce physiological replication-independent endogenous DNA double-strand breaks (Phy-RIND-EDSB) or youth-associated genomic stable DNA gaps (Youth-DNA-GAPs), thereby conferring enhanced resistance to DNA damage to the host genome. Background Art

[0002] It is hypothesized that endogenous DNA damage, associated with loss of epigenetic marks, contributes to the deterioration of health in the elderly and in patients with many non-communicable diseases (NCDs) (1). In addition, DNA damage can also be caused by various external and internal factors, such as heat, ultraviolet light, free radicals, methylating agents, and other mutagenic compounds. DNA damage can cause mutations, leading to carcinogenesis or birth defects. To prevent mutations, cells have a DNA damage response (DDR) to detect, signal, halt cell proliferation, and / or repair cellular DNA damage. However, excessive interference with the DDR may lead to metabolic dysfunction, poor growth, cell aging, including senescence, and death or apoptosis (2,3).

[0003] A large number of studies have been conducted worldwide to treat various diseases by manipulating DNA repair in cells. For example, U.S. Patent No. 9359605 teaches a method for treating solid tumor lung cancer by inhibiting BRCA2 and RAD51 (which are DNA double-strand break repair proteins). Similarly, International Patent Application No. PCT / EP2014 / 057904 describes a polynucleotide-based molecule that can inhibit the poly-(ADP-ribose) polymerase (PARP) mechanism in cancer treatment. Adam et al. further proposed a possible method for preventing mitochondrial dysfunction in human subjects by administering a fruit extract of the genus Elaeagnus in International Patent Application No. PCT / US2014 / 015110.

[0004] Considering that genomic instability in cells can also be driven by a reduction in epigenomic modifications (1), any attempt to promote epigenetic editing (i.e., adding epigenetic marks to cells) may reduce DNA damage. Through appropriate epigenetic modifications, the deterioration of cellular function can be restored (1). One of the epigenetic marks known to be effective against DNA damage is Phy-RIND-EDSBs or Youth-DNA-GAPs (1,4). Therefore, it is necessary to find ways to promote the formation of Youth-DNA-GAPs, which will inevitably improve the clinical conditions of diseases related to biological aging DNA and / or accumulated DNA damage. Summary of the Invention

[0005] The present disclosure aims to provide a composition capable of restoring cell vitality and / or preventing cell DNA damage. More specifically, the disclosed composition facilitates the expression or transfection of Box A of HMGB1 protein in cells administered with the disclosed composition to obtain improved genome stabilization effect.

[0006] A further object of the present disclosure relates to compositions for restoring the vitality of biological senescent cells, restoring cell growth and healing processes in subjects who have suffered potential DNA damage, such as burns, or have low levels of Youth-DNA-GAP, including the elderly and individuals with diabetes mellitus (DM).

[0007] Another object of the present invention is to provide a method for preventing nuclear DNA damage. More specifically, the method comprises expressing and / or presenting a molecularly engineered HMGB1 protein, particularly a Box A domain, to form one or more Youth-DNA-GAPs within the genome, thereby providing resistance to DNA damage formation.

[0008] One aspect of the present disclosure relates to a vector capable of expressing a peptide in a cell to prevent DNA damage in the cell nucleus, the vector comprising a polynucleotide sequence as shown in SEQ ID No. 1 or SEQ ID No. 2 encoding the peptide.

[0009] One main aspect of the present disclosure relates to a method for producing Youth-DNA-GAPs to prevent DNA damage in a cell nucleus, the method comprising: transfecting a cell with an agent comprising a peptide as shown in SEQ ID No. 3 and / or a vector comprising a polynucleotide sequence as shown in SEQ ID No. 1 or SEQ ID No. 2 encoding the peptide in the cell. Preferably, the vector is used to express the encoded peptide in the cell after the transfection step, wherein the encoded peptide forms one or more complexes including Youth-DNA-GAP in the cell, which complexes are capable of rejuvenating DNA in the cell nucleus and / or preventing DNA damage.

[0010] Therefore, the disclosed method may further comprise overexpressing the peptide encoded by the polynucleotide sequence residing in the transfection vector.

[0011] According to further embodiments of the disclosed methods, prevention of DNA damage is achieved by reducing the DNA damage response.

[0012] In several embodiments, prevention of DNA damage is achieved by increasing the resistance of the cell to DNA damaging agents.

[0013] Another aspect of the present disclosure relates to a method for improving healing of injured tissue in a subject, comprising the steps of:

[0014] contacting an agent comprising the peptide shown in SEQ ID No. 3 and / or a vector comprising the polynucleotide sequence shown in SEQ ID No. 1 encoding the peptide with a damaged tissue composed of a plurality of cells; and

[0015] The cells of the injured tissue are transfected with the peptide and / or the vector, wherein the vector is used to express the encoded peptide in the cells after the transfection step, wherein the encoded peptide forms one or more complexes in the cells, and the one or more complexes can improve the healing of the injured tissue by enhancing the growth of the cells.

[0016] For many embodiments, the subject is a DM patient, who generally tends to have lower youth-DNA-GAP formation in cells and therefore suffers from reduced healing rates of injured tissues. The disclosed methods improve the healing rate of injured tissues in these subjects by forming youth-DNA-GAPs.

[0017] In further embodiments, the injured tissue is caused by a burn.

[0018] Another aspect of the present disclosure relates to a local or systemic pharmaceutical composition for rejuvenating DNA to reduce DNA damage. The composition generally comprises a vector comprising a polynucleotide sequence as shown in SEQ ID No. 1 or SEQ ID No. 2 encoding a peptide.

[0019] According to another aspect of the present disclosure, a method for rejuvenating senescent cells, preventing DNA damage, and enhancing the healing process in mammals is disclosed. Preferably, the method comprises locally or systemically administering an agent having an expression vector containing a polynucleotide sequence as shown in SEQ ID No. 1 or SEQ ID No. 2 encoding a peptide. To enhance insertion or uptake of the expression vector by target cells, the expression vector can be linked to a carrier such as a cell-penetrating peptide or a nanoemulsion.

[0020] Further aspects of the present disclosure include a pharmaceutical composition for rejuvenating DNA and / or reducing DNA damage in mammalian cells, comprising:

[0021] A biological component, the biological component being one of (i) a vector comprising an expressible polynucleotide sequence encoding a peptide as shown in SEQ ID No. 1 or SEQ ID No. 2 or (ii) a peptide as shown in SEQ ID No. 3; and

[0022] A carrier system is chemically linked to the biological component to facilitate entry of the biological component into the cell when the composition is contacted with the cell, wherein the carrier system is a cell penetrating peptide, a nanoemulsion, etc.

[0023] Brief Description of the Figures

[0024] Figure 1 Figures 1 and 2 show low levels of Youth-DNA-GAPs and EDSB in examples of biologically senescent cells in elderly individuals and cells from patients with DM. (A) shows the correlation between EDSB and age. (B) shows the levels of each EDSB in individuals without DM and with DM. (C) shows the EDSB levels in age-matched and sex-matched individuals without DM (normal) and with DM. The average levels of EDSB are shown as histograms, and the error bars represent the SEM.

[0025] Figure 2This figure illustrates the generation of Youth-DNA GAPs by Box A of HMGB1, a consequence of DSB generation by HMGB1 restriction enzyme activity. (A) Graph showing the increase in the percentage of EDSBs in HeLa DNA reacted with HMGB1 at different ratios in the presence or absence of T4 polymerase. (B) Graph showing the response of HMDNA from HK2 and HeLa cells to EDSBs generated by HMGB1, with greater EDSB generation in DNA incubated with HMGB1. Experiments were performed in triplicate, and scale bars represent standard errors.

[0026] Figure 3 Images showing the generation of Youth-DNA-GAPs by HMGB1's Box A. Electron micrographs of HK-2 cells show the results of DNA damage in situ ligation followed by cell transfection for proximity ligation assay (DI-PLA). (A) HMGB1 transfection and (B) Box A transfection yield positive signals in (A) and (B), while (C) Box B transfection, (D) Box BC transfection, (E) random peptide sequence control plasmid transfection, and (F) no transfection were performed. (C)-(F) show no signal.

[0027] Figure 4 Graphs showing the generation of Youth-DNA-GAP by HMGB1 indicated by Box A of HMGB1, and showing the results regarding the percentage of EDSB input in DNA immunoprecipitation (DIP), (A) HEK293 and HeLa cell lines in the presence of 8-hydroxy-2'-deoxyguanosine (8-OHdG), (B) HEK293 cells transfected with Box A, HMGB1, and a control plasmid in the presence of 8-OHdG, (C) HEK293 cells transfected with Box A, HMGB1, and a control plasmid in the presence of 7-methylguanosine, with boxes representing the interquartile range (25th to 75th percentiles) and the midline representing the 50th percentile (whiskers represent minimum and maximum values. *P < 0.05, **P < 0.01, ***P < 0.0001)

[0028] Figure 5 is a diagram indicating the potential application of Box A of HMGB1 in reducing DNA damage, and shows the results regarding 8-OHdG levels (8-OHdGs ng / ml) and apurinic / apyrimidinic site (AP-site) levels (AP-site / 100,000 bp) in HMGB1 and Box A-overexpressing HEK293 and HK-2 cells (n=9 each), (A) is 8-OHdG, (B) is AP sites in HMGB1 and Box A-overexpressing cells (*P<0.05, **P<0.01, ***P<0.0001).

[0029] Figure 6 Box A shows the results of HMGB1 in reducing the following aspects, among which (A) is related to p-ATM (Ser1981), p53, p21, p16 INK4A and γH2AX expression levels in HK2 cells after treatment with 2,500 ng / μl of HMGB1, Box A, or PC for 48 h (blots were reprobed with β-actin to confirm equal sample loading); and (B) is a graphical representation showing the corresponding results of (A), in which protein types are plotted against relative protein levels (data were compared by one-way ANOVA and Dunnett's multiple comparison test, *P value < 0.05, **P value < 0.01, ***P value < 0.001).

[0030] Figure 7 This is a graphical representation showing the results of Box A of HMGB1 in promoting cellular resistance to DNA damaging agents in the presence of Box A and / or HMGB1, wherein (A) relates to the results obtained by treating cells transfected with scrambled plasmid, Box A, and HMGB1 with H2O2 for 24 hours, and (B) relates to the results obtained by treating cells transfected with scrambled plasmid, Box A, and HMGB1 with MMS for 24 hours, wherein the values ​​are the mean ± SD of 12 independent determinations. The values ​​were compared by one-way ANOVA and Dunnett's multiple comparison test (*P value < 0.05, **P value < 0.01, ***P value < 0.001).

[0031] Figure 8 The results of the effect of Box A of HMGB1 on promoting cell growth are shown, wherein figures (A), (B), (C) and (D) show the cell proliferation data in the MTT assay 4 days after overexpression of Box A in HEK 293 cells, overexpression of HMGB1 in HEK 293 cells, overexpression of Box A in HK-2 cells, and overexpression of HMGB1 in HK-2 cells (C and D), respectively; (n=9 for each group), *P<0.05, **P<0.01, ***P<0.0001.

[0032] Figure 9(A) Images and (B) Graphs showing the results of Box A regarding the effect of HMGB1 on wound closure and healing in grouped diabetic rats, wherein the wound areas of each group on days 3, 5, 7, 10, and 14 were measured using NIH ImageJ analysis tools and compared with the measured values ​​obtained on day 0, and the wound closure rate in Box A of the HHMGB1 plasmid-treated group was significantly improved compared with the plasmid control-treated group or the NSS-treated control group (*P<0.05, **P<0.01, and ***P<0.001, N=8 for each group).

[0033] Figure 10 The results show that HMGB1 Box A promotes the healing and contracture of second-degree burn wounds, wherein (A) is an image of second-degree burn wounds in rats treated daily with HMGB1 Box A and a control group treated with saline. Wounds treated with HMGB1 Box A exhibit greater contraction and less inflammation than those in the control group. (B) is a graphical representation further demonstrating that the healing effect of HMGB1 Box A on second-degree burn wounds in the treatment group was significantly enhanced compared to the control group. P < 0.001, *P < 0.05, **P < 0.01, and ***P < 0.001, N = 8 per group.

[0034] Figure 11 Figure 2 shows the effect of Box A of HMGB1 in reversing senescence. HK2 cells were pretreated with 2.5 μM etoposide for 72 h to induce cellular senescence, and then transfected with Box A and scramble control plasmids and incubated for 48 h. (A) is a bright field image captured to show cell morphology and density (scale bar = 50 μm), and (B) is a Western blot gel image illustrating the expression of p16 in HK2 cells pretreated with different methods. INK4A and γH2AX expression levels, while β-actin was reprobed to confirm equal sample loading.

[0035] Figure 12 is a graph showing the results of HK2 cells exposed to the DNA damaging agent H2O2, treated with the cell-penetrating peptide IMT-P8-BoxA peptide and a control, indicating that IMT-P8-BoxA promotes cellular resistance to DNA damaging agents and prevents H2O2-induced DNA damage, wherein, HK2 cells were pretreated with IMT-P8-BoxA peptide, and then the cells were incubated with H2O2 for 24 hours, and then the survival rate was assessed by MTT assay 48 hours later and compared by one-way analysis of variance and Dunnett's multiple comparison test (*P<0.05, **P<0.01, ***P<0.001).

[0036] Figure 13SEQ ID No. 1 of the DNA sequence of Box A of HMGB1, SEQ ID No. 2 of the DNA sequence of HMGB1, and SEQ ID No. 3 of the peptide sequence of Box A of HMGB1 are shown.

[0037] Figure 14 is a flow chart showing one embodiment of the disclosed method for rejuvenating DNA and preventing DNA damage.

[0038] Detailed description

[0039] The present disclosure may be embodied in other specific forms without departing from its structure, method, or other essential characteristics as broadly described herein and hereinafter claimed. The described embodiments are to be considered in all respects as illustrative only and not restrictive. The scope of the present disclosure is, therefore, indicated by the appended claims rather than by the foregoing description. All variations that come within the meaning and range of equivalence of the claims are intended to be included within their scope.

[0040] As used herein, the term "polynucleotide" or "nucleic acid" refers to mRNA, RNA, cRNA, cDNA or DNA. The term generally refers to oligonucleotides greater than 30 nucleotide residues in length.

[0041] As used herein, the term "gene" may refer to a functionally significant DNA sequence. It may be a naturally occurring nucleic acid sequence, or a recombinant nucleic acid sequence derived from a natural source or a synthetic construct. The term "gene" may also refer to, for example, but not limited to, cDNA and / or mRNA that is directly or indirectly encoded or derived from a genomic DNA sequence.

[0042] Unless otherwise indicated, the terms "complex," "biocomplex," and "Youth-DNA-GAP" are used interchangeably throughout this specification to refer to epigenetic biomarkers for genome stability in cells.

[0043] As used herein, the term "aging cell" and "aging cells" may refer to cells with deteriorated functional properties due to the aging process or senescence induction or accumulation of DNA damage. More specifically, "aging cells" refer to cells with Youth-DNA-GAP less than 0.3% of the control EDSB PCR, and / or accumulation of senescence-associated β-galactosidase cells greater than 50%, and / or accumulation of DNA damage, wherein there are more than 3.5 γ-H2AX foci / cell or more than 7 8-OHDG / 10 6 dG.

[0044] According to one aspect of the present disclosure, a method for preventing DNA damage in a cell nucleus is disclosed. Preferably, a reagent comprising a vector containing a polynucleotide sequence encoding a peptide as set forth in SEQ ID No. 1 or SEQ ID No. 2 is administered to a cell, the vector being used to express the encoded peptide within the cell; and the encoded peptide is overexpressed in the cell. Preferably, the overexpressed peptide forms one or more complexes (or Youth-DNA-GAPs) within the cell, which are capable of revitalizing DNA in the cell nucleus and preventing DNA damage. More specifically, the peptides encoded by SEQ ID No. 1 or SEQ ID No. 2, respectively, are HMGB1 protein and Box A of HMGB1 protein. Those skilled in the relevant art will appreciate that the polynucleotide sequences set forth in SEQ ID No. 1 and SEQ ID No. 2 can be further modified to enhance expression, for example, or to improve compatibility with specific host cell types. These modifications may result in the modified polynucleotide sequence retaining only approximately 70% to 90% of the sequence set forth in SEQ ID No. 1 or SEQ ID No. 2. Preferably, such modifications should not depart from the scope of Box A related to HMGB1 and / or HMGB1-based compositions, as well as methods or reagents for preventing DNA damage, accelerating external wound healing, and treating non-infectious diseases associated with low Youth-DNA-GAP production in a subject, etc., encompassed by the present disclosure.

[0045] For many embodiments, the administering step may involve administering the agent to the subject topically, enterally, and / or parenterally, although topical administration is preferred due to its less invasive nature. The agents of the present invention may be administered in various forms depending on the route of administration to achieve the desired effect. In those embodiments in which the agent is administered topically, the agent comprises a vector comprising a polynucleotide sequence encoding the HMBG1 protein or a peptide encoding Box A of the HMBG1 protein, as shown in SEQ ID No. 1 or SEQ ID No. 2, respectively. In particular, for the expression plasmid used, the vector is preferably nano-coated in the form of a nanoemulsion to facilitate its adsorption to cells or tissues surrounding the wound, thereby expressing Box A of the HMBG1 protein or HMGB1 protein within the cells via the vector and subsequently forming a complex capable of preventing DNA damage. More specifically, the present disclosure discovered that HMGB1 or Box A of HMGB1 can generate Youth-DNA-GAP in cells, conferring significant resistance to a wide range of DNA-damaging agents. The known deoxyribose phosphate lyase activity and DNA bending ability of the HMGB1 protein may play a role in the generation of Youth-DNA-GAP. The HMGB1 gene contains two DNA-binding domains (Box A and Box B) and an acidic tail. To date, the present disclosure has only discovered that Box A proteins or HMGB1 proteins with Box A domains appear to confer cells with the ability to resist DNA-damaging agents and limit DNA damage. Therefore, the vectors used in the disclosed methods for preventing DNA damage in the cell nucleus can contain one or more regulatory sequences operable with SEQ ID No. 1 or SEQ ID No. 2. For many embodiments, peptide transfection systems, such as cell-penetrating peptides, can also be used to deliver the vectors into cells.

[0046] According to another aspect of the present disclosure, a method for preventing DNA damage in a cell nucleus is disclosed. The method basically comprises: transfecting the cell with an agent comprising a peptide as shown in SEQ ID No. 3 and / or a vector comprising a polynucleotide sequence as shown in SEQ ID No. 1 or SEQ ID No. 2 for encoding the peptide in the cell. Preferably, the vector is used to express the encoded peptide in the cell after the transfection step, wherein the encoded peptide forms one or more complexes including Youth-DNA-GAP in the cell, which complex is capable of revitalizing the DNA in the cell nucleus and / or preventing DNA damage. In several embodiments, the vector may have a promoter region that initiates transcription and translation of the encoded peptide only in the presence of a promoter entity, thereby regulating the expression of the peptide at a predetermined time, period, level and / or in cells of a specific tissue.

[0047] For many embodiments, the method of preventing DNA damage can further include overexpressing the encoded peptide by transfecting the cells with a predetermined amount of an expression vector, or upregulating peptide expression using a predetermined concentration of a promoter entity. Furthermore, in some embodiments, prevention of DNA damage and reduction of the DNA damage response are achieved by generating Youth-DNA-GAPs, as shown in some of the examples provided below. Alternatively, generating Youth-DNA-GAPs can also promote cellular resistance to DNA-damaging agents.

[0048] On the other hand, the present disclosure relates to a method for improving the healing of injured tissue in a subject, comprising the following steps: contacting an agent comprising a peptide as shown in SEQ ID No. 3 and / or a vector comprising a polynucleotide sequence as shown in SEQ ID No. 1 or SEQ ID No. 2 encoding the peptide with an injured tissue composed of a plurality of cells; and transfecting the cells of the injured tissue with the peptide and / or the vector. Similarly, the vector is used to express the encoded peptide in the cells after the transfection step. In particular, the encoded peptide forms one or more complexes in the cells, which can improve the healing of the injured tissue by correcting the delayed healing of DNA-damaged cells or biologically aged cells. When the subject is a diabetic mammal or a DM patient, the method of the present disclosure can significantly improve the healing rate of the injured subject. For some embodiments, the injured tissue is caused by a burn.

[0049] According to another aspect of the present disclosure, a pharmaceutical composition for preventing DNA damage is disclosed. The composition comprises a vector containing a polynucleotide sequence encoding Box A protein or HMGB1 as shown in SEQ ID No. 1 or SEQ ID No. 2. The vector may incorporate a regulatory sequence or region for regulating the expression of the encoded protein. The vector may be encapsulated or linked to a carrier system such as a cell-penetrating peptide or nanoemulsion to effectively transport the vector into cells. Depending on the applicable embodiment, the composition may be administered topically or systemically. The present disclosure found a significant inverse correlation between Youth-DNA-GAP and the elderly or DM patients, both of whom are known to have biologically aged DNA. Conversely, DNA damage is common in age-related non-communicable diseases (NCDs). Specifically, Youth-DNA-GAPs can prevent DNA damage. As Youth-DNA-GAPs decrease in the elderly and diabetic patients, these patients have increased endogenous DNA damage and elevated DDR, leading to unhealthy cell function. The disclosed compositions incorporate plasmids or vectors for expressing, or more preferably overexpressing, Box A of the HMGB1 protein and / or the HMGB1 protein, which have been found to be a means of generating Youth-DNA-GAP in cells.

[0050] Furthermore, the present disclosure demonstrates that Box A of HMGB1 stabilizes the human genome better than HMGB1. The present inventors believe that Box A of the HMGB1 peptide can enter the cell nucleus and form its Youth-DNA-GAPs. Box A of the HMGB1 peptide possesses all known functions of HMGB1 required for Youth-DNA-GAP formation. In addition to entering the cell nucleus and generating Youth-DNA-GAPs, HMGB1 is known to have other functions within Box A that interact with various extracellular and intracellular enzymes and components. The present disclosure hypothesizes that the diverse functions of the HMGB1 protein result in its reduced efficiency in producing complexes or Youth-DNA-GAPs. In this regard, some embodiments of various aspects of the present disclosure intentionally exclude the expression of Box B and the C-terminus of the HMGB1 protein, and instead utilize Box A of HMGB1 specifically to prevent DNA damage and / or rejuvenate damaged cells, in addition to its extracellular functions.

[0051] As previously described, the inventors further disclose that the formed complex can be used to monitor and prevent genomic instability, which is a contributing factor to health deterioration in many disease states. Therefore, another aspect of the present disclosure relates to methods for rejuvenating cells and improving wound healing rates in biologically aged subjects. The methods comprise topically or systemically administering an agent with a carrier containing a peptide encoding SEQ ID No. 1 or a polynucleotide sequence as set forth in SEQ ID No. 1. Preferably, the agent initiates overexpression of the encoded peptide in biologically aged cells, such that the overexpressed peptide ultimately leads to the formation of a Youth-DNA-GAP or complex, which improves genomic stability in the affected cells and inhibits at least one or more DDR enzymatic reactions. In particular, Phy-RIND-EDSB or Youth-DNA-GAP is a unique epigenomic marker in humans that is progressively reduced in the elderly and patients with diabetes. The present disclosure discloses that HMGB1 or Box A of the HMGB1 protein is an effective tool for generating Youth-DNA-GAP in cells. With the formation of Youth-DNA-GAPs, the topically applied composition can increase the stability of DNA chains in long-distance cis, reduce endogenous DNA damage and DDR, and improve the resistance of cells to DNA damaging agents. Therefore, the disclosed composition rejuvenates cells in biologically aging subjects and promotes wound healing by overexpressing HMGB1 and / or Box A of HMGB1. Preferably, the subject is a biologically aging mammal. The disclosed composition can be used to address health problems related to poor cell growth, cell aging, and delayed healing in age-related non-communicable diseases (including the elderly and diabetic patients).

[0052] The inventors of the present disclosure have discovered a genome stabilization biomarker, Youth-DNA-GAP, or complex, that may form in the presence of HMGB1 protein or Box A of HMGB1 protein. Notably, Youth-DNA-GAP is an epigenetic marker, not a marker of DNA damage. Epigenetic marks are generated by cellular enzyme activity. Epigenetic markers, such as Youth-DNA-GAPs, are shown herein, through the examples provided below, to be beneficial in injured and / or aging cells. The disclosed examples also demonstrate that Youth-DNA-GAPs generated by HMGB1 or Box A of HMGB1 stabilize the genome; therefore, Youth-DNA-GAPs are epigenetic markers.

[0053] In addition, another aspect of the present disclosure is to produce a pharmaceutical composition for preventing DNA damage, increasing resistance to DNA damage, enhancing cell growth, improving healing of injured tissue, reducing endogenous DNA damage and / or reducing the damage response of DNA in mammals. The composition comprises: a peptide and a vector. The peptide comprises at least 70% of the amino acid sequence shown in SEQ ID No. 3; the vector is chemically linked to the peptide and used to introduce the linked peptide into cells of a predetermined tissue type of a mammal to obtain the beneficial results for the mammal as described above. The vector can be a cell-penetrating peptide, a nanoemulsion, etc., which allows the linked peptide to exert one or more of the mentioned beneficial results in an immediate or almost immediate manner in cells contacted with the disclosed composition, compared to other disclosed embodiments using expression vectors.

[0054] The following examples are intended to further illustrate the present invention and are not intended to limit the present invention to the specific examples described therein.

[0055] Example 1

[0056] It has been reported that youth-DNA-GAP levels are lower in biologically aged individuals, elderly people, and individuals with DM. In particular, the hemoglobin A1C (HbA1C) levels of 120 patients were assessed and then divided into a non-DM (80 samples) group and a DM (40 samples) group. All subjects were recruited from the Tambon Health Promoting Hospital Service, Nakhon Si Thammarat, Thailand between 2015 and 2016. The participants were aged between 15 and 80 years. All subjects voluntarily participated in the study. The study was reviewed and approved by the Human Rights Ethical Review Committee for Research Involving Human Subjects of Walai Lak University, Nakhon Si Thammarat, Thailand. Written informed consent was obtained from each participant. In yeast, youth-DNA-GAPs decrease chronologically in aged yeast, and decreased youth-DNA-GAPs drive the biological aging process (4). The present disclosure found that the youth-DNA-GAP levels were lower in elderly participants (r = -0.4726, P < 0.0001) ( Figure 1 A). It is known that diabetic patients have an accelerated cellular aging process (5). The present disclosure also found that the level of Youth-DNA-GAP in WBC of diabetic patients was lower than that in WBC of non-diabetic patients ( Figure 1 B and sex-age adjustment Figure 1 C). Thus, reduced levels of Youth-DNA-GAP in humans have been found in populations known to exhibit biological DNA aging. These data lead the present disclosure to hypothesize that, as in yeast, a reduction in Youth-DNA-GAP promotes DNA damage and subsequent deterioration of cellular function.

[0057] Example 2

[0058] The present invention found that HMGB1 produces Youth-DNA-GAPs because the HMGB1 protein has a lytic enzyme activity. To produce HMGB1 protein, HMGB1 cDNA (NM_001313893.1) (6) was generated in the pRSET A vector (Thermo Fisher Scientific, MA, USA). The vector was constructed by GeneArt TM Gene Synthesis (Thermo Fisher Scientific, MA, USA) was performed. Sequence fidelity was confirmed by Sanger sequencing. The HMGB1 vector was then transformed into BL21(DE3) pLysS competent cells (Promega, WI, USA) for protein production.

[0059] DNA from two human immortalized kidney cell lines, HEK293 and HK-2, and a cervical cancer cell line, HeLa, were co-cultured with 2 μg of HMGB1 protein. The cells were incubated at 37°C for 16 hours in a total volume of 50 μl of PBS (New England Biolabs, MA, USA). As a control, 2 μg of HeLa DNA was incubated with purified EGFP protein or AluI (New England Biolabs). DNA to HMGB1 protein dosage ratios were 5:1, 4:1, 3:1, 2:1, and 1:1.

[0060] To measure Phy-RIND-EDSBs or Youth-DNA-GAPs caused by HMGB1 cleavage activity, EDSB PCR was performed as previously described (7). The present disclosure found that purified HMGB1 protein can digest DNA in a dose-dependent manner ( Figure 2 A- Figure 2 B) Most of the DSBs generated by HMGB1 have blunt ends ( Figure 2 A).

[0061] Example 3

[0062] The present disclosure discovered that Box A of HMGB1 produces Youth-DNA-GAP and thus colocalizes with Youth-DNA-GAP. The present disclosure determined the colocalization between Youth-DNA-GAP and proteins from expression plasmids and DI-PLA (8). This study used full-length human HMGB1, Box A, Box B, Box BC and random peptide sequence control (PC) expression plasmids. The present disclosure transformed the commercial pcDNA3.1 Flag insertion expression vector (Invitrogen, Carlsbad, USA) into Escherichia coli (DH5α) host cells. Plasmid DNA was isolated using the Qiagen Plasmid Miniprep Kit (Qiagen, Switzerland) according to the manufacturer's instructions. Cells were transfected with plasmids (final plasmid concentration, 2,500 ng / ml) using Lipofectamine 3000 (transfection reagent) (Invitrogen, Carlsbad, USA) and cultured in an incubator for 24-48 hours.

[0063] In addition, the present disclosure performs DI-PLA between Flag and DSB as described previously (8). DI-PLA is performed by InSitu Orange Starter Kit Mouse / Rabbit(DUO92102)( The samples were incubated at room temperature for 15 minutes before analysis using a fluorescence or confocal microscope using 20× and 40× objectives.

[0064] Plasmid protein localization was observed at each red dot using DI-PLA technique ( Figure 3 ). DI-PLA results showed that HMGB1 and Box A bound DNA near each Youth-DNA-GAP, while Box B and BC molecules did not. Figure 3 A) and Box A plasmids ( Figure 3 Positive signal in cells transfected with Box B ( Figure 3 C), Box BC( Figure 3 D), control plasmid ( Figure 3 E) cells and untransfected cells ( Figure 3 F) No DI-PLA signal was shown.

[0065] Example 4

[0066] The present disclosure concludes that Box A of HMGB1 generates Youth-DNA-GAP to prevent DNA damage. If Youth-DNA-GAPs prevent DNA damage, then Youth-DNA-GAPs and DNA damage should rarely coexist. The present disclosure uses antibodies against DNA damage to perform DIP (9) and compares the EDSB concentration of DIP DNA with the concentration of input DNA. First, HMW DNA was prepared from cells transfected with full-length human HMGB1, Box A, Box B, Box BC and PC expression plasmids. Second, EDSB linker-ligated HMW DNA was prepared as described previously (7). Then, the EDSB of DIP DNA was compared with the input DNA using the EDSB PCR protocol as described previously (7). Figure 4 A shows the % EDSB PCR of HeLa and HEK293 cell lines input. The present disclosure then measured the cis coexistence between 8-OHdG or 7-methylguanosine and EDSB in cells transfected with HMGB1 and HMGB1 expression plasmids Box A and negative control plasmids ( Figure 4 B and C). The genomes containing DNA damage of all tested cells were significantly deficient in EDSB, among which the genomes containing DNA damage of cells transfected with HMGB1 Box A and HMGB1 expression plasmids had less EDSB than those of cells transfected with the negative control plasmid ( Figure 4 B and C). Therefore, Box A of HMGB1 and HMGB1 expression plasmid increased the proportion of Youth-DNA-GAP in cells.

[0067] Example 5

[0068] The present invention found that Box A of HMGB1 can reduce endogenous DNA damage. Specifically, DNA from cells transfected with full-length human HMGB1, Box A, and PC expression plasmids was extracted by phenol-chloroform method and resuspended in sterile dH2O. Subsequently, OxiSelect TM The oxidative DNA damage ELISA kit (Cell Bio Labs, Inc., San Diego, USA) was used to measure the 8-OHdG level in DNA. The AP site level was determined by OxiSelect TM The oxidative DNA damage quantification kit (CellBio Labs, Inc., San Diego, USA) was used to determine the oxidative DNA damage. The present invention transfected HMGB1 and Box A expression plasmids into HEK293 and HK2 cell lines and found that both plasmids led to a reduction in several types of endogenous DNA damage, including 8-OHdG and AP sites ( Figure 5 A- Figure 5B) Therefore, the endogenous DNA damage reduction function of HMGB1 belongs to the Box A domain.

[0069] Example 6

[0070] The present invention found that Box A of HMGB1 reduced DDR. In particular, protein lysates from cells transfected with full-length human HMGB1, Box A, and PC expression plasmids were prepared using RIPA buffer (Sigma Chemical, St. Louis, MO, USA) and a protease inhibitor cocktail (Pierce Biotechnology, Rockford, IL, USA) and analyzed by a BCA protein assay kit from Pierce Biotechnology (Rockford, IL, USA). Standard Western blots were prepared and incubated overnight with specific primary antibodies against p-ATM (Ser1981), p53, p21, p16INK4A, phosphor-γ-H2AX (Ser139), and β-actin. The immune complexes were detected by Immobilon Western Chemiluminescent HRP Substrate (Merck, DA, Germany) and exposed by an Azure c300 imaging system (Azure Biosystems, CA, USA).

[0071] The cellular response to DNA damage is regulated by the DDR signaling pathway, which consists of a cascade of protein kinases that promote phosphorylation within the DDR network (10). To determine the effects of HMGB1 and Box A plasmids on DDR, the present disclosure evaluated the protein expression levels of γH2AX, p-ATM (ser1981), p53, p21, and p16INK4A (11, 12). The present disclosure found that the expression levels of DDR signaling pathway proteins were reduced in HMGB1- and Box A-transfected cells ( Figure 6 ).

[0072] Example 7

[0073] The present disclosure reveals that Box A of HMGB1 promotes cell proliferation. To study cell proliferation after transfection with HMGB1 and Box A plasmids, MTT reagent (5 mg / ml) was used daily for 4 days after inoculation.

[0074] ( The transfected cell lines were evaluated and the expression of proteins was measured at 570 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). It was observed that the cell proliferation rates of HEK293 and HK-2 cells overexpressing HMGB1 and Box A were significantly higher than those of control cells ( Figure 8 ).

[0075] Example 8

[0076] The present disclosure also hypothesizes that overexpression of Box A and HMGB1 genes increases the resistance of cells to DNA damage. To demonstrate this possibility, cells transfected with Box A, HMGB1, and scrambled plasmids of HMGB1 were treated with DNA damaging agents, including H2O2 and methanesulfonate (MMS). In particular, the MTT assay was used to assess cell survival under treatment with DNA damaging agents. Cells were seeded in 96-well plates at 4,000 cells per 100 μl (40,000 cells / ml). 24 hours after plasmid transfection, the cells were plated with 400 μl of MMS containing increasing concentrations of MMS ( Missouri, USA) (0-2 mM) medium was treated with cells for one hour, and then hydrogen peroxide (H2O2) ( Missouri, USA)) (0-250 μM) were cultured in a CO2 incubator for 24 hours. Then, the medium containing MMS or H2O2 was replaced with normal working medium. Cell growth was measured by MTT assay 48 hours after treatment. The data are expressed as the percentage of cell survival, where the survival rate of the control group (medium without DNA damaging agent) was arbitrarily set as 100%. Treatment of cells with DNA damaging agents showed that the cell survival percentage of cells overexpressing Box A and HMGB1 was significantly higher than that of scrambled cells ( Figure 7 AB). Interestingly, Box A overexpression protected cells better than HMGB1 ( Figure 7 Furthermore, MTT and cell counting assays demonstrated that increasing HMGB1Box A expression in cells is an effective and safe method for preventing DNA damage.

[0077] Example 9

[0078] The present disclosure hypothesizes that overexpression of Box A and HMGB1 genes will improve the healing process in individuals suffering from conditions of accelerated biological aging (e.g., DM patients). The animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the Faculty of Medicine, Chulalongkorn University (Approval No. 006 / 2561, September 2018). Male Wistar rats (6 weeks old, 150-180 g) were randomly divided into two groups and injected intraperitoneally with a single dose of 65 mg / kg body weight of STZ ( Missouri, USA-Aldrich, USA), dissolved in 50 mM sodium citrate buffer (Alfa Aesar, USA) and 50 mM sodium citrate buffer (2 mL / kg body weight) (13). Seven days after STZ induction, rats with STZ-induced FBS greater than 250 mg / dL were designated as the diabetic group, and rats with FBS less than 150 mg / dL were designated as the non-diabetic group.

[0079] Two pairs of full-thickness excisional wounds were created on the back of rats using an 8-mm biopsy punch and splinted with a silicone ring (14). Diabetic and non-diabetic rats were further divided into three groups and treated with nano-coated Box A of HMGB1 plasmid, nano-coated PC, and NSS, respectively. The nano-coated pcDNA 3.1(+) plasmid control was used as an untreated control, and NSS was represented as a standard wound dressing in this study. Non-diabetic and diabetic wounds were dressed daily and treated with each type of intervention for 14 days. The wound area was measured on days 0, 3, 5, 7, and 14 after treatment and reported as the percentage of wound closure using the following formula: percentage of wound closure = [(wound area on day 0 - wound area on day n) / wound area on day 0] × 100 (day n represents day 3, 5, 7, or 14). After 14 days of complete healing process, all rats were sacrificed, the wound area was excised, and immediately collected in 10% formalin buffer for histology and immunohistochemical 8-OHdG determination.

[0080] After wound collection, the wounds were fixed in 10% neutral buffered formalin for at least 48 hours. The tissues were then dehydrated and paraffin-embedded, and then sectioned at 3 μm thickness using a microtome. Tissue sections were then stained with H&E and Giemsa for histopathology and immune cell infiltration, respectively. Histopathological evaluation was performed and interpreted blindly by two pathologists. Tissue granulation and re-epithelialization were assessed in the observed healing wound areas and reported as an overall histological score, which included 1 = normal tissue, 2 = mature fibroblasts, 3 = immature fibroblasts, 4 = mild inflammation, and 5 = granulation tissue.

[0081] Three micron paraffin-embedded sections were dewaxed and then incubated with Proteinase K (DAKO, CA) for 2 minutes for antigen retrieval. Tissue sections were treated with a 1:8,000 dilution of polyclonal goat anti-8-OHdG (Merck Millipore), followed by an HRP-conjugated anti-goat secondary antibody (DAKO, CA). Wound sections were also counterstained with hematoxylin. The present disclosure tested the efficacy of HMGB1 plasmid-encapsulated Ca-P nanoparticles (Box A) in promoting wound healing in a murine DM wound model. The results obtained from the experimental murine DM are reported in Table 1 below.

[0082] To investigate the effects of Box A / Ca-P treatment with HMGB1 plasmids on diabetic wound closure, 8-mm splinted excisional wounds were topically treated with Box A, PC, or NSS with HMGB1 plasmids once daily for 14 days. To deliver the plasmids into target cells, each type of plasmid was coated with a nanoparticle solution as previously described by Zhao et al. (2014) with some modifications prior to topical administration (15). The most effective ratio of plasmid to nanoparticle solution for transfection was 5 μg of plasmid in 100 μl of nanoparticle solution. Briefly, the Ca-P nanoparticle solution consisted of a mixture of 50 μl of 0.5 M calcium chloride (CaCl2) solution (Merck Millipore, USA) and 5 μg of plasmid DNA, and a mixture of 50 μl of 0.01 M sodium carbonate (Na2CO3) solution (Merck Millipore, USA) and 0.01 M sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O) solution (Merck Millipore, USA) was prepared. A 3 molar ratio of CO32- / PO4 (31:1) was used. First, the plasmid DNA-calcium complex was prepared by mixing 16 μl of CaCl2 solution and plasmid DNA and adjusting the final volume to 50 μl using sterile dH2O. Then, the plasmid DNA-calcium complex was added to a mixture of 50 μl of Na2CO3 and NaH2PO4·H2O solution (16 μl) and sterile dH2O (34 μl). The nanoparticle-coated plasmid solution was prepared before use.

[0083] Table 1. Body weight and fasting blood glucose levels in non-diabetic and diabetic rats at the end of the study. Seven days after STZ induction, pre-treatment fasting blood glucose levels were measured; levels >250 mg / dL were defined as diabetes, and rats meeting this criterion were included in the diabetic group. Non-diabetic rats (FBS <150 mg / dL) were injected with citrate buffer and served as a control group. Following injury, diabetic wounds were treated daily with NSS, Nano-PC, or Nano-BoxA and compared to untreated non-diabetic wounds. Post-treatment FBS was confirmed at the end of the study.

[0084]

[0085]

[0086] Abbreviations: Non-DM: non-diabetic group; DM: diabetic group; DM+NSS: diabetic wounds treated with normal saline; DM+NanoPC: diabetic wounds treated with plasmid-controlled nanoparticles; DM+NanoBox A: diabetic wounds treated with nanoparticles containing the Box A plasmid. Data are expressed as mean ± SEM. ***P < 0.001, significant difference compared with the non-DM group.

[0087] Representative images of diabetic wounds at 0, 3, 5, 7, 10, and 14 days after wounding showed that the area of ​​diabetic wounds treated with Box A of the HMGB1 plasmid was smaller compared with those treated with PC or NSS ( Figure 9 A). Treatment with HMGB1 plasmid Box A showed increased wound closure compared with the control group, especially on days 5 to 7 (P < 0.0001) ( Figure 9 B). In diabetic wound sections, HMGB1-treated Box A improved the mean histological score, with a large number of mature fibroblasts and less inflammation (overall grade), and significantly reduced 8-OHdG levels by immunohistochemical staining (P < 0.001) compared with sections treated with PC or NSS (Table 2). These experiments support that HMGB1 Box A promotes wound healing, especially in DM patients with low levels of Youth-DNA-GAP.

[0088] Table 2. Histological parameters of diabetic wounds on day 14 after daily NSS (N=8), PC (N=8), or Box A (N=8) treatment. Histological scores were graded, including overall grade (1=normal tissue, 2=abundant mature fibroblasts, 3=abundant immature fibroblasts, 4=mild inflammation, 5=granulation tissue), fibroblasts (0=absent, 1=immature, and 2=mature), fibrosis (0=absent, 1=present), and neovascularization and inflammatory infiltrate (0=absent, 1=mild, 2=moderate, 3=abundant). Anti-8-OHdG staining was also assessed (0=absent, 1=mild, 2=moderate, 3=abundant).

[0089]

[0090] Abbreviations: NSS treatment; saline treatment group, PC treatment group; plasmid control treatment group; Box A treatment group; HMGB1 plasmid Box A treatment group.

[0091] Data are expressed as mean ± SEM. *P < 0.05 and **P < 0.01, significant difference compared with the NSS-treated group; P<0.05 significant difference compared with the PC treatment group; P < 0.01 and P<0.001 significant difference compared with the PC treatment group.

[0092] Example 10

[0093] The present disclosure hypothesizes that overexpression of Box A and HMGB1 genes will improve the healing process in individuals exposed to DNA damaging agents (e.g., hyperthermia). Animal experimental protocols followed the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health (NIH Publication No. 8023, revised 1978). All animal experiments were performed in accordance with the Chulalongkorn University Animal Care and Use Committee (approval number: 003 / 2562 on 03 / 2019). This study used a one-way ANOVA to calculate the sample size using the program G*Power3.1, with α error = 0.05, power = 0.95, effect size f = 1, number of groups = 4, and the results were approximately 32 wounds from 16 rats used for the experiment (16). Sixteen 8-week-old male Wistar rats (150-180 g) were obtained from the Namura Laboratory Animal Center (Bangkok, Thailand). The rats were acclimated for 7 days under a controlled 12-hour light / dark cycle and fed standard food and water ad libitum. To inflict second-degree burns, rats were anesthetized with isoflurane and the dorsal skin was shaved. Two second-degree burn wounds were created on the back of each rat using a 10 mm wide aluminum rod heated to 100°C. The rats were further divided into four groups and treated daily with normal saline (NSS), a scramble-flag plasmid treatment group (plasmid control group), a calcium phosphate nanoparticle treatment group (control group), and a Box A plasmid treatment group. The nanocoated pcDNA3.1(+)(scramble) plasmid control group and the calcium phosphate nanoparticle group were used as untreated controls, and NSS represented the standard wound dressing in this study. The wound area was measured using the NIH ImageJ analysis tool at 0, 7, 14, 21, and 28 days after injury and reported as the percentage of wound contracture relative to day 0. All rats were euthanized after 28 days, and the wound tissue was excised and immediately collected in 10% formalin buffer for further evaluation. In the Box A group of HMGB1 protein, the burn wound closure rate was significantly improved from the 7th day to the 28th day after injury compared with the wound closure rates of the saline, scramble plasmid and calcium phosphate nanoparticle treatment groups ( Figure 10 ), particularly from day 10 to day 21 (P < 0.001). In contrast, no significant differences were observed between wounds treated with the plasmid control group, calcium phosphate nanoparticles, and saline at any time point.

[0094] Example 11

[0095] The present disclosure reveals the efficiency of Box A of HMGB1 in rejuvenating biologically aged cells by showing the reversal effect of Box A on 2.5 μM etoposide-induced cellular senescence. HK2 cells were pretreated with 2.5 μM etoposide for 72 h. Etoposide was used to induce cellular senescence as previously described (17). After 72 h, cells were transfected with HMGB1 or PC plasmids of Box A (final concentration of plasmid, 2,500 ng / ml) using Lipofectamine 3000 and incubated for 48 h. Representative cell images showed that the effect of etoposide pretreatment on cell density had characteristics of senescent cells, such as enlarged and flattened cell shape, and loss of proliferation potential, compared with the control, scramble, and Box A transfected groups. β-galactosidase (SA-β-gal) was assessed as previously described using a SA-β-gal staining kit (Cell Signaling Technology, Beverly, MA, USA) according to the manufacturer's instructions (18). The number of SA-β-gal positive cells decreased significantly after transfection with BoxA plasmid, while the number of β-gal positive cells remained at a high level after reversion with scramble plasmid ( Figure 11 A).

[0096] In addition, the present disclosure detects p16 INK4A (a senescence-related cell cycle inhibitor) and γH2AX protein expression in HK2 cells (19). The results showed that 2.5 μM etoposide increased p16 compared with PC, HMGB1 BoxA-transfected cells and control group. INK4A and γH2AX expression in p16 cells receiving BoxA of HMGB1 after etoposide treatment INK4A and γH2AX levels were significantly lower in the etoposide-treated group ( Figure 11 B).

[0097] Example 12

[0098] The genome stabilization effect of Box A of HMBG1 peptide and peptide transfection system (such as cell penetrating peptide) (20). Here, IMT-P8 is a cell penetrating peptide (21). To evaluate the effect of IMT-P8-BoxA peptide (Genscript, Piscataway, NJ, USA), cells were treated with culture medium containing 0.25 μM concentration of IMT-P8-BoxA peptide for 2 hours. Afterwards, the cells were washed with 1XPBS and treated with hydrogen peroxide (H2O2) (Sigma Chemical, St. Louis, MO, USA) for 24 hours. Then, the cells were replaced with normal culture medium and cultured at 37°C for 48 hours. Finally, cell viability was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Figure 12 It was shown that cells treated with IMT-P8-BoxA peptide had increased cell survival after incubation with H2O2 compared to control cells.

[0099] This disclosure reports the first genome stability biomarker, Youth-DNA-GAPs, and a drug, Box A of HMGB1, which holds promise for monitoring and preventing genomic instability that leads to health deterioration in many disease conditions. First, we report that Phy-RIND-EDSB or Youth-DNA-GAP is a unique human epigenomic marker that is reduced in the elderly and patients with diabetes. Second, we demonstrate that HMGB1 generates Youth-DNA-GAPs. Third, Youth-DNA-GAPs increase the stability of DNA chains in cis over long distances, such that Box A of HMGB1 can reduce endogenous DNA damage as well as the DDR and increase cellular resistance to DNA-damaging agents. Box A of HMGB1 is used to repair delayed wound healing and burns in diabetic rats. Finally, Box A of HMGB1 is used to rejuvenate senescent cells.

[0100] A previous study showed that the negative correlation between Youth-DNA-GAP and biological aging was very strong in yeast (4). Here, in humans, the elderly and DM patients also showed a strong association, both of which are known to have biologically aged DNA. DNA damage is common in the elderly and age-related non-communicable diseases (22,23). Therefore, Youth-DNA-GAPs may be an effective biomarker for determining the biological age of NCD patients. Here, the present disclosure shows that the genome stabilization function of HMGB1 is mediated by Youth-DNA-GAPs. HMGB1 has the ability to bend DNA and stabilize double-stranded DNA to prevent denaturation (24). These two properties support the findings of the present disclosure that Youth-DNA-GAP is generated by HMGB1. In addition, similar to EDSB generated by topoisomerase (25), Youth-DNA-GAP can stabilize the eukaryotic genome by alleviating torsional forces, thereby stabilizing double-stranded DNA to prevent denaturation. Box A of HMGB1 not only reduces endogenous DNA damage but also increases the resistance of cells to DNA damaging agents. The reduction of torsional forces by the gap structure of Youth-DNA-GAPs should increase DNA stability. Interestingly, Box A stabilizes the genome more effectively than the entire HMGB1 protein. One possible explanation is that HMGB1 is a multifunctional protein both in the nucleus and extracellularly (26). The presence of Box B and the C terminus should separate the molecule from other roles, such as inflammation. Nevertheless, the exclusion of Box B and the C terminus helps to clarify the role of Box A of HMGB1 on genome stability.

[0101] It is well known that overall DSB repair can reduce Youth-DNA-GAPs after a DSB induction event (4). Therefore, the mechanism of delayed genome instability in cells exposed to DNA damaging agents may involve a reduction in Youth-DNA-GAPs, which may explain why Box A of HMGB1 promotes burn wound healing. The present disclosure shows that molecularly engineered Box A of HMGB1 can stabilize eukaryotic genomes by generating Youth-DNA-GAPs. The present disclosure demonstrates that the genome stabilizing properties of Box A of HMGB1 lead to many applications, including reducing endogenous DNA damage, reducing DDR, increasing cellular resistance to DNA damaging agents, promoting cell growth, promoting tissue healing processes in individuals with DM, promoting burn tissue healing processes and regeneration of aged cells. If any other non-infectious diseases are caused by low levels of Youth-DNA-GAPs, the present disclosure may potentially use Box A of HMGB1 as a genome stabilizing molecule to treat such non-infectious diseases.

[0102] It should be understood that the present invention can be embodied in other specific forms and is not limited to the only embodiment described above. However, modifications and equivalents of the disclosed concepts that are readily apparent to those skilled in the art are intended to be included within the scope of the appended claims.

[0103] References

[0104] 1.A.Mutirangura,Is global hypomethylation a nidus for molecular pathogenesis of age-related noncommunicablediseases? Epigenomics 11,577-579(2019).

[0105] 2. HLOu, B.Schumacher, DNA damage responses and p53 in the aging process. Blood 131, 488-495 (2018).

[0106] 3. I. Shimizu, Y. Yoshida, M. Suda, T. Minamino, DNA damage response and metabolic disease. Cell Metab 20, 967-977 (2014).

[0107] 4.J.Thongsroy,M.Patchsung,M.Pongpanich,S.Settayanon,A.Mutirangura,Reduction in replication-independent endogenous DNA double-strand breakspromotes genomic instability during chronological aging in yeast.FASEB J,fj201800218RR(2018).

[0108] 5. JEMorley, Diabetes and aging: epidemiologic overview. Clin Geriatr Med 24, 395-405, v (2008).

[0109] 6.Ferrari,S.,Finelli,P.,Rocchi,M.and Bianchi,M.E.(1996)The activegene that encodes human high mobility group 1protein(HMG1)contains intronsand maps to chromosome 13.Genomics,35,367-371.

[0110] 7.W.Pornthanakasem,N.Kongruttanachok,C.Phuangphairoj,C.

[0111] Suyarnsestakorn,T.Sanghangthum,S.Oonsiri,W.Ponyeam,T.Thanasupawat,O.Matangkasombut,A.Mutirangura,LINE-1methylation status of endogenous DNAdouble-strand breaks.Nucleic Acids Res 36,3667-3675(2008).

[0112] 8.A.Galbiati,F.d'Adda di Fagagna,DNA Damage In Situ Ligation Followedby Proximity Ligation Assay(DI-PLA).Methods Mol Biol 1896,11-20(2019).

[0113] 9.P.M.Lizardi,Q.Yan,N.Wajapeyee,Methyl-Cytosine-BasedImmunoprecipitation for DNA Methylation Analysis.Cold Spring Harb Protoc2017,pdb prot094854(2017).

[0114] 10.A.Marechal,L.Zou,DNA damage sensing by the ATM and ATRkinases.Cold Spring Harb Perspect Biol 5,(2013).

[0115] 11.A.Sharma,K.Singh,A.Almasan,Histone H2AX phosphorylation:a markerfor DNA damage.Methods Mol Biol 920,613-626(2012).

[0116] 12.K.Evangelou,J.Bartkova,A.Kotsinas,I.S.Pateras,M.Liontos,G.Velimezi,M.Kosar,T.Liloglou,I.P.Trougakos,L.Dyrskjot,C.L.Andersen,M.Papaioannou,Y.Drosos,G.Papafotiou,Z.Hodny,B.Sosa-Pineda,X.R.Wu,A.Klinakis,T.Orntoft,J.Lukas,J.Bartek,V.G.Gorgoulis,The DNA damage checkpoint precedesactivation of ARF in response to escalating oncogenic stress duringtumorigenesis.Cell Death Differ 20,1485-1497(2013).

[0117] 13.A.J.King,The use of animal models in diabetes research.Br JPharmacol 166,877-894(2012).

[0118] 14.J.M.Davidson,F.Yu,S.R.Opalenik,Splinting Strategies toOvercomeConfounding Wound Contraction in Experimental Animal Models.Adv WoundCare(New Rochelle)2,142-148(2013).

[0119] 15.D.Zhao,C.Q.Wang,R.X.Zhuo,S.X.Cheng,Modification ofnanostructuredcalcium carbonate for efficient gene delivery.Colloids Surf B Biointerfaces118,111-116(2014).

[0120] 16.Cai EZ,Ang CH,Raju A,Tan KB,Hing EC,Loo Y,et al.Creation ofconsistent burn wounds:a rat model.Archives of plastic surgery.2014;41:317-24.

[0121] 17.Zhou,L.,Chen,X.,Liu,T.,Gong,Y.,Chen,S.,Pan,G.,...He,F.(2015).Melatonin reverses H2 O2-induced premature senescence in mesenchymal stemcells via the SIRT1-dependent pathway.J Pineal Res,59(2),190-205.doi:10.1111 / jpi.12250

[0122] 18.Hall,B.M.,Balan,V.,Gleiberman,A.S.,Strom,E.,Krasnov,P.,Virtuoso,L.P.,...Gudkov,A.V.(2017).p16(Ink4a)and senescence-associated beta-galactosidase can be induced in macrophages as part of a reversible responseto physiological stimuli.Aging(Albany NY),9(8),1867-1884.doi:10.18632 / aging.101268

[0123] 19.Casella,G.,Munk,R.,Kim,K.M.,Piao,Y.,De,S.,Abdelmohsen,K.,&Gorospe,M.(2019).Transcriptome signature of cellular senescence.Nucleic Acids Res,47(14),7294-7305.doi:10.1093 / nar / gkz555

[0124] 20.Milletti F(August 2012).Cell-penetrating peptides:classes,origin,and current landscape.Drug Discovery Today.17(15–16):850–60.

[0125] 21.Gautam,A.,Nanda,J.S.,Samuel,J.S.,Kumari,M.,Priyanka,P.,Bedi,G.,...Raghava,G.P.(2016).Topical Delivery of Protein and Peptide Using NovelCell Penetrating Peptide IMT-P8.Sci Rep,6,26278.doi:10.1038 / srep26278

[0126] 22.J.P.Soares,A.Cortinhas,T.Bento,J.C.Leitao,A.R.Collins,I.Gaivao,M.P.Mota,Aging and DNA damage in humans:a meta-analysis study.Aging(AlbanyNY)6,432-439(2014).

[0127] 23.M.Milic,A.Frustaci,A.Del Bufalo,J.Sanchez-Alarcon,R.Valencia-Quintana,P.Russo,S.Bonassi,DNA damage in non-communicable diseases:A clinicaland epidemiological perspective.Mutat Res 776,118-127(2015).

[0128] 24.M.J.McCauley,J.Zimmerman,L.J.Maher,3rd,M.C.Williams,HMGB bindingto DNA:single and double box motifs.J Mol Biol 374,993-1004(2007).

[0129] 25.S.S.Teves,S.Henikoff,DNA torsion as a feedback mediator oftranscription and chromatin dynamics.Nucleus 5,211-218(2014).

[0130] 26.I.Ugrinova,E.Pasheva,HMGB1 Protein:A Therapeutic Target Inside andOutside the Cell.Adv Protein Chem Struct Biol 107,37-76(2017).

Claims

1. A vector capable of overexpressing a peptide in a cell to prevent DNA damage in the cell nucleus, the vector comprising a polynucleotide sequence consisting of SEQ ID No. 1 encoding the peptide.

2. A pharmaceutical composition for rejuvenating DNA and / or reducing DNA damage in mammalian cells, comprising: A biological component, the biological component being one of (i) a vector comprising an expressible polynucleotide sequence consisting of SEQ ID No. 1 encoding a peptide or (ii) a peptide consisting of SEQ ID No. 3; and A carrier system is chemically linked to the biological component to facilitate entry of the biological component into the cell when the composition is contacted with the cell, wherein the carrier system is a cell penetrating peptide or a nanoemulsion.

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