HOXA3 treatment method for accelerating wound healing of senile non-diabetic mouse

Abstract

Chronic wounds are characterized by a persistent, overinflamed environment which hinders the progression of regenerative wound closure. Such chronic wounds are particularly common in diabetic patients, typically requiring distal limb amputation, but also occur in non-diabetic elderly patients. HoxA3 is a member of a somatotype modeling and main regulatory transcription factor homologous frame family, and when the HoxA3 is locally applied as a plasmid wrapped in hydrogel, it has been proved that wound closure of diabetic mice can be accelerated through inducible expression of HoxA3. We now provide an independent repetition of these basis in vivo diabetes wound closure studies, and extend it through minimum dose threshold estimation. Furthermore, the natural healing speeds of the wounds of the elderly non-diabetic mouse and the young diabetic mouse are observed to be similar, which provides power for testing local HoxA3 plasmids in the elderly non-diabetic mouse, and the wound healing is observed to be accelerated again. In these short-term studies, we do not observe any serious adverse reaction with naked eyes or through local histology. The local HoxA3 is an attractive transformation candidate target of a chronic wound whether the local HoxA3 is used as a plasmid or a future alternative form.

Description

[0001] field

[0002] The processes, methods, and systems of the present disclosure relate to compositions, methods, and systems for accelerating wound healing in subjects with impaired wound healing (eg, elderly subjects).

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Patent Application No. 63 / 477,354, filed on December 27, 2022, entitled “HOXA3 Therapeutic Approaches to Accelerate Wound Healing in Aged Non-diabetic Mice,” under 35 U.S.C. §119(e), which is incorporated herein by reference in its entirety.

[0005] Sequence Listing

[0006] This application contains a sequence listing that was submitted electronically and is hereby incorporated by reference in its entirety.

[0007] background

[0008] Different patient populations suffer from impaired wound healing, which can lead to complications. In some cases, patients with wounds from these populations may require significantly longer recovery times, such as from weeks to months, which increases the risk and severity of complications. In some cases, these patient populations include elderly patients and those with diabetes.

[0009] Treatments for diabetic foot ulcers (DFUs) vary, but their effectiveness is difficult to assess due to small study sizes and a lack of head-to-head comparisons. Given the progress in amputation rates, DFU treatment remains inadequate. The mainstay of treatment remains focused on basic wound care, such as cleaning, debridement, disinfection, and dressing. Commercially available dressings composed of biomaterials, such as human placental tissue, human amnion and chorion membranes, umbilical cord lining, and even bovine collagen and shark cartilage, typically achieve closure rates of 30–70% at 12 weeks, compared to 20–30% in control groups. However, in a 2016 clinical trial, an amniotic tissue graft called Epifix achieved closure in 31 of 32 wounds at 12 weeks, compared to 18 of 35 wounds in the control group. However, these treatments require human or animal tissue. Biologically active therapies, including topical patches containing rhEGF or PDGF growth factors, have only halved the 12-week failure rate, similar to most human tissue grafts.

[0010] Normal wound healing proceeds through a series of overlapping phases, beginning with coagulation, followed by inflammation initiated by neutrophils and macrophages to control introduced pathogens, re-epithelialization by keratinocytes, subepithelial angiogenesis, wound contracture, and scar remodeling of the provisional extracellular matrix. Multiple factors contribute to impaired wound healing in DFUs, including reduced growth factor production, angiogenesis, macrophage regenerative function, and keratinocyte and fibroblast migration and proliferation. Specifically, the wound environment must shift from an initial inflammatory phase to a regenerative phase for healing to occur. However, the etiology of wound repair defects is thought to differ between populations in different patient populations, making it unlikely that a single therapy will successfully treat different populations.

[0011] What are needed are therapeutic compositions and methods for effectively treating and / or assisting wound healing, particularly in high-risk populations.

[0012] Summary of the Invention

[0013] Disclosed herein are compositions, methods, and systems for enhancing wound healing in patients in need thereof. In one aspect, a method for enhancing wound repair in a patient is disclosed, comprising identifying a wound in the patient, administering a composition comprising HOXA3 to the wound, contacting at least one cell or tissue with HOXA3, and allowing the wound to heal. In many embodiments, the patient can be elderly and / or human. In many embodiments, HOXA3 is a coding sequence, such as mRNA encoding HOXA3, and / or the composition can include methylcellulose. The method can also include administering HOXA3 after the administering step.

[0014] In another aspect, a system for enhancing wound repair in a patient is disclosed, comprising an applicator and a composition comprising HOXA3. In some embodiments, HOXA3 is a coding sequence, such as an mRNA encoding HOXA3. In some embodiments, the applicator is solid or semisolid and may comprise methylcellulose and / or may be a patch configured to contact a wound. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The wound measurement procedure is described. Wounds are measured manually using a digital caliper, taking four measurements and averaging them. The first measurement is aligned end-to-end, and subsequent measurements are aligned at 45 degrees to each previous measurement to achieve uniform coverage. The final average is recorded as the size of a given wound on a given day.

[0016] Figure 2Figure 3: HOXA3-mediated wound closure in diabetic mice. ... (A) Negative control (green fluorescent protein, GFP) treated wounds measured 76% of the original wound size 9 days after wound formation, while HoxA3-treated wounds measured 63% of the open wound size. Percent closure is defined as the measured diameter divided by the initial wound diameter (i.e., 6 mm). (B) Representative images of wounds 9 days after wound induction. HoxA3 mice were largely re-epithelialized, with clearer-looking skin compared to the scab-forming control group. Statistical significance was determined at p < 0.05 by mixed-effects analysis of variance (ANOVA).

[0017] Figure 3 Histopathology scoring of fibroblast density, granulation tissue formation, and percent complete epithelialization (this is a global count, so there are no error bars) for diabetic wound healing at day 9 showed statistically significant support for HoxA3. Definitions of the histopathology scoring criteria are listed in the Methods section.

[0018] Figure 4 Representative histology of wounds in diabetic mice treated with HoxA3 plasmid patches for 9 days. (A) Example of H&E wound staining, highlighting newly formed blood vessels (black arrows). (B) Fibroblast density near the wound. (C) Epithelialization of the wound site. In the GFP-treated group, red brackets indicate open lesions, while black lines highlight infiltration of immune debris. In the HOXA3-treated group, blue lines highlight fully healed wounds. Healthy tissue is highlighted by the black dashed line, and healthy hair follicles are marked by black arrows.

[0019] Figure 5 Figure 2 shows a study of different doses and different dosing frequencies of HOXA3 in diabetic mice. (A) Male mice, n=5, were treated with a wound as described, with a wafer replaced three times and once, and a non-treated control. Treatment was performed weekly starting from wound induction. Because the three-treatment group was found to have an advantage, a second study was performed using this protocol to change the plasmid content. (B) In this study, male mice (n=5) were induced with wounds and treated with 25 μg or lower doses replaced weekly for three weeks (i.e., a total of three times, which was the best treatment obtained in the study of different dosing frequencies). Data represent mean values; error bars represent SEM.

[0020] Figure 6Results are shown for mice that were wounded and observed for 7 days. Cohorts included aged wild-type mice (18 months), young wild-type mice (12 weeks old), or young db / db mice (12 weeks old). Young mice recovered approximately 80% (i.e., the wound diameter measured was 80% smaller than the initial wound size of 6 mm), while aged and db / db mice recovered only approximately 20% on day 7. (B) Bar graph analysis of the last day of wound measurement. ns not significant, ****p<0.0001, statistics determined by one-way ANOVA. Data represent mean values; error bars represent SEM.

[0021] Figure 7 Presents results showing that HoxA3 accelerates wound healing in aged, non-diabetic mice. Males (n=10) and females (n=10) aged 18 months were double punch wounded. Panel A shows weekly measurements of wound size. Wounds healed within 3 weeks. Panel B shows that at 2 weeks, the extent of healing was significantly greater in HoxA3 mice, both males and females. Panel C shows that blood glucose was ~180 mg / dL at 3 weeks. For all graphs, ns not significant, ****p<0.0001, statistical data determined by one-way ANOVA. Data represent mean; error bars represent SEM.

[0022] Figure 8 The genetic sequence of one embodiment of human wild-type HoxA3 used in plasmid studies (with the amino-terminal translated peptide sequence of the unexpressed genetic sequence SEQ ID No: 4) (accession numbers NM_030661 and O43365; see uniprot.org / uniprot / O43365, SEQ ID No: 1) is shown, with a Myc tag and a His tag (SEQ ID No: 5) inserted at the carboxyl terminus.

[0023] Figure 9 Shown is a time course line graph of an in vitro scratch assay, in which a layer of human dermal microvascular endothelial cells (DMEC) fills an artificial scratch in a monolayer over time. The bar graph highlights the portion of the scratch that remains open at 36 hours under various treatment regimens, indicating that co-incubation with human recombinant HoxA3 protein (previously with the addition of TAT or PenSV40 cell-penetrating peptide motifs to the HoxA3 protein molecule) accelerates closure.

[0024] Figure 10Shown are histograms of oil deposition per cell under different treatments, as well as micrographs of treated cell populations from the Raw 246.7 human macrophage cell line after co-incubation with oxidized low-density lipoprotein (Ox-LDL). The histograms highlight that co-incubation with recombinant human HoxA3 protein (previously with the addition of either TAT or PenSV40 cell-penetrating peptide motifs) prevents Ox-LDL uptake.

[0025] Figure 11 The figure shows that HoxA3(m), a recombinant protein derived from a modified sequence of wild-type human HoxA3, closes diabetic wounds faster than HoxA3(wt). Panel A shows the average wound closure trajectory in a diabetic mouse skin wound model; Panel B shows the average area under the curve (AUC) of the wounds along the trajectory.

[0026] Figure 12 An amino acid sequence comparison of HoxA3 and HoxB3 (Panel A; SEQ ID NO: 1 vs. SEQ ID NO: 2) and an amino acid sequence comparison of HoxA3 and BoxD3 (Panel B; SEQ ID ID: 1 vs. SEQ ID NO: 3) are shown. The percent sequence identity (49% and 60%, respectively) and sequence similarity (59% and 70%, respectively) are also shown. Sequences: HoxA3, see uniprot.org / uniprot / 043365; HoxB3, see uniprot.org / uniprot / P14651; HoxD3, see uniprot.org / uniprot / P31249.

[0027] Figure 13 Protein production schemes for TAT-HOXA3(m) and TAT-HOXA2(wt) are shown.

[0028] Figure 14 Purification and verification of protein isolation are shown (TAT-HOXA3 (wt) is shown).

[0029] FIG15 shows the results of a scratch defect assay on primary hDMECs (top) and primary keratinocytes (bottom) from a 55-year-old patient.

[0030] Figure 16 shows the results of a primary human monocyte polarization assay. Detailed Description of the Invention

[0032] Disclosed herein are compositions, methods, and systems for treating patients suffering from impaired wound healing. In many embodiments, treatment may involve applying a composition comprising HOXA3 to a wound. In many embodiments, HOXA3 may be administered as a protein or nucleic acid encoding sequence, such as a DNA plasmid or mRNA sequence. The disclosed HOXA3 may be wild-type HOXA3 or a modified HOXA3, in one embodiment, mammalian HOXA3. Modified HOXA3 may comprise one or more amino acid deletions, substitutions, and / or additions, or may comprise one or more moieties, such as moieties that add functionality to HOXA3. The disclosed moieties may aid in cellular targeting, stability, resistance to degradation, and the like. In one embodiment, the modification is a cell-penetrating peptide or CPP. In some embodiments, the HOXA3 composition is administered via a solid or semisolid carrier, such as a film or patch impregnated with HOXA3, and / or HOXA3 may be added or reapplied after the carrier is applied to the wound. In many embodiments, the disclosed compositions, methods, and systems can enhance wound healing compared to untreated wounds.

[0033] Impaired wound healing occurs in various patient populations. Current treatments for accelerating wound healing, suitable for diverse patient populations, include debridement and hyperbaric oxygen therapy. However, these treatments require specialized equipment and highly trained staff, and may necessitate prolonged hospitalization. Meanwhile, several topical treatments, such as wound dressings, are available for home use. Most of these deliver antimicrobial agents directly to the site of infection to reduce bacterial burden, promote wound protection, and promote healing. However, research demonstrating actual efficacy in accelerating wound healing is insufficient. Furthermore, multiple mechanisms contribute to wound healing, and these treatments address symptoms rather than the underlying mechanisms of chronic wounds. Some therapies, such as EPIFIX and REGRANEX, are based on growth factors (i.e., recombinant platelet-derived growth factor; PDGF), which are essential for wound healing. However, these treatments are costly, and recombinant proteins like REGRANEX do not persist in the harsh, protease-rich environment of chronic wounds. Furthermore, although REGRANEX is the only growth factor therapy approved by the US Food and Drug Administration for impaired wound healing (in patients with diabetes), its use is limited by its high cost and adverse side effects, including a possible increased risk of malignancy. In conclusion, a therapeutic approach is needed to address the multifactorial nature of impaired wound healing.

[0034] Diabetic foot ulcers (DFUs) are a common and devastating complication of diabetes. Due to impaired wound healing, they can take weeks to months to heal. As of 2018, 10.5% of the US population had diabetes. In a given year, 1-4% of diabetic patients in the US experience a DFU, a statistic that has remained unchanged over the past four decades. Of these DFUs, 14-24% will require lower limb amputation. Consequently, approximately 80,000 DFU amputations occur in the US annually. In addition to reduced quality of life, each amputation in the US results in hospitalization costs of $12,000-16,000.

[0035] Homeobox protein A3 (HOXA3) is a transcription factor that promotes diabetic wound healing. Mace et al. used a methylcellulose local gene delivery patch to deliver a HOXA3 expression plasmid to wounds in young Leprodb / Lepradb (db / db) diabetic mice. Application of the HOXA3 expression plasmid significantly improved wound closure in db / db mice compared with a negative control, a vector plasmid. In a large-diameter wound model (2.5 cm), HOXA3 treatment increased wound closure, detectable as early as 7 days, and resulted in closure of all wounds within 42 days, compared with 77 days in untreated mice. Further rigorous in vitro and in vivo studies demonstrated enhanced neovascularization, keratinocyte migration, and endothelial cell invasion and migration compared with a negative vector control. The mechanisms underlying the wound healing ability of HOXA3 have also been preliminarily investigated. Al-Sadoun et al. demonstrated that macrophages transduced with HOXA3 inhibited M1 polarization and promoted M2 polarization by regulating Pu.1 / Spi1 and Stat6, compared with a negative control, mCherry transduction. Furthermore, Mace et al. demonstrated that HOXA3 treatment mobilized and recruited endothelial progenitor cells and attenuated inflammatory pathways compared with control mice, and Mahdipour et al. demonstrated that HOXA3 promoted the differentiation of hematopoietic progenitor cells into proangiogenic Gr-1+CD11b+ myeloid cells, stimulating angiogenesis. Taken together, these published in vitro and in vivo data provide evidence that HOXA3 may influence angiogenesis, re-epithelialization, and pro-healing macrophage polarization.

[0036] Wild-type aged mice appear to respond to wounds with impaired healing. However, the cause of this impairment has not been fully characterized. Applicants show herein that HoxA3 is an effective wound healing therapy in aged mammals.

[0037] Although the Mace and Boudreau laboratories have previously demonstrated the ability of a HoxA3 plasmid (or similar homologs such as HoxB3, HoxD3, etc.) to accelerate diabetic wound closure in vivo, one skilled in the art would not reasonably expect that HoxA3-based therapies would have similar efficacy in non-diabetic aged mice. Applicants' ability to treat wounds in aged mice was unexpected. Previous in vitro studies have shown that HoxA3 promotes macrophage polarization to M2 gene expression, stimulates endothelial cell motility, and stimulates keratinocyte motility.

[0038] The applicants hypothesized that these factors may also be affected during healthy aging, which could be termed "inflammaging." However, the effectiveness of HoxA3 in non-diabetic aging wounds is unclear, given that hyperglycemia itself is known to be detrimental to wound healing through the formation of advanced glycation end products (AGEs), which are not direct targets of HoxA3.

[0039] Chronic wounds, both diabetic and non-diabetic, are a significant source of morbidity and cost, affecting 1–2% of the general population in developed countries. Among individuals aged 65 years and older, the prevalence of chronic ulcers, pressure ulcers, and DFUs is 2.3%, 1.8%, and 0.7%, respectively. The very elderly and ill are at greatest risk, with nursing home patients particularly susceptible to pressure injuries, with a prevalence of 2.5%. Despite the severity of this problem, no new treatments have been approved for chronic wounds since 1997 (beccamin gel).

[0040] The wounds in this study were sterile wounds to achieve controlled, uniform injury. Although clinical reports vary, microbial biofilms have been observed in approximately 70% of chronic wounds. It may be necessary to address infection issues before treatment with HoxA3, but this has not been tested. The wounds in this study were acute but benefited from HOXA3 treatment. Therefore, in one embodiment, the disclosed HoxA3 therapeutic composition can be administered by the patient (or others) at home. In addition, in many embodiments, the disclosed treatment methods can be used for administration to common / everyday wounds in older or elderly individuals. In many embodiments, the disclosed compositions and treatment methods can be used for early intervention, such as treatment of pressure sores in medical, long-term care, or assisted living facilities.

[0041] Applicants note that inflammaging may be a cumulative process that may become more prevalent / pronounced at very advanced ages. However, this process occurs continuously and may also be accelerated in patients with other health risk factors. Therefore, Applicants hypothesize that HoxA3 treatment may be particularly beneficial for treating wounds in elderly patients, while still being effective in middle-aged patients who have already begun the inflammaging process and / or have other risk factors. Without wishing to be limited by theory, Applicants speculate that while the use of HoxA3 may be less effective in inflammatory wounds in younger patients (compared to the elderly and other high-risk populations), stump wounds may also be effectively treated with the compositions and methods of the present invention. Specifically, even in the absence of microbial involvement, stump wounds may become chronic due to wear and tear of prosthetic devices, leading to the formation of a highly inflammatory environment.

[0042] In some embodiments, applicants use plasmids expressing HOXA3 to achieve a sustained effect on wound healing. In some cases, the plasmid may not integrate into the host genome. In other embodiments, the HOXA3 protein can be administered to the wound. In many embodiments, the dosage and dosing schedule may vary depending on the type of composition used due to differences in degradation, cellular uptake, nuclear trafficking, and intracellular half-life.

[0043] patient

[0044] The disclosed methods can effectively treat a variety of patient populations. In some embodiments, the patient being treated suffers from a chronic or long-lasting wound, for example, the patient can be selected from the group consisting of the elderly, diabetics, and amputees. The patient may be in a healthcare facility. In some embodiments, the disclosed patient is at home and can self-administer the disclosed compositions and treatment methods. In many embodiments, the wound or injury is diabetic-related, age-related, chronic, or acute. In many embodiments, the wound or injury may be associated with an area of inflammation, such as an amputation stump wound.

[0045] As defined herein, an "elderly" patient may be about 60 years of age or older, such as about 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 years of age and older.

[0046] In some embodiments, the disclosed compositions can be used to treat patients with conditions other than wounds or injuries. In one embodiment, the condition may be atherosclerosis, and treatment may help prevent the formation of foam cells. In these embodiments, the disclosed compositions can be systemically administered to patients and / or included in a coating of a medical device (e.g., a stent) for administration. In stent-based embodiments, HOX can be eluted from the stent and absorbed by macrophages at or near coronary artery lesions (e.g., atherosclerotic plaques). In embodiments, when systemically administered, treatment may be more convenient, and most of the therapeutic composition is still expected to reach the target cells, particularly where the HOX therapeutic composition comprises lipid nanoparticles (LNPs), because these particles may be preferentially absorbed by macrophages.

[0047] target cells

[0048] The disclosed compositions and methods can target various mammalian cells. In most embodiments, the target cells are located at, near, or around a wound or injury, such as in the skin, epithelium, epidermis, dermis, fat layer, blood vessels, etc. In some embodiments, the target cells are one or more of neutrophils, macrophages, epithelial cells, keratinocytes, and fibroblasts.

[0049] Drug administration

[0050] Various dosages and dosing schedules are contemplated for treating wounds using the disclosed methods. In many embodiments, wounds can be treated 1-7 times per week or more, for example, 1X or 3X per week, or once every two weeks. The amount of the composition can vary depending on the type of composition and the form of HOXA3 (e.g., protein, plasmid, and / or mRNA, etc.). In some embodiments, where HOXA3 is in the form of a plasmid, the amount can vary from about 1 μg to about 25 μg.

[0051] HOX

[0052] Disclosed herein are various compositions and methods comprising a HOX, such as a HOX amino acid sequence having at least about 45% identity to HoxA3. In some embodiments, the HOX amino acid sequence can have about 80% identity to HoxA3, HoxB3, HoxC3, or HoxD3, which have been shown to affect wound healing in diabetic mice (Hansen, S. et al., Am. J. of Path., Vol. 163, No. 6, 2003). In many embodiments, the HOX is mammalian, such as human. In many embodiments, the HOX is HOXA3, such as Figure 8As shown. In various embodiments, HOX can be wild-type / natural or non-natural / modified HOX. For example, wild-type or modified mammalian HOXA3. Modified HOXA3 can be in various forms. In one embodiment, the modified HOXA3 can comprise one or more amino acid deletions, substitutions and / or additions, or HOXA3 can comprise one or more parts, such as parts that can add function to HOXA3. In one embodiment, the modified HOXA3 can comprise one or more substitutions selected from L175D, E176K and E178K (nomenclature refers to HOXB4; HOXA3 nomenclature is L206D, E207K and E209K). As used herein, HOXA3(m) can refer to a protein, or a nucleic acid sequence encoding a protein comprising the amino acid substitutions L175D, E176K and E178K. The disclosed parts can help HOXA3 cell targeting, stability, resistance to degradation, etc. In one embodiment, the modification is a cell penetrating peptide or CPP to enhance cellular uptake and / or nuclear transport of the molecule.

[0053] HOXA3 composition

[0054] The disclosed compositions, methods, and systems include the step of contacting at least one mammalian cell or tissue located at or near a wound with a formulation comprising HOXA3. The formulation may include, but is not limited to, pharmaceutically acceptable carriers, solids, semisolids, methylcellulose, nanoparticles, poly(lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplexes, liposomes, polymers, carbohydrates (including monosaccharides), cationic lipids, fibrin gels, fibrin hydrogels, fibrin glues, fibrin sealants, fibrinogen, thrombin, rapidly eliminated lipid nanoparticles (reLNPs), and combinations thereof. In many embodiments, as described by Truzzi et al. (In vivo Biodistribution of Respirable Solid Lipid Nanoparticles Surface-Decorated with a Mannose-Based Surfactant: A Promising Tool for Pulmonary Tuberculosis Treatment. Nanomaterials 2020; 10:568-83), which is incorporated herein by reference in its entirety, LNPs can help target target cells, such as macrophages, that can preferentially take up the LNPs.

[0055] Wounds can be treated with HOXA3 in various amounts and forms. In some embodiments, HOXA3 can be administered in the form of a protein, peptide, or coding sequence. In embodiments where HOXA3 is administered in the form of a coding sequence, the coding sequence can be a nucleic acid, such as RNA or DNA. In one embodiment, the coding sequence is RNA, such as mRNA.

[0056] The HoxA3 formulation applied to the wound can take various forms. In many embodiments, the formulation is a transcription construct. In some embodiments, the transcription construct can be a nucleic acid sequence encoding HoxA3, or a protein or peptide having at least about 45% identity to HoxA3, for example, greater than about 45%, 50%, 60%, 70%, 80%, 90%, or 95%, and less than 100%, 90%, 80%, 70%, 60%, or 50% identity. In some embodiments, the nucleic acid can be an RNA sequence, such as an mRNA sequence. In some embodiments, the transcription construct can be a virus, plasmid, or other suitable vector. In one embodiment, the transcription construct can be a plasmid, which can be loaded onto a solid or semisolid carrier prior to application to the wound. In one example, a patch can be impregnated with the transcription construct, such as a plasmid. In these embodiments, the patch can be impregnated with 1-100 μg of the transcription construct.

[0057] HOXA3 formulations can be delivered to cells and tissues using a nucleic acid coding sequence. The coding sequence can be RNA or DNA. When the coding sequence is DNA, the nucleic acid can be delivered in a lipid nanoparticle composition, a viral vector, or a plasmid. When the coding sequence is mRNA, the mRNA can be delivered in an RNA-based viral particle or lipid nanoparticle composition. In some embodiments, lipid nanoparticles can be prepared by mixing a solution containing preformed lipid nanoparticles with a solution containing mRNA to form mRNA lipid nanoparticles.

[0058] Messenger RNA (mRNA) is becoming an increasingly useful active ingredient in treating a variety of diseases. mRNA-based therapies involve administering messenger RNA to patients in need of the therapy. In most cases, the mRNA is delivered to the patient's cells, where it encodes the protein encoded by the mRNA. Lipid nanoparticles are often used to facilitate this delivery. In most cases, the lipid nanoparticles encapsulate the therapeutic mRNA for efficient in vivo delivery of the mRNA.

[0059] In some embodiments, altering the lipid composition may affect the intracellular delivery and / or expression of mRNA coding sequences to various types of mammalian cells and tissues (e.g., mammalian epithelial cells). In other embodiments, the disclosed particles, lipid nanoparticles, or viral particles may include one or more sequences, tags, receptors, etc. that help target the particles to appropriate cells, such as epithelial cells.

[0060] Disclosed herein are viral vectors comprising one or more nucleic acids encoding HOXA3. Adeno-associated virus (AAV) is a small virus that infects humans and belongs to the genus Parvoviridae (Parvoviridae). AAV is a replication-defective, non-enveloped virus of approximately 20 nm that contains a linear, single-stranded DNA (ssDNA) genome of approximately 4.8 kilobases (kb). AAV is designed to deliver various genes to cells and tissues. In some embodiments, AAV includes one or more nucleic acid sequences encoding peroxidase.

[0061] applicator

[0062] An applicator can be used to contain or carry a HOXA3 formulation. In some embodiments, the applicator is a solid or semisolid. In many embodiments, the disclosed HOXA3 formulation can be added directly to the applicator. In one embodiment, the applicator is a patch or film that can be configured to conform to and / or contact cells or tissue at or near a wound. In some embodiments, one or more doses can be administered directly and / or repeatedly to the applicator. In one embodiment, the disclosed applicator can be made at least in part of methylcellulose. Alternatively, the disclosed HOXA3 composition can be incorporated into a cream or ointment for topical administration.

[0063] Enhance wound repair

[0064] The disclosed treatment methods can enhance wound repair in a patient compared to a similar untreated wound in a matched patient. In some embodiments, at a time point following injury and / or initiation of treatment, the healing rate in treated patients can be 20% to 400% higher. In some embodiments, the time point can be from about 1 to about 4 weeks, for example, about 4 weeks. In one embodiment, for example, wound healing in treated patients can exceed about 70%, 80%, 90%, or 95%, compared to about 50% healing in untreated patients within two weeks.

[0065] The disclosed treatment methods may result in wounds healing faster than untreated wounds. In some embodiments, treated wounds may heal about one to two weeks earlier than the size of the wound. In one embodiment, a wound of about 6 mm may heal more than one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen days earlier than a 6 mm untreated wound, and less than about two weeks, thirteen, twelve, ten, nine, eight, seven, six, five, four, three, or two days earlier.

[0066] Accelerated wound healing can be measured by various methods. In one embodiment, as described above, it can be measured by macroscopically measuring the various lengths / dimensions of the wound. In these embodiments, the measurement method can be as follows: Figure 1 In other cases, the measurement can be performed by microscopic examination. In these embodiments, the examination can be one or more of vascular density, epithelial cell layer, inflammation, and granulation tissue formation. In various embodiments, at a given time point, the treated wound can re-epithelialize by about 400% or more compared to the untreated wound. In one example, about 80% of the treated wound and about 20% of the untreated wound can re-epithelialize at this time point. As described above, the time point can be about 1 to about 4 weeks.

[0067] The disclosed methods of treatment can be applied in various forms and in various administration arrangements. In some embodiments, the disclosed doses can be administered immediately after injury, or several hours or days after injury. In many embodiments, the dose can be repeated to achieve 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses. When the composition is a plasmid administered by an applicator, such as a patch or film, the patch or film can contain about 0.01 to 50 μg, such as 2.0 μg, 1.0 μg, 5 μg, or 25 μg. Example

[0068] Example 1 - HoxA3 accelerates wound healing in elderly patients

[0069] As described below, the applicant found that the wound healing response of old wild-type mice and young diabetic mice was similar. The applicant hypothesized that HoxA3 could effectively accelerate wound healing and created a study to test this hypothesis. In these studies, mice were wounded as described above and received three doses of 25μg HoxA3 treatment (one treatment at the time of injury and once a week thereafter). Male and female mice were tested separately (n=10). The size of the mice's wounds was observed weekly with a digital caliper. All wounds healed after 21 days. There was no significant difference in wound closure at the time of injury or 1 week after injury. However, at 2 weeks after injury, the treated wounds consistently healed by more than 90%, while the untreated wounds consistently healed by only about 50%. At 3 weeks, the mice were sacrificed and blood glucose was tested, and the results showed no significant difference between the two groups. All groups were non-diabetic patients.

[0070] Figure 7 HoxA3 treatment has been shown to accelerate wound healing in aged, non-diabetic mice. In these studies, male (n=10) and female (n=10) aged (18 months) mice were double punch wounded. Wound size was measured weekly. Wounds healed within 3 weeks. HoxA3 mice showed significantly better healing at 2 weeks in both male and female mice. Blood glucose levels measured at 3 weeks were ~180 mg / dL. For all graphs, ns not significant, ****p<0.0001, statistical data determined by one-way ANOVA. Data represent mean; error bars represent SEM.

[0071] Example 2 - Topical HOXA3 plasmid accelerates wound healing in diabetic mice at the microscopic level.

[0072] During the acclimation period, diabetic mice (db / db) were randomly assigned to undergo punch injury and subsequent wound size monitoring until day 10 after injury, at which time the mice were humanely sacrificed. One animal died during the acclimation period, leaving Group 1 (control) with 9 animals and Group 2 (treatment) with 10 animals. Treatment consisted of applying GFP (control) or HOXA3 plasmid patches (25 μg DNA plasmid in 1% methylcellulose, applied as a 50 μL patch after drying on wax paper for 5 hours) to the wounds on days 0, 2, 4, 7, and 9. Splints and sutures were changed as needed throughout the study.

[0073] The primary outcome was histology, but wounds of three representative animals from each group were measured on days 0, 2, 4, 7, and 9. All animals were weighed on days 0, 2, 4, 7, and 9. On day 10, each group was humanely sacrificed, and the wound areas were harvested. One wound from each animal was fixed in 10% neutral buffered formalin, while the other was snap-frozen. The fixed wounds were embedded in paraffin blocks for microscopic examination and scoring by a blinded pathologist.

[0074] On day 9, the final recorded data point before sacrifice, the HoxA3 cohort closed rapidly from approximately 85% patency to approximately 65% patency, while the control group remained at approximately 80% patency. Although day 9 was the first day of statistical significance, this fact was incidental because the results were not tabulated until after sacrifice, and day 10 was chosen simply as a midway point for untreated healing based on a previous pilot study (not shown).

[0075] Anonymous wounds were scored by a board-certified pathologist analyzing 400X fields of view for each wound. No statistical differences were observed in vascular density, maximum epithelial thickness, or inflammation; however, HoxA3 significantly increased granulation tissue formation, with nearly 80% of HoxA3 wounds completely re-epithelialized compared to only 20% of the control group ( Figure 3 ).

[0076] Figure 3 The results of histopathology scoring for diabetic wound healing at day 9, fibroblast density, granulation tissue formation, and percent complete epithelialization of the cohort (this is a global count, so there are no error bars) are shown to be statistically significant in favor of HoxA3. Definitions of the histopathology scoring criteria are also provided in the Materials and Methods section. Figure 4 The top row shows the marked labeling of distinct blood vessels containing red blood cells (RBCs) and areas of tissue granulation in the HoxA3 samples, which were absent in the GFP group. Figure 4 B The second row shows the density of fibroblasts marked by the abundant presence of extracellular matrix material. Figure 4 C shows re-epithelialized HoxA3 wounds versus open control wounds.

[0077] A dose study was conducted to determine the minimum frequency and dose of plasmid administration required to elicit accelerated healing. The resulting minimum threshold was intended to design additional experiments to improve the therapy by determining the optimal dynamic range. First, mice were wounded and treated with a 25ug plasmid patch, and wound size was monitored every two days with a digital caliper. Mice received a total of three doses (once per week), a single dose, or an untreated control. A total of three doses (i.e., weekly) were effective, while a single dose was ineffective ( Figure 5 A).

[0078] The effect of the dose was then assessed by varying the plasmid content in the gel patch over a three-dose regimen. Observations were performed daily to improve temporal resolution. Cohorts included no treatment, 25 μg patches (similar to the most effective dosing frequency approach), and intermediate doses, including 0.2 μg, 1 μg, and 5 μg. Figure 5 As shown in B, unlike the others, 25 μg was almost completely cured on the 15th day.

[0079] like Figure 5 As shown in Figure B, unlike the others, 25 μg showed almost complete healing by day 15. Specifically, Figure A shows the results for male mice (n=5) that were injured as described and treated with the patch, with patch changes including weekly changes (three times total) and once upon injury, as well as a non-treated control. The second study used a weekly schedule to vary the plasmid content. In this study, male mice (n=5) were injured and treated with 25 μg or lower doses.

[0080] Local plasmid patch of HoxA3 accelerates wound healing in non-diabetic aged mice

[0081] Aging is associated with the phenomenon of “inflammaging” because with age, the immune system of older mammals enters a state of increasingly chronic, low-level immune activation. It is well known that wounds in elderly humans heal more slowly than in young people. This effect is known to be mediated in part by macrophages and is exacerbated in diabetic patients. In a pilot study evaluating the relative wound healing rates of aged wild-type mice, young wild-type mice, and young diabetic mice (12 weeks of age), young wild-type mice healed rapidly, taking approximately one week. However, by day 7, young db / db and aged wild-type mice had similar healing rates of only 20%. Applicants hypothesized that due to the similar trajectories observed in these two groups in this pilot experiment, aged non-diabetic mice might benefit from the HoxA3 plasmid.

[0082] Figure 6 Results are shown for mice that were wounded and observed for 7 days. The cohorts in this study included old wild-type mice (18 months), young wild-type mice 12 weeks of age, or young db / db mice (12 weeks of age). Young mice recovered approximately 80%, while old mice and db / db mice only recovered approximately 20% at day 7. Figure B shows a bar graph analysis of the last day of wound measurement. ns not significant, ****p<0.0001, statistics determined by one-way ANOVA. Data represent mean values; error bars represent SEM.

[0083] Example 3 - Relative Wound Closure of DMEC Treated with Various HOXA3 Interventions

[0084] Figure 9Results from an in vitro scratch / migration assay are shown, comparing HOXA3 fused with either the HIV-TAT cell penetrating motif, the Penatrin cell penetrating motif, or no cell penetrating motif. Briefly, a scratch is made to disrupt adherent cells, and migration back to the scratch area is measured as an indicator of wound closure. Untreated DMECs are less mobile and therefore fill the scratch more slowly than DMECs treated with HOXA3 protein. HoxA3 proteins harbor either the HIV-TAT cell penetrating peptide (CPP) motif or the PenatrinSV40 CPP. TAT was more effective at doses comparable to PenatrinSV40. TAT-HoxA3 showed dose dependence, as the 200nM dose resulted in higher migration than the 50nM dose, which itself outperformed the closure rate of untreated cells.

[0085] Primary dermal microvascular endothelial cells, "DMEC" cells (ATCC, PCS-110-010), were seeded in 96-well plates at a density of 12,000 cells / well. Cells were cultured overnight in the supplier's recommended medium to allow for cell adhesion. The medium was removed, and fresh medium containing the listed HOXA3 treatments (or vehicle control) was added to the growth medium and incubated for 4 hours. After 4 hours of incubation, the cells were scratched with a P200 pipette tip. Wound size was measured over time using the IncuCyte S3 Dynamic Scratch Wound Module.

[0086] Example 4 - 264.7 macrophages treated with CPP-HOXA3 (WT) show a reduction in the foam cell phenotype

[0087] Figure 10 Shown is a study of foam cell formation in HOXA3-treated and untreated macrophages. Briefly, Raw 264.7 cells (a macrophage lineage) were treated with 100 μg / mL oxidized low-density lipoprotein (oxidized LDL) concurrently with the listed HOXA3 treatments (or vehicle control). Cells were co-cultured with lipids and HOXA3 in DMEM for 48 hours. After the 4-hour incubation, the culture medium was removed from the cells, which were then washed with PBD and stained with Oil Red O.

[0088] Untreated macrophages accumulated oil deposits, indicative of a foam cell (FC) phenotype. Co-incubation with HoxA3 protein, either with the HIV-TAT cell-penetrating peptide (CPP) motif or the Penatrin-SV40 CPP motif, mitigated the formation of oil deposits and preserved the morphology of non-oxLDL-treated cells. This effect was dose-dependent, as 200 nM of TAT-HoxA3 was superior to 50 nM. Each dose of HIV-TAT was more effective than Penatrin-SV40. However, a 200 nM dose of PenSV40-HoxA3 was also effective. These results suggest that HoxA3 protein exposure prevents macrophages from becoming foam cells after oxidized LDL treatment.

[0089] Oil Red O working solution was prepared by dissolving Oil Red O powder (Sigma-Aldrich Cat#MAK194) in 100% isopropanol at a concentration of 30 mg / mL. Formalin (10%) was pipetted onto the cells and gently swirled to mix. The cells were incubated with formalin for 30 minutes. The formalin was then discarded and the cells were washed twice with water, followed by the addition of 60% isopropanol and the cells were incubated for 5 minutes. After discarding the 60% isopropanol, the Oil Red O working solution was evenly covered on the cells and allowed to incubate for 10-20 minutes. The Oil Red O solution was then discarded and the cells were washed 3 times with water until no excess stain was visible. Hematoxylin was added to the cells and incubated for 1 minute, followed by the discarding of the hematoxylin and the washing of the cells 3 times with water. The cells were then covered with water and observed under a 40X objective.

[0090] Example 5 - Changes in wound closure over time using WT or mutant HOXA3

[0091] The HOXA3 plasmid was modified to produce a modified HOXA3 protein with the following substitutions: L175D, E176K, and E178K. The resulting plasmid was designated the HOXA3(m) plasmid. As described above, the ability of the HOXA3(m) plasmid to promote wound closure in diabetic mice was compared with wild-type HOXA3. Figure 11 Results from these studies are presented, demonstrating that despite using the best-known wild-type HoxA3 dosing regimen from applicants' previous studies, wound closure was approximately 13% faster, as measured by mean wound diameter at each time point.

[0092] Materials and methods

[0093] method

[0094] Diabetic Mice: Male B6.Cg-m+ / +Leprodb / J mice, 8-12 weeks old, were purchased from Jackson Laboratories (strain code 000697) at the start of the study. Mice were individually housed and acclimated on site for approximately one week prior to study activity. Mice were housed at 20-24°C (68-74°F) with a 12-hour light-dark cycle and 30-70% humidity. Mice were given ad libitum access to water and food throughout the study. Prior to surgical injury, mice were randomly divided into two treatment groups based on blood glucose levels.

[0095] Aged mice: Male and female C57BL / 6J mice (77 weeks old) were purchased from Jackson Laboratory. Mice were individually housed and acclimated to the site approximately one week prior to study activity. Mice were maintained as described above. Prior to surgical injury, mice were randomly assigned to two treatment groups based on blood glucose levels.

[0096] Injury: All mice underwent two 6 mm full-thickness wounds (through the lipid membrane) created on the shoulder using a hole punch. These wounds were splinted with a silicone splint, non-absorbable sutures, and surgical glue to prevent skin contracture. An example video of the wounding procedure is available in the supplementary data. Animals received nonsteroidal anti-inflammatory drugs once daily for three days after the injury.

[0097] Treatment: Methylcellulose patches were prepared by mixing 25 μg (or lower amounts, if indicated) of HoxA3 plasmid DNA in 1% methylcellulose and spotting 50 μL drops onto PARAFILM using a positive-displacement pipette. An example video of patch formation is provided in the Supplementary Data. Prior to application, the patches were allowed to air dry for up to 5 hours to achieve a solid, uniform consistency. Plasmid patches were prepared prior to treatment. The sequence of the HoxA3 plasmid is provided in the Supplementary Information.

[0098] Wound size measurement: Wounds were measured using a digital caliper under isoflurane anesthesia. Figure 1 Measurements were taken for each wound in four directions and averaged for individual animals. Two wounds from the same animal were generally unrelated and therefore considered statistically independent.

[0099] Histology: The wounds and surrounding areas were excised from the mice and fixed in 10% neutral buffered formalin for 48 hours. The wounds were then dehydrated by immersion in the following solutions: 70% ethanol (2 changes, 1 hour each), 85% ethanol (2 changes, 1 hour each), 95% ethanol (2 changes, 1 hour each), 100% ethanol (2 changes, 1 hour each), Histoclear xylene substitute for 3 hours, and liquid paraffin for 3 hours (3 changes, 1 hour each). The tissue was placed in a mold filled with liquid paraffin and allowed to solidify on ice. The paraffin blocks were stored at room temperature until sectioning. The tissue was sectioned at a thickness of 5 μm to adhere to microscope slides. The samples were placed on a slide warmer at 38°C until the moisture disappeared. The paraffin was melted at 65°C for 20 minutes before dewaxing and subsequent staining.

[0100] Masson's trichrome staining: After dewaxing and rehydrating the slides with distilled water, they were immediately incubated with Bouin's solution at 55°C for 60 minutes. After the 60-minute incubation with Bouin's solution, the slides were allowed to cool for 10 minutes. After equilibration to room temperature, the tissue was rinsed with water until completely transparent. The slides were then incubated with hematoxylin for 5 minutes and immediately rinsed with tap water for 2 minutes. The slides were then incubated with a Biebrisch scarlet and acid fuchsin solution for 15 minutes and immediately washed with tap water. The slides were then incubated with a phosphomolybdic acid / phosphotungstic acid solution for 15 minutes. Without washing, the slides were incubated with analgin blue solution for 10 minutes and then washed with deionized water. The slides were then incubated with 1% acetic acid for 5 minutes. Following the acetic acid incubation, the slides were rapidly cleared with two washes of 95% ethanol, followed by a rapid exchange with xylene substitute, and finally mounted with toluene mounting medium.

[0101] H+E staining: The slides were incubated twice with Histoclear (a xylene substitute) for 5 minutes, then dewaxed with two changes of 100% ethanol and two changes of 95% ethanol (3 minutes and 2 minutes respectively). The slides were then rehydrated with tap water for 1 minute. The slides were then stained with hematoxylin for 2 minutes and then rinsed twice with tap water for 45 seconds each. The slides were then placed in the blue reagent for 15 seconds. After the blue reagent treatment, the slides were washed twice with tap water for 30 seconds. The slides were then washed in 100% ethanol for 10 seconds and then incubated with Eosin Y for 3 minutes. After incubation with Eosin Y, the slides were dehydrated with 100% ethanol, changing the ethanol 4 times during the process. The slides were then transparentized with histoclear twice for 3 minutes each. The slides were then mounted with toluene mounting medium.

[0102] Histopathology Scoring: Anonymous specimens were scored by a third-party, board-certified pathologist. Histological sections were scored using the following criteria: Vascular density, the number of blood vessels containing visible blood cells and lined by endothelium; Maximum epithelial thickness, the number of epidermal cell layers in wounds or open lesions; Fibroblast density, the density of cells and surrounding extracellular matrix as measured by Masson's trichrome staining; and Granulation tissue formation, the presence of new blood vessels. Overall inflammation was also assessed. Values for vascular density and maximum epithelial thickness are total counts, while other criteria are scored on a 1-5 scale for relative severity.

[0103] Statistics and Data: Anonymized raw data were sent to a third-party statistician. Each figure is accompanied by relevant statistical analyses. Data annotation and statistical analyses were performed using GraphPad Prism 9.

[0104] Example 6 - HOXA3 protein production

[0105] TAT-HOXA3(m) and TAT-HOXA2(wt) were produced, purified and tested in culture. In some cases, the production of HOXA3 in bacterial systems can be difficult, so Applicants expressed the disclosed proteins in 293T cells.

[0106] Briefly, transfected cells were harvested, lysed, and clarified, and HOXA3 protein was purified sequentially using metal affinity, cation exchange, and size exclusion. Figure 13 The protein production steps are provided in more detail.

[0107] Using chromatography, Figure 14 Protein purity was confirmed by gel electrophoresis (bottom left) and gel electrophoresis (top right). Fractions collected and pooled from size exclusion chromatography were analyzed by gel electrophoresis, showing a strong band at the correct molecular weight of ~50 kD. It is noteworthy that Western blot analysis of the samples was performed after protein concentration, freezing, and thawing. This means that after freeze / thaw, there is a large amount of protein with the correct molecular weight.

[0108] The biological activity of HOXA3 protein produced in 293 cells was confirmed in a relevant model cell type: primary human microvascular endothelial cells (hDMEC) derived from the skin of a 55-year-old patient. Aged hDMEC were found to grow much slower than young hDMEC, which required several cycles of seeding optimization. Ultimately, 8,000 cells were seeded into 6-well plates and allowed to attach overnight before the start of the experiment. Scratch defects were created in the monolayer using a p200 pipette tip, and closure was monitored over time. Images of the scratched area were acquired at 4-hour intervals.

[0109] To quantify wound closure rates, the density of confluent wounds was measured over time using the IncuCyte S3 Kinetic Scratch Assay Module.

[0110] Untreated cells migrated and filled the gaps slower than HOXA3-treated DMECs in a dose-dependent manner ( FIG. 15 ).

[0111] Example 7 - Effect of HOXA3 on Macrophage Polarization in Elderly People

[0112] Human peripheral blood-derived monocytes were cultured in ImmunoCult™-SF macrophage medium supplemented with M-CSF for 6 days to differentiate into M0 macrophages, and then stimulated with LPS / IFN-γ for approximately 48 hours to generate M1 macrophages. The effects of TAT-HOXA3(wt) and TAT-HOXA3(m) on the production of IL-6, IL-12p70, and TNFα cytokines by LPS / IFN-γ-activated M1 macrophages were evaluated by preincubating M-CSF-derived M0 macrophages with the test article for 30 minutes and then adding the stimulant for 2 days. The secretion of IL-6, IL-12p70, and TNFα in the cell culture supernatant was characterized using the MESOSCALE Discovery Panels. The bar graph in Figure 16 shows the results for TAT-HOXA3(m), which are very similar to those obtained with TAT-HOXA3(wt).

[0113] Cell Thawing, Plating, and M0 Differentiation (Day 0): On the day of culture setup (Day 0), frozen human peripheral blood mononuclear cells from a single donor were thawed, washed, and viability assessed using a Nexcelom Cellometer Auto 2000 Cell Profiler and AO / PI viability staining as described above. Cells were resuspended in ImmunoCult™-SF Macrophage Medium containing 50 ng / mL M-CSF (Shenandoah, Cat#100-03-10UG) at a concentration of 1 x 10 6 cells / mL and at 1x 10 5 Cells were seeded at a density of 100 μL / well in 96-well tissue culture treated plates (Costar, Cat#3595) to initiate the culture. Each condition was cultured in triplicate.

[0114] Medium Replacement and Replenishment (Day 3): On Day 3, prepare fresh ImmunoCult™-SF Macrophage Medium containing 50 ng / mL M-CSF. Fill each well with 50 μL (50% volume) of freshly prepared medium, mix gently, and return the wells to the incubator for an additional 3 days at 37°C, 5% CO2.

[0115] Macrophage stimulation and test article treatment (Day 6): Solvent control and test article working stock solutions (5X stocks) were prepared as described in Example 6 and added to the appropriate Day 6 treatment medium (40 μL / well). The solvent was 50 mM HEPES (pH 7.5 @ 4°C), 500 mM NaCl, and 5% glycerol. The cells were incubated at 37°C, 5% CO₂, and allowed to rest for 30 minutes in the presence of the test article or solvent. After a 30-minute rest, 10 μL / well of a 20X stock solution containing LPS (Sigma, Cat#L6529) and IFN-γ (Shenandoah, Cat#100-77-20UG) was added to the appropriate wells to a final concentration of 10 ng / mL LPS and 50 ng / mL IFN-γ in a final volume of 200 μL / well. The cultures were incubated at 37°C, 5% CO₂ for 2 days. For unstimulated control cultures (see the leftmost bar in the graph of Figure 16), 50 μL / well of ImmunoCult™ SF Macrophage Medium was added instead of LPS / IFN-γ and the test article or solvent. All conditions were repeated three times.

[0116] MSD supernatant collection (Day 8) On Day 8, after 2 days of stimulation, the supernatant was transferred to a new 96-well round-bottom polypropylene plate (Costar, Cat#3879), centrifuged at 1500 rpm for 5 minutes to remove the supernatant, and then the supernatant was carefully transferred to two new 96-well round-bottom plates in two ~90 μL aliquots. The supernatant plate was sealed with Parafilm and stored at -80°C until ready for MSD analysis.

[0117] One replicate of the 40 μg / mL and 10 μg / mL conditions of TAT-HOXA3 (wt) had severe bacterial contamination and was therefore not collected for downstream analysis. MESO SCALE DISCOVERY analysis of cytokine expression in culture supernatants A custom V-Plex Proinflammatory Factor Panel Kit (MSD, Cat# K151A9H-1, K151QWD-1) was used for cytokine quantification of IL-6, IL-12p70, and TNFα. Calibrators, wash buffer, read buffer, and detection antibody solution were prepared according to the kit instructions. For IL-6, IL-12p70, and TNFα, culture supernatant samples were brought to room temperature on the day of the assay and performed at a 1:2 dilution. Since the TNFα sample quantified above the standard curve at a 1:2 dilution, the TNFα assay was repeated at a 1:50 dilution in PBS containing 1% BSA. Samples and calibrators were processed according to the kit instructions. In brief, the plate was prepared by washing three times with wash buffer (PBS + 0.05% Tween-20 [SIGMA, Cat# P1379]), and then the supernatant and calibrator were added to the plate and incubated with shaking at room temperature for 2 hours. The plate was then washed three times with wash buffer, and then the detection antibody was added and incubated with shaking at room temperature for 2 hours. The plate was then washed three times with wash buffer, 2X read buffer T was added, and the plate was read on a MESOQUICKPLEX SQ 120 instrument. Analysis and calculation, the concentration of IL-6, IL-12p70 and TNFα was determined by interpolating the instrument response of each sample to the appropriate standard curve using MSDDiscovery Workbench 4.0 software, and then applying the sample dilution factor. The effect of the solvent control was determined by calculating the absolute cytokine expression of each solvent condition relative to the standard control (medium only) and expressed as the multiple change of the standard control. The effect of each test article was determined by calculating the absolute cytokine expression of each test article condition relative to the closest matching solvent control and expressed as the multiple change of the solvent control. Solvent and test sample conditions were plotted in GraphPad Prism version 9.1.

[0118] Compared to vehicle and buffer controls, IL-12p70 was inhibited in a dose-dependent manner in HOXA3(m) (shown) and HOXA3(wt) (not shown). At higher concentrations of drug (essentially HOXA3(m) or solvent, which alone was found to be effective at high concentrations), IL12-p70 secretion was inhibited even with vehicle, suggesting vehicle toxicity, but at these doses, the inhibition of IL12-p70 in HOXA3-treated samples compared to vehicle samples remained statistically significant. IL-6 expression was not affected by TAT-HOXA3(m) (shown) or TAT-HOXA(wt) (not shown). TNFα secretion is difficult to interpret because the ratio of TNFα secretion in HOXA3-treated cells to solvent was reduced at higher doses, but the absolute secretion was unaffected. Secretion curves are shown in Figures 16A, B, and C. Note that for each image, the leftmost bar shows secretion from unstimulated cells that released essentially no chemokine in the presence of solvent or HOXA3, and the black bar to the right of unstimulated cells shows secretion from cells that were stimulated but without solvent or HOXA3.

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[0169] Although a number of embodiments have been disclosed, other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description. It will be apparent that the present invention is capable of modification in various obvious respects, all without departing from the spirit and scope of the present invention. The detailed description is, therefore, to be construed as illustrative in nature and not restrictive.

[0170] All references disclosed herein, whether patent or non-patent, are hereby incorporated by reference in their entirety, as if each reference were fully incorporated herein by reference. In the event of a conflict between a reference and the specification, the specification (including definitions) will control.

[0171] While the present disclosure has been described with a certain degree of particularity, it is to be understood that the disclosure has been made by way of example and that changes in detail or structure may be made without departing from the spirit of the disclosure as defined in the appended claims.

[0172] SEQ ID NO: 1

[0173] HOXA3 - available at uniprot.org / uniprot / 043365

[0174] MQKATYYDSSAIYGGYPYQAANGFAYNANQQPYPASAALGADGEYHRPACSLQSPSSAGGHPKAHELSEACLRTLSAPPSQPPSLGEPPLHPPPPQAAPPAPQPPQPAPQPPAPTPAAPPPPSSASPPQNASNNPTPANAAKSPLLNSPTVAKQIFPWMKESRQNTKQKTSSSSSGESCAGDKSPPGQASSKRARTAYTSAQLVELEKEFHFNRYLCRPRRVEMANLLNLTERQIKIWFQNRRMKYKKDQKGKGMLTSSGGQSPSRSPVPPGAGGYLNSMHSLVNSVPYEPQSPPPFSKPPQGTYGLPPASYPASLPSCAPPPPPQKRYTAAGAGAGGTPDYDPHAHGLQGNGSYGTPHIQGSPVFVGGSYVEPMSNSGPALFGLTHLPHAASGAMDYGGAGPLGSGHHHGPGPGEPHPTYTDLTGHHPSQGRIQEAPKLTHL

[0175] SEQ ID NO:2

[0176] HOXB3 - Available from uniprot.org / uniprot / Pl4651

[0177] MQKATYYDNAAAALFGGYSSYPGSNGFGFDVPPQPPFQAATHLEGDYQRSACSLQSLGNAAPHAKSKELNGSCMRPGLAPEPLSAPPGSPPPSAAPTSATSNSSNGGGPSKSGPPKCGPGTNSTLTKQIFPWMKESRQTSKLKNNSPGTAEGCGGGGGGGGGGGSGGSGGGGGGGGGGDKSPPGSAASKRARTAYTSAQLVELEKEFHFNRYLCRPRRVEMANLLNLSERQIKIWFQNRRMKYKKDQKAKGLASSSGGPSPAGSPPQPMQSTAGFMNALHSMTPSYESPSPPAFGKAHQNAYALPSNYQPPLKGCGAPQKYPPTPAPEYEPHVLQANGGAYGTPTMQGSPVYVGGGGYADPLPPPAGPSLYGLNHLSHHPSGNLDYNGAPPMAPSQHHGPCEPHPTYTDLSSHHAPPPQGRIQEAPKLTHL

[0178] SEQ ID NO:3

[0179] HOXD3 - uniprot.org / uniprot / P31249

[0180] MLFEQGQQALELPECTMQKAAYYENPGLFGGYGYSKTTDTYGYSTPHQPYPPPAAASSLDTDYPGSACSIQSSAPLRAPAHKGAELNGSCMRPGTGNSQGGGGGSQPP GLNSEQQPPQPPPPPPPTLPPSSPTNPGGGVPAKKPKGGPNASSSSATISKQIFPWMKESRQNSKQKNSCATAGESCEDKSPPGPASKRVRTAYTSAQLVELEKEFHFN RYLCRPRRVEMANLLNLTERQIKIWFQNRRMKYKKDQKAKGILHSPASQSPERSPPLGGAAGHVAYSGQLPPVPGLAYDAPSPPAFAKSQPNMYGLAAYTAPLSSCLP QQKRYAAPEFEPHPMASNGGGFASANLQGSPVYVGGNFVESMAPASGPVFNLGHLSHPSSSASVDYSCAAQIPGNHHHGPCDPHPTYTDLSAHHSSQGRLPEAPKLTHL

[0181] SEQ ID NO:4

[0182] Unexpressed leader peptide sequence

[0183] NYDSLGDPSWLVKLGTELGS

[0184] SEQ ID NO:5

[0185] Myc terminal peptide sequence with His tag

[0186] GPFEQKLISEEDLNMHTGHHHHHH

[0187] SEQ ID NO:6

[0188] Gene nucleotide sequence of expression plasmid

[0189]

Claims

1. A method for enhancing wound repair in a patient, comprising: Identify the patient's wounds; applying a composition comprising HOXA3 to the wound; and Heal the wound.

2. The method according to claim 1, characterized in that The HOXA3 is selected from a nucleic acid sequence and an amino acid sequence.

3. The method according to claim 1 or 2, characterized in that The HOXA3 is mRNA encoding HOXA3.

4. The method according to any one of claims 1 to 3, characterized in that The HOXA3 is contained in a plasmid.

5. The method according to any one of claims 1 to 4, characterized in that The HOXA3 includes a portion for penetrating cells.

6. The method according to any one of claims 1 to 5, characterized in that The HOXA3 is at least about 49% identical to wild-type HOXA3.

7. The method according to any one of claims 1 to 6, characterized in that The composition includes lipid nanoparticles comprising HOXA3.

8. The method according to any one of claims 1 to 7, characterized in that The patient is a human.

9. The method according to any one of claims 1 to 8, characterized in that The patient is selected from one or more of a patient who is at least about 60 years old, suffers from diabetes, and suffers from immunosenescence.

10. The method according to any one of claims 1 to 9, characterized in that The patient is an amputee.

11. A system for enhancing wound repair in a patient, comprising: medication dispenser; and Compositions comprising HOXA3.

12. The system according to claim 11, characterized in that The HOXA3 is a coding sequence.

13. The system according to claim 11 or 12, characterized in that The HOXA3 is mRNA encoding HOXA3.

14. The system according to any one of claims 11-12, characterized in that The applicator is solid or semi-solid.

15. The system according to any one of claims 11 to 14, characterized in that The applicator includes methylcellulose.

16. The system according to any one of claims 11 to 15, characterized in that The applicator is a patch configured to contact the wound.

17. The method according to any one of claims 11 to 16, characterized in that The HOXA3 is selected from a nucleic acid sequence and an amino acid sequence.

18. The method according to any one of claims 11 to 17, characterized in that The HOXA3 is mRNA encoding HOXA3.

19. The method according to any one of claims 11 to 18, characterized in that The HOXA3 is contained on a plasmid.

20. The method according to any one of claims 11 to 19, characterized in that The HOXA includes a moiety for penetrating cells.

21. The method according to any one of claims 11 to 20, characterized in that The HOXA3 is at least 49% identical to wild-type HOXA3.

22. The method according to any one of claims 11 to 22, characterized in that The composition includes lipid nanoparticles comprising HOXA3.

23. A composition for treating atherosclerotic lesions, comprising: HOXA3.

24. A composition for aiding wound closure in a subject suffering from a wound, comprising: HOXA3.

25. The composition according to claim 24, characterized in that The HOXA3 is selected from a wild type or a modified type.

26. The composition according to claim 24 or 25, characterized in that The HOXA3 is a modified HOXA3.

27. The composition according to any one of claims 24 to 26, characterized in that The HOXA3 is modified to include one or more substitutions selected from the group consisting of L175D, E176K, and E178K.

28. The composition of any one of claims 24-27, comprising a cell penetrating moiety.

29. The composition according to any one of claims 24 to 28, for use in treating one or more of diabetic wounds, senile wounds, stump wounds and atherosclerosis.

30. The composition of claim 29, for use in treating diabetic wounds.

31. The composition of claim 29 for use in treating senile wounds.

32. The composition of claim 29, for use in treating stump wounds.

33. The composition of claim 29, for use in treating atherosclerosis.

34. A composition for accelerating wound repair in a subject in need thereof, comprising: a modified HoxA3 molecule; and Pharmaceutically acceptable carrier.

35. The composition according to claim 34, characterized in that The modified HoxA3 molecule is selected from the group consisting of proteins, nucleic acids, mRNAs, plasmids and vectors.

36. The composition according to claim 34 or 35, characterized in that The modified HoxA3 molecule comprises, or encodes, a protein having one or more substitutions selected from the group consisting of L175D, E176K, and E178K.

37. The composition according to any one of claims 34 to 36, characterized in that The modified HoxA3 includes a cell-penetrating portion.

38. The composition according to any one of claims 34 to 37, characterized in that The modified HoxA3 is contained in lipid nanoparticles.

39. The composition according to any one of claims 34 to 38, characterized in that The subject is a human.

40. The composition according to any one of claims 34 to 39, characterized in that The subject is selected from one or more of a patient who is at least about 60 years old, suffers from diabetes, and suffers from immunosenescence.

41. The composition according to any one of claims 34 to 40, characterized in that The composition is contained in an applicator, and wherein the applicator is solid or semi-solid.

42. The composition according to claim 41, characterized in that The applicator includes methylcellulose.

43. The composition according to claim 41, characterized in that The applicator is a patch configured to contact the wound.

44. The method according to any one of claims 1 to 43, wherein: The HOX is selected from the group consisting of HoxA3, HoxD3, HoxB3 and HoxC3.

45. The method or composition according to claim 44, characterized in that The Hox amino acid sequence includes one or more amino acid substitutions at L175D, E176K, and E178K of HoxA3, or at equivalent positions in HoxD3, HoxB3, or HoxC3.

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