HoxA3 treatment to promote wound healing in non-diabetic aging mice

Abstract

Chronic wounds are characterized by a persistent hyperinflammatory environment that inhibits progression toward regenerative wound closure. Such chronic wounds are particularly prevalent in diabetic patients, often necessitating distal limb amputation, but they also occur in non-diabetic and elderly patients. Inducible expression of HoxA3, a homeobox family member and master regulatory transcription factor for body patterning, has been shown to promote wound closure in diabetic mice when administered topically as a plasmid encapsulated in a hydrogel. Here, we provide independent reproducibility of basal in vivo studies of diabetic wound closure and further expand on these studies while minimizing the estimated minimum dose threshold. Furthermore, we observed similarities in spontaneous wound healing rates between non-diabetic aged mice and diabetic young mice, which motivated us to test the topical application of HoxA3 plasmids in non-diabetic aged mice, where accelerated wound healing was also observed. In these short studies, no significant adverse effects were observed macroscopically or by local histology. Local administration of HoxA3, either as a plasmid or as a future alternative therapy, is a noteworthy translational option for chronic wounds.

Description

[Technical Field]

[0001] The disclosed processes, methods, and systems are directed to compositions, methods, and systems for promoting wound healing in subjects with impaired wound healing, such as, by way of example, elderly subjects.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 63 / 477,354, entitled "HOXA3 Treatment Methods for Accelerating Wound Healing Aged Non-Diabetic Mice," filed December 27, 2022, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing This application contains an electronically submitted Sequence Listing, which is hereby incorporated by reference in its entirety.

[0004] background Various patient populations suffer from impaired wound healing, which can lead to complications. In some cases, patients with wounds belonging to these populations require significantly longer recovery times, such as weeks to months, and are at increased risk and severity of complications. In some cases, these patient populations include aging patients and diabetic patients.

[0005] Treatment of diabetic foot ulcers (DFUs) spans multiple modalities, but efficacy is difficult to assess due to small study sizes and a lack of direct comparisons. As evidenced by the trend in existing amputation rates, DFU treatment remains inadequate. Primary treatment remains focused on basic wound care, such as cleansing, debridement, antiseptics, and bandaging. Commercially available dressings composed of biomaterials such as human placental tissue, human amniotic membrane and chorion, umbilical cord membrane, and bovine collagen and shark cartilage generally achieve 30–70% closure at 12 weeks, whereas control closure rates range from 20–30%. In a 2016 clinical trial, amniotic membrane tissue grafts, commercially available as Epifix, closed 31 of 32 wounds at 12 weeks, compared with only 18 of 35 control wounds. These treatments require human or animal tissue. Biologically defined active treatments include rhEGF or PDGF growth factors embedded in topical patches, but results only halve the 12-week failure rate, which is similar to most human tissue grafts.

[0006] Normal wound healing progresses through a series of overlapping stages, beginning with coagulation, followed by an inflammatory response initiated by neutrophils and macrophages to control introduced pathogens, followed by re-epithelialization by keratinocytes, subepithelial angiogenesis, wound contraction, and provisional extracellular matrix scar repair. Impaired wound healing in DFUs is due to multiple factors, including reduced growth factor production, angiogenesis, macrophage regenerative function, and keratinocyte and fibroblast migration and proliferation. For healing to progress, the wound periphery must specifically transition from the initial inflammatory stage to the regenerative stage. However, the etiology of defective wound repair in different patient populations is thought to differ between populations, and therefore, a single treatment cannot be expected to successfully treat multiple populations.

[0007] What is needed are therapeutic compositions and methods for effectively treating wounds and / or assisting wound healing, particularly in at-risk populations.

[0008] overview Disclosed herein are compositions, methods, and systems for promoting wound healing in a patient in need thereof. In one aspect, a method for enhancing wound repair in a patient is disclosed, comprising identifying a wound in the patient, applying 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 may be elderly and / or human. In many embodiments, the HoxA3 is a coding sequence, e.g., mRNA encoding HoxA3, and / or the composition may comprise methylcellulose. The method of the present invention may further comprise the step of reapplying HoxA3 after the applying step.

[0009] In another aspect, a system for promoting wound repair in a patient is disclosed, comprising an applicator and a composition comprising HoxA3. In some embodiments, the HoxA3 is a coding sequence, e.g., an mRNA encoding HoxA3. In some embodiments, the applicator is solid or semi-solid and can include methylcellulose and / or can be a patch configured to contact the wound. [Brief explanation of the drawings]

[0010] [Figure 1]

[0033] Figure 1 shows the wound measurement procedure. Wounds were measured manually using digital calipers. Four measurements were taken and the resulting values ​​were averaged. The first measurement was aligned from head to tail, and subsequent measurements were adjusted by 45 degrees from the previous measurement to ensure even coverage. The final average value was recorded as the desired wound size for a given day. [Figure 2]Figure 1 shows that HoxA3 promotes diabetic wound closure. (A) The size of wounds treated with a negative control (green fluorescent protein, GFP) measured 76% of the original wound size 9 days after wounding, while the size of wounds treated with HoxA3 measured 63% of the open wound size. Percent closure is defined as the measured diameter divided by the original wound diameter (i.e., 6 mm). (B) Representative images of wounds 9 days after wounding. Compared to controls with scabs, HoxA3 mice generally had re-epithelialized, cleaner skin appearance. Statistical significance was determined by mixed-effects analysis of variance (ANOVA) at p<0.05. [Figure 3] Figure 1 shows histopathology scores for diabetic wound healing over 9 days. Fibroblast density, granulation tissue formation, and complete epithelialization rate for the cohort (these are global counts, so there are no error bars) show statistical significance in favor of HoxA3. Definitions of histopathology score criteria are listed in the Methods section. [Figure 4] Representative histological images from a 9-day study of HoxA3 plasmid patches in diabetic mouse wounds. (A) Example of a hematoxylin-eosin stained wound highlighting newly formed blood vessels (black arrows). (B) Fibroblast density near the wound site. (C) Epithelialization of the wound site. The red GFP-treated bracket indicates an open wound, and the black line highlights immune debris infiltration. In the HoxA3-treated specimen, the blue line highlights a fully healed wound. Healthy tissue is highlighted by the black dotted line, and healthy hair follicles are indicated by the black arrows. [Figure 5]Figure 1 shows a variable-dose and variable-frequency study of HoxA3 in diabetic mice. (A) Male mice (n=5) were wounded as described above and treated with a wafer change ranging from one change to three. Controls were untreated. Treatments were weekly from the time of wound induction. Because the three-time treatment group demonstrated superiority, a second study was conducted using this protocol to vary the plasmid content. (B) In this study, male mice (n=5) were wounded and treated with either 25 μg or lower doses, with weekly changes for three weeks (i.e., a total of three changes, which was the superior treatment compared to the variable-frequency study). Data represent the mean, and error bars represent the standard error of the mean (SEM). [Figure 6] Wounded mice were observed for 7 days. Cohorts included aged wild-type mice (18 months old), 12-week-old young wild-type mice, or young db / db mice (12 weeks old). By day 7, young mice showed approximately 80% recovery (i.e., the measured wound diameter was 80% smaller than the original wound size of 6 mm), while aged and db / db mice showed only approximately 20% recovery. (B) Column analysis of the final day of wound measurement. ns indicates "not significant," **** indicates "p<0.0001," and statistics were determined by one-way analysis of variance. Data represent the mean, and error bars indicate the standard error of the mean (SEM). [Figure 7] These figures show that HoxA3 promotes wound healing in non-diabetic aged mice. Male (n = 10) and female (n = 10) aged (18-month-old) mice were wounded using a dual punch. Panel A shows the results of weekly measurements of wound size. Wounds were healed by week 3. Panel B shows that at week 2, HoxA3 mice showed significantly better healing in both male and female mice. Panel C shows that at week 3, blood glucose levels were measured at 180 mg / dL or lower. For all figures, ns indicates "not significant," **** indicates "p<0.0001," and statistics were determined by one-way analysis of variance. Data represent the mean, and error bars indicate the standard error of the mean (SEM). [Figure 8]1 represents the gene sequence of one embodiment of human wild-type HoxA3 (Accession numbers: NM_030661 and O43365; uniprot.org / uniprot / O43365; see SEQ ID NO: 1) used in the plasmid survey (with the amino-terminal translated peptide sequence of the unexpressed gene sequence, SEQ ID NO: 4), and with Myc and His tags at the carboxy terminus (SEQ ID NO: 5). [Figure 9] FIG. 1 shows a time course line graph of an in vitro scratch assay in which a layer of human dermal capillary endothelial cells (DMEC) fills an artificially created scratch in a monolayer over time, and a bar graph highlighting the area of ​​scratches left open for 36 hours in various treatment protocols, demonstrating that incubation with human recombinant HoxA3 protein (with either TAT or PenSV40 cell-penetrating peptide motifs prepended to the HoxA3 protein molecule) promotes closure. [Figure 10] Bar graph of oil deposits per cell for various treatments and photomicrographs of treated cell populations from the Raw246.7 human macrophage line after co-incubation with oxidized low-density lipoprotein (Ox-LDL). The histogram highlights that co-incubation with human recombinant HoxA3 protein (either TAT or PenSV40 cell-penetrating peptide motifs prepended to the HoxA3 protein molecule) prevents uptake of Ox-LDL. [Figure 11] Figure 1 shows graphs demonstrating that HoxA3(m), a recombinant protein derived from a modified sequence of wild-type human HoxA3, closes diabetic wounds more rapidly than HoxA3(wt). Panel A shows the mean wound closure process in a diabetic mouse model in which skin wounds were created, and Panel B shows the mean AUC of the wound versus the process. [Figure 12]Figure 1 shows an alignment of the amino acid sequences of HoxA3 vs. HoxB3 (Panel A; SEQ ID NO: 1 vs. SEQ ID NO: 2) and HoxA3 vs. HoxD3 (Panel B; SEQ ID NO: 1 vs. SEQ ID NO: 3). Also shown are the percentage identities (49% and 60%, respectively) and sequence identity (59% and 70%, respectively). Sequences: HoxA3, see uniprot.org / uniprot / O43365; HoxB3, see uniprot.org / uniprot / P14651; HoxD3, see uniprot.org / uniprot / P31249. [Figure 13] FIG. 1 shows the protein production protocols for TAT-HoxA3(m) and TAT-HoxA3(wt). [Figure 14] FIG. 1 shows purification and validation of protein isolation (TAT-HoxA3(wt) shown). [Figure 15] FIG. 1 shows the results of a scratch wound assay on primary hDMECs (top) and primary keratinocytes (bottom) taken from a 55-year-old patient. [Figure 16] FIG. 1 shows the results of an assay for the polarization of primary human monocytes.

[0011] Detailed Description Disclosed herein are compositions, methods, and systems for treating wound healing disorders in patients with wounds. In many embodiments, treatment can include applying a composition containing HoxA3 to the wound. In many embodiments, HoxA3 can be applied as a protein or nucleic acid coding sequence, such as a DNA plasmid or mRNA sequence. The disclosed HoxA3 can be wild-type HoxA3 or a modified HoxA3, for example, a mammalian HoxA3. The modified HoxA3 can include one or more amino acid deletions, substitutions, and / or additions, or the HoxA3 can include one or more moieties, such as moieties that can add functionality to HoxA3. The disclosed moieties can aid in HoxA3 cellular targeting, stability, resistance to degradation, etc. In one embodiment, the modification is a cell-penetrating peptide or CPP. In some embodiments, the HoxA3 compositions are applied via a solid or semi-solid support, e.g., a wafer or patch, that can be impregnated with HoxA3, and / or the HoxA3 can be added or re-added after the support is applied to the wound. In many embodiments, the disclosed compositions, methods, and systems can promote wound healing compared to untreated wounds.

[0012] Impaired wound healing is common in various patient populations. Current treatments that promote wound healing and are applicable to various patient populations include debridement and hyperbaric oxygen therapy. However, these treatments require specialized equipment and skilled staff and may require prolonged hospitalization. Alternatively, topical treatments, such as wound dressings, that can be used at home also exist. Most of these deliver antimicrobial agents directly to the site of infection, reducing bacterial load and promoting wound protection and healing. However, there is insufficient research to demonstrate their actual effectiveness in promoting wound healing. Furthermore, multiple mechanisms contribute to wound healing, and such treatments treat symptoms rather than the underlying mechanisms of chronic wounds. Some therapeutic agents, such as Epifix and Regranex, are based on growth factors (i.e., recombinant platelet-derived growth factor; PDGF), which are key to wound healing. However, these treatments are expensive, and recombinant proteins, such as Regranex, do not persist in the harsh, protease-rich environment of chronic wounds. Furthermore, although Regranex is the only FDA-approved growth factor treatment for impaired wound healing (in diabetic patients), its use is limited by its high cost and side effects, including occasional increased malignant potential. In summary, there is a need for therapeutic agents that can address the multifactorial nature of impaired wound healing.

[0013] Diabetic foot ulcers (DFUs) are a common and serious complication of diabetes and can take weeks to months to heal due to impaired wound healing. As of 2018, approximately 10.5% of the US population had diabetes. 1% to 4% of the US diabetic population experienced a DFU in a given year, a statistic that has remained stable over the past 20 years. Of DFUs, 14% to 24% require amputation of the lower extremity. Accordingly, approximately 80,000 DFU amputations are performed annually in the US. In addition to the reduced quality of life, the hospitalization cost per amputation in the US is $12,000 to $16,000.

[0014] 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-expressing plasmid to wounds in young Leprdb / Leprdb (db / db) diabetic mice. Application of the HoxA3-expressing plasmid significantly improved the rate of wound closure in db / db mice compared with vehicle plasmid as a negative control. In a large-diameter (2.5 cm) wound model, treatment with HoxA3 significantly improved the rate of wound closure as early as day 7, with complete wound closure achieved in 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 negative vehicle controls. The mechanisms underlying the wound-healing ability of HoxA3 were also previously investigated. Al Sadoun et al. showed that macrophages transduced with HoxA3 inhibited M1 polarization and promoted M2 polarization compared with the negative control, mCherry transduction, by regulating Pu.1 / Spi1 and Stat6. Furthermore, Mace et al. demonstrated that HoxA3 treatment recruited and recruited endothelial progenitor cells while attenuating 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, which stimulate neovascularization. These published in vitro and in vivo data provide evidence that HoxA3 can influence macrophage polarization to promote angiogenesis, re-epithelialization, and healing.

[0015] Wild-type aged mice exhibit impaired wound healing in response to injury. However, the pathogenesis of this impairment remains poorly understood. In this disclosure, Applicants demonstrate that HoxA3 is an effective wound healing treatment for aging mammals.

[0016] Although the ability of HoxA3 (or its paralogs, such as HoxB3 and HoxD3) plasmids to promote diabetic wound closure in vivo was previously demonstrated by the Mace and Boudreau research groups, one skilled in the art could not reasonably have predicted that HoxA3-based therapy would have similar efficacy in non-diabetic aged mice. Applicant's ability to treat wounds in aged mice is unexpected. Previously, HoxA3 has been shown in vitro to promote macrophage polarization toward M2 gene expression, stimulate endothelial cell motility, and stimulate keratinocyte motility.

[0017] The applicant hypothesized that these factors may also be affected during healthy aging, which is known as "inflammaging." However, because hyperglycemia itself is known to be detrimental to wound healing due to the formation of advanced glycation endproducts (AGEs), which are not directly targeted by HoxA3, the effectiveness of HoxA3 in non-diabetic aging wounds was unclear.

[0018] Chronic wounds, both diabetic and nondiabetic, are a significant cause of morbidity and cost, affecting 1% to 2% of the general population in developed countries. Among older adults (≥65 years), the prevalence of chronic ulcers, pressure ulcers, and DFUs is 2.3%, 1.8%, and 0.7%, respectively. The very elderly and critically ill are at greatest risk, and nursing home patients are particularly susceptible to pressure ulcers, with a prevalence of 2.5%. Despite the seriousness of the problem, no new treatments for chronic wounds have been approved since 1997 (becaplermin gel).

[0019] The wounds in this study were created as sterile wounds to achieve controlled, uniform injury. Clinical reports vary, but the presence of microbial biofilms is observed in approximately 70% of chronic wounds. Prior to treatment with HoxA3, resolution of infection may be necessary, but this has not been tested. Although the wounds in this study were acute, treatment with HoxA3 was effective. Thus, in one embodiment, the disclosed HoxA3 therapeutic composition can be applied by the patient (or another person) at home. In many embodiments, the disclosed treatments may be useful for administration to wounds commonly or routinely used by elderly individuals. In many embodiments, the disclosed compositions and treatments may be useful in early intervention for pressure ulcers, for example, in healthcare facilities, long-term care facilities, or assisted living facilities.

[0020] Applicants note that symptoms of inflammaging accumulate and may become more frequent or pronounced at extremes of old age. However, the process continues and may even be accelerated in patients with other health risk factors. Therefore, applicants hypothesize that while HoxA3 treatment may be particularly beneficial for treating wounds in elderly patients, it may also be effective in middle-aged patients who are entering the inflammaging process and / or have other risk factors. Without wishing to be bound by theory, applicants speculate that while the use of HoxA3 in inflammatory wounds in younger patients may be less effective (compared to elderly or other at-risk populations), residual limb wounds may also be effectively treated with the compositions and methods of the present invention. Specifically, residual limb wounds may become chronic due to prosthetic wear, even in the absence of microbial involvement, resulting in a hyperinflammatory environment.

[0021] In some embodiments, applicants have used HoxA3 expression plasmids to achieve sustained effects on wound healing. In some cases, the plasmids may not be integrated into the host genome. In other embodiments, HoxA3 protein can be applied to the wound. In many embodiments, dosage and schedule may vary based on the type of composition used due to differences in degradation, cellular uptake, nuclear transport, and intracellular half-life.

[0022] patient The disclosed methods can effectively treat a variety of patient groups. In some embodiments, the patient being treated suffers from a chronic or long-lasting wound; for example, the patient may be selected from elderly patients, diabetic patients, and amputee patients. The patient may be in a medical facility. In some embodiments, the disclosed patient may be at home and self-administer or perform the disclosed compositions and treatments. In many embodiments, the wound or injury is diabetes-related, age-related, chronic, or acute. In many embodiments, the wound or injury may be associated with an inflamed area, for example, a stump wound.

[0023] An "aging" patient, as defined herein, may be about 60 years of age or older, for example, about 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and older.

[0024] In some embodiments, the disclosed compositions can also be used in patients with conditions other than wounds or injuries. In one embodiment, the condition can be atherosclerosis, and treatment can prevent foam cell formation. In these embodiments, the disclosed compositions can be administered systemically to the patient and / or included in a coating on a medical device, such as a stent. In stent-based embodiments, Hox may be eluted from the stent and taken up by macrophages at or near a coronary lesion, such as an atherosclerotic plaque. In systemic administration embodiments, treatment may be more convenient, and more of the therapeutic composition can be expected to reach the target cells, particularly if the Hox therapeutic composition includes liponanoparticles (LNPs), as these particles are preferentially taken up by macrophages.

[0025] target cell The disclosed compositions and methods can target a variety of mammalian cells. In most embodiments, the target cells are present at, near, or surrounding an internal wound or injury, such as within the skin, epidermis, epidermis, fat layer, blood vessels, etc. In some embodiments, the target cells are one or more of neutrophils, macrophages, epithelial cells, keratinocytes, and fibroblasts.

[0026] dosage Various dosages and schedules are contemplated for treating wounds using the disclosed methods. In many embodiments, wounds may be treated 1 to 7 or more times per week, for example, once or three times per week, or once every two weeks. The amount of composition may vary depending on the type of composition and the form of HoxA3 (e.g., protein, plasmid, and / or mRNA). In some embodiments, when the HoxA3 form is a plasmid, the amount may vary from about 1 μg to about 25 μg.

[0027] Hox Disclosed herein are various compositions and methods comprising a Hox amino acid sequence having at least about 45% identity to Hox, e.g., HoxA3. In some embodiments, the Hox amino acid sequence may 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, e.g., human. In many embodiments, the Hox is HoxA3, e.g., as shown in Figure 8. In various embodiments, the Hox can be wild-type / naturally occurring or non-naturally occurring / modified Hox. For example, the Hox is wild-type or modified mammalian HoxA3. The modified HoxA3 can be in various forms. In one embodiment, the modified HoxA3 may contain one or more amino acid deletions, substitutions, and / or additions, or the HoxA3 may contain one or more moieties, such as moieties that can add functionality to HoxA3. In one embodiment, the modified HoxA3 may contain one or more substitutions selected from L175D, E176K, and E178K (nomenclature based on HoxB4; HoxA3 nomenclature is L206D, E207K, and E209K). As used herein, HoxA3(m) may refer to a protein or a nucleic acid sequence encoding a protein containing the amino acid substitutions L175D, E176K, and E178K. The disclosed moieties may aid in HoxA3 cellular targeting, stability, resistance to degradation, etc. In one embodiment, the modification is a membrane-permeable peptide or CPP that facilitates cellular uptake and / or nuclear transport of molecules.

[0028] HoxA3 composition The disclosed compositions, methods, and systems include contacting at least one mammalian cell or tissue located at or near a wound with a formulation comprising HoxA3, which formulation can include, but is not limited to, a pharmaceutically acceptable carrier, a solid, a semi-solid, methylcellulose, a nanoparticle, a poly(lactic-co-glycolic acid) (PLGA) microsphere, a lipidoid, a lipoplex, a liposome, a polymer, a carbohydrate (including a monosaccharide), a cationic lipid, a fibrin gel, a fibrin hydrogel, a fibrin glue, a fibrin sealant, fibrinogen, thrombin, a rapidly clearing lipid nanoparticle (reLNP), and combinations thereof. In many embodiments, LNPs may be useful for targeting target cells, such as macrophages, that can preferentially take up the LNPs, as described in 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 by reference in its entirety.

[0029] Wounds can be treated with various amounts and various forms of HoxA3. In some embodiments, HoxA3 can be applied as protein, peptide, or coding sequence. In the embodiment where HoxA3 is added as a coding sequence, the coding sequence can be a nucleic acid, for example, RNA or DNA. In one embodiment, the coding sequence is RNA, for example, mRNA.

[0030] The HoxA3 formulation applied to the wound can take a variety of 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, e.g., greater than about 45%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95%, and less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% identity. In some embodiments, the nucleic acid can be an RNA sequence, e.g., an mRNA sequence. In some embodiments, the transcription construct can be a virus, a plasmid, or other suitable vector. In one embodiment, the transcription construct can be a plasmid, which can be applied to a solid or semi-solid support prior to application to the wound. In one example, the transcription construct, e.g., a plasmid, can be impregnated into a wafer. In these embodiments, the wafer can be impregnated with 1 to 100 μg of the transcription construct.

[0031] The HoxA3 formulation can be delivered to cells and tissues as a nucleic acid coding sequence. The coding sequence can be RNA or DNA. If the coding sequence is DNA, the nucleic acid can be delivered in a lipid nanoparticle composition, a viral vector, or a plasmid. If the coding sequence is mRNA, the mRNA can be delivered in an RNA-based viral particle or lipid nanoparticle composition. In some embodiments, the lipid nanoparticles can be prepared by mixing a solution containing preformed lipid nanoparticles with a solution containing mRNA to form mRNA lipid nanoparticles.

[0032] Messenger RNA (mRNA) is becoming an increasingly useful active ingredient for the treatment of various diseases. mRNA-based therapies involve the administration of messenger RNA to patients in need of treatment. In most cases, the mRNA is delivered to the patient's cells, where it encodes the protein encoded by the mRNA. To facilitate this delivery, lipid nanoparticles are commonly used. In most cases, lipid nanoparticles encapsulate the therapeutic mRNA for efficient in vivo delivery of the mRNA.

[0033] In some embodiments, altering the lipid composition can affect the intracellular delivery and / or expression of mRNA coding sequences in various types of mammalian cells and tissues (e.g., mammalian epithelial cells). In other embodiments, the disclosed particles, lipid nanoparticles, or viral particles can include one or more sequences, tags, receptors, etc. that assist in targeting the particles to appropriate cells, e.g., epithelial cells.

[0034] Disclosed herein is a viral vector comprising one or more nucleic acids encoding HoxA3. Adeno-associated virus (AAV) is a small virus that infects humans and belongs to the genus Dependoparvovirus (family Parvoviridae). AAV is a replication-defective, non-enveloped virus measuring approximately 20 nm, containing a linear, single-stranded DNA (ssDNA) genome of approximately 4.8 kilobases (kb). AAV has been designed to deliver various genes to cells and tissues. In some embodiments, AAV comprises a nucleic acid sequence encoding one or more peroxidases.

[0035] Applicator An applicator can be used to contain or hold the HoxA3 formulation. In some embodiments, the applicator is solid or semi-solid. In many embodiments, the disclosed HoxA3 formulation may be added directly to the applicator. In one embodiment, the applicator is a patch or wafer, which can be configured to adhere to and / or contact cells or tissue at or near the wound. In some embodiments, one or more doses can be applied and / or reapplied directly to the applicator. In one embodiment, the disclosed applicator can be fabricated at least in part from methylcellulose. Alternatively, the disclosed HoxA3 composition can be incorporated into a cream or ointment for topical application.

[0036] Promoting wound repair The disclosed treatment methods may result in accelerated wound repair in a patient compared to a similar untreated wound in a control patient. In some embodiments, the treated patient may achieve 20% to over 400% healing at a time point after injury and / or after initiation of treatment. In some embodiments, this time point may be from about 1 week to about 4 weeks, for example, about 4 weeks. In one embodiment, for example, a treated patient's wound may be about 70%, 80%, 90%, or even 95% healed at 2 weeks, while an untreated patient's wound may only be about 50% healed.

[0037] The disclosed treatment methods may cause wounds to heal more quickly than untreated wounds. In some embodiments, treated wounds may heal approximately one day to two weeks faster, depending on the size of the wound. In one embodiment, a wound of approximately 6 mm in diameter may heal more than one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, or thirteen days, and less than two weeks, thirteen days, twelve days, eleven days, ten days, nine days, eight days, seven days, six days, five days, four days, three days, or two days faster than an untreated wound of approximately 6 mm in diameter.

[0038] Accelerated wound healing can be measured in various ways. In one embodiment, as disclosed above, the measurement can be by macroscopic measurement of various lengths / dimensions of the wound. In these embodiments, the measurement method can be that described in FIG. 1. In other cases, the measurement can be 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 certain time point, greater than about 400% of the treated wound can be re-epithelialized compared to the untreated wound. In one example, at that time point, about 80% of the treated wound and about 20% of the untreated wound can be re-epithelialized. As disclosed above, that time point can be from about 1 week to about 4 weeks.

[0039] The disclosed therapeutic methods can be applied in a variety of forms and with a variety of application schedules. In some embodiments, the disclosed doses can be applied immediately after wounding, or several hours or days after wounding. In many embodiments, dosing can be repeated to achieve 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses. When the composition is a plasmid applied via an applicator such as a patch or wafer, the patch or wafer can contain about 0.01 to 50 μg of the plasmid, e.g., 2.0 μg, 1.0 μg, 5 μg, or 25 μg.

[0040] Example Example 1 - HoxA3 promotes wound healing in aging patients As disclosed below, Applicants observed similarities in the wound healing responses of wild-type aged mice and diabetic young mice. Applicants hypothesized that HoxA3 might be effective in promoting wound healing and designed studies to test this hypothesis. In these studies, mice were wounded as described above and treated with three doses of 25 μg of HoxA3 (treatments were administered once at the time of wounding and once weekly thereafter). Male and female mice were studied separately (n=10). Mice were observed weekly using digital calipers to measure wound size. All wounds closed on day 21. No significant differences were observed in wound closure at the time of wounding or one week after wounding. However, two weeks after wounding, treated wounds consistently healed to over 90%, whereas untreated wounds consistently healed to only approximately 50%. After three weeks, the mice were sacrificed and blood glucose levels were examined, revealing no significant differences between the groups. None of the groups were diabetic.

[0041] Figure 7 shows that treatment with HoxA3 promotes wound healing in non-diabetic aged mice. In these studies, wounds were created using a dual punch in male (n = 10) and female (n = 10) aged (18-month-old) mice. Wound size was measured weekly. Wounds healed in 3 weeks. By 2 weeks, HoxA3 mice, both male and female, showed significant healing. Blood glucose levels measured at week 3 were below 180 mg / dL. For all figures, ns indicates "not significant," **** indicates "p < 0.0001," and statistics were determined by one-way analysis of variance. Data represent the mean, and error bars indicate the standard error of the mean (SEM).

[0042] Example 2 - Topical application of HoxA3 plasmid promotes wound healing at the microscopic level in diabetic mice Diabetic mice (db / db) were randomized to undergo punch wounds during the acclimation period, subsequently monitored for wound size, and sacrificed on day 10 post-injury to ensure safety. One mouse died during the acclimation period, leaving nine mice in Group 1 (control) and ten mice in Group 2 (treatment). Treatment included GFP (control) or HoxA3 plasmid wafers (25 μg of DNA plasmid in 1% methylcellulose, dried on wax paper for 5 hours, then applied as a 50 μL patch) on days 0, 2, 4, 7, and 9. Splints and sutures were replaced as needed throughout the study.

[0043] Although the intended primary outcome was histology, wounds were measured on three mice representing each group on days 0, 2, 4, 7, and 9. All mice were weighed on days 0, 2, 4, 7, and 9. On day 10, each group was euthanized without suffering, and the wound sites were harvested. For each mouse, one wound was fixed in 10% neutral buffered formalin and the other was flash-frozen. Fixed wounds were embedded in paraffin blocks for microscopic examination and scoring by a blinded pathologist.

[0044] At day 9, the last recorded data point before sacrifice, the HoxA3 cohort rapidly closed from approximately 85% open to approximately 65% ​​open, while the control group remained approximately 80% open. Day 9 was the first day of statistical significance, but this fact was coincidental because results were not tabulated until after sacrifice and day 10 was simply chosen as the midpoint of untreated healing based on a previous pilot experiment (not shown).

[0045] Anonymized wounds were analyzed and scored by a board-certified pathologist at 400x magnification. While no statistically significant differences were observed in vascular density, maximum epithelial thickness, or inflammation, HoxA3 dramatically promoted granulation tissue formation, with nearly 80% of HoxA3-treated wounds completely re-epithelialized compared with only 20% in the control group (Figure 3).

[0046] Figure 3 shows histopathology scores for diabetic wound healing over 9 days. Fibroblast density, granulation tissue formation, and complete epithelialization rate for the cohort (these are global counts; no error bars are present) were statistically significantly favored by HoxA3. The definition of histopathology scoring criteria in the Materials and Methods section is also shown. The first column in Figure 4 annotates patent vessels with red blood cells (RBCs) and granulation formation in the HoxA3 sample, which are absent in the GFP sample. The second column, Figure 4B, shows the density of fibroblasts, which indicates a high concentration of extracellular matrix material. Figure 4C shows the HoxA3 reepithelialized wound and the open control wound.

[0047] A dose study was conducted to determine the minimum administration frequency and dosage of the plasmid required to induce early healing. The resulting minimum threshold was intended to be used to design additional experiments with improved treatments by determining the optimal dynamic range. Mice were wounded and treated with a 25 μg plasmid patch. Wound size was measured every two days using a digital caliper. Mice received a total of three administrations (once weekly), a single administration, or no treatment as a control. Three administrations (once weekly) were effective, but a single administration was not (Figure 5A).

[0048] Next, we evaluated the effect of dose by varying the plasmid content in the gel patch over three doses. Observations were performed daily to improve temporal resolution. Cohorts included untreated mice, mice treated with 25 μg wafers (corresponding to the most effective dosing frequency approach), and mice treated with intermediate doses including 0.2 μg, 1 μg, and 5 μg. As shown in Figure 5B, the 25 μg treatment, unlike the other treatments, resulted in almost complete healing by day 15.

[0049] As shown in Figure 5B, the 25 μg dose, unlike the other treatments, resulted in almost complete healing by day 15. Specifically, Figure A shows the results of male mice (n=5) with wounds created as described above and treated with wafers once a week (3 times total, with one change at the time of wounding), as well as a no-treatment control. A second study to vary the plasmid content was performed using a once-a-week protocol. In this study, male mice (n=5) were wounded and treated with 25 μg or lower doses. HoxA3 topical plasmid patches promote wound healing in non-diabetic aging mice.

[0050] Aging is associated with a phenomenon known as "inflammatory aging." This occurs because the immune system of aging mammals gradually shifts with age toward a state of increasingly chronic, low-level immune activation. Wound healing is known to be slower in aging human patients than in younger individuals. 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 wild-type aged mice, wild-type young mice, and diabetic young mice (12 weeks old), wild-type young mice healed rapidly, taking approximately one week. In contrast, db / db young mice and wild-type aged mice were similarly only 20% healed by day 7. Based on the similar progression of the two populations observed in this pilot study, applicants hypothesized that non-diabetic aged mice might benefit from HoxA3 plasmid.

[0051] Figure 6 shows the results of wounded mice observed for 7 days. In this study, cohorts included wild-type aged mice (18 months old), 12-week-old wild-type young mice, or db / db young mice (12 weeks old). By day 7, the young mice showed approximately 80% recovery, while the aged and db / db mice showed only approximately 20% recovery. Figure B shows a column plot analysis of the final day of wound measurement. ns indicates "not significant," **** indicates "p<0.0001," and statistics were determined by one-way analysis of variance. Data represent the mean, and error bars indicate the standard error of the mean (SEM).

[0052] Example 3 - Relative wound closure of DMEC treated with various HoxA3 interventions Figure 9 shows the results of an in vitro scratch / motility assay in which HoxA3 was fused to the cell penetration motifs of HIV-TAT or Penatrin, or not fused to the cell penetration motif. Briefly, a scratch was created to disrupt adherent cells, and then cell motility back into the scratch area was measured as an indicator of wound closure. Untreated DMECs were less motile and therefore filled the scratch more slowly than DMECs treated with HoxA3 protein. HoxA3 protein contained either the HIV-TAT cell-penetrating peptide (CPP) motif or the Penatrin SV40 CPP. TAT was more effective than Penatrin SV40 at a dose equivalent to that of Penatrin SV40. TAT-HoxA3 showed a dose-dependent increase in motility at a dose of 200 nM, exceeding that at a dose of 50 nM, and the 50 nM dose itself was superior to untreated cells.

[0053] Primary dermal microvascular endothelial cells (DMEC cells) (ATCC, PCS-110-010) were seeded at a density of 12,000 cells per well in 96-well plates. Cells were cultured overnight in the manufacturer's recommended medium to allow attachment. The medium was removed, and the growth medium was replenished with fresh medium containing the listed HoxA3 therapeutic agent (or vehicle control) and incubated for 4 hours. After 4 hours of incubation, wells were scratched with the tip of a P200 pipette. Wound size over time was measured using an IncuCyte S3 with a kinetic scratch wound module.

[0054] Example 4 - Reduction of foam cell phenotype exhibited by 264.7 macrophage cells treated with CPP-HoxA3(WT) Figure 10 shows a study of foam cell formation in macrophages treated or untreated with HoxA3. Briefly, Raw264.7 cells, a macrophage lineage, were treated with 100 μg / mL of oxidized low-density lipoprotein (LDLP) simultaneously with the listed HoxA3 treatments (or vehicle control). Cells were cultured with lipids and HoxA3 in DMEM for a total of 48 hours. After the 4-hour incubation, the media was removed from the cells and washed with PBD before staining with Oil Red O.

[0055] Untreated macrophages showed accumulation of oil deposits, indicative of a foam cell (FC) phenotype. Co-incubation of HoxA3 protein with either the HIV-TAT cell-penetrating peptide (CPP) motif or the Penatrin-SV40 CPP motif attenuated the formation of oil deposits, allowing cells to retain the morphology of OxLDL-untreated cells. The effect was dose-dependent, with 200 nM TAT-HoxA3 being superior to 50 nM. Per dose, HIV-TAT was more effective than Penatrin-SV40. However, 200 nM Penatrin-SV40-HoxA3 was also effective. These results indicate that exposure to HoxA3 protein prevents macrophages from transforming into foam cells in response to treatment with oxidized LDL.

[0056] Oil Red O working solution was prepared by dissolving Oil Red O powder (Sigma-Aldrich Cat# MAK194) at a concentration of 30 mg / mL in 100% isopropyl alcohol. Formalin (10%) was pipetted onto the cells and gently mixed by swirling. The cells were incubated in formalin for 30 minutes. The formalin was then discarded, and the cells were washed twice with water. 60% isopropanol was then added and the cells were incubated for 5 minutes. After discarding the 60% isopropanol, Oil Red O working solution was evenly coated onto the cells and incubated for 10-20 minutes. The Oil Red O solution was then discarded, and the cells were washed three times with water until no excess staining was observed. Hematoxylin was added to the cells and incubated for 1 minute. The hematoxylin was then discarded, and the cells were washed three times with water. The cells were then covered with water and observed under a 40x objective.

[0057] Example 5 - Wound closure over time with WT or mutant HoxA3 The HoxA3 plasmid was modified to generate a modified HoxA3 protein with substitutions L175D, E176K, and E178K. The resulting plasmid is referred to as the HoxA3(m) plasmid. The ability of the HoxA3(m) plasmid to promote wound closure in diabetic mice was compared to that of wild-type HoxA3 described above. Figure 11 shows the results of these studies, demonstrating approximately 13% faster wound closure times, as measured by the average wound diameter at each time point, despite using the best-known wild-type HoxA3 administration protocol from Applicants' previous studies.

[0058] material and method method Diabetic mice: B6.Cg-m + / + Leprdb / J male mice (strain code: 000697), 8–12 weeks old at the start of the study, were obtained from Jackson Laboratories. Mice were housed singly and allowed to acclimate locally for approximately 1 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 had free access to water and food throughout the study. Prior to surgical wound creation, mice were randomly assigned to two treatment groups based on blood glucose levels.

[0059] Aging mice: C57BL / 6J male and female mice (77 weeks old) were obtained from Jackson Laboratories. Mice were individually housed and allowed to acclimate on-site for approximately one week prior to study activity. Mice were housed as described above. Prior to surgical wound creation, mice were randomly assigned to two treatment groups based on blood glucose levels.

[0060] Wounding: All mice underwent two 6 mm full-thickness wounds at the shoulder using a punch. These wounds were splinted with silicone splints, non-absorbable sutures, and surgical adhesive to prevent skin contracture. A sample video of the wound creation is included in the accompanying data. Mice were treated with a non-steroidal anti-inflammatory drug once daily for three days after wound creation.

[0061] Treatment: Methylcellulose wafers were prepared by mixing 25 μg (or less) of HoxA3 plasmid DNA into 1% methylcellulose and depositing 50 μL droplets onto parafilm using a forced-displacement pipette. A sample video of this wafer preparation is included in the accompanying data. Wafers were allowed to air dry for up to 5 hours before application, allowing them to reach a solid consistency. Plasmid wafers were prepared immediately prior to treatment. The sequence of the HoxA3 plasmid is provided in the Supplementary Information.

[0062] Wound size measurements: Wounds were measured under isoflurane anesthesia using digital calipers. Measurements of each wound were taken according to the following four orientations (see Figure 1) and averaged for a single mouse. Duplicate wounds in the same mouse were generally uncorrelated and therefore treated as statistically independent.

[0063] Histology: Wounds and surrounding areas were excised from mice and fixed in 10% neutral-buffered formalin for 48 hours. Wounds were then dehydrated by immersion in the following solutions: 70% ethanol (two hourly changes), 85% ethanol (two hourly changes), 95% ethanol (two hourly changes), 100% ethanol (two hourly changes), Histoclear xylene substitute for 3 hours, and liquid paraffin for 3 hours (a total of three hourly changes). The tissues were placed in molds containing liquid paraffin wax and allowed to solidify on ice. Paraffin blocks were stored at room temperature until sectioning. Tissues were cut at 5 μm thickness and attached to microscope slides. Samples were placed on a 38°C slide warmer until moisture evaporated. The paraffin wax was melted at 65°C for 20 minutes for deparaffinization, followed by staining.

[0064] Mason's Trichome: Immediately after deparaffinizing and rehydrating the slides with distilled water, they were incubated in Boyne's solution at 55°C for 60 minutes. After the 60-minute incubation in Boyne's solution, the slides were cooled for 10 minutes. After equilibrating to room temperature, the tissue was rinsed with water until completely transparent. The slides were then incubated in hematoxylin for 5 minutes and then immediately rinsed with tap water for 2 minutes. The slides were then incubated in Biebrich scarlet / acid fuchsin solution for 15 minutes and then immediately rinsed with tap water. The slides were then incubated in phosphomolybdic / phosphotungstic acid solution for 15 minutes and then immediately rinsed with tap water. Without washing, the slides were incubated in analine blue solution for 10 minutes and then washed with deionized water. The slides were then incubated in 1% acetic acid solution for 5 minutes. After incubation in acetic acid, slides were rapidly cleared with two changes of 95% ethanol followed by a rapid exchange into a xylene substitute. Slides were mounted in toluene mounting fluid.

[0065] H+E staining: Slides were deparaffinized by incubating them twice (5 minutes each) in Histoclear (a xylene substitute), two changes of 100% ethanol, and two changes of 95% ethanol (3 minutes and 2 minutes, respectively). Slides were then rehydrated in tap water for 1 minute. Slides were then stained with hematoxylin for 2 minutes and then immediately rinsed twice in tap water for 45 minutes each. Slides were then immersed in cyanide reagent for 15 seconds. After the cyanide reagent, slides were washed twice in tap water for 30 seconds each. Slides were then washed in 100% ethanol for 10 seconds, followed by a 3-minute incubation in eosin Y. After incubation in eosin Y, slides were dehydrated in four changes of 100% ethanol. Slides were cleared by successive incubations in Histoclear for 3 minutes each. Slides were mounted in toluene mounting solution.

[0066] Histopathological Scoring: De-identified samples were scored by a third-party board-certified pathologist. Criteria for pathological scoring of histological slides were as follows:

[0067] Vascular density: the number of endothelialized blood vessels containing visible blood cells.

[0068] Maximum epidermal thickness: The number of cell layers of the epidermis in the area of ​​a wound or open lesion.

[0069] Fibroblast density: determined by cell density measured with Mason's Trichrome and the surrounding extracellular matrix.

[0070] Granulation tissue formation: The presence of newly formed blood vessels.

[0071] Global inflammation was also assessed. Vascular density and maximum epithelial thickness were measured as total counts, while other measures were scored as a relative severity score ranging from 1 to 5.

[0072] Statistics and Data: Anonymized raw data were sent to a third-party statistician. Each figure is annotated with the relevant statistical analysis. Annotated data preparation and statistical analysis were performed using GraphPad Prism 9.

[0073] Example 6 - HoxA3 Protein Production TAT-HoxA3(m) and TAT-HoxA3(wt) were produced, purified, and tested in culture. Because production of HoxA3 in bacterial systems can be difficult in some cases, Applicants expressed the disclosed proteins in 293T cells.

[0074] Briefly, transfected cells were harvested, lysed, and clarified. HoxA3 protein was purified sequentially using metal affinity, cation exchange, and size exclusion. The protein production procedure is detailed in Figure 13.

[0075] Protein purity was confirmed using chromatography (Figure 14, bottom left corner) and gel electrophoresis (Figure 14, top right corner). Collected and pooled fractions from size exclusion chromatography were analyzed by gel electrophoresis, which showed a strong band at the correct molecular weight of approximately 50 kDa or less. It is important to note that Western blotting of the samples was performed after concentrating, freezing, and thawing the protein. This means that a significant amount of protein at the correct molecular weight is present after freeze / thaw.

[0076] The bioactivity of HoxA3 protein produced in 293 cells was confirmed in a relevant model cell line: primary dermal human microvascular endothelial cells (hDMECs) derived from a 55-year-old patient. Elderly hDMECs were found to grow significantly slower than younger hDMECs, requiring several cycles of seeding optimization. Finally, 8,000 cells were seeded into a 6-well plate and allowed to adhere overnight before the start of the assay. A scratch wound was created in the monolayer using a p200 pipette tip, and closure was then monitored over time. Images of the scratch area were captured at regular intervals over a 4-hour period.

[0077] To quantify the rate of wound closure, the density of the "wound" was measured at confluency over time. Analysis was completed using an IncuCyte S3 with a Kinetic Scratch Wound Analysis module.

[0078] In a dose-dependent manner, untreated cells migrated and filled the void more slowly than HoxA3-treated DMEC (FIG. 15).

[0079] Example 7 - Effect of HoxA3 on polarization of aged human macrophages Human peripheral blood-derived monocytes were differentiated into M0 macrophages by culturing them in ImmunoCult™-SF Macrophage Medium supplemented with M-CSF for 6 days 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 IL-6, IL-12p70, and TNFα cytokine production by LPS / IFN-γ-activated M1 macrophages were assessed by preincubating M-CSF-derived M0 macrophages with test substances for 30 minutes before adding stimuli for 2 days of activation. Secretion of IL-6, IL-12p70, and TNFα in cell culture supernatants was characterized using 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).

[0080] Cell Thawing, Plating, and MO Differentiation (Day 0): At the initiation of culture (Day 0), frozen human peripheral blood monocytes from a single donor were thawed, washed, and assessed for cell viability using a Cell Profiler (Nexcelom Cellometer Auto 2000 Cell Profiler) and AO / PI dead cell labeling reagents as outlined above. Cells were plated at 1 x 10 in ImmunoCult™-SF Macrophage Medium containing 50 ng / mL M-CSF (Shenandoah, Cat# 100-03-10UG). 6 Resuspend the cells at 1 × 10 cells / mL and plate them in a 96-well tissue culture-treated plate (Costar, Cat# 3595). 5 Cells were plated per well (100 μL / well). Triplicate cultures were set up for each condition.

[0081] Medium replenishment and supplementation (Day 3): Fresh ImmunoCult™-SF Macrophage Medium containing 50 ng / mL M-CSF was prepared on Day 3. Each well was covered with 50 μL (50% volume) of freshly prepared medium, mixed gently, and returned to the incubator for an additional 3 days at 37°C and 5% CO2.

[0082] Macrophage stimulation and test article treatment (Day 6): Vehicle control and test article working stock solutions (5x stock) were prepared as described in Example 6 and added to the appropriate day 6 treatment cultures (40 μL / well). The solvent was 50 mM HEPES (pH 7.5 at 4°C), 500 mM NaCl, and 5% glycerol. Cells were incubated at 37°C and 5% CO2 for 30 minutes in the presence of test article or vehicle. After 30 minutes, 10 μL / well of a 20x diluted stock solution of LPS (Sigma, Cat# L6529) and IFN-γ (Shenandoah, Cat# 100-77-20UG) were added to the appropriate wells to achieve final concentrations of 10 ng / mL LPS and 50 ng / mL IFN-γ in a final volume of 200 μL / well. Cultures were incubated at 37°C and 5% CO2 for 2 days. For unstimulated control cultures (see the leftmost bar in Figure 16 panels), 50 μL / well of ImmunoCult™ SF Macrophage Medium was added in place of LPS / IFN-γ and test substance or vehicle. All conditions were set up in triplicate.

[0083] 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) and centrifuged at 1500 rpm for 5 minutes to clarify the supernatant, which was then carefully transferred in 2-90 μL aliquots to a new 96-well round-bottom plate. The supernatant plate was sealed with parafilm and stored at -80°C until ready for MSD analysis.

[0084] One replicate of the 40 μg / mL and 10 μg / mL conditions for TAT-HoxA3(wt) was heavily contaminated with bacteria and was not collected for downstream analysis. For MESO SCALE DISCOVEY analysis of cytokine expression in culture supernatants, the V-Plex Proinflammatory 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 L-6, IL-12p70, and TNFα, on the day of measurement, culture supernatant samples were brought to room temperature and assayed at a 1:2 dilution. Because the TNFα sample showed concentrations above the standard curve at a 1:2 dilution, the TNFα assay was repeated using a 1:50 dilution in PBS containing 1% BSA. Samples and calibrators were processed according to the kit instructions. Briefly, plates were prepared by washing three times with wash buffer (PBS + 0.05% Tween-20 [SIGMA, Cat# P1379]). Then, supernatants and calibrators were added to the plates and incubated at room temperature for 2 hours with shaking. The plates were then washed three times with wash buffer, and then detection antibodies were added and incubated at room temperature for 2 hours with shaking. The plates were then washed three times with wash buffer, 2x read buffer T was added, and the plates were read on a MESO WUICKPLEX SQ 120 Instrument. Analysis and Calculations: IL-6, IL-12p70, and TNFα concentrations were determined using MSD Discovery Workbench 4.0 software by interpolating the instrument response of each sample to the appropriate standard curve and then applying the sample dilution factor. The effect of the solvent control was determined by calculating the absolute value of cytokine expression levels for each solvent condition relative to the standard control (medium only) and expressed as a ratio of change relative to the standard control. The effect of each test substance was determined by calculating the absolute value of cytokine expression relative to the best-matched vehicle control and expressed as a percentage of the change from the standard control. Vehicle and test substance conditions were graphed using GraphPad Prism version 9.1.

[0085] IL-12p70 showed dose-dependent suppression in response to HoxA3(m) (shown) and HoxA3(wt) (not shown) relative to vehicle and buffer controls. At higher drug concentrations (primarily HoxA3(m) or vehicle, which was found to be effective at higher concentrations), IL-12p70 secretion was suppressed relative to vehicle, suggesting vehicle toxicity; however, the suppression of IL-12p70 between HoxA3-treated and vehicle samples was still statistically significant at these doses. IL-6 expression was unaffected by TAT-HoxA3(m) (shown) or TAT-HoxA(wt) (not shown). Interpretation of TNFα secretion was difficult because the ratio of TNFα secretion with HoxA3 treatment to vehicle decreased at higher doses, but absolute secretion levels were unaffected. Secretion profiles are shown in Figure 16, panels A, B, and C. In each figure, the leftmost bar represents secretion from unstimulated cells, essentially showing no chemokine release due to either vehicle or HoxA3. Note that the black bar to the right of the bar showing secretion from unstimulated cells shows secretion upon stimulation, but not due to vehicle or HoxA3.

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[0087] While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description. As will become apparent, the present invention can be modified in various obvious aspects without departing from the spirit and scope of the present invention. Accordingly, the detailed description is to be regarded as illustrative in nature and not as restrictive.

[0088] All references disclosed herein, whether patent or non-patent, are hereby incorporated by reference as if each were incorporated by citation in its entirety. In the event of a conflict between a reference and the specification, the specification, including definitions, will control.

[0089] Although the present disclosure has been described with a certain degree of particularity, it is understood that the disclosure has been made for purposes of illustration and that changes in detail or structure may be made without departing from the spirit of the disclosure as defined in the appended claims. [Table 1-1] [Table 1-2] [Table 1-3]

Claims

1. 1. A method of promoting wound repair in a patient, comprising: Identifying the patient's wound; applying a composition containing HoxA3 to said wound; and allowing the wound to heal; A method comprising:

2. The method of claim 1 , wherein the HoxA3 is selected from a nucleic acid sequence and an amino acid sequence.

3. The method according to claim 1 or 2, wherein the HoxA3 is an mRNA encoding HoxA3.

4. The method of any one of claims 1 to 3, wherein the HoxA3 is contained on a plasmid.

5. The method of claim 1 , wherein the HoxA3 comprises a portion for penetrating cells.

6. 6. The method of claim 1, wherein the HoxA3 is at least about 49% identical to wild-type HoxA3.

7. The method of claim 1 , wherein the composition comprises lipid nanoparticles comprising the HoxA3.

8. The method of any one of claims 1 to 7, wherein the patient is a human.

9. 9. The method of any one of claims 1 to 8, wherein the patient is selected from one or more of: at least about 60 years old, diabetic, and suffering from immunosenescence.

10. 10. The method of any one of claims 1 to 9, wherein the patient is an amputee.

11. 1. A system for promoting wound repair in a patient, comprising: an applicator; and a composition containing HoxA3; Including, the system.

12. The system of claim 11 , wherein the HoxA3 is a coding sequence.

13. The system according to claim 11 or 12, wherein the HoxA3 is an mRNA encoding HoxA3.

14. 13. The system of claim 11 or 12, wherein the applicator is solid or semi-solid.

15. 15. The system of any one of claims 11 to 14, wherein the applicator comprises methylcellulose.

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

17. 17. The method of any one of claims 11 to 16, wherein the HoxA3 is selected from a nucleic acid sequence and an amino acid sequence.

18. 18. The method of any one of claims 11 to 17, wherein the HoxA3 is an mRNA encoding HoxA3.

19. 19. The method of any one of claims 11 to 18, wherein the HoxA3 is contained on a plasmid.

20. 20. The method of any one of claims 11 to 19, wherein the HoxA3 comprises a moiety for penetrating a cell.

21. 21. The method of any one of claims 11 to 20, wherein the HoxA3 is at least about 49% identical to wild-type HoxA3.

22. 23. The method of any one of claims 11 to 22, wherein the composition comprises lipid nanoparticles comprising the HoxA3.

23. A composition for treating atherosclerotic lesions, comprising HoxA3.

24. A composition for assisting wound closure in a subject having a wound, comprising HoxA3.

25. 25. The composition of claim 24, wherein the HoxA3 is selected from wild-type HoxA3 or modified HoxA3.

26. 26. The composition of claim 24 or 25, wherein the HoxA3 is a modified HoxA3.

27. 27. The composition of any one of claims 24 to 26, wherein the HoxA3 is modified to include one or more substitutions selected from L175D, E176K, and E178K.

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

29. 30. A composition according to any one of claims 24 to 28 for use in the treatment of one or more of diabetic wounds, ageing wounds, stump wounds and atherosclerosis.

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

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

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

33. 30. The composition of claim 29 for use in the treatment of atherosclerosis.

34. 1. A composition for use in promoting wound repair in a subject in need thereof, comprising: A modified HoxA3 molecule; and Pharmaceutically Acceptable Carriers A composition comprising:

35. 35. The composition of claim 34, wherein the modified HoxA3 molecule is selected from a protein, a nucleic acid, an mRNA, a plasmid, and a vector.

36. 36. The composition of claim 34 or 35, wherein the modified HoxA3 molecule comprises or encodes a protein comprising one or more substitutions selected from L175D, E176K, and E178K.

37. 37. The composition of any one of claims 34 to 36, wherein the modified HoxA3 comprises a cell-penetrating domain.

38. 38. The composition of any one of claims 34 to 37, wherein the modified HoxA3 is contained in a lipid nanoparticle.

39. 39. The composition of any one of claims 34 to 38, wherein the subject is a human.

40. 40. The composition of any one of claims 34 to 39, wherein the subject is selected from one or more of: at least about 60 years old, has diabetes, and suffers from immunosenescence.

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

42. 42. The composition of claim 41, wherein the applicator comprises methylcellulose.

43. 42. The composition of claim 41, wherein the applicator is a patch configured to contact the wound.

44. 44. Any one of claims 1 to 43, wherein Hox is selected from HoxA3, HoxD3, HoxB3, or HoxC3.

45. 45. The method or composition of claim 44, wherein the amino acid sequence of Hox comprises one or more amino acid substitutions at L175D, E176K and E178K in HoxA3, or the equivalent positions in HoxD3, HoxB3, or HoxC3.

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