ABCB5-targeted stem cell reprogramming and Anti-aging strategies

Abstract

Methods and compositions for rejuvenating and reprogramming stem cells are disclosed. The methods involve administering to a subject an ABCB5 targeted composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload comprised of an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor in an effective amount to reprogram and rejuvenate ABCB5+ stem cells in the subject. The compositions include anti-ABCB5 antibody conjugated to a therapeutic payload comprising an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor.

Description

[0001] ABCB5-TARGETED STEM CELL REPROGRAMMING AND ANTI-AGING STRATEGIES

[0002] RELATED APPLICATION

[0003] This application claims the benefit of the filing date under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 592,353, filed on October 23, 2023, the entire contents of which is incorporated herein by reference in its entirety.

[0004] FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under Grant number EY025794 awarded by The National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] ABCB5 is a multidrug resistance (MDR) mediator expressed in diverse human malignancies, where it is specifically overexpressed on therapy-resistant tumor subpopulations previously found to represent CSC. ABCB5 confers cancer cell drug resistance to chemotherapeutic agents such as doxorubicin and 5 -fluorouracil (5-FU). ABCB5+ stem cells are also found in normal tissues and have a role in immune modulation, tissue regeneration and aging. Regenerative medicine involves the repair, regeneration, maintenance, and replacement of tissues and organs using exogenous materials such as scaffolds. The scaffolds may be seeded with cells, such as primary cells or stem cells, and numerous factors to encourage tissue growth. However, a number of challenges remain in the design of appropriate material for regenerative medicine and tissue engineering.

[0008] SUMMARY

[0009] Stem cell therapeutics is a rapidly advancing field of medicine that utilizes or targets stem cells for the treatment of various diseases and injuries. Stem cells are cells that have not yet terminally differentiated and thus have the potential to develop into different cell types in the body. Additionally, a unique characteristic of stem cells is their ability to self-renew, thus maintaining a pool of undifferentiated cells that can replenish differentiated cells as they die. For example, limbal stem cells (LSCs), which are located in the corneal limbus of the eye and play a crucial role in maintaining the health and function of the cornea, must be periodically replaced for persistent support of corneal epithelial cells, which are necessary for vision. LSCs replenish themselves via self-renewal. Some populations of cells are relatively short-lived and turn over quickly, while stem cells are long-lived and critical for maintaining populations of more short-lived progenitor cells or terminally differentiated cells. In certain disease states, replacing cells that are dying, degenerating, or malfunctioning using stem cells represents a promising avenue of therapy. To achieve this, stem cells can be targeted for epigenetic reprogramming to promote a particular cellular phenotype. However, current techniques to induce epigenetic reprogramming in specific cell populations target most cells generally and indiscriminately. This poses a problem where therapies targeting long-lived stem cells are necessary or beneficial, as targeting methods often may also or even preferentially affect shortlived cells, which proliferate and differentiate more readily.

[0010] Accordingly, in some aspects, the present disclosure provides a composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload comprising an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor.

[0011] In some embodiments the therapeutic payload comprises a nanoparticle comprising the epigenetic reprogramming factor or factors or nucleic acid encoding an epigenetic reprogramming factor or factors. In some embodiments the nanoparticle is a polymeric nanoparticle. In some embodiments the nanoparticle is a lipid nanoparticle. In some embodiments the nanoparticle is a liposome.

[0012] In some embodiments the epigenetic reprogramming factors comprise OCT-4, SOX2, and / or KLF4. In some embodiments the epigenetic reprogramming factors comprise OSK (OCT-4, SOX2, and KLF4). In some embodiments the OSK is linked directly to the anti- ABCB5 antibody.

[0013] In some embodiments the nanoparticle is linked to the anti-ABCB5 antibody.

[0014] In some embodiments the therapeutic payload comprises a viral vector comprising the nucleic acid encoding an epigenetic reprogramming factor or factors. In some embodiments the viral vector is selected from a lentivirus vector, a retrovirus vector, an adenovirus vector, an alphavirus vector, a vaccinia virus vector, and an adeno-associated virus vector (AAV). In some embodiments the viral vector is AAV. In some embodiments the viral vector is linked to the anti-ABCB5 antibody through a linker and wherein the linker is an endonuclease or an endonuclease domain. In some embodiments the linker is a HUH-endonuclease or a HUH- endonuclease domain. In some embodiments the nucleic acid encoding the epigenetic reprogramming factor comprises a nucleotide sequence encoding OCT-4, a nucleotide sequence encoding SOX2, and / or a nucleotide sequence encoding KLF4.

[0015] In some embodiments the nucleic acid encoding the epigenetic reprogramming factors comprises a regulatory element. In some embodiments the regulatory element is an inducible promoter or enhancer. In some embodiments the inducible promoter or enhancer is an ABCB5 promoter or an ABCB5 enhancer. In some embodiments, the ABCB5 promoter comprises a sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, the ABCB5 promoter comprises a sequence of SEQ ID NO: 1. In some embodiments, the ABCB5 enhancer comprises a sequence having at least 90% identity to SEQ ID NO: 2. In some embodiments, the ABCB5 enhancer comprises a sequence of SEQ ID NO: 2.

[0016] In some embodiments a pharmaceutical composition comprising any of the compositions disclosed herein with a pharmaceutically acceptable excipient is provided.

[0017] In some aspects a method comprising administering to a subject an ABCB5 targeted composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload comprised of an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor or factors in an effective amount to reprogram and rejuvenate ABCB5+ stem cells in the subject is provided.

[0018] A method comprising administering to a subject a nucleic acid comprising a nucleotide sequence comprising an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding an epigenetic reprogramming factor, in an effective amount to reprogram and rejuvenate ABCB5+ stem cells in the subject.

[0019] In some embodiments, the nucleotide sequence comprising an inducible ABCB5 regulatory element comprises a sequence of SEQ ID NO: 1 or 2.

[0020] In some aspects a method comprising administering to ex vivo cell cultures containing ABCB5+ cells an ABCB5 targeted composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload comprised of an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor or factors in an effective amount to reprogram and rejuvenate ABCB5+ stem cells is provided.

[0021] In some embodiments the method is a method of treating a degenerative disease. In some embodiments the degenerative disease is a degenerative disease of the eye. In some embodiments the degenerative disease of the eye is a retinal disorder or corneal disorder. In some embodiments the retinal disorder is aged-related macular degeneration (AMD) or glaucoma. In some embodiments the comeal disorder is limbal stem cell deficiency (LSCD). In some embodiments the degenerative disease is a degenerative disease of CNS.

[0022] In some embodiments the ABCB5+ stem cells are limbal ABCB5+ stem cells. In some embodiments the ABCB5+ stem cells are dermal ABCB5+ stem cells.

[0023] In some embodiments the method is a method of treating aging. In some embodiments the method of treating aging comprises treatment of skin tissue or intestinal tissue. In some embodiments the method of treating aging comprises treatment ABCB5+ stem cell dysfunction- associated inflammaging.

[0024] In some embodiments the subject is a stem cell recipient.

[0025] In some embodiments the ABCB5 targeted composition is any of the compositions disclosed herein. In some embodiments the nucleic acid comprises a viral vector. In some embodiments the nucleic acid comprises a plasmid. In some embodiments the nucleic acid comprises a DNA or mRNA encoding an epigenetic reprogramming factor.

[0026] In some aspects a method for enhancing stem cell transplantation comprising administering to a subject an ABCB5 targeted composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload comprised of an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor in an effective amount to reprogram and rejuvenate ABCB5+ stem cells in the subject, and optionally wherein the subject is a stem cell donor and the administration is performed prior to donation to a stem cell recipient. In some embodiments the subject is a stem cell recipient and the administration is performed after donation to the recipient is provided.

[0027] In some embodiments the ABCB5 targeted composition is any one of the compositions disclosed herein. In some embodiments the nucleic acid comprises a viral vector. In some embodiments the nucleic acid comprises a plasmid. In some embodiments the nucleic acid comprises a DNA or mRNA encoding an epigenetic reprogramming factor.

[0028] In some aspects a method for preparing a population of cells, the method comprises: isolating primary cells comprised of ABCB5+ cells from human tissue; culturing the isolated primary cells in culture medium until the cells produce enough progeny to reach greater than 60% confluence of mixed cells; harvesting the mixed cells; culturing the harvested mixed cells; contacting the harvested mixed cells with an ABCB5 targeted composition comprising an anti- ABCB5 antibody conjugated to a therapeutic payload comprised of an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor. In some embodiments the nucleic acid comprises a viral vector. In some embodiments the nucleic acid comprises a plasmid. In some embodiments the nucleic acid comprises a DNA or mRNA encoding an epigenetic reprogramming factor.

[0029] Some aspects relate to a population of ABCB5+ cells, the population comprising an exogenous nucleic acid comprising a nucleotide sequence comprising an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding an epigenetic reprogramming factor. In some embodiments, the nucleotide sequence comprising an inducible ABCB5 regulatory element comprises a sequence of SEQ ID NO: 1 or 2.

[0030] In some embodiments administration of the composition increases OCT-4, SOX2, and / or KLF4 expression in the ABCB5+ harvested mixed cells relative to ABCB5+ harvested mixed cells to which the composition is not administered. In some embodiments the ABCB5 targeted composition is any one of the compositions disclosed herein.

[0031] Some aspects relate to a nucleic acid comprising an ABCB5 regulatory element, wherein the ABCB5 regulatory element is a nucleic acid having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to comprises a sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the ABCB5 regulatory element consists essentially of a sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 shows the decline of the percentage of ABCB5+ stem cells present in the human dermis as age increases.

[0034] FIG. 2 shows the effect of OSK (OCT-4, SOX2, and KLF4) reprogramming targeted to ABCB5-expressing stem cells on ABCB5 expression. ABCB5+ stem cells were untransduced, transduced with a green fluorescent protein (GFP) reporter correlating to ABCB5 expression, or transduced with the GFP reporter and OSK. Transducing ABCB5+ stem cells with OSK lead to an increase in ABCB5 expression, as indicated by a significant increase in GFP, as compared to untransduced stem cells or stem cells transduced only with the GFP reporter.

[0035] FIG. 3 is a schematic of a composition provided herein. Shown is an anti-ABCB5 monoclonal antibody linked to an adeno-associated virus (AAV) vector via an HUH- endonuclease tag. The AAV vector contains an OSK construct, which enters the target cell upon binding of the anti-ABCB5 antibody to ABCB5 on the surface of the target cell. After binding, the AAV will fuse with the target cell, releasing the OSK construct to induce OCT-4, SOX2, and KLF4 expression in the target cell. FIG. 4 contains flow cytometry plots illustrating the expression of green fluorescent protein (GFP) in transfected melanoma cells with (bottom row) or without (top row) ABCB5 treatment with the anti-ABCB5 antibody 3C2-1D12. GFP expression was driven by various ABCB5 regulatory elements, with CMV as a constitutively expressed positive control unrelated to ABCB5 expression.

[0036] FIG. 5 shows GFP expression in primary human corneal epithelial cells, driven by an inducible ABCB5b promoter, the ABCB5 -lOkb enhancer, or CMV as a constitutive positive control. The solid, unfilled curve represents GFP expression with the Shieldl inducible construct; the filled curve represents the Shieldl- control.

[0037] FIG. 6 shows GFP expression driven by Shieldl inducible ABCB5 regulatory elements in ABCB5+ derived skin cells. Top left is an example flow cytometry plot, the bottom left is the mean fluorescence intensity (MFI) of GFP expression in Shieldl- (left bars) or Shield 1+ (right bars) populations, and the right graphs show GFP fluorescence in bulk cell populations (left column) versus ABCB5+ enriched subpopulations (right column). For graphs on the right, the solid, unfilled curve represents GFP expression in Shieldl+ cells and the fill curve represents GFP expression in Shieldl- cells.

[0038] FIGs. 7A-7B illustrate the plasmids used to induce transcription of the stem cell epigenetic reprogramming OSK factors (OCT-4, SOX2, and KLF-4) and downstream effect on ABCB5 expression. A schematic of the two-plasmid system is shown on top of, and schematics for ABCB5 regulatory-element driven constructs and control constructs are shown on the left (FIG. 6A). OCT-4, SOX2, and KLF-4 protein expression resulting from the different constructs are shown in the western blot on the bottom right (FIG. 6A). Cells transfected with plasmids containing the ABCB5 TFRR driving expression of the OSK factors showed a robust increase in ABCB5 mRNA 7 days after administration of doxycycline to induce transcription (FIG. 6B, left). The cell surface expression of ABCB5 was also increased in transfected cells at 48 hours and 7 days post-induction of the OSK reprogramming factors (FIG. 6B, right).

[0039] FIGs. 8A-8F relate to the generation of an ABCB5-GFP reporter line. The construct used to encode a transgene driving GFP expression under an ABCB5 exon is shown in FIG. 8A. FIGs. 8B-8C shows MFI of GFP reporter expression and ABCB5 expression in ABCB5-gene knock-in skin cells created with CRISPR and the construct of FIG. 8A (exemplary flow plot and histogram in FIG. 8B; summary in FIG. 8C). FIG. 8D is an immunoprecipitation blot of endogenous ABCB5 using an anti-myc antibody (clone 9E10). FIGs. 8E (no puromycin) and 8F (with puromycin) show selection of ABCB5+ cells using puromycin. FIGs. 9A-9C show the plasmids and sequences used to drive OSK expression using an ABCB5 TFRR domain (FIG. 9A), the ABCB5 -lOkb Enhancer (FIG. 9B), or a Tet-on 3G domain (FIG. 9C).

[0040] FIGs. 10A-10E relate to reprogramming of dermal mesenchymal stem cells (DMSCs) following OSK treatment. AL10-AL13 each represent a different human donor sample. FIG. 10A showed MFI fold increase of ABCB5 expression. FIGs. 10B-10C shows expression of Oct4, Sox2, and KLF4 (FIG. 10B) or Sox2 and KLF4 (FIG. IOC) in transfected DMSCs. FIGs. 10D-10E show Oct4 (FIG. 10D) or Sox2 and KLF4 (FIG. 10E) in transfected DMSCs 7 days post-transfection.

[0041] DETAILED DESCRIPTION

[0042] Stem cell therapy is an innovative medical approach that harnesses the unique properties of stem cells to treat a wide range of diseases and injuries. Stem cells, which can be derived from adult tissues, possess the ability to differentiate into other cell types in the body. This regenerative potential makes them valuable for repairing damaged tissues and organs, as well as addressing conditions such as cardiovascular disease, neurodegenerative disorders, and autoimmune conditions. Stem cell therapy can involve transplantation of stem cells reprogrammed in vitro or the reprogramming of stem cells in vivo to promote a more functional phenotype.

[0043] A method for reprogramming adult stem cells is via the introduction of epigenetic reprogramming factors that are important for maintaining pluripotency and self-renewal in embryonic stem cells. For example, the transcription factors octamer-binding transcription factor 4 (OCT-4), SRY-box 2 (SOX2), Kruppel-like factor 4 (KLF4), called the OSK factors, are often used in combination to reprogram stem cells. Typically, epigenetic reprogramming factors are targeted to cells generally. It has been discovered herein that targeting epigenetic reprogramming factors to ABCB5+ stem cells has unexpected therapeutic advantages. For instance, it is demonstrated herein that aged cells can be rejuvenated, taking on properties of younger ABCB5+ stem cells, while avoiding off target effects.

[0044] Provided herein are compositions capable of targeting ABCB5+ stem cells and delivering an epigenetic reprogramming factor to the ABCB5+ stem cells. Also provided are methods for administering ABCB5 targeted compositions to a subject. Such compositions and methods can be used to epigenetically reprogram ABCB5+ stem cells to restore them to a younger phenotype and to treat aging or age-related disease.

[0045] Stem cells

[0046] Stem cells are a heterogenous population of cells that are characterized by their abilities to both self-renew and to differentiate into various specialized cell types throughout the body. They are fundamental to development, tissue repair, and maintenance of health and cognition through an organism’s life. Stem cells can be broadly categorized into long-lived stem cells, which include tissue-specific adult stem cells that help maintain and repair tissue over a lifetime, and more short-lived stem cells, which includes progenitor cells that divide and differentiate more rapidly than long-lived stem cell populations. As an individual ages, the function and fitness of long-lived stem cells declines. This presents a complication commonly associated with stem cell therapies, as adult stem cell dysfunction frequently results in (1) a scarcity of the target stem cell population that increases with age, and (2) a failure of stem cells to engraft following transplantation. Stem cell therapy that preferentially targets long-lived stem cells for epigenetic reprogramming and restores the fitness of such populations is therefore an attractive concept, especially if employed in vivo.

[0047] Stem cell therapy

[0048] Stem cell therapy is a growing field that involves the use of stem cells to treat various medical conditions and promote tissue regeneration. In some embodiments, stem cells are administered to a subject with a degenerative disease. The degenerative disease may be a neurodegenerative disease (e.g., Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, or multiple sclerosis), a cardiovascular disease (e.g., congestive heart failure or ischemic heart disease), an ophthalmic disease (e.g., corneal damage, glaucoma, or age-related macular degeneration), a lung disease (e.g., chronic obstructive pulmonary disease), a liver disease (e.g., cirrhosis or hepatitis), a kidney disease (e.g., chronic kidney disease), a muscular disease (e.g., Duchenne muscular dystrophy or Becker muscular dystrophy), or another disease. There are a number of therapeutic modalities or approaches employed in stem cell therapy, including:

[0049] Autologous stem cell therapy, wherein stem cells are derived from the patient's own body, typically from bone marrow or adipose tissue or skin. These cells are collected, processed, and then reintroduced into the patient's body to promote healing and tissue regeneration. Allogeneic stem cell therapy, in which stem cells are obtained from a donor, typically a healthy individual. These cells are carefully matched to minimize the risk of rejection. Allogeneic stem cell therapy allows for the availability of a larger pool of stem cells for treatment.

[0050] Embryonic stem cell therapy, which uses stem cells derived from embryos that can differentiate into any cell type in the body.

[0051] Induced pluripotent stem cell therapy, in which induced pluripotent stem cells (iPSCs) are generated by reprogramming adult cells, such as skin cells, to a pluripotent state, similar to embryonic stem cells. iPSCs have the potential to differentiate into various cell types and can be derived from the patient's own cells, minimizing the risk of rejection.

[0052] Mesenchymal stem cell therapy, which involves mesenchymal stem cells (MSCs), a type of adult stem cell found in various tissues, including bone marrow, adipose tissue, umbilical cord tissue, and skin. MSCs have immunomodulatory properties and can differentiate into different cell types.

[0053] Gene-modified stem cell therapy, in which stem cells are genetically modified to enhance their therapeutic properties or to correct specific genetic defects. For example, gene editing techniques like CRISPR-Cas9 can be used to edit the genes of stem cells, enabling the correction of genetic mutations before transplantation.

[0054] ABCB5+ stem cells

[0055] Long-lived stem cells can be distinguished by markers expressed on their cell surface. A protein that has been implicated in the maintenance and function of long-lived stem cells is ATP-binding cassette sub-family B member 5 (ABCB5). ABC transporters are involved in the transport of structurally diverse molecules ranging from small ions, sugars, and peptides to more complex organic molecules, and ABCB5 surface expression is used to identify long-lived stem cells that have the capacity to self-renew and to differentiate into mature cells of multiple adult cell lineages. “ABCB5+ stem cells” or “ABCB5+ cells,” as used herein, refers to such cells. In some embodiments the ABCB5+ stem cells are limbal ABCB5+ stem cells. In some embodiments the ABCB5+ stem cells are dermal ABCB5+ stem cells.

[0056] ABCB5+ stem cells have a role in tissue regeneration and aging. Regenerative medicine involves the repair, regeneration, maintenance, and replacement of tissues and organs optionally using exogenous materials such as scaffolds. Methods for enhancing tissue regeneration and repair and disruption of aging are disclosed herein. It has been discovered that an ABCB5 targeted composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload comprised of epigenetic reprogramming factors is useful in targeted treatments that promote healthy aging and / or reverse the effects of age-related diseases. While unidirectionally progressing organismal aging has generally been thought to be irreversible, recent work has now challenged this dogma, by showing that partial in vivo epigenetic reprogramming of somatic cells using an AAV-OSK gene therapy (AAV: Adeno-Associated Virus; OSK: OCT-4, SOX2, KLF4) is capable of reversing the age of cells in the retina, rejuvenating old cells to a “younger” youthful transcriptome and methylome, and resulting in restoration of visual function in old mice and mice with glaucoma, an age-related disease (Lu et al. Nature, 2020; Yang et al. Cell, 2023). While this technology represents a significant advance in the field, targeted delivery in a way that avoids off-target side effects has been challenging.

[0057] The methods disclosed herein provide a targeted delivery or induction of expression utilizing expression of the gene ABCB5, which encodes a cell surface receptor that marks tissuespecific stem / progenitor cells in multiple mammalian tissues (including, for instance the eye - retina and corneal limbus, skin - dermis, intestine - basal crypts, and testis) required for normal development, maintenance, and regeneration (Ksander et al Nature 2014, Schatton et al Cell Reports 2015). ABCB5 is down-regulated or lost during aging. It is demonstrated herein that AAV / OSK reprogramming targeted to ABCB5 antibody-purified ABCB5 / GFP reporter stem cells enhances ABCB5 expression (FIG. 1). It is also shown herein that use of ABCB5 regulatory elements, such as promoters, enhancers, and transcription factor regulatory regions enhances GFP and OSK factor expression in ABCB5-expressing cells, without targeting delivery of GFP or OSK factors to cells expressing ABCB5 on their surface (FIGs. 3-7). In some aspects, the disclosure provides a population of ABCB5+ cells comprising an exogenous nucleic acid comprising a nucleotide sequence comprising an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding an epigenetic reprogramming factor.

[0058] These findings show that declining adult stem cell function is an important part of the aging process and critically involved in atrophy- and inflammaging-mediated tissue susceptibility to age-related disease, and that stem cell-targeted epigenetic reprogramming can serve to reverse and / or prevent such age-related tissue dysfunctions. A novel strategy for sustained rejuvenation of aging tissues through reversal of adult stem cell aging, using the targeted delivery of epigenetic factors guided by an anti-ABCB5 antibody is provided herein. Targeted therapy allows for the selective epigenetic reprograming of ABCB5-expressing stem cells, thereby restoring aged and injured tissue, such as for example, RPE (retinal pigment epithelial) cells that are critical for normal retinal development, maintenance, and regeneration in two highly prevalent, blindness-causing retinal disorders, e.g., aged-related macular degeneration (AMD) and glaucoma. The disclosed therapeutics may be used for broad application in the development of anti-aging strategies directed at other tissues that contain ABCB5 stem cells, including but not limited to, skin, corneal limbus, intestine, testis, and ABCB5+ stem cell dysfunction-associated inflammaging that contributes to CNS degenerative diseases.

[0059] Epigenetic Reprogramming

[0060] The methods disclosed herein provide a stem cell-targeted solution to two of the major biological and clinical challenges of promoting healthy aging and treating age-related diseases. By using a therapeutic composition comprised of an anti-ABCB5 antibody linked to a therapeutic payload comprised of epigenetic reprogramming factors, an enhanced therapy with fewer potential side effects may be achieved. For instance, the epigenetic reprogramming factors in the therapeutic composition may be delivered in the form of adeno-associated virus OCT-4 / SOX2 / KLF4 (AAV / OSK) gene therapy, described in more detail below. The ABCB5+ stem cell-targeted AAV / OSK epigenetic reprogramming avoids the problem of untargeted in vivo AAV / OSK epigenetic reprogramming. Despite recent successes in restoring old or injured somatic cells through untargeted reprogramming, this prior approach may represent only a temporary fix for cells that turnover either rapidly or slowly or may preferentially target shortlived stem cells over long-lived stem cells. A more sustained or even permanent solution to the problem of aging will likely require efficient reprogramming and rejuvenation of old, long-lived stem cells, resulting in an endless supply of “younger” progeny cells. The methods disclosed herein address these challenges by enhancing the stem cells specifically at the damaged or aging tissue.

[0061] An exemplary therapeutic composition is shown in FIG. 2. ABCB5 monoclonal antibody (mAb)-AAV / OSK nanoparticles are constructed using an engineered AAV bearing a HUH-tag, which forms a covalent bond to single- stranded DNA oligos conjugated to mAbs. The ABCB5 mAb clone 3C2-1D12 (FDA-approved for clinical trial-grade ABCB5+ stem cell selection (or alternatively other ABCB5 antibodies such as fully human high-affinity mAbs) may be linked in this manner to an AAV. Additionally, the therapeutic payload may also be targeted and delivered specifically to ABCB5+ stem cells using an anti-ABCB5 mAb- conjugated liposome or other non-viral nanoparticle formulations to achieve stem cell epigenetic reprogramming and restoration of youthful stem cell function and disease attenuation / rever s al .

[0062] OSK therapy

[0063] There are several factors used to reprogram differentiated adult cells into stem cells, or to promote stem cell function and / or survival. Such factors often include one or more transcription factors that induce or repress the expression of genes that promote a stem cell phenotype. Some transcription factors that are used to reprogram differentiated cells into stem cells or to promote stem cell survival, function, and / or proliferation include octamer-binding transcription factor 4 (OCT-4), SRY-box 2 (SOX2), Kriippel-like factor 4 (KLF4), Nanog, Lin28, and Myc, among other transcription factors.

[0064] OCT-4, SOX2, and KLF4 are often used in combination to epigenetically reprogram stem cells and are known as the OSK factors. To induce stem cell reprogramming, OSK factors or nucleic acids encoding the OSK factors are introduced into stem cells via cell-targeting vectors. Once inside the cell, expression of the OSK factors results in activation of a complex network of genes involved in pluripotency, cell cycle regulation, and epigenetic remodeling. Additionally, expression of the OSK factors results in the silencing of lineage- specific genes, which further promotes the pluripotency and self-renewal properties of reprogrammed stem cells. Stem cells reprogrammed with the OSK factors exhibit a “younger” stem cell phenotype, meaning they exhibit higher functionality and self-renewal capacity than aged stem cells. Sequences for and methods of using the OSK factors are known in the art and described elsewhere (Scholer et al. EMBO J, 1989; Pan et al. Cell Res, 2002; Okita et al. Nature, 2007; US Patent Publication No. US 2012 / 745,436)

[0065] In some embodiments, the epigenetic reprogramming factor comprises OCT-4, SOX2, and / or KLF4. In some embodiments, the epigenetic reprogramming factor comprises OSK. In some embodiments, the OSK is linked directly to an anti-ABCB5 antibody. In some embodiments, the nucleic acid encoding the epigenetic reprogramming factor comprises a sequence with at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 5, SEQ ID NO: 6, and / or SEQ ID NO: 7. In some embodiments, the nucleic acid encoding the epigenetic reprogramming factor comprises a sequence of SEQ ID NO: 5, SEQ NO: 6, and / or SEQ ID NO: 7.

[0066] In some embodiments, the nucleic acid encoding the epigenetic reprogramming factor comprises a regulatory element. In some embodiments, the nucleic acid comprises a sequence with at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequence of SEQ ID NO: 4. In some embodiments, the nucleic acid comprises a sequence of SEQ ID NO: 4.

[0067] In some embodiments, the nucleic acid encoding the epigenetic reprogramming factor comprises a nucleotide sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a T2A linker. In some embodiments, the nucleotide sequence encoding the cleavable linker comprises a sequence of at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) of the sequence SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the cleavable linker comprises the sequence of SEQ ID NO: 8. In some embodiments, the cleavable linker is a P2A linker. In some embodiments, the nucleotide sequence encoding the cleavable linker comprises of at least 90% sequence identity(e.g„ 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) of the sequence of SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the cleavable linker comprises the sequence of SEQ ID NO: 9.

[0068] Compositions of epigenetic factors linked to ABCB 5 antibodies

[0069] Provided here are compositions and methods for targeting ABCB5+ stem cells for epigenetic reprogramming. In some aspects, the disclosure provides a composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload comprising an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor. Antibodies that bind and target cell surface ABCB5 are known and have been described in the literature. Some anti-ABCB5 antibodies are commercially available. The anti-ABCB5 antibodies useful according to the disclosure target ABCB5 on the surface of stem cells to deliver the therapeutic payload and produce a therapeutic outcome, such as improved stem cell function, restoration of aged or injured cells, and / or reduction of allogeneic rejection or teratoma formation.

[0070] Antibodies, also known as immunoglobulins, are glycoproteins produced by B cells. Using a unique and highly evolved system of recognition, antibodies can recognize a target and tag a target epitope, foreign entity, cancer cell or invading microbe for attack by the immune system thereby neutralizing its effect. The production of antibodies is the main function of the humoral immune system. Antibodies are secreted by a plasma cell which is a type of white blood cell. Provided herein are antibodies capable of binding ABCB5, as well as nucleic acids encoding said antibodies, and uses thereof for therapeutic, research, and diagnostic purposes when linked to therapeutic payload. Thus, the present disclosure provides antibodies that bind to ABCB5. In some embodiments, the anti-ABCB5 antibody binds cell surface-displayed ABCB5 on an extracellular loop. Alternatively, or in addition, the anti-ABCB5 antibody may have low binding affinity to denatured ABCB5 or does not bind to the denatured ABCB5.

[0071] An antibody (interchangeably used in plural form) is an immunoglobulin molecule capable of specific binding to a target antigen (e.g., ABCB5 in the present disclosure), through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0072] A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding. The VH and VL regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Thus, an antibody variable region consists of a “framework” region interrupted by three “antigen binding sites”. The antigen binding sites are defined using various terms: (i) Complementarity Determining Regions (CDRs), three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3), are based on sequence variability; (ii) “Hypervariable regions,” “HVR,” or “HV,” three in the VH (Hl, H2, H3) and three in the VL (LI, L2, L3), refer to the regions of an antibody variable domains which are hypervariable in structure as defined by Chothia and Lesk. “Framework” or “framework sequences” are the remaining sequences of a variable region other than those defined to be antigen binding sites. Because the antigen binding sites can be defined by various terms as described above, the exact amino acid sequence of a framework depends on how the antigen-binding site was defined.

[0073] The antibodies described herein can be murine, rat, human, primate, porcine, or any other origin (including chimeric or humanized antibodies). Such antibodies are non-naturally occurring, i.e., would not be produced in an animal without human act (e.g., immunizing such an animal with a desired antigen or fragment thereof).

[0074] Any of the antibodies described herein can be either monoclonal or polyclonal. A “monoclonal antibody” refers to a homogenous antibody population and a “polyclonal antibody” refers to a heterogeneous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made.

[0075] In one example, the antibody used in the methods described herein is a humanized antibody, optionally a humanized version of 3C2-1D12. Humanized antibodies refer to forms of non-human (e.g. murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have Fc regions modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation.

[0076] In another example, the antibody described herein is a chimeric antibody, which can include a heavy constant region and a light constant region from a human antibody. Chimeric antibodies refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human. In some embodiments, amino acid modifications can be made in the variable region and / or the constant region.

[0077] Antibody heavy and light chain constant regions are well known in the art, e.g., those provided in the IMGT database (www.imgt.org) or at www.vbase2.org / vbstat.php., both of which are incorporated by reference herein.

[0078] In some embodiments the antibody is an antigen binding fragment. As used herein, the term “antigen binding fragment” refers to an antibody fragment such as, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv) 2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), a single domain antibody (sdab) an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment binds. According to particular embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment (i.e., portion of the heavy chain which is included in the Fab fragment). According to other particular embodiments, the antigen-binding fragment comprises Fab and F(ab'). In yet another example, the antibody described herein can be a single-domain antibody, which interacts with the target antigen via only one single variable domain such as a single heavy chain domain (as opposed to traditional antibodies, which interact with the target antigen via heavy chain and light chain variable domains). A single-domain antibody can be a heavychain antibody (VHH) which contains only an antibody heavy chain and is devoid of light chain. In additional to a variable region (for example, a VH), a single-domain antibody may further comprise a constant region, for example, CHI, CH2, CH3, CH4, or a combination thereof.

[0079] In some embodiments, the antibodies and antigen binding fragments thereof comprise a fragment crystallizable (Fc) region. The Fc region is the tail region of an antibodies and antigen binding fragments thereof which contains constant domains (e.g., CH2 and CH3); the other region of the antibodies and antigen binding fragments thereof being the Fab region which contains a variable domain (e.g., VH) and a constant domain (e.g., CHI), the former of which defines binding specificity.

[0080] As described herein, antibodies can comprise a VH domain. In some embodiments, the VH domain further comprises one or more constant domains (e.g., CH2 and / or CH3) of an Fc region and / or one or more constant domains (e.g., CHI) of a Fab region. In some embodiments, each of the one or more constant domains (e.g., CHI, CH2, and / or CH3) can comprise or consist of portions of a constant domain. For example, in some embodiments, the constant domain comprises 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less of the corresponding full sequence.

[0081] Alternatively, an antibody described herein may comprise up to 5 (e.g., 4, 3, 2, or 1) amino acid residue variations in one or more of the CDR regions of one of the antibodies known in the art and / or exemplified herein and binds the same epitope of antigen with substantially similar affinity (e.g., having a KD value in the same order). In one example, the amino acid residue variations are conservative amino acid residue substitutions. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0082] In some embodiments, the anti-ABCB5 antibodies described herein specifically bind to the corresponding target antigen or an epitope thereof. An antibody that “specifically binds” to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and / or with greater affinity with a particular target antigen than it does with alternative targets. An antibody “specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (ABCB5) or an antigenic epitope therein is an antibody that binds this target antigen with greater affinity, avidity, more readily, and / or with greater duration than it binds to other antigens or other epitopes in the same antigen. It is also understood with this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. In some examples, an antibody that “specifically binds” to a target antigen or an epitope thereof may not bind to other antigens or other epitopes in the same antigen (e.g., binding not detectable in a conventional assay).

[0083] In some embodiments, the antibodies described herein specifically bind to ABCB5 of a specific species (e.g., human ABCB5) as relative to ABCB5 from other species. For example, the antibodies described herein may specifically binds to human ABCB5 as relative to mouse ABCB5. In other embodiments, the antibodies described herein may cross-react with human ABCB5 and one or more ABCB5 from a non-human species (e.g., a non-human primate such as macaque or pig). In some embodiments, the antibodies cross-react with human, macaque, and pig ABCB5 with similar binding affinity but have significantly lower binding affinity to mouse ABCB5.

[0084] In some embodiments, an anti-ABCB5 antibody as described herein has a suitable binding affinity for the target antigen (e.g., human ABCB5) or antigenic epitopes thereof. As used herein, “binding affinity” refers to the apparent association constant or KA, which is the ratio of association and dissociation constants, K-on and K-off, respectively. The KA is the reciprocal of the dissociation constant (KD). The anti-ABCB5 antibody described herein may have a binding affinity (KD) of at least 10-8, 10-9, 10-10 M, or lower for the target antigen or antigenic epitope. An increased binding affinity corresponds to a decreased value of KD. Higher affinity binding of an antibody for a first antigen relative to a second antigen can be indicated by a higher KA (or a smaller numerical value KD) for binding the first antigen than the KA (or numerical value KD) for binding the second antigen. In such cases, the antibody has specificity for the first antigen (e.g., a first protein in a first conformation or mimic thereof) relative to the second antigen (e.g., the same first protein in a second conformation or mimic thereof; or a second protein). In some embodiments, the anti-ABCB5 antibodies described herein have a higher binding affinity (a higher KA or smaller KD) to ABCB5 as compared to the binding affinity to another membrane protein (e.g., ABCB1, ABCB4 and ABCB11). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 2.5, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1,000, 5,000, 10,000 or 105 fold. In some embodiments, any of the anti-ABCB5 antibodies may be further affinity matured to increase the binding affinity of the antibody to the target antigen or antigenic epitope thereof.

[0085] Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance (SPR), fluorescent activated cell sorting (FACS) or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are: HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20) and PBS buffer (lOmM PO4-3, 137 mM NaCl, and 2.7 mM KC1). These techniques can be used to measure the concentration of bound proteins as a function of target protein concentration. The concentration of bound protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following equation: [Bound] = [Free] / (Kd+ [Free]).

[0086] In some embodiments the anti-ABCB5 antibodies are antibodies, fragmentz or derivatives thereof disclosed in US 2023 / 0203153, which is herein incorporated by reference in its entirety for all the disclosure of anti-ABCB5 antibodies. In some embodiments the ABCB5 antibody is 3C2-1D12 or derivatives thereof such as those described in U.S. Patent No. 8,697,072, which is hereby incorporated by reference for disclosure related to ABCB5 antibodies and fragments and derivatives thereof.

[0087] Nucleic acids encoding epigenetic reprogramming factor may be delivered to ABCB5 + cells. In some embodiments, the disclosure provides a composition wherein the therapeutic payload comprises a nucleic acid encoding an epigenetic reprogramming factor. In some embodiments, the nucleic acid encoding the epigenetic reprogramming factor comprises a nucleotide sequence encoding OCT-4, a nucleotide sequence encoding SOX2, and / or a nucleotide sequence encoding KLF4. The nucleic acid may be, for instance, a vector such as a viral vector encoding the epigenetic reprogramming factor. The vector, in some embodiments may be linked to a targeting moiety that targets ABCB5 + cells, such as an antibody or antibody fragment. The nucleic acid may also be, for instance, a nucleotide sequence encoding one or more epigenetic reprogramming factors and under the control of a regulatory element, such as an inducible promoter. An inducible promoter may limit the expression of the epigenetic reprogramming factor to specific cells, such as ABCB5+ cells.

[0088] For certain compositions and methods of the present disclosure, it is preferred to target a protein or a nucleic acid encoding the protein to particular cells. In such instances, a vehicle used for delivering the nucleic acid or protein according into a cell can have a targeting molecule attached thereto. For example, a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or incorporated within the nucleic acid delivery vehicle. Vehicles can include viral vectors or nanoparticles. In some aspects, a composition provided herein comprises an anti-ABCB5 antibody conjugated via a linker to a viral vector and wherein the viral vector comprises one or more nucleotide sequences encoding an epigenetic reprogramming factor. In some embodiments, the viral vector is selected from a lentivirus vector, a retrovirus vector, an adenovirus vector, an alphavirus vector, a vaccinia virus vector, and an adeno-associated virus vector (AAV).

[0089] Retroviral vectors

[0090] One method of introducing exogenous genetic material into the ABCB5+stem cells is by targeting the cells using replication-deficient retroviruses. Replication-deficient retroviruses are capable of directing synthesis of all virion proteins, but are incapable of making infectious particles. Accordingly, these genetically altered retroviral vectors have general utility for high- efficiency transduction of genes in cultured cells. Retroviruses have been used extensively for transferring genetic material into cells. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell line with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with the viral particles) are provided in the art. The major advantage of using retroviruses is that the viruses insert efficiently a single copy of the gene encoding the therapeutic agent into the host cell genome, thereby permitting the exogenous genetic material to be passed on to the progeny of the cell when it divides. In addition, gene promoter sequences in the LTR region have been reported to enhance expression of an inserted coding sequence in a variety of cell types. The major disadvantages of using a retrovirus expression vector are (1) insertional mutagenesis, i.e., the insertion of the therapeutic gene into an undesirable position in the target cell genome which, for example, leads to unregulated cell growth and (2) the need for target cell proliferation in order for the therapeutic gene carried by the vector to be integrated into the target genome. Despite these apparent limitations, delivery of a therapeutically effective amount of a therapeutic agent via a retrovirus can be efficacious if the efficiency of transduction is high and / or the number of target cells available for transduction is high.

[0091] Alphavirus vectors

[0092] Alphavirus vectors are a class of viral vectors derived from alphaviruses, a group of positive-sense RNA viruses. These vectors have been engineered for use in various applications, particularly in vaccine development and gene therapy. Alphavirus vectors are safe and versatile, making them valuable in the development of vaccines against infectious diseases and for the delivery of therapeutic genes in biomedical research.

[0093] Vaccinia virus vectors

[0094] Vaccinia virus vectors are a type of viral vector derived from the vaccinia virus, which is a member of the poxvirus family. These vectors have been extensively used in biotechnology and vaccine development. Vaccinia virus vectors are known for their large genome capacity, which allows them to carry and express multiple exogenous genes.

[0095] Adenoviral vectors

[0096] Another viral candidate useful as an expression vector for targeting ABCB5+stem cells is the adenovirus, a double- stranded DNA virus. Like the retrovirus, the adenovirus genome is adaptable for use as an expression vector for gene transduction, i.e., by removing the genetic information that controls production of the virus itself. Because the adenovirus functions usually in an extrachromosomal fashion, the recombinant adenovirus does not have the theoretical problem of insertional mutagenesis. More recently it has been reported that certain adenoviral sequences confer intrachromosomal integration specificity to carrier sequences, and thus result in a stable transduction of the exogenous genetic material. Adeno-associated viral vectors

[0097] Adeno-associated viral (AAV) vectors are a type of virally derived vector used to deliver small payloads of therapeutic formulations, e.g., genetic material, with low immunogenicity and a favorable safety profile. AAV vectors are derived from adeno-associated viruses, which are small, non-pathogenic viruses that can integrate into the genome of another organism (a “host”), e.g., a mammal, to induce gene expression in the cell or tissue to which the AAV is delivered. Provided herein in some embodiments is a composition comprising an anti-ABCB5 antibody is fused to an AAV vector. In the use of adeno-associated viruses as vectors, the endogenous viral genome is modified (e.g., the viral nucleic acids are removed) and the therapeutic formulation (e.g., therapeutic nucleic acids) to be delivered is inserted. In some embodiments, the AAV vector comprises a nucleotide sequence encoding octamer-binding transcription factor 4 (OCT- 4), a nucleotide sequence encoding SRY-box 2 (SOX2), and a nucleotide sequence encoding Kriippel-like factor 4 (KLF4).

[0098] Viral particles

[0099] Viral particles, also called virions consist of a viral genome surrounded by a capsid. Like other viral vectors, the genomes of viral particles can be engineered to comprise nucleic acids encoding the genes of interest to be expressed in target cells.

[0100] Other Nucleic acids

[0101] Other nucleic acids used for delivery of specific nucleic acid sequences to cells are known in the art. For instance, the nucleic acid may be a DNA, RNA, mRNA encoding the epigenetic reprogramming factor.

[0102] Nanoparticles

[0103] Nanoparticles are small particles or structures with dimensions typically ranging from 1 to 100 nanometers. Nanoparticles can be composed of various materials, including metals, polymers, ceramics, or biological molecules, and they exhibit unique properties and behaviors due to their small size and high surface area-to-volume ratio. Nanoparticles can also be liposomes, which have one or more lipid bilayers surrounding an aqueous pocket, or lipid nanoparticles, which have a single lipid bilayer that encapsulates an aqueous or a non-aqueous pocket. The size and physical properties of nanoparticles make them a useful tool to facilitate delivery of therapeutic payloads to cells. In some aspects, an anti-ABCB5 antibody is conjugated to a nanoparticle comprising an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor. In some embodiments, the nanoparticle is a polymeric nanoparticle. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the nanoparticle is a liposome.

[0104] Where liposomes are employed to deliver the nucleic acids of the invention, proteins which bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and / or to facilitate uptake. Such proteins include proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like. Polymeric delivery systems also have been used successfully to deliver nucleic acids into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acids.

[0105] Thus, as will be apparent to one of ordinary skill in the art, a variety of suitable vectors are available for transferring exogenous genetic material into ABCB5+stem cells. The selection of an appropriate vector to deliver a therapeutic agent for a particular condition amenable to gene replacement therapy and the optimization of the conditions for insertion of the selected expression vector into the cell, are within the scope of one of ordinary skill in the art without the need for undue experimentation.

[0106] Regulatory Elements

[0107] The selection and optimization of a particular expression nucleic acid for expressing an epigenetic reprogramming factor may also utilize the inclusion of one or more appropriate regulatory elements, e.g., a promoter or an enhancer. Naturally-occurring constitutive promoters control the expression of essential cell functions. As a result, a gene under the control of a constitutive promoter is expressed under all conditions of cell growth. Exemplary constitutive promoters include the promoters for the following genes which encode certain constitutive or “housekeeping” functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR) (Scharfmann et al., Proc. Natl. Acad. Sci. USA 88:4626-4630 (1991)), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, the actin promoter (Lai et al., Proc. Natl. Acad. Sci. USA 86: 10006- 10010 (1989)), and other constitutive promoters known to those of skill in the art. In addition, many viral promoters function constitutively in eukaryotic cells. These include: the early and late promoters of SV40; the long terminal repeats (LTRS) of Moloney Leukemia Virus and other retroviruses; and the thymidine kinase promoter of Herpes Simplex Virus, among many others. Accordingly, any of the above-referenced constitutive promoters can be used to control transcription of a heterologous gene insert.

[0108] In some embodiments, a composition provided herein comprises a viral vector that further comprises a regulatory element associated with the one or more nucleotide sequences encoding the epigenetic reprogramming factor. In some embodiments, the regulatory element is an inducible promoter or enhancer. In some embodiments, the inducible promoter or enhancer is an ABCB5 promoter or an ABCB5 enhancer.

[0109] In some aspects, a nucleic acid provided herein comprises an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding an epigenetic reprogramming factor. By using ABCB5 regulatory elements, such as an ABCB5 promoter or ABCB5 enhancer, epigenetic reprogramming factors can be selectively expressed in the cells that endogenously use such regulatory elements (e.g., ABCB5+ stem cells). In some embodiments, the inducible ABCB5 regulatory element is an ABCB5 promoter. In some embodiments, the nucleic acid comprises a sequence of at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid comprises a sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid comprises a sequence of at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequence of SEQ ID NO: 4. In some embodiments, the nucleic acid comprises a sequence of SEQ ID NO: 4. In some embodiments, the inducible ABCB5 regulatory element is an ABCB5 enhancer. In some embodiments, the nucleic acid comprises a sequence of at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid comprises a sequence of SEQ ID NO: 2. .

[0110] Genes that are under the control of inducible promoters or enhancers are expressed only or to a greater degree, in the presence of an inducing agent, (e.g., transcription under control of the metallothionein promoter is greatly increased in presence of certain metal ions). In some embodiments, the inducible component of the ABCB5 regulatory element comprises a sequence of at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequence of SEQ ID NO: 3. In some embodiments, the inducible component of the ABCB5 regulatory element comprises a sequence of SEQ ID NO: 3. For the purpose of this discussion an “enhancer” is simply any nontranslated DNA sequence which works contiguous with the coding sequence (in cis) to change the basal transcription level dictated by the promoter. In some embodiments, the inducible ABCB5 regulatory element is a chemically-inducible ABCB5 regulatory element. Inducible promoters or enhancers include responsive elements (REs) which stimulate transcription when their inducing factors are bound. For example, there are REs for serum factors, steroid hormones, and cyclic AMP. There are also a number of chemical agents used to control gene expression. Examples of such chemical agents include, but are not limited to, isopropyl P-D-l -thiogalactopyranoside, tetracycline, doxycycline, tamoxifen, dexamethasone, and retinoic acid. Promoters or enhancers containing a particular RE can be chosen in order to obtain an inducible response and in some cases, the RE itself may be attached to a different promoter, thereby conferring inducibility to the recombinant gene. Thus, by selecting the appropriate promoter or enhancer (constitutive versus inducible; strong versus weak), it is possible to control both the existence and level of expression of a therapeutic agent in targeted ABCB5+ stem cell. Selection and optimization of these factors for delivery of a therapeutically effective dose of a particular therapeutic agent is deemed to be within the scope of one of ordinary skill in the art without undue experimentation, taking into account the above-disclosed factors and the clinical profile of the subject.

[0111] The promoter or enhancer characteristically has a specific nucleotide sequence necessary to initiate transcription or facilitate expression. Promoters and enhancers may also be used in combination to obtain the desired gene transcription activity. Preferably, the exogenous genetic material is introduced into the ABCB5+stem cell genome immediately downstream from the promoter so that the promoter and coding sequence are operatively linked so as to permit transcription of the coding sequence. A preferred retroviral expression vector includes an exogenous regulatory element to control transcription of the inserted exogenous gene. Such exogenous promoters and enhancers include both constitutive and inducible promoters and enhancers. The selection of a suitable promoter, enhancer, selection gene and / or signal sequence is deemed to be within the scope of one of ordinary skill in the art without undue experimentation .

[0112] In some embodiments, administration of a therapeutically effective amount of a composition increases OCT-4, SOX2, and / or KLF4 expression in ABCB5+ stem cells above a control level, wherein the control level is OCT-4, SOX2, and / or KLF4 expression in an ABCB5+ stem cell that does not receive the composition or prior to receiving the composition. Linkers

[0113] In some embodiments, an anti-ABCB5 antibody is fused to an AAV vector via a linker. There are a number of linker types that can fuse or conjoin two or more biological or chemical materials. The linker can be a glycine-serine linker, a linker with a protease cleavage site, a linker with a restriction enzyme site, an endonuclease or endonuclease domain linker, or another type of linker. In some embodiments, the linker is an endonuclease or an endonuclease domain. In some embodiments, the linker is a HUH-endonuclease or a HUH-endonuclease domain.

[0114] Diseases

[0115] In some aspects, the compositions and methods provided by the present disclosure may be useful in treating aging or various diseases. In some aspects, the present disclosure provides a method comprising administering to a subject an ABCB5 targeted composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload comprised of an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor in an effective amount to reprogram and rejuvenate ABCB5+stem cells in the subject. In some embodiments, the subject is a stem cell recipient. In some embodiments, the ABCB5 targeted composition is a composition described herein.

[0116] In some aspects, the present disclosure provides a method comprising administering to a subject a nucleic acid comprising a nucleotide sequence comprising an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding an epigenetic reprogramming factors, in an effective amount to reprogram and rejuvenate ABCB5+ stem cells in the subject.

[0117] Due to their ability to differentiate into lineage- specific cell types, stem cells are an attractive therapy in the treatment of degenerative disorders or injury. One approach in such treatments is to harvest cells from a subject and reprogram the cells in vitro before transplanting them back into the subject (autologous transplantation) or into a different subject (allogeneic transplantation). A major drawback of this technique is the scarcity of target stem cell populations in adults, as well as decreased functionality of adult stem cells that result in poor engraftment. Another approach is to target stem cells in vivo for epigenetic reprogramming, but current techniques target most cells indiscriminately, which may result in preferentially reprogramming short-lived progenitor or differentiated cells, which proliferate and turn over more rapidly, over long-lived stem cells. The targeting of long-lived stem cells is beneficial or necessary in the treatment of aging or in the treatment of disorders in which the dysfunction of long-lived stem cells is implicated. In some embodiments, a method provided herein is a method of treating aging. In some embodiments, the method of treating aging comprises treatment of skin tissue or intestinal tissue.

[0118] Ocular degenerative diseases

[0119] Ocular degenerative diseases encompass a group of progressive eye conditions that lead to the deterioration of the structures within the eye, often resulting in vision loss or impairment. These diseases can affect various parts of the eye, including the retina, lens, or optic nerve, and are typically chronic and irreversible in nature. Retinal disorders encompass a range of conditions that affect the retina, the light-sensitive tissue at the back of the eye, leading to vision problems. One such retinal disorder is age-related macular degeneration (AMD), a progressive eye disease that primarily affects older adults and can lead to severe central vision loss. It occurs when the macula, the central part of the retina, deteriorates over time, impacting the ability to see fine details and perform tasks like reading and recognizing faces.

[0120] Another degenerative eye disorder is glaucoma, which is characterized by increased intraocular pressure, which can damage the optic nerve and lead to irreversible vision loss if left untreated. It often develops slowly without noticeable symptoms in its early stages, making regular eye exams crucial for early detection and management.

[0121] In some embodiments, a method provided herein is a method of treating a degenerative disease. In some embodiments, the degenerative disease is a degenerative disease of the eye. In some embodiments, the degenerative disease of the eye is a retinal disorder. In some embodiments, the retinal disorder is age-related macular degeneration (AMD) or glaucoma.

[0122] In some embodiments, the degenerative disease of the eye is a corneal disorder, for example, limbal stem cell deficiency.

[0123] CNS degenerative diseases

[0124] Degenerative diseases of the central nervous system (CNS) comprise a group of disorders characterized by the gradual and progressive degeneration of nerve cells in the brain and spinal cord. These conditions, which include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease, among others, result in the deterioration of cognitive, motor, or sensory functions, often leading to profound disability. In some embodiments, provided herein is a method of treating a degenerative disease. In some embodiments, the degenerative disease is a degenerative disease of the CNS.

[0125] Inflammaging

[0126] Inflammaging is a phenomenon in which chronic, low-grade inflammation increases with age. Inflammaging represents a state of chronic, smoldering inflammation within the body, even in the absence of infection or injury, and is believed to play a role in the aging process and the development of age-related diseases. Inflammaging is associated with a wide range of conditions, including cardiovascular disease, diabetes, neurodegenerative diseases, and frailty, as well as a weakened immune system. In some embodiments, a method provided herein is a method of treating aging. In some embodiments, the method of treating comprises treatment of ABCB5+stem cell dysfunction-associated inflammaging.

[0127] In addition to use in compositions and methods disclosed herein, the anti-ABCB5 antibodies may be used to isolate ABNCB5+ stem cells from human tissue. The isolated cells may treated with epigenetic reprogramming factors in vitro or ex- vivo to rejuvenate the cells. Those cells may then be delivered to the same or different human patient for therapeutic or antiaging purposes.

[0128] As used herein, a subject is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.

[0129] Additional Embodiments

[0130] The following are additional embodiments of the invention:

[0131] 1. A method comprising administering to a subject an ABCB5 targeted composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload comprised of an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor in an effective amount to reprogram and rejuvenate ABCB5+ stem cells in the subject.

[0132] 2. A method comprising administering to a subject a nucleic acid comprising a nucleotide sequence comprising an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding an epigenetic reprogramming factor, in an effective amount to reprogram and rejuvenate ABCB5+ stem cells in the subject. 3. The method of embodiment 1 or 2, wherein the method is a method of treating a degenerative disease.

[0133] 4. The method of embodiment 3, wherein the degenerative disease is a degenerative disease of the eye.

[0134] 5. The method of embodiment 4, wherein the degenerative disease of the eye is a retinal disorder or corneal disorder including limbal stem cell deficiency (LSCD).

[0135] 6. The method of embodiment 5, wherein the retinal disorder is aged-related macular degeneration (AMD) or glaucoma.

[0136] 7. The method of embodiment 3, wherein the degenerative disease is a degenerative disease of CNS.

[0137] 8. The method of embodiment 1 or 2, wherein the method is a method of treating aging.

[0138] 9. The method of embodiment 8, wherein the method of treating aging comprises treatment of skin tissue or intestinal tissue.

[0139] 10. The method of embodiment 8, wherein the method of treating aging comprises treatment ABCB5+ stem cell dysfunction-associated inflammaging.

[0140] 11. The method of embodiment 1 or 2, wherein the subject is a stem cell recipient.

[0141] 12. The method of any of the above embodiments, wherein the ABCB5 targeted composition is a composition of any of the claims.

[0142] 13. A method for enhancing stem cell transplantation comprising administering to a subject an ABCB5 targeted composition comprising a nucleic acid comprising a nucleotide sequence comprising an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding an epigenetic reprogramming factor in an effective amount to reprogram and rejuvenate ABCB5+ stem cells in the subject, and wherein the subject is a stem cell donor and the administration is performed prior to donation or a stem cell recipient and the administration is performed after donation.

[0143] 14. A method for enhancing stem cell transplantation comprising administering to a subject an ABCB5 targeted composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload comprised of an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor in an effective amount to reprogram and rejuvenate ABCB5+ stem cells in the subject, and wherein the subject is a stem cell donor and the administration is performed prior to donation or a stem cell recipient and the administration is performed after donation.

[0144] 15. The method of embodiment 13 or 14, wherein the ABCB5 targeted composition is a composition of any of the composition claims.

[0145] 16. A method for preparing a population of cells, the method comprising: isolating primary cells comprised of ABCB5+ cells from human tissue; culturing the isolated primary cells in culture medium until the cells produce enough progeny to reach greater than 60% confluence of mixed cells; harvesting the mixed cells; culturing the harvested mixed cells; contacting the harvested mixed cells with an ABCB5 targeted composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload comprised of an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor.

[0146] 17. The method of embodiment 16, wherein administration of the composition increases OCT- 4, SOX2, and / or KLF4 expression in the ABCB5+harvested mixed cells relative to ABCB5+harvested mixed cells to which the composition is not administered.

[0147] 18. The method of embodiment 16 or 17, wherein the ABCB5 targeted composition is a composition of any of the claims.

[0148] 19. A population of ABCB5+ cells, comprising an exogenous nucleic acid comprising a nucleotide sequence comprising an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding an epigenetic reprogramming factor. 20. The population of cells of embodiment 19, wherein at least 99% of the population of cells is comprised of ABCB5+ cells.

[0149] 21. The population of cells of embodiment 19, wherein the nucleic acid comprises a viral vector.

[0150] 22. The population of cells of embodiment 21, wherein the viral vector is selected from a lentivirus vector, a retrovirus vector, an adenovirus vector, an alphavirus vector, a vaccinia virus vector, and an adeno-associated virus vector (AAV).

[0151] 23. The population of cells of embodiment 21, wherein the viral vector is AAV.

[0152] 24. The population of cells of embodiment 20, comprising a nanoparticle encapsulating the nucleic acid.

[0153] 25. The population of cells of embodiment 24, wherein the nanoparticle is a polymeric nanoparticle.

[0154] 26. The population of cells of embodiment 24, wherein the nanoparticle is a lipid nanoparticle.

[0155] 27. The population of cells of embodiment 24, wherein the nanoparticle is a liposome.

[0156] 28. The population of cells of any of embodiments 20-27, wherein the nucleic acid encoding the epigenetic reprogramming factor comprises a nucleotide sequence encoding OCT-4, a nucleotide sequence encoding SOX2, and / or a nucleotide sequence encoding KLF4.

[0157] 29 The population of cells of any of embodiments 20-27, wherein the inducible ABCB5 regulatory element is an ABCB5 promoter.

[0158] 30. The population of cells of any of embodiments 20-27, wherein the inducible ABCB5 regulatory element is an ABCB5 enhancer. 31. The population of cells of any of embodiments 20-27, wherein the inducible ABCB5 regulatory element is a chemically-inducible ABCB5 regulatory element.

[0159] EXAMPLES

[0160] Example 1. Identification of human ABCB5 gene-regulatory sequences.

[0161] Using a combination of the ENCODE database and the UCSC Genome Browser, a limited and previously uncharacterized number of potential ABCB5 gene regulation regions were identified, including enhancers, promoters, and transcription factor regulatory regions.

[0162] Enhancers: Two putative enhancers around approximate regions of -50kb and -lOkb, relative to the ABCB5 (NM_001163941) transcriptional start site (TSS) were identified. These regions corresponded to described peaks of H3K27Ac described on the ENCODE / UCSC Genome browser. H3K27Ac often corresponds with regions of active gene expression such as enhancers and promoters.

[0163] Promoters: Based on known transcription start sites of ABCB5 isoforms (e.g. NM_001163941, and NM_178559) putative proximal promoters were identified as being regions up to -1.5kb upstream of these .

[0164] Transcription Factor Regulatory Regions (TFRRs): Based upon regions of observed transcription factor binding enrichment peaks from the ENCODE database, regions out with classic promoter-regions that may be responsible for regulation of ABCB5 were identified. One particular region downstream of the TSS for isoform NM_178559 was selected for testing. Sequences are shown in the Table below.

[0165] Example 2. GFP expression driven by inducible ABCB5 regulatory elements.

[0166] Cloned regions of ABCB5 driving expression of Shield 1 -dependent inducible GFP, were transfected into G3361 melanoma cells. Relative activity of the regions is depicted in the “GFP gate” (FIG. 4). CMV was used as a constitutively active positive control, unrelated to ABCB5 expression. Activity was increased after 72h blockade of ABCB5 using antibody 3C2-1D12. Highest fold-increase was detected in TFRR (0.21% to 1.17% -> 5.5 fold). Enhancers fold increase were similar (1.27% to 2.68% -> 2.1 fold for -lOkb; 0.27% to 0.73% -> 2.7 fold for - 50kb). Promoters were least affected (0.24 to 0.36% ->1.5 fold for beta; 0.22% to 0.31% ->1.4 fold for gamma). The experiment was repeated in primary human corneal epithelial cells (FIG. 5) and ABCB5+ skin-derived cells (FIG. 6) with similar results. Example 3. A two-plasmid system for inducing OSK expression.

[0167] Using a 2-plasmid system, ABCB5 gene components were be used to drive expression of rejuvenation transcription factors OCT4, SOX2, and KLF4 (OSK) in a population of skin cells. The constitutively active EEF1A promoter was used as positive control (similar to CMV previously shown, EEF1A is active in all cells regardless of ABCB5 status). The Tet-3G system is inducible, requiring the addition of doxycycline to proceed, allowing for temporal control of induction. The combination of the OSK plasmid and the plasmids under control of the ABCB5 regulatory elements led to OSK expression (FIG. 7A) and downstream upregulation of ABCB5 at both the transcript (FIG. 7B, left) and protein (FIG. 7B, right) level, indicating that induction of OSK reprogramming creates a positive feedback loop whereby ABCB5 expression is also increased.

[0168] Example 4. OSK expression in ABCB5 knock-in skin cells.

[0169] ABCB5-gene knock-in GFP-reporter skin cells (created using CRISPR) were used to show that using constitutively-expressed OSK rejuvenation factors leads to increased amounts of ABCB5+ stem cells. This was also shown by cell-surface flow-cytometry using anti-ABCB5 antibody 3C2-1D12 (“3C2”), immunoprecipitation using an anti-Myc antibody (FIGs. 8A-8D). Addition of the antibiotic puromycin allowed for selection of ABCB5+ cells (FIGs. 8E-8F).

[0170] Example 5. Design of OSK expression-driving plasmids.

[0171] Three plasmids were designed for use in transfection to induce OSK expression in cells (FIGs. 9A-9C). The sequences are shown in Table 1 below.

[0172] Example 6. ABCB5-targeting OSK plasmids induce epigenetic reprogramming in ABCB5+ stem cells.

[0173] The epigenetic reprogramming factors OSK (Oct4 / Sox3 / Klf4) have been used to reprogram differentiated somatic cells into pluripotent stem cells, but whether OSK factors can reprogram ABCB5+ stem cells (for example, to rejuvenate aged ABCB5+ stem cells) had not previously been interrogated. This Example tested whether the OSK factors can be used to impact ABCB5 expression of human dermal mesenchymal stem cells (DMSCs) and whether OSK expression is sustained following transfection of DMSCs. Methods

[0174] ABCB5+ dermal mesenchymal stem cells (DMSCs) from 3 donors were infected for 7 days with an AAV2 virus at 4 x 104p.f.u. The AAV2 virus was designed to employ custom gene-driven expression of Tet3G (gene elements included an EFla constitutively active promoter, an ABCB5 -lOkb Enhancer, or an ABCB5 gene element TFRR), with a second AAV2 virus encoding TRE3-OSK (Oct4 / Sox2 / Klf4). 100 p.f.u. of Adenovirus 5 (null plasmid - Vector Biolab #1240) was added to aid OSK expression, and lug / ml doxycycline was added to activate Tet3G.

[0175] As negative controls, AAV2 expression of CMV-GFP or EFla-TdTomato were similarly used (also in the presence of doxyxycline which was inert to these controls).

[0176] Media was subsequently changed every 3 days to replenish doxycycline (no new virus was added after initial dose).

[0177] AAV2 virus and custom- generated viral plasmids were created to the specifications described in this Example by Vector Builder.

[0178] Results

[0179] A specific protocol for expression of OSK in ABCB5+ DMSCs was developed and tested. Oct4 overexpression was highly efficient after 7 days (FIGs. 10D-10E) (. Highest using EFla promoter (739 fold vs. control), and slightly less with the ABCB5 gene elements, the - lOkb Enhancer (395 fold vs. control), and the TFRR (618 fold vs. control). For Sox2 and KLF4, overexpression was still robustly detected for EFla promoter (25.8 fold and 14 fold vs. control), -lOkb Enhancer (11.9 fold and 13.7 fold vs. control), and TFRR (and 28.4 fold vs. control) (FIGs. 10A-10C).

[0180] Using the 3C2-1D12 antibody, relatively more ABCB5 was observed in OSK-expressed samples using gene elements from EFla (6.34% positivity), ABCB5 - lOkb enhancer (6.6%), and ABCB5 TFRR (6.61%) gene elements versus compared to 3 different controls, no AAV (5.84%), CMV-GFP (4.22%), EFla-tdTomato (5.56%), after 1 week (Tables 2 and 3). This represents over 20% more cell-surface ABCB5 in all cases when compared to average control (5.21%), and over 25% more ABCB5 when using ABCB5-specific gene elements.

[0181] Taken together, the data show that DMSCs, in particular ABCB5+ DMSCs, can be targeted for epigenetic reprogramming using the OSK factors, that the OSK factors increase ABCB5 expression, and that expression of the OSK factors is sustained for at least a week following transfection. The results indicate that reprogramming ABCB5+ stem cells using the OSK factors may provide a method by which aged ABCB5+ stem cells can be repaired or rejuvenated.

[0182] Table 1. Plasmid sequences

[0183] Table 2. ABCB5 expression following treatment with OSK

[0184] The present disclosure provides, in some embodiments, pharmaceutical compositions comprising a pharmaceutically acceptable excipient. A "pharmaceutically acceptable excipient," after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be "acceptable" also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include, but are not limited to, colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate.

[0185] A composition may be administered by any route that results in a therapeutically effective outcome. These include, but are not limited, to injection, intradermal, intramuscular, intranasal, intraoral, intraocular, intraperitoneal, intravenous, topical, and / or subcutaneous administration. The present disclosure provides methods of administering to a subject an ABCB5-targeted composition. In some aspects, a method provided herein comprises administering to a subject an ABCB5 targeted composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload comprised of an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor in an effective amount to reprogram and rejuvenate ABCB5+stem cells in the subject.

[0186] The specific “therapeutically effective amount,” prophylactically effective, or otherwise appropriate dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the cell therapy arts.

[0187] This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload comprising an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor.

2. The composition of claim 1, wherein the therapeutic pay load comprises a nanoparticle comprising the epigenetic reprogramming factor or nucleic acid encoding an epigenetic reprogramming factor.

3. The composition of claim 2, wherein the nanoparticle is a polymeric nanoparticle.

4. The composition of claim 2, wherein the nanoparticle is a lipid nanoparticle.

5. The composition of claim 2, wherein the nanoparticle is a liposome.

6. The composition of any one of claims 1-5, wherein the epigenetic reprogramming factor comprises OCT-4, SOX2, and / or KLF4.

7. The composition of claim 6, wherein the epigenetic reprogramming factor comprises OSK (OCT-4, SOX2, and KLF4).

8. The composition of claim 7, wherein the OSK is linked directly to the anti-ABCB5 antibody.

9. The composition of claim 7, wherein the nanoparticle is linked to the anti-ABCB5 antibody.

10. The composition of claim 1, wherein the therapeutic payload comprises a viral vector comprising the nucleic acid encoding an epigenetic reprogramming factor.

11. The composition of claim 10, wherein the viral vector is selected from a lentivirus vector, a retrovirus vector, an adenovirus vector, an alphavirus vector, a vaccinia virus vector, and an adeno-associated virus vector (AAV).

12. The composition of claim 11, wherein the viral vector is AAV.

13. The composition of claim 10, wherein the viral vector is linked to the anti-ABCB5 antibody through a linker and wherein the linker is an endonuclease or an endonuclease domain.

14. The composition of claim 13, wherein the linker is a HUH-endonuclease or a HUH- endonuclease domain.

15. The composition of any one of claims 1-14, wherein the nucleic acid encoding the epigenetic reprogramming factor comprises a nucleotide sequence encoding OCT-4, a nucleotide sequence encoding SOX2, and / or a nucleotide sequence encoding KLF4.

16. The composition of any one of claims 1-15, wherein the nucleic acid encoding the epigenetic reprogramming factor comprises a regulatory element.

17. The composition of claim 16, wherein the regulatory element is an inducible promoter or enhancer.

18. The composition of claim 17, wherein the inducible promoter or enhancer is an ABCB5 promoter or an ABCB5 enhancer.

19. The composition of claim 18, wherein the ABCB5 promoter comprises a sequence having at least 90% identity to SEQ ID NO: 1.

20. The composition of claim 18 or 19, wherein the ABCB5 promoter comprises a sequence of SEQ ID NO: 1.

21. The composition of claim 18, wherein the ABCB5 enhancer comprises a sequence having at least 90% identity to SEQ ID NO: 2.

22. The composition of claim 18 or 21, wherein the ABCB5 enhancer comprises a sequence of SEQ ID NO: 2.

23. A pharmaceutical composition comprising the composition of any one of claims 1-22 and a pharmaceutically acceptable excipient.

24. A nucleic acid comprising a nucleotide sequence comprising an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding an epigenetic reprogramming factor.

25. The nucleic acid of claim 24, wherein the nucleic acid comprises a viral vector.

26. The nucleic acid of claim 25, wherein the viral vector is selected from a lentivirus vector, a retrovirus vector, an adenovirus vector, an alphavirus vector, a vaccinia virus vector, and an adeno-associated virus vector (AAV).

27. The nucleic acid of claim 25, wherein the viral vector is AAV.

28. The nucleic acid of claim 24, comprising a nanoparticle encapsulating the nucleic acid.

29. The nucleic acid of claim 28, wherein the nanoparticle is a polymeric nanoparticle, a lipid nanoparticle or a liposome.

30. The nucleic acid of any one of claims 24-29, wherein the nucleic acid encoding the epigenetic reprogramming factor comprises a nucleotide sequence encoding OCT-4, a nucleotide sequence encoding SOX2, and / or a nucleotide sequence encoding KLF4.

31. The nucleic acid of any one of claims 24-30, wherein the nucleic acid comprises the sequence of SEQ ID NO: 4 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 4.

32. The nucleic acid of any one of claims 24-30, wherein the inducible ABCB5 regulatory element is an ABCB5 promoter and / or an ABCB5 enhancer.

33. The nucleic acid of any one of claims 24-32, wherein the inducible ABCB5 regulatory element is a chemically-inducible ABCB5 regulatory element.

34. A method comprising administering to a subject an ABCB5 targeted composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload comprised of an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor in an effective amount to reprogram and rejuvenate ABCB5+ stem cells in the subject, optionally wherein the ABCB5+ stem cells are limbal ABCB5+ stem cells or dermal ABCB5+ stem cells.

35. A method comprising administering to a subject a nucleic acid comprising a nucleotide sequence comprising an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding an epigenetic reprogramming factor, in an effective amount to reprogram and rejuvenate ABCB5+ stem cells in the subject, optionally wherein the ABCB5+ stem cells are limbal ABCB5+ stem cells or dermal ABCB5+ stem cells.

36. The method of claim 35, wherein the nucleotide sequence comprising an inducible ABCB5 regulatory element comprises a sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

37. A population of ABCB5+ cells, comprising an exogenous nucleic acid comprising a nucleotide sequence comprising an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding an epigenetic reprogramming factor, optionally wherein at least 99% of the population of cells is comprised of ABCB5+ cells.

38. The population of cells of claim 37, wherein the nucleotide sequence comprising an inducible ABCB5 regulatory element comprises a sequence of SEQ ID NO: 1 or 2.

39. A composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload comprising an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor, wherein the nucleic acid comprises a sequence of SEQ ID NO: 1 or 2.

40. A nucleic acid comprising a ABCB5 regulatory element, wherein the ABCB5 regulatory element is a nucleic acid having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to comprises a sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

41. The nucleic acid of claim 40, wherein the ABCB5 regulatory element consists essentially of a sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

Images