Stem cell reprogramming and anti-aging strategies targeting abcb5

Abstract

Methods and compositions for revitalizing and reprogramming stem cells are disclosed. The method involves administering an effective amount of an ABCB5-targeting composition to a subject to reprogram and revitalize ABCB5+ stem cells in the subject. The ABCB5-targeting composition comprises an anti-ABCB5 antibody conjugated to a therapeutic payload, the therapeutic payload comprising an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor. The composition comprises an anti-ABCB5 antibody conjugated to a therapeutic payload, the therapeutic payload comprising an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor.

Description

[0001] Related applications

[0002] This application claims the filing date interest in U.S. Provisional Application No. 63 / 592,353, filed October 23, 2023, pursuant to 35 USC § 119(e), the entire contents of which are incorporated herein by reference.

[0003] Federally funded research

[0004] This invention was made with government support under grant number EY025794 from the National Institutes of Health. The government holds certain rights to this invention. Background Technology

[0005] ABCB5 is a multidrug resistance (MDR) mediator expressed in a variety of human malignancies, with specific overexpression in previously identified treatment-resistant tumor subpopulations representing cancer cells (CSCs). ABCB5 confers drug resistance to chemotherapeutic agents such as doxorubicin and 5-fluorouracil (5-FU). ABCB5+ stem cells are also present in normal tissues and play roles in immune regulation, tissue regeneration, and aging. Regenerative medicine involves the repair, regeneration, maintenance, and replacement of tissues and organs using exogenous materials such as scaffolds. Scaffolds can be seeded with cells (e.g., primary cells or stem cells) and various factors that promote tissue growth. However, many challenges remain in designing suitable materials for regenerative medicine and tissue engineering. Summary of the Invention

[0006] Stem cell therapy is a rapidly developing field of medicine that utilizes stem cells or targets stem cells to treat a variety of diseases and injuries. Stem cells are undifferentiated cells and therefore have the potential to develop into different cell types in the body. Another unique characteristic of stem cells is their self-renewal capacity, thus maintaining a pool of undifferentiated cells that can replenish differentiated cells as they die. For example, limbal stem cells (LSCs), located in the limbus of the eye and playing a crucial role in maintaining the health and function of the cornea, must be periodically replaced to continuously support the corneal epithelial cells essential for vision. LSCs replenish themselves through self-renewal. Some cell populations are relatively short-lived and renew rapidly, while stem cells are long-lived and essential for maintaining shorter-lived progenitor or terminally differentiated cell populations. In certain disease states, using stem cells to replace dying, degenerating, or dysfunctional cells represents a promising therapeutic approach. To achieve this, stem cells can be targeted for epigenetic reprogramming to promote specific cell phenotypes. However, current techniques for inducing epigenetic reprogramming in specific cell populations often indiscriminately target the majority of cells. This poses a problem when targeted therapy for long-lived stem cells is necessary or beneficial, because targeting methods can often, or even preferentially, affect short-lived cells that are more prone to proliferation and differentiation.

[0007] Therefore, in some aspects, this disclosure provides compositions comprising an anti-ABCB5 antibody conjugated to a therapeutic payload, said therapeutic payload comprising an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor.

[0008] In some embodiments, the therapeutic payload comprises nanoparticles containing epigenetic reprogramming factors or nucleic acids encoding one or more epigenetic reprogramming factors. In some embodiments, the nanoparticles are polymer nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the nanoparticles are liposomes.

[0009] In some embodiments, the epigenetic reprogramming factor includes OCT-4, SOX2, and / or KLF4. In some embodiments, the epigenetic reprogramming factor includes OSK (OCT-4, SOX2, and KLF4). In some embodiments, OSK is directly linked to an anti-ABCB5 antibody.

[0010] In some implementations, the nanoparticles are linked to anti-ABCB5 antibodies.

[0011] In some embodiments, the therapeutic payload comprises a viral vector containing nucleic acids encoding one or more epigenetic reprogramming factors. In some embodiments, the viral vector is selected from lentiviral vectors, retroviral vectors, adenovirus vectors, alphavirus vectors, vaccinia virus vectors, and adeno-associated virus (AAV) vectors. In some embodiments, the viral vector is an AAV. In some embodiments, the viral vector is linked to an anti-ABCB5 antibody via a adapter, and the adapter is a nuclease or a nuclease domain. In some embodiments, the adapter is a HUH-nuclease or a HUH-nuclease domain.

[0012] In some implementations, the nucleic acid encoding the epigenetic reprogramming factor includes a nucleotide sequence encoding OCT-4, a nucleotide sequence encoding SOX2, and / or a nucleotide sequence encoding KLF4.

[0013] In some embodiments, the nucleic acid encoding the epigenetic reprogramming factor contains a regulatory element. In some embodiments, the regulatory element is an inducible promoter or enhancer. In some embodiments, the inducible promoter or enhancer is the ABCB5 promoter or ABCB5 enhancer. In some embodiments, the ABCB5 promoter contains a sequence having at least 90% identity with SEQ ID NO: 1. In some embodiments, the ABCB5 promoter contains the sequence of SEQ ID NO: 1. In some embodiments, the ABCB5 enhancer contains a sequence having at least 90% identity with SEQ ID NO: 2. In some embodiments, the ABCB5 enhancer contains the sequence of SEQ ID NO: 2.

[0014] In some embodiments, pharmaceutical compositions are provided that comprise any of the compositions disclosed herein and pharmaceutically acceptable excipients.

[0015] In some aspects, a method is provided comprising administering to a subject an effective amount of a composition targeting ABCB5 to reprogram and revitalize ABCB5+ stem cells in the subject, said ABCB5-targeting composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload, said therapeutic payload comprising an epigenetic reprogramming factor or a nucleic acid encoding one or more epigenetic reprogramming factors.

[0016] A method comprising administering an effective amount of nucleic acid to a subject to reprogram and revitalize ABCB5+ stem cells in the subject, said nucleic acid comprising a nucleotide sequence containing an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding epigenetic reprogramming factors.

[0017] In some embodiments, the nucleotide sequence containing the inducible ABCB5 regulatory element comprises the sequence of SEQ ID NO: 1 or 2.

[0018] In some aspects, a method is provided comprising administering an effective amount of an ABCB5-targeting composition to an ex vivo cell culture containing ABCB5+ cells to reprogram and revitalize ABCB5+ stem cells, said ABCB5-targeting composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload, said therapeutic payload comprising an epigenetic reprogramming factor or a nucleic acid encoding one or more epigenetic reprogramming factors.

[0019] In some embodiments, the method is a method for treating degenerative diseases. In some embodiments, the degenerative disease is an ocular degenerative disease. In some embodiments, the ocular degenerative disease is a retinal disease or a corneal disease. In some embodiments, the retinal disease is age-related macular degeneration (AMD) or glaucoma. In some embodiments, the corneal disease is limbal stem cell deficiency (LSCD). In some embodiments, the degenerative disease is a CNS degenerative disease.

[0020] In some implementations, ABCB5+ stem cells are limbal ABCB5+ stem cells. In some implementations, ABCB5+ stem cells are dermal ABCB5+ stem cells.

[0021] In some implementations, the method is a treatment for aging. In some implementations, the treatment for aging includes treating skin tissue or intestinal tissue. In some implementations, the treatment for aging includes treating inflammatory aging associated with ABCB5+ stem cell dysfunction.

[0022] In some implementations, the recipients are stem cell recipients.

[0023] In some embodiments, the composition targeting ABCB5 is any composition 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 DNA or mRNA encoding an epigenetic reprogramming factor.

[0024] In some aspects, methods for enhancing stem cell transplantation are provided, comprising administering to a subject an effective amount of an ABCB5-targeting composition to reprogram and revitalize ABCB5+ stem cells in the subject, said ABCB5-targeting composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload, said therapeutic payload comprising an epigenetic reprogramming factor or nucleic acid encoding an epigenetic reprogramming factor, 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.

[0025] In some embodiments, the composition targeting ABCB5 is any composition 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 DNA or mRNA encoding an epigenetic reprogramming factor.

[0026] In some aspects, a method for preparing a cell population includes: isolating primary cells containing ABCB5+ cells from human tissue; culturing the isolated primary cells in a culture medium until the cells produce sufficient progeny to achieve a confluence of mixed cells greater than 60%; harvesting the mixed cells; culturing the harvested mixed cells; and contacting the harvested mixed cells with a composition targeting ABCB5, said composition containing an anti-ABCB5 antibody conjugated to a therapeutic payload, said therapeutic payload containing an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor.

[0027] 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 DNA or mRNA encoding an epigenetic reprogramming factor.

[0028] Some aspects involve ABCB5+ cell populations containing exogenous nucleic acids comprising a nucleotide sequence containing an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding epigenetic reprogramming factors. In some embodiments, the nucleotide sequence containing the inducible ABCB5 regulatory element comprises the sequence of SEQ ID NO: 1 or 2.

[0029] In some embodiments, application of the composition increases the expression of OCT-4, SOX2, and / or KLF4 in ABCB5+ harvested mixed cells compared to untreated ABCB5+ harvested mixed cells. In some embodiments, the composition targeting ABCB5 is any of the compositions disclosed herein.

[0030] Some aspects involve nucleic acids containing the 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 with a sequence containing SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the ABCB5 regulatory element consists essentially of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Attached Figure Description

[0031] Figure 1 The study showed that the percentage of ABCB5+ stem cells in human dermis decreases with age.

[0032] Figure 2 The effects of OSK (OCT-4, SOX2, and KLF4) reprogramming on ABCB5 expression in stem cells targeting ABCB5 were demonstrated. ABCB5+ stem cells were categorized as untransduced, transduced with a green fluorescent protein (GFP) reporter associated with ABCB5 expression, or transduced with both a GFP reporter and OSK. Compared to untransduced stem cells or stem cells transduced with only a GFP reporter, OSK transduction of ABCB5+ stem cells resulted in increased ABCB5 expression, as indicated by the significant increase in GFP expression.

[0033] Figure 3 This is a schematic diagram of the composition provided herein. It illustrates an anti-ABCB5 monoclonal antibody linked to an adeno-associated virus (AAV) vector via a HUH-endonuclease tag. The AAV vector contains an OSK construct, which enters the target cell after the anti-ABCB5 antibody binds to ABCB5 on the target cell surface. Following binding, the AAV fuses with the target cell, releasing the OSK construct to induce the expression of OCT-4, SOX2, and KLF4 in the target cell.

[0034] Figure 4 Includes flow cytometry plots showing the expression of green fluorescent protein (GFP) in transfected melanoma cells treated with anti-ABCB5 antibody 3C2-1D12 (bottom row) or untreated (top row) with ABCB5. GFP expression is driven by multiple ABCB5 regulatory elements, with CMV serving as a positive control for constitutive expression independent of ABCB5 expression.

[0035] Figure 5GFP expression in primary human corneal epithelial cells is shown, driven by the inducible ABCB5b promoter, the ABCB5-10 kb enhancer, or CMV as a constitutive positive control. Solid, unfilled curves represent GFP expression using the Shield1 inducible construct; filled curves represent Shield1-control.

[0036] Figure 6 This diagram illustrates GFP expression driven by Shield1-inducible ABCB5 regulatory elements in ABCB5+ derived skin cells. The top left is an exemplary flow cytometry plot, the bottom left is the mean fluorescence intensity (MFI) of GFP expression in the Shield1- (left bar) or Shield1+ (right bar) populations, and the right panel shows GFP fluorescence in the overall cell population (left column) relative to the ABCB5+-enriched subpopulation (right column). In the right-hand panel, solid, unfilled curves represent GFP expression in Shield1+ cells, and filled curves represent GFP expression in Shield1- cells.

[0037] Figures 7A to 7B The plasmids used to induce OSK factor (OCT-4, SOX2, and KLF-4) transcription in stem cells and their downstream effects on ABCB5 expression are shown. A schematic diagram of the dual plasmid system is shown at the top, and schematic diagrams of the ABCB5 regulatory element-driven construct and the control construct are shown on the left. Figure 6 A). The expression of OCT-4, SOX2, and KLF-4 proteins generated from different constructs is shown in the lower right Western blot (A). Figure 6 A). Seven days after administration of doxycycline to induce transcription, cells transfected with a plasmid containing ABCB5 TFRR, which drives OSK factor expression, showed a robust increase in ABCB5 mRNA. Figure 6 B, left). At 48 hours and 7 days after induction of the OSK reprogramming factor, cell surface expression of ABCB5 was also increased in transfected cells. Figure 6 B, right).

[0038] Figures 8A to 8F This involves the generation of ABCB5-GFP reporter lines. The constructs used to encode transgenes driving GFP expression under the ABCB5 exon are described in... Figure 8A As shown in the image. Figures 8B to 8C It shows the use of CRISPR and Figure 8A The MFI (Exemplary flow cytometry and histogram) of ABCB5 gene knock-in into skin cells created by the construct is used to measure GFP reporter expression and ABCB5 expression. Figure 8B In summary Figure 8C middle). Figure 8D It is an immunoprecipitation blot of endogenous ABCB5 using antimyc antibody (clone 9E10). Figure 8E (Without puromycin) and 8F (with puromycin) show the use of puromycin to select ABCB5+ cells.

[0039] Figures 9A to 9C The diagram shows the use of the ABCB5 TFRR structural domain ( Figure 9A ), ABCB5-10 kb enhancer ( Figure 9B ) or Tet-on 3G architecture ( Figure 9C The plasmids and sequences that drive OSK expression.

[0040] Figures 10A to 10E This involves the reprogramming of dermal mesenchymal stem cells (DMSCs) following OSK treatment. AL10 to AL13 each represent different human donor samples. Figure 10A The MFI fold increase of ABCB5 expression is shown. Figures 10B to 10C This shows Oct4, Sox2, and KLF4 in transfected DMSCs. Figure 10B ) or Sox2 and KLF4 ( Figure 10C The expression of ). Figures 10D to 10E This shows Oct4 in DMSCs transfected 7 days post-transfection. Figure 10D ) or Sox2 and KLF4 ( Figure 10E ). Detailed Implementation

[0041] Stem cell therapy is an innovative medical approach that utilizes the unique properties of stem cells to treat a wide range of diseases and injuries. Stem cells (which can be derived from adult tissues) have the ability to differentiate into other cell types within the body. This regenerative potential makes them valuable for repairing damaged tissues and organs, and for addressing conditions such as cardiovascular disease, neurodegenerative diseases, and autoimmune diseases. Stem cell therapy can involve transplanting reprogrammed stem cells in vitro or reprogramming stem cells in vivo to promote a more functional phenotype.

[0042] Methods for reprogramming adult stem cells involve introducing epigenetic reprogramming factors important for maintaining the pluripotency and self-renewal of embryonic stem cells. For example, octamer-binding transcription factor 4 (OCT-4), SRY box 2 (SOX2), and Krüppel-like factor 4 (KLF4), known as OSK factors, are often used in combination to reprogram stem cells. Typically, epigenetic reprogramming factors broadly target cells. This paper has shown that targeting epigenetic reprogramming factors to ABCB5+ stem cells has unexpected therapeutic advantages. For example, this paper demonstrates that senescent cells can be revitalized, exhibiting characteristics of younger ABCB5+ stem cells, while avoiding off-target effects.

[0043] This article provides compositions capable of targeting ABCB5+ stem cells and delivering epigenetic reprogramming factors to ABCB5+ stem cells. A method for administering the ABCB5-targeting composition to a subject is also provided. 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 diseases.

[0044] stem cells

[0045] Stem cells are heterogeneous cell populations characterized by their ability to self-renew and differentiate into a variety of specialized cell types throughout the body. They are essential for development, tissue repair, and the maintenance of health and cognition throughout an organism's life. Stem cells can be broadly classified into long-lived stem cells (including tissue-specific adult stem cells that help maintain and repair tissues throughout life) and shorter-lived stem cells (including progenitor cells that divide and differentiate more rapidly than long-lived stem cell populations). As an individual ages, the function and adaptability of long-lived stem cells decline. This leads to complications commonly associated with stem cell therapy, as dysfunction of adult stem cells often results in (1) an increasing scarcity of the target stem cell population with age, and (2) failure of stem cells to engraft after transplantation. Therefore, stem cell therapy that prioritizes epigenetic reprogramming of long-lived stem cells and restores the adaptability of these populations is an attractive concept, especially if used in vivo.

[0046] stem cell therapy

[0047] Stem cell therapy is an evolving field that involves using stem cells to treat a variety of medical conditions and promote tissue regeneration. In some implementations, stem cells are administered to subjects suffering from degenerative diseases. These degenerative diseases can be neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease, Huntington's disease, or multiple sclerosis), cardiovascular diseases (such as congestive heart failure or ischemic heart disease), ophthalmic diseases (such as corneal damage, glaucoma, or age-related macular degeneration), lung diseases (such as chronic obstructive pulmonary disease), liver diseases (such as cirrhosis or hepatitis), kidney diseases (such as chronic kidney disease), muscle diseases (such as Dichenne muscular dystrophy or Becker muscular dystrophy), or another disease. Various treatment modalities or methods are employed in stem cell therapy, including:

[0048] Autologous stem cell therapy involves stem cells derived from the patient's own body, typically from bone marrow, adipose tissue, or skin. These cells are collected, processed, and then reintroduced into the patient to promote healing and tissue regeneration.

[0049] Allogeneic stem cell therapy involves obtaining stem cells 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 acquisition of a large stem cell bank for treatment.

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

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

[0052] Mesenchymal stem cell therapy 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 possess immunomodulatory properties and can differentiate into different cell types.

[0053] Genetically modified stem cell therapy involves stem cells being genetically modified to enhance their therapeutic properties or correct specific genetic defects. For example, gene-editing technologies such as 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. Proteins involved in the maintenance and function of long-lived stem cells are ATP-binding cassette sub-family Bmember 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 with the ability to self-renew and differentiate into mature cells of various adult cell lineages. As used herein, “ABCB5+ stem cells” or “ABCB5+ cells” refers to such cells. In some embodiments, ABCB5+ stem cells are limbal ABCB5+ stem cells. In some embodiments, ABCB5+ stem cells are dermal ABCB5+ stem cells.

[0056] ABCB5+ stem cells play a role in tissue regeneration and aging. Regenerative medicine involves the optional use of exogenous materials (e.g., scaffolds) to repair, regenerate, maintain, and replace tissues and organs. Methods for enhancing tissue regeneration and repair, as well as interrupting aging, are disclosed in this paper. It has been found that compositions containing anti-ABCB5 antibodies conjugated to therapeutic payloads containing epigenetic reprogramming factors can be used for targeted therapies to promote healthy aging and / or reverse the effects of age-related diseases. While unidirectional aging of the body is generally considered irreversible, recent work has now challenged this dogma: demonstrating that partial in vivo epigenetic reprogramming of somatic cells using AAV-OSK gene therapy (AAV: adeno-associated virus; OSK: OCT-4, SOX2, KLF4) can reverse cellular senescence in the retina, revitalizing senescent cells to a “younger” transcriptome and methylome, and leading to restoration of visual function in aged 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 advancement in the field, targeted delivery in a way that avoids off-target side effects has remained challenging.

[0057] The methods disclosed in this paper utilize the expression of the gene ABCB5 to provide targeted delivery or expression induction. ABCB5 encodes a cell surface receptor on tissue-specific stem cells / progenitor cells essential for normal development, maintenance, and regeneration in various mammalian tissues, including, for example, the eye-retina and limbus, skin-dermis, intestine-basal recess, and testis (Ksander et al., Nature 2014; Schatton et al., Cell Reports 2015). ABCB5 is downregulated or lost during aging. This paper demonstrates that AAV / OSK reprogramming targeting ABCB5 / GFP reporter stem cells purified with an ABCB5 antibody enhances ABCB5 expression. Figure 1 This article also shows that using ABCB5 regulatory elements (e.g., promoters, enhancers, and transcription factor regulatory regions) enhances the expression of GFP and OSK factors in cells expressing ABCB5 without targeting GFP or OSK factors to cells expressing ABCB5 on their surface. Figure 3 (to 7). In some aspects, this disclosure provides ABCB5+ cell populations comprising exogenous nucleic acids containing nucleotide sequences of an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding epigenetic reprogramming factors.

[0058] These findings suggest that the decline in adult stem cell function is a significant part of the aging process and is closely associated with susceptibility to age-related diseases mediated by atrophy and inflammatory aging. Furthermore, epigenetic reprogramming targeting stem cells can be used to reverse and / or prevent such age-related tissue dysfunction. This paper presents a novel strategy for the sustained revitalization of aging tissues by reversing adult stem cell aging through targeted delivery of epigenetic factors guided by anti-ABCB5 antibodies. Targeted therapy allows for the selective epigenetic reprogramming of ABCB5-expressing stem cells, thereby restoring aging and damaged tissues, such as RPE (retinal pigment epithelial) cells, which are crucial for the normal development, maintenance, and regeneration of the retina in two highly prevalent, blinding retinal diseases: age-related macular degeneration (AMD) and glaucoma. The disclosed treatment can be widely applied in the development of anti-aging strategies targeting other tissues containing ABCB5 stem cells (including, but not limited to, skin, limbus, intestine, and testis), as well as inflammatory aging associated with ABCB5+ stem cell dysfunction leading to CNS degenerative diseases.

[0059] Epigenetic reprogramming

[0060] The methods disclosed in this paper offer stem cell-targeted solutions to two major biological and clinical challenges: promoting healthy aging and treating age-related diseases. Enhanced therapy with fewer potential side effects can be achieved by using therapeutic compositions comprising anti-ABCB5 antibodies linked to a therapeutic payload containing epigenetic reprogramming factors. For example, the epigenetic reprogramming factors in the therapeutic composition can be delivered in the form of adeno-associated virus OCT-4 / SOX2 / KLF4 (AAV / OSK) gene therapy, which is described in more detail below. AAV / OSK epigenetic reprogramming targeting ABCB5+ stem cells avoids the problems associated with in vivo non-targeted AAV / OSK epigenetic reprogramming. While recent successes have been made in restoring senescent or damaged somatic cells through non-targeted reprogramming, such existing approaches may only represent temporary repair of rapidly or slowly regenerating cells, or may preferentially target short-lived stem cells relative to long-lived stem cells. More durable or even permanent solutions to aging problems will likely require the effective reprogramming and revitalization of senescent long-lived stem cells to generate a continuous supply of “younger” progeny cells. The method disclosed in this paper addresses these challenges by specifically enhancing stem cells at damaged or aging tissues.

[0061] Exemplary therapeutic compositions in Figure 2 As shown in the diagram, ABCB5 monoclonal antibody (mAb)-AAV / OSK nanoparticles are constructed using a modified AAV carrying a HUH tag, which forms a covalent bond with a single-stranded DNA oligonucleotide conjugated to the mAb. 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) can be linked to the AAV in this manner. Alternatively, anti-ABCB5 mAb conjugated liposomes or other non-viral nanoparticle formulations can be used to target and specifically deliver therapeutic payloads to ABCB5+ stem cells to achieve stem cell epigenetic reprogramming and restoration of function in young stem cells, as well as disease mitigation / reversal.

[0062] OSK treatment

[0063] Several factors exist that can be used to reprogram differentiated adult cells into stem cells, or to promote stem cell function and / or survival. Such factors typically include one or more transcription factors that induce or inhibit the expression of genes that promote stem cell phenotypes. Some transcription factors 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), Krüppel-like factor 4 (KLF4), Nanog, Lin28, and Myc, as well as other transcription factors.

[0064] OCT-4, SOX2, and KLF4 are frequently used in combination for epigenetic reprogramming of stem cells and are collectively known as OSK factors. To induce stem cell reprogramming, OSK factors or nucleic acids encoding OSK factors are introduced into stem cells via cell-targeting vectors. Once inside the cells, OSK factor expression leads to the activation of a complex network of genes involved in pluripotency, cell cycle regulation, and epigenetic remodeling. Additionally, OSK factor expression 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 OSK factors exhibit a “younger” stem cell phenotype, meaning they demonstrate higher functionality and self-renewal capacity than senescent stem cells. The sequences of OSK factors and methods of using 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. 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, OSK is directly linked to an anti-ABCB5 antibody. In some embodiments, the nucleic acid encoding the epigenetic reprogramming factor comprises a sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with 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 the sequences of SEQ ID NO: 5, SEQ NO: 6, and / or SEQ ID NO: 7.

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

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

[0068] Composition of epigenetic factors linked to ABCB5 antibody

[0069] Compositions and methods for targeting ABCB5+ stem cells for epigenetic reprogramming are provided herein. In some aspects, this disclosure provides compositions comprising an anti-ABCB5 antibody conjugated to a therapeutic payload, said therapeutic payload comprising an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor. Antibodies that bind to and target ABCB5 on the cell surface are known and have been described in the literature. Some anti-ABCB5 antibodies are commercially available. Anti-ABCB5 antibodies useful according to this disclosure target ABCB5 on the surface of stem cells to deliver a therapeutic payload and produce therapeutic outcomes, such as improved stem cell function, restoration of senescent or damaged 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 recognition system, antibodies can recognize and tag target epitopes, foreign entities, cancer cells, or invading microorganisms for attack by the immune system, thereby neutralizing their effects. Antibody production is a major function of the humoral immune system. Antibodies are secreted by plasma cells (a type of white blood cell).

[0071] This document provides antibodies capable of binding to ABCB5, nucleic acids encoding said antibodies, and their use in conjunction with therapeutic payloads for therapeutic, research, and diagnostic purposes. Therefore, this disclosure provides antibodies that bind to ABCB5. In some embodiments, the anti-ABCB5 antibody binds to ABCB5 displayed on the cell surface at an extracellular loop. Alternatively or supplementally, the anti-ABCB5 antibody may have low binding affinity for denatured ABCB5 or may not bind to denatured ABCB5.

[0072] An antibody (used interchangeably in the plural form) is an immunoglobulin molecule capable of specifically binding to a target antigen (e.g., ABCB5 in this disclosure) through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. The term “antibody” as used herein encompasses not only full-length (i.e., full-length) polyclonal or monoclonal antibodies, but also their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), single chains (scFv), mutants thereof, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, biantibodies, nanobodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified conformation of an immunoglobulin molecule containing an antigen recognition site having the desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include any class of antibodies, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and antibodies need not be of any particular class. Immunoglobulins can be classified into different classes based on the antibody amino acid sequence of their heavy chain constant domains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are designated as α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well-known.

[0073] A typical antibody molecule contains 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 hypervariable regions, also known as complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Therefore, the antibody variable region is composed of framework regions interspersed with three antigen-binding sites. Antigen binding sites are defined using several terms: (i) complementarity-determining regions (CDRs), which have three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3), based on sequence variability; and (ii) "hypervariable regions," "HVRs," or "HVs," which have three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3), referring to the hypervariable regions of the antibody variable domain in structures defined by Chothia and Lesk, etc. A "framework" or "frame sequence" is the remaining sequence of the variable region other than that defined as an antigen binding site. Because antigen binding sites can be defined using various terms as described above, the exact amino acid sequence of a frame depends on how the antigen binding site is defined.

[0074] The antibodies described herein may be of mouse, rat, human, primate, pig, or any other origin (including chimeric or humanized antibodies). Such antibodies are not naturally occurring, i.e., they will not be produced in animals without human intervention (e.g., immunization of such animals with the desired antigen or fragment thereof).

[0075] Any antibody described herein may be monoclonal or polyclonal. "Monoclonal antibody" refers to a group of homologous antibodies, and "polyclonal antibody" refers to a group of heterologous antibodies. These terms do not limit the source of the antibody or the method of its preparation.

[0076] In one instance, the antibody used in the methods described herein is a humanized antibody, optionally a humanized form of 3C2-1D12. A humanized antibody is a form of non-human (e.g., mouse) antibody that is a specific chimeric immunoglobulin, an immunoglobulin chain, or an antigen-binding fragment containing a minimal sequence derived from a non-human immunoglobulin. In most cases, the humanized antibody is a human immunoglobulin (receptor antibody) in which residues from the complementarity-determining region (CDR) of the receptor are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit, possessing the desired specificity, affinity, and capability. In some cases, Fv frame region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may include residues not present in the recipient antibody or the introduced CDR or frame sequence but included to further refine and optimize antibody performance. Generally, humanized antibodies will contain substantially all of at least one and typically two variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are those of human immunoglobulin common sequences. Humanized antibodies will also preferably contain at least a portion of immunoglobulin constant regions or domains (Fc), typically those of human immunoglobulins. 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, or six) altered relative to the original antibody, which are also referred to as being “derived” from one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation.

[0077] In another example, the antibody described herein is a chimeric antibody, which may comprise a heavy constant region and a light constant region derived from a human antibody. A chimeric antibody is an antibody having a variable region or a portion thereof from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions derived from an antibody of one mammal (e.g., a non-human mammal such as a mouse, rabbit, or rat), while the constant portion is sequence homologous to that of an antibody derived from another mammal (e.g., a human). In some embodiments, amino acid modifications may be made in the variable and / or constant regions.

[0078] The constant regions of the antibody heavy and light chains are well known in the art, for example, those available in the IMGT database (www.imgt.org) or in www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0079] In some implementations, the antibody is an antigen-binding fragment. As used herein, the term "antigen-binding fragment" refers to an antibody fragment such as a biantibody, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized biantibody (ds biantibody), single-chain antibody molecule (scFv), single-domain antibody (sdab), scFv dimer (bivalent biantibody), multispecific antibody formed from a portion of an antibody containing one or more CDRs, camelized single-domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain the complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen that binds to a parent antibody or a fragment of a parent antibody. According to some specific embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment (i.e., a portion of the heavy chain contained in the Fab fragment). According to other specific embodiments, the antigen-binding fragment comprises Fab and F(ab').

[0080] In another example, the antibody described herein can be a single-domain antibody that interacts with the target antigen only through a single variable domain (e.g., a single heavy chain domain) (as opposed to conventional antibodies that interact with the target antigen through both heavy and light chain variable domains). A single-domain antibody can be a heavy chain antibody (VHH) that contains only the antibody heavy chain and lacks the light chain. In addition to the variable region (e.g., VH), a single-domain antibody may also contain constant regions, such as CH1, CH2, CH3, CH4, or combinations thereof.

[0081] In some embodiments, the antibody and its antigen-binding fragment include a fragment crystallizable (Fc) region. The Fc region is the tail region of the antibody and its antigen-binding fragment, which contains constant structural domains (e.g., CH2 and CH3); another region of the antibody and its antigen-binding fragment is the Fab region, which contains variable structural domains (e.g., VH) and constant structural domains (e.g., CH1), the former defining the binding specificity.

[0082] As described herein, antibodies may comprise a VH domain. In some embodiments, the VH domain may further comprise one or more constant domains of the Fc region (e.g., CH2 and / or CH3) and / or one or more constant domains of the Fab region (e.g., CH1). In some embodiments, each of the one or more constant domains (e.g., CH1, CH2, and / or CH3) may comprise a portion of or consist of a portion 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-length sequence.

[0083] Alternatively, the antibodies described herein may contain up to five (e.g., four, three, two, or one) amino acid residue variations in one or more CDR regions of one or more of the antibodies known in the art and / or exemplified herein, and bind the same antigenic epitopes with substantially similar affinity (e.g., having equivalent KD values). In one instance, the amino acid residue variation is a conserved amino acid residue substitution. As used herein, “conserved amino acid substitution” means an amino acid substitution that does not alter the relative charge or size characteristics of the protein to which the substitution is made. Variants may be prepared according to methods for altering polypeptide sequences known to those skilled in the art, methods described, for example, in references compiling such methods, such as, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions between amino acids in 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.

[0084] In some embodiments, the anti-ABCB5 antibody described herein binds specifically to the corresponding target antigen or its epitope. "Specifically binding" to an antigen or epitope is a well-known term in the art. A molecule is said to exhibit "specific binding" if its reaction with a particular target antigen is more frequent, faster, longer-lasting, and / or has a higher affinity than its reaction with an alternative target. An antibody is said to "specifically bind" to a target antigen or epitope if it binds with a higher affinity, stronger affinity, easier binding, and / or longer duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (ABCB5) or an epitope therein binds to that target antigen with a higher affinity, stronger affinity, easier binding, and / or longer duration than it binds to other antigens or other epitopes of the same antigen. It can also be understood under this definition that, for example, an antibody that specifically binds to a first target antigen may specifically or preferentially bind to a second target antigen or may not specifically or preferentially bind to a second target antigen. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. In some instances, antibodies that "specifically bind" to a target antigen or its epitope may not bind to other antigens or other epitopes of the same antigen (e.g., bindings that are undetectable in routine assays).

[0085] In some embodiments, the antibodies described herein bind specifically to ABCB5 of a specific species (e.g., human ABCB5) relative to ABCB5 from other species. For example, the antibodies described herein can bind specifically to human ABCB5 relative to mouse ABCB5. In other embodiments, the antibodies described herein can cross-react with human ABCB5 and one or more ABCB5s from non-human species (e.g., non-human primates such as macaques or pigs). In some embodiments, the antibodies cross-react with human, macaque, and pig ABCB5s with similar binding affinity, but have a significantly lower binding affinity for mouse ABCB5.

[0086] In some embodiments, the anti-ABCB5 antibody, as described herein, has a suitable binding affinity to the target antigen (e.g., human ABCB5) or its epitope. As used herein, “binding affinity” refers to the apparent association constant, or KA, which is the ratio of the association constant to the dissociation constant (K-on and K-off, respectively). KA is the reciprocal of the dissociation constant (KD). The binding affinity (KD) of the anti-ABCB5 antibody to the target antigen or epitope described herein may be at least 10⁻⁸, 10⁻⁹, 10⁻¹⁰ M or lower. Increased binding affinity corresponds to a decreased KD value. Higher affinity binding of the antibody to the first antigen relative to the second antigen can be indicated by a higher KA (or a smaller numerical KD) for binding the first antigen than for binding the second antigen. In such cases, the antibody is specific to the first antigen (e.g., the first protein or its mimicry in a second conformation; or the second protein) relative to the second antigen (e.g., the same first protein or its mimicry in a first conformation). In some embodiments, the anti-ABCB5 antibodies described herein exhibit a higher binding affinity (higher KA or lower KD) to ABCB5 compared to their binding affinity to another membrane protein (e.g., ABCB1, ABCB4, and ABCB11). The difference 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 anti-ABCB5 antibody may be further affinity-matured to increase its binding affinity to the target antigen or its epitope.

[0087] 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 fluorescence assays). Exemplary conditions for evaluating binding affinity are: HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20) and PBS buffer (10 mM PO4-3, 137 mM NaCl, and 2.7 mM KCl). These techniques can be used to measure the concentration of the bound protein as a function of the target protein concentration. The concentration of the bound protein ([bound]) is generally correlated with the concentration of the free target protein ([free]) by the following formula: [bound] = [free] / (Kd + [free]).

[0088] In some embodiments, the anti-ABCB5 antibody is the antibody, fragment thereof, or derivative thereof disclosed in US 2023 / 0203153, which is incorporated herein by reference in its entirety for all disclosures related to the anti-ABCB5 antibody. In some embodiments, the ABCB5 antibody is 3C2-1D12 or a derivative thereof, such as those described in U.S. Patent No. 8,697,072, which is incorporated herein by reference for all disclosures relating to the ABCB5 antibody, its fragments, and derivatives thereof.

[0089] Nucleic acids encoding epigenetic reprogramming factors can be delivered to ABCB5+ cells. In some embodiments, this disclosure provides compositions in which a 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 can be, for example, a vector encoding an epigenetic reprogramming factor, such as a viral vector. In some embodiments, the vector can be linked to a targeting portion targeting ABCB5+ cells, said targeting portion being, for example, an antibody or antibody fragment. The nucleic acid can also be, for example, a nucleotide sequence encoding one or more epigenetic reprogramming factors and controlled by a regulatory element, such as an inducible promoter. An inducible promoter can restrict the expression of the epigenetic reprogramming factor to specific cells, such as ABCB5+ cells.

[0090] For certain compositions and methods of this disclosure, it is preferable to target a protein or nucleic acid encoding the protein to a specific cell. In such cases, the carrier for delivering the nucleic acid or protein into the cell may have a targeting molecule linked thereto. For example, a molecule (e.g., an antibody specific to a surface membrane protein on a target cell or a ligand for a receptor on a target cell) may bind to or be incorporated into the nucleic acid delivery carrier. The carrier may include a viral vector or nanoparticles. In some aspects, the compositions provided herein comprise an anti-ABCB5 antibody conjugated to a viral vector via a linker, 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 lentiviral vectors, retroviral vectors, adenovirus vectors, alphavirus vectors, vaccinia virus vectors, and adeno-associated virus vectors (AAV).

[0091] Retroviral vector

[0092] Introducing exogenous genetic material into ABCB5 +One approach in stem cell therapy involves targeting cells using replication-deficient retroviruses. Replication-deficient retroviruses can direct the synthesis of all virosomal proteins but cannot produce infectious particles. Therefore, these genetically modified retroviral vectors have general utility for the efficient transduction of genes in cultured cells. Retroviruses have been widely used to transfer genetic material into cells. Standard protocols for generating replication-deficient retroviruses are provided in the art, comprising the steps of incorporating exogenous genetic material into a plasmid, transfecting a packaging cell line with the plasmid, generating recombinant retroviruses from the packaging cell line, collecting viral particles from tissue culture medium, and infecting target cells with the viral particles.

[0093] The main advantage of using retroviruses is that these viruses efficiently insert a single copy of the gene encoding a therapeutic agent into the host cell genome, thereby allowing the foreign genetic material to be passed on to the cell's offspring during cell division. Furthermore, gene promoter sequences in the LTR region have been reported to enhance the expression of the inserted coding sequence in a variety of cell types. The main disadvantages of using retroviral expression vectors are (1) insertional mutagenesis, i.e., the therapeutic gene is inserted into an undesirable location in the target cell genome, which, for example, leads to unregulated cell growth, and (2) the need for target cell proliferation to integrate the therapeutic gene carried by the vector into the target genome. Despite these apparent limitations, delivery of therapeutically effective amounts of therapeutic agents via retroviruses can be effective if transduction efficiency is high and / or the number of target cells available for transduction is large.

[0094] Alphavirus vector

[0095] Alphavirus vectors are a class of viral vectors derived from alphaviruses (the positive-sense RNA virus group). These vectors have been engineered for a variety of 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 delivering therapeutic genes in biomedical research.

[0096] vaccinia virus vector

[0097] Vaccinvirus vectors are a type of viral vector derived from vacciniaviruses (members of the Poxviridae family). These vectors have been widely used in biotechnology and vaccine development. Vaccinvirus vectors are known for their large genome capacity, which allows them to carry and express a variety of exogenous genes.

[0098] Adenovirus vector

[0099] Another option is to target ABCB5. +Adenoviruses, double-stranded DNA viruses, are viral candidates for stem cell expression vectors. Like retroviruses, adenovirus genomes are well-suited for use as expression vectors for gene transduction, i.e., by removing the genetic information controlling the virus's own production. Since adenoviruses typically function extrachromosomally, recombinant adenoviruses do not present the theoretical problems of insertional mutagenesis. Recent reports indicate that certain adenovirus sequences confer intrachromosomal integration specificity to vector sequences, thereby enabling stable transduction of exogenous genetic material.

[0100] Adeno-associated virus vector

[0101] Adeno-associated virus (AAV) vectors are a type of virus-derived vector used to deliver small payloads of therapeutic agents (e.g., genetic material) with low immunogenicity and a favorable safety profile. AAV vectors are derived from adeno-associated viruses, small, non-pathogenic viruses that integrate into the genome of another organism (“host”) (e.g., a mammal) to induce gene expression in the cells or tissues to which the AAV is delivered. In some embodiments, compositions are provided herein comprising an anti-ABCB5 antibody fused to an AAV vector. When using an adeno-associated virus as a vector, the endogenous viral genome is modified (e.g., viral nucleic acid is removed) and the therapeutic agent to be delivered (e.g., therapeutic nucleic acid) 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 Krüppel-like factor 4 (KLF4).

[0102] Virus particles

[0103] Viral particles, also known as virions, consist of a viral genome encased in a capsid. Like other viral vectors, the genome of a viral particle can be modified to contain nucleic acids encoding a target gene to be expressed in target cells.

[0104] Other nucleic acids

[0105] Other nucleic acids used to deliver a specific nucleic acid sequence into a cell are known in the art. For example, nucleic acids can be DNA, RNA, or mRNA encoding epigenetic reprogramming factors.

[0106] Nanoparticles

[0107] Nanoparticles are small particles or structures typically ranging in size from 1 to 100 nanometers. Nanoparticles can be composed of a variety of materials, including metals, polymers, ceramics, or biomolecules, and exhibit unique properties and behaviors due to their small size and high surface area to volume ratio. Nanoparticles can also be liposomes having one or more lipid bilayers surrounding an aqueous pocket, or lipid nanoparticles having a single lipid bilayer encapsulating an aqueous or non-aqueous pocket. The size and physical properties of nanoparticles make them useful tools for facilitating the delivery of therapeutic payloads to cells. In some aspects, anti-ABCB5 antibodies are conjugated to nanoparticles containing epigenetic reprogramming factors or nucleic acids encoding epigenetic reprogramming factors. In some embodiments, the nanoparticles are polymer nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the nanoparticles are liposomes.

[0108] When using liposomes to deliver the nucleic acids of the present invention, proteins that bind to surface membrane proteins associated with endocytosis can be incorporated into the liposome formulation to target and / or promote uptake. Such proteins include proteins or fragments thereof that are tropist to specific cell types, antibodies against proteins undergoing internalization in circulation, proteins that target intracellular localization and increase intracellular half-life, etc. As known to those skilled in the art, polymer delivery systems have also been successfully used to deliver nucleic acids into cells. Such systems even allow for oral delivery of nucleic acids.

[0109] Therefore, it will be apparent to those skilled in the art that a variety of suitable vectors can be used to transfer exogenous genetic material into ABCB5. + In stem cells, the selection of a suitable vector for delivering therapeutic agents appropriate for specific conditions requiring gene replacement therapy and the optimization of conditions for inserting the selected expression vector into cells are within the skill of a person of ordinary skill in the art, without the need for excessive experimentation.

[0110] Adjustment element

[0111] The selection and optimization of specific nucleic acids for expressing epigenetic reprogramming factors can also utilize one or more suitable regulatory elements, such as promoters or enhancers. Naturally occurring constitutive promoters control the expression of essential cellular functions. Therefore, genes under the control of constitutive promoters are expressed under all cell growth conditions. Exemplary constitutive promoters include promoters for genes encoding certain constitutive or "housekeeping" functions, such as 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, actin promoter (Lai et al., Proc. Natl. Acad. Sci. USA 86:10006-10010 (1989)), and other constitutive promoters known to those skilled in the art. Additionally, many viral promoters function constitutively in eukaryotic cells. These include: early and late promoters of SV40; long terminal repeats (LTRS) of Moloney leukemia virus and other retroviruses; and the thymidine kinase promoter of herpes simplex virus, etc. Therefore, any of the constitutive promoters mentioned above can be used to control the transcription of heterologous gene inserts.

[0112] In some embodiments, the compositions provided herein comprise a viral vector that further comprises a regulatory element associated with a nucleotide sequence encoding one or more epigenetic reprogramming factors. 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 ABCB5 enhancer.

[0113] In some aspects, the nucleic acid provided herein comprises an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding epigenetic reprogramming factors. By using an ABCB5 regulatory element, such as an ABCB5 promoter or an ABCB5 enhancer, epigenetic reprogramming factors can be selectively expressed in cells endogenously using 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 having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with respect to the sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid comprises a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 4 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity). In some embodiments, the nucleic acid comprises the 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 having at least 90% sequence identity with the sequence of SEQ ID NO: 2 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity). In some embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 2.

[0114] Genes controlled by inducible promoters or enhancers are expressed only or to a greater extent in the presence of an inducer (e.g., transcription controlled by metallothionein promoters is significantly enhanced in the presence of certain metal ions). In some embodiments, the inducible component of the ABCB5 regulatory element comprises a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 3 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity). In some embodiments, the inducible component of the ABCB5 regulatory element comprises the sequence of SEQ ID NO: 3. For the purposes of this discussion, an "enhancer" is simply any non-translated DNA sequence that operates sequentially with a coding sequence (cis) to alter the basal transcriptional level determined 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) that stimulate transcription when their inducing factors are bound. For example, REs exist for serum factors, steroid hormones, and cyclic AMPs. Numerous chemical agents exist for controlling gene expression. Examples of such agents include, but are not limited to, isopropyl β-D-1-thiogalactoside, tetracycline, doxycycline, tamoxifen, dexamethasone, and retinoic acid. Promoters or enhancers containing specific REs can be selected to obtain inducible responses, and in some cases, the RE itself can be linked to different promoters to confer inducibility to recombinant genes. Therefore, by selecting appropriate promoters or enhancers (constitutive vs. inducible; strong vs. weak), both the presence and expression levels of therapeutic agents in the targeted ABCB5+ stem cells can be controlled. Considering the factors disclosed above and the clinical characteristics of the subjects, the selection and optimization of these factors for delivering specific therapeutic agents at therapeutically effective doses are considered to be within the scope of those skilled in the art without the need for excessive experimentation.

[0115] Promoters or enhancers are characterized by specific nucleotide sequences necessary to initiate transcription or promote expression. Promoters and enhancers can also be used in combination to achieve the desired gene transcriptional activity. Preferably, exogenous genetic material is introduced into ABCB5 immediately downstream of the promoter. + In stem cell genomes, promoters and coding sequences are efficiently linked, thereby allowing transcription of the coding sequence. Preferred retroviral expression vectors include exogenous regulatory elements for controlling transcription of the inserted exogenous gene. Such exogenous promoters and enhancers include both constitutive promoters and enhancers and inducible promoters and enhancers. The selection of suitable promoters, enhancers, selectable genes, and / or signaling sequences is considered to be within the scope of those skilled in the art without requiring excessive experimentation.

[0116] In some embodiments, administration of a therapeutically effective amount of the composition increases the expression of OCT-4, SOX2, and / or KLF4 in ABCB5+ stem cells to above control levels, wherein the control levels are the expression of OCT-4, SOX2, and / or KLF4 in ABCB5+ stem cells that have not received the composition or have not received the composition.

[0117] connector

[0118] In some embodiments, the anti-ABCB5 antibody is fused to the AAV vector via a linker. Many types of linkers exist that can fuse or conjoin two or more biological or chemical substances. Linkers can be glycine-serine linkers, linkers with protease cleavage sites, linkers with restriction enzyme sites, endonuclease or endonuclease domain linkers, or other types of linkers. In some embodiments, the linker is an endonuclease or endonuclease domain. In some embodiments, the linker is a HUH endonuclease or HUH endonuclease domain.

[0119] disease

[0120] In some aspects, the compositions and methods provided by this disclosure can be used to treat aging or various diseases. In some aspects, this disclosure provides a method comprising administering an effective amount of an ABCB5-targeting composition to a subject to reprogram and revitalize ABCB5+ stem cells in the subject, said ABCB5-targeting composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload, said therapeutic payload comprising an epigenetic reprogramming factor or nucleic acid encoding an epigenetic reprogramming factor. In some embodiments, the subject is a stem cell recipient. In some embodiments, the ABCB5-targeting composition is the composition described herein.

[0121] In some aspects, this disclosure provides a method comprising administering an effective amount of nucleic acid to a subject to reprogram and revitalize ABCB5+ stem cells in the subject, said nucleic acid comprising a nucleotide sequence containing an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding epigenetic reprogramming factors.

[0122] Stem cells are attractive therapeutic agents for treating degenerative conditions or injuries due to their ability to differentiate into lineage-specific cell types. One approach to such treatment involves obtaining cells from a subject, reprogramming them in vitro, and then 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 and the poor engraftment caused by decreased functionality of adult stem cells. Another approach is to target stem cells in vivo for epigenetic reprogramming, but existing techniques indiscriminately target most cells, which can lead to preferential reprogramming of short-lived progenitor cells or differentiated cells relative to long-lived stem cells, which proliferate and renew more rapidly. Targeting long-lived stem cells is beneficial or necessary in treating aging or conditions involving dysfunction of long-lived stem cells. In some embodiments, the methods provided herein are methods for treating aging. In some embodiments, methods for treating aging include treating skin tissue or intestinal tissue.

[0123] ocular degenerative diseases

[0124] Degenerative eye diseases encompass a group of progressive eye conditions that cause degeneration of structures within the eye, typically resulting in vision loss or impairment. These diseases can affect multiple parts of the eye, including the retina, lens, or optic nerve, and are usually chronic and irreversible in nature. Retinal diseases cover 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 disease is age-related macular degeneration (AMD), a progressive eye disease that primarily affects older adults and can cause severe central vision loss. It occurs as the macula (the central part of the retina) degenerates over time, affecting the ability to see fine details and perform tasks such as reading and recognizing faces.

[0125] Another degenerative eye disease is glaucoma, characterized by elevated intraocular pressure. If left untreated, it can damage the optic nerve and lead to irreversible vision loss. It typically develops slowly and without noticeable symptoms in its early stages, making regular eye exams crucial for early detection and management.

[0126] In some embodiments, the methods provided herein are methods for treating degenerative diseases. In some embodiments, the degenerative disease is an ocular degenerative disease. In some embodiments, the ocular degenerative disease is a retinal condition. In some embodiments, the retinal condition is age-related macular degeneration (AMD) or glaucoma.

[0127] In some implementations, ocular degenerative diseases are corneal diseases, such as limbal stem cell deficiency (LSCD).

[0128] CNS degenerative diseases

[0129] Central nervous system (CNS) degenerative diseases encompass a group of conditions characterized by the gradual and progressive degeneration of nerve cells in the brain and spinal cord. These conditions (including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease) lead to the decline of cognitive, motor, or sensory functions, often resulting in severe disability. In some embodiments, methods for treating degenerative diseases are provided herein. In some embodiments, the degenerative disease is a CNS degenerative disease.

[0130] Inflammatory aging

[0131] Inflammatory aging is a phenomenon characterized by increased chronic, low-grade inflammation with age. It represents a state of chronic, smoldering inflammation in the body, even in the absence of infection or injury, and is thought to play a role in the aging process and the development of age-related diseases. Inflammatory aging is associated with a wide variety of conditions and a weakened immune system, including cardiovascular disease, diabetes, neurodegenerative diseases, and frailty. In some embodiments, the methods provided herein are methods for treating aging. In some embodiments, the treatment methods include treatment with ABCB5. + Inflammatory aging associated with stem cell dysfunction.

[0132] In addition to the compositions and methods disclosed herein, anti-ABCB5 antibodies can be used to isolate ABCB5+ stem cells from human tissues. The isolated cells can be treated in vitro or ex vivo with epigenetic reprogramming factors to revitalize them. These cells can then be delivered to the same or different patients for therapeutic or anti-aging purposes.

[0133] The subjects used in this article are humans, non-human primates, cattle, horses, pigs, sheep, goats, dogs, cats, or rodents.

[0134] Other implementation plans

[0135] The following are other embodiments of the present invention:

[0136] 1. A method comprising administering an effective amount of an ABCB5-targeting composition to a subject to reprogram and revitalize ABCB5+ stem cells in the subject, said ABCB5-targeting composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload, said therapeutic payload comprising an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor.

[0137] 2. A method comprising administering an effective amount of nucleic acid to a subject to reprogram and revitalize ABCB5+ stem cells in the subject, said nucleic acid comprising a nucleotide sequence containing an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding epigenetic reprogramming factors.

[0138] 3. The method described in implementation scheme 1 or 2, wherein the method is a method for treating degenerative diseases.

[0139] 4. The method described in Implementation Scheme 3, wherein the degenerative disease is an ocular degenerative disease.

[0140] 5. The method of implementation scheme 4, wherein the ocular degenerative disease is a retinal disease or a corneal disease, including limbal stem cell deficiency (LSCD).

[0141] 6. The method of implementation scheme 5, wherein the retinal condition is age-related macular degeneration (AMD) or glaucoma.

[0142] 7. The method of implementation scheme 3, wherein the degenerative disease is a CNS degenerative disease.

[0143] 8. The method described in implementation scheme 1 or 2, wherein the method is a method for treating aging.

[0144] 9. The method of implementation scheme 8, wherein the method of treating aging includes treating skin tissue or intestinal tissue.

[0145] 10. The method of implementation scheme 8, wherein the method of treating aging includes treating inflammatory aging associated with ABCB5+ stem cell dysfunction.

[0146] 11. The method of implementation scheme 1 or 2, wherein the subject is a stem cell recipient.

[0147] 12. The method of any one of the above embodiments, wherein the composition targeting ABCB5 is the composition of any one of the claims.

[0148] 13. A method for enhancing stem cell transplantation, comprising administering to a subject an effective amount of a composition targeting ABCB5 to reprogram and revitalize ABCB5+ stem cells in the subject, the composition targeting ABCB5 comprising a nucleic acid containing a nucleotide sequence of an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding an epigenetic reprogramming factor, and wherein the subject is a stem cell donor and the administration is performed prior to donation, or the subject is a stem cell recipient and the administration is performed after donation.

[0149] 14. A method for enhancing stem cell transplantation, comprising administering to a subject an effective amount of an ABCB5-targeting composition to reprogram and revitalize ABCB5+ stem cells in the subject, the ABCB5-targeting composition comprising an anti-ABCB5 antibody conjugated to a therapeutic payload, the therapeutic payload comprising an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor, and wherein the subject is a stem cell donor and the administration is performed prior to donation, or the subject is a stem cell recipient and the administration is performed after donation.

[0150] 15. The method of embodiment 13 or 14, wherein the composition targeting ABCB5 is the composition of any one of the composition claims.

[0151] 16. A method for preparing a cell population, the method comprising:

[0152] Isolate primary cells containing ABCB5+ cells from human tissue;

[0153] The isolated primary cells were cultured in a culture medium until the cells produced enough progeny to achieve a confluence of more than 60% of the mixed cells;

[0154] Harvest the mixed cells;

[0155] Cultured and harvested mixed cells;

[0156] The harvested mixed cells are contacted with a composition targeting ABCB5, the composition containing an anti-ABCB5 antibody conjugated to a therapeutic payload, the therapeutic payload containing an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor.

[0157] 17. The method of embodiment 16, wherein, relative to ABCB5 without the application of the composition... + Harvested mixed cells, when treated with the composition, showed increased ABCB5 levels. + OCT-4, SOX2 and / or KLF4 expression in harvested mixed cells.

[0158] 18. The method of embodiment 16 or 17, wherein the composition targeting ABCB5 is the composition of any one of the claims.

[0159] 19. An ABCB5+ cell population containing exogenous nucleic acids, said exogenous nucleic acids comprising a nucleotide sequence containing an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding epigenetic reprogramming factors.

[0160] 20. The cell population described in embodiment 19, wherein at least 99% of the cell population consists of ABCB5+ cells.

[0161] 21. The cell population described in Implementation Scheme 19, wherein the nucleic acid comprises a viral vector.

[0162] 22. The cell population described in Implementation Scheme 21, wherein the viral vector is selected from lentiviral vectors, retroviral vectors, adenovirus vectors, alphavirus vectors, vaccinia virus vectors, and adeno-associated virus vectors (AAV).

[0163] 23. The cell population described in Implementation Scheme 21, wherein the viral vector is AAV.

[0164] 24. The cell population described in embodiment 20, comprising nanoparticles encapsulating the nucleic acid.

[0165] 25. The cell population described in embodiment 24, wherein the nanoparticles are polymer nanoparticles.

[0166] 26. The cell population described in embodiment 24, wherein the nanoparticles are lipid nanoparticles.

[0167] 27. The cell population described in embodiment 24, wherein the nanoparticles are liposomes.

[0168] 28. The cell population according to any one of embodiments 20 to 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.

[0169] 29. The cell population according to any one of embodiments 20 to 27, wherein the inducible ABCB5 regulatory element is an ABCB5 promoter.

[0170] 30. The cell population according to any one of embodiments 20 to 27, wherein the inducible ABCB5 regulatory element is an ABCB5 enhancer.

[0171] 31. The cell population according to any one of embodiments 20 to 27, wherein the inducible ABCB5 regulatory element is a chemically induced ABCB5 regulatory element.

[0172] Example

[0173] Example 1. Identification of the regulatory sequence of the human ABCB5 gene

[0174] Using a combination of the ENCODE database and the UCSC genome browser, a limited number of previously uncharacterized potential ABCB5 gene regulatory regions were identified, including enhancer, promoter, and transcription factor regulatory regions.

[0175] enhancer Two putative enhancers were identified around regions of approximately -50 kb and -10 kb relative to the transcriptional start site (TSS) of ABCB5 (NM_001163941). These regions correspond to the peak of H3K27Ac described on the ENCODE / UCSC Genome Browser. H3K27Ac typically corresponds to regions of active gene expression, such as enhancers and promoters.

[0176] promoter Based on known transcription start sites of ABCB5 isotypes (e.g., NM_001163941 and NM_178559), the presumed proximal promoters were identified as regions up to -1.5 kb upstream of these sites.

[0177] Transcription Factor Regulatory Region (TFRR) Based on regions of transcription factor binding enrichment peaks observed in the ENCODE database, regions outside the classic promoter region potentially responsible for ABCB5 regulation were identified. A specific region downstream of the TSS of isotype NM_178559 was selected for testing. The sequence is shown in the table below.

[0178] Example 2. GFP expression driven by the inducible ABCB5 regulatory element

[0179] The ABCB5 clonal region driving Shield1-dependent inducible GFP expression was transfected into G3361 melanoma cells. The relative activity of the region is described in “GFP gating”. Figure 4 CMV was used as a constitutive activity positive control unrelated to ABCB5 expression. Activity increased 72 hours after ABCB5 blockade with antibody 3C2-1D12. The highest fold increase (0.21% to 1.17%) was detected in the TFRR. (5.5-fold). The fold increase of enhancers is similar (1.27% to 2.68% for -10 kb). 2.1 times; for -50 kb, 0.27% to 0.73% 2.7 times). The promoter is minimally affected (0.24% to 0.36% for β). 1.5 times; for γ, 0.22% to 0.31%. 1.4 times). In primary human corneal epithelial cells ( Figure 5) and ABCB5+ skin-derived cells ( Figure 6 The experiment was repeated in another location, and similar results were obtained.

[0180] Example 3. A dual-plasmid system for inducing OSK expression

[0181] Using a 2-plasmid system, the ABCB5 genomic fraction was used to drive the expression of revitalized transcription factors OCT4, SOX2, and KLF4 (OSK) in a skin cell population. The constitutively active EEF1A promoter was used as a positive control (similar to the previously shown CMV, EEF1A was active in all cells regardless of ABCB5 status). The Tet-3G system is inducible and requires the addition of doxycycline, allowing for time-controlled induction. Combination of the OSK plasmid with a plasmid under the control of ABCB5 regulatory elements resulted in OSK expression (…). Figure 7A ); and ABCB5 in transcripts ( Figure 7B (left) and protein ( Figure 7B The downstream upregulation of both (right) indicates that OSK reprogramming induces a positive feedback loop, thereby increasing ABCB5 expression.

[0182] Example 4. OSK expression in ABCB5 knock-in skin cells

[0183] ABCB5 gene knock-in GFP reporter skin cells (created using CRISPR) were used to demonstrate that constitutively expressed OSK rejuvenation factor resulted in an increased amount of ABCB5+ stem cells. This was also demonstrated by cell surface flow cytometry using the anti-ABCB5 antibody 3C2-1D12 (“3C2”) and immunoprecipitation using the anti-Myc antibody. Figures 8A to 8D The addition of the antibiotic puromycin allows for the selection of ABCB5+ cells (…). Figures 8E to 8F ).

[0184] Example 5. Design of plasmids to drive OSK expression

[0185] Three plasmids were designed for transfection to induce OSK expression in cells. Figures 9A to 9C The sequences are shown in Table 1 below.

[0186] Example 6. Induction of epigenetic reprogramming in ABCB5+ stem cells by OSK plasmids targeting ABCB5.

[0187] 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 (e.g., revitalize senescent ABCB5+ stem cells) has not been previously investigated. This embodiment tests whether OSK factors can be used to affect ABCB5 expression in human dermal mesenchymal stem cells (DMSCs) and whether OSK expression persists after DMSC transfection.

[0188] method

[0189] ABCB5+ dermal mesenchymal stem cells (DMSCs) from three donors were used in a 4 × 10⁻⁶ regimen. 4 AAV2 virus infection with pfu for 7 days. The AAV2 virus was engineered to drive Tet3G expression with a custom gene (genetic elements including the EF1a constitutively active promoter, the ABCB5-10 kb enhancer, or the ABCB5 gene element TFRR), and a second AAV2 virus encoding TRE3-OSK (Oct4 / Sox2 / Klf4). 100 pfu of adenovirus 5 (empty plasmid - Vector Biolab #1240) was added to assist OSK expression, and 1 μg / ml doxycycline was added to activate Tet3G.

[0190] As a negative control, AAV2 expression of CMV-GFP or EF1a-TdTomato was similarly used (also in the presence of doxycycline, which is inert to these controls).

[0191] The culture medium was then changed every 3 days to supplement doxycycline (no new virus was added after the initial dose).

[0192] The AAV2 virus and the custom-generated viral plasmids were created by Vector Builder according to the specifications described in this embodiment.

[0193] result

[0194] A specific protocol for OSK expression in ABCB5+ DMSCs was developed and tested. Oct4 overexpression was highly efficient after 7 days. Figures 10D to 10E The highest expression was observed with the EF1a promoter (739-fold increase relative to control), while slightly lower expression was observed with the ABCB5 gene element, the -10 kb enhancer (395-fold increase relative to control), and TFRR (618-fold increase relative to control). For Sox2 and KLF4, robust overexpression was still detected against the EF1a promoter (25.8-fold and 14-fold increase relative to control), the -10 kb enhancer (11.9-fold and 13.7-fold increase relative to control), and TFRR (28.4-fold increase relative to control). Figures 10A to 10C ).

[0195] Using the 3C2-1D12 antibody, after 1 week, compared with 3 different controls (no AAV (5.84%), CMV-GFP (4.22%), and EF1a-tdTomato (5.56%)), a relatively higher proportion of ABCB5 was observed in OSK expression samples using gene elements derived from EF1a (6.34% positive), ABCB5-10 kb enhancer (6.6%), and ABCB5 TFRR (6.61%) gene elements (Tables 2 and 3). This represents more than 20% more ABCB5 on the cell surface in all cases compared with the mean control (5.21%), and more than 25% more ABCB5 when using ABCB5-specific gene elements.

[0196] In summary, the data indicate that DMSCs, particularly ABCB5+ DMSCs, can be targeted for epigenetic reprogramming using OSK factors. OSK factors enhance ABCB5 expression, and OSK factor expression persists for at least one week post-transfection. These results suggest that reprogramming ABCB5+ stem cells using OSK factors provides a method for repairing or revitalizing senescent ABCB5+ stem cells.

[0197] Table 1. Plasmid sequences

[0198]

[0199] Table 2. ABCB5 Expressions After OSK Processing

[0200] Table 3. Anti-ABCB5 antibody MFI (subtracting isotype background staining)

[0201]

[0202] In some embodiments, this disclosure provides pharmaceutical compositions comprising pharmaceutically acceptable excipients. The “pharmaceutically acceptable excipient” does not cause undesirable physiological effects after or during administration to a subject. The carrier in the pharmaceutical composition must also be “acceptable” in the sense that it is compatible with the active ingredient and can stabilize the active ingredient. One or more solubilizers may be used as pharmaceutical carriers for delivering the active agent. Some examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible carriers, excipients, additives, and diluents to obtain compositions usable as dosage forms. Some examples of other carriers include, but are not limited to, colloidal silica, magnesium stearate, cellulose, and sodium dodecyl sulfate.

[0203] The composition can be administered via any route that produces a therapeutically effective outcome. These include, but are not limited to, injection, intradermal, intramuscular, intranasal, intraoral, intraocular, intraperitoneal, intravenous, topical, and / or subcutaneous administration. This disclosure provides a method of administering a composition targeting ABCB5 to a subject. In some aspects, the method provided herein includes administering an effective amount of a composition targeting ABCB5 to a subject to reprogram and revitalize ABCB5+ stem cells in the subject, said composition targeting ABCB5 comprising an anti-ABCB5 antibody conjugated to a therapeutic payload comprising an epigenetic reprogramming factor or a nucleic acid encoding an epigenetic reprogramming factor.

[0204] The specific “therapeutic effective dose,” prophylactic effective dose, or other appropriate dose level for any particular subject will depend on a number of factors, including the condition being treated and its severity; the activity of the specific compound used; the specific ingredients used; the patient’s age, weight, general health, sex, and diet; the timing, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors known in the field of cell therapy.

[0205] The invention is not limited in its application to the details of the arrangement and construction of the components set forth in the following description or illustrated in the accompanying drawings. The invention is capable of other embodiments and can be practiced or implemented in a variety of ways. Furthermore, the wording and terminology used herein are for descriptive purposes and should not be considered limiting. The use of “comprising,” “including,” or “having,” “containing,” “involving,” and / or variations thereof herein is intended to cover the items listed below and their equivalents, as well as additional items.

Claims

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

3. The composition of claim 2, wherein the nanoparticles are polymer nanoparticles.

4. The composition of claim 2, wherein the nanoparticles are lipid nanoparticles.

5. The composition of claim 2, wherein the nanoparticles are liposomes.

6. The composition of any one of claims 1 to 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 directly linked to the anti-ABCB5 antibody.

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

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

11. The composition of claim 10, wherein the viral vector is selected from lentiviral vectors, retroviral vectors, adenovirus vectors, alphavirus vectors, vaccinia virus vectors, and adeno-associated virus vectors (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 via a linker, and wherein the linker is a nuclease or a nuclease 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 to 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 to 15, wherein the nucleic acid encoding the epigenetic reprogramming factor comprises a regulatory element.

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

18. The composition of claim 17, wherein the inductive 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 with SEQ ID NO:

1.

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

1.

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

2.

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

2.

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

24. Nucleic acid, which contains a nucleotide sequence containing an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding epigenetic reprogramming factors.

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 lentiviral vectors, retroviral vectors, adenovirus vectors, alphavirus vectors, vaccinia virus vectors, and adeno-associated virus vectors (AAV).

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

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

29. The nucleic acid of claim 28, wherein the nanoparticles are polymer nanoparticles, lipid nanoparticles, or liposomes.

30. The nucleic acid of any one of claims 24 to 29, wherein the nucleic acid encoding an 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 to 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 with SEQ ID NO:

4.

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

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

35. A method comprising administering an effective amount of nucleic acid to a subject to reprogram and revitalize ABCB5+ stem cells in the subject, the nucleic acid comprising a nucleotide sequence containing an inducible ABCB5 regulatory element and one or more nucleotide sequences encoding an epigenetic reprogramming factor, 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 containing the inducible ABCB5 regulatory element comprises the sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

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

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

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

40. 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 with a sequence comprising SEQ ID NO: 1 or SEQ ID NO:

2.

41. The nucleic acid of claim 40, wherein the ABCB5 regulatory element is substantially composed of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

Images