Use of regenerative factors in organ transplantation

Abstract

Materials and methods for restoring an ex vivo organ prior to transplantation of the organ into a subject in need thereof are provided. The methods and materials are useful for treating, improving, restoring, and / or regenerating organs, such as kidney and liver, the organ is aged and / or characterized by organ injury caused by ischemic injury, toxic injury, perfusion injury, hypertension injury, immune injury, physical injury, steatosis, virus-induced hepatitis, alcohol or fibrosis irrelevant to any known cause. Materials and methods provided may increase the available library of donor organs, such as kidneys and livers. In addition, the provided materials and methods can treat organs that are then autotransplanted.

Description

[0001] References to sequence lists submitted electronically via EFS-WEB

[0002] The sequence list (name: 4967_023PC01_Seqlisting_ST26.XML; size: 166,421 bytes; and creation date: January 19, 2024), which was submitted electronically with this application, is incorporated herein by reference in its entirety.

[0003] Cross-references to related applications

[0004] This application claims priority to European Patent Application No. 23382042.2, filed on January 19, 2023, which is hereby incorporated by reference in its entirety. Background Technology

[0005] At any given time, hundreds of thousands of patients around the world are waiting for organ transplants and are unable to receive them due to the scarcity of suitable organs. In an aging population, the potential number of organs available for transplantation increases, but inadequate organ quality often prevents their use. This need is particularly acute for common chronic diseases affecting organs such as the kidneys and liver.

[0006] Chronic kidney disease (CKD) is a global health burden due to an aging population and the high and increasing prevalence of diabetes and hypertension worldwide. Aging is associated with decreased glomerular filtration rate in the kidneys, increased vascular and interstitial lesions, and increased susceptibility to acute kidney injury (e.g., ischemia-reperfusion injury) (Weinstein, JR and Anderson, S. Advances in Chronic Kidney Disease, Vol. 17, 302–307, 2010). Transplantation is currently the best alternative therapy for end-stage renal disease, but many patients are unable to receive timely transplants due to organ scarcity. In addition, 20%–30% of organs collected from donors are rejected for transplantation due to insufficient quality, increasing scarcity and delaying life-saving transplants. In the United States, donor organ rejection rates are particularly high among marginal donors (Aubert et al., JAMA Int. Med. 179(10):1365–74, 2019). The demand for kidneys and other organs is not being met, and one possibility is to expand the donor bank to include donor organs that are currently considered to be of insufficient quality, such as organs from older people who are often considered to be of insufficient quality due to a history of hypertension or other conditions (Ojo, AO et al., American Journal of Transplantation vol.4 27–37, 2004).

[0007] Chronic liver disease is a global burden due to population aging and the high and increasing incidence of pathologies such as hepatitis, hepatocellular carcinoma (HCC), and cirrhosis worldwide. Transplantation is currently the best alternative therapy for end-stage liver disease and HCC, but many patients cannot receive timely transplants due to organ scarcity. Expanding the donor bank to include livers currently considered of insufficient quality, for example, by repairing functional impairments in older livers to include older donors, could address the current shortage of donor livers.

[0008] Somatic cell reprogramming to a pluripotent state has been achieved by overexpressing Yamanaka factors (Oct-3 / 4, Sox2, Klf4, and c-Myc [OSKM]). However, the reprogramming process is lengthy and complex, and complete reprogramming can induce tumor development (Takahashi, K. et al., Cell 131, 861–872, 2007; Takahashi, K. and Yamanaka, S., Cell 126, 663–676, 2006; Polo, JM et al., Cell 151, 1617–1632, 2012; Abad, M. et al., Nature 502, 340–345, 2013). Therefore, alternative approaches are needed to reverse the consequences of donor organ aging.

[0009] Partial reprogramming has emerged as an alternative to full reprogramming and a potential treatment for age-related bodily effects. However, challenges remain in translating reprogramming from the laboratory to the clinic, particularly for the kidneys and organs. For example, viral vectors do not deliver therapeutic agents to the kidneys when administered systemically because most vectors are absorbed by the liver, infection is often inefficient, and / or vector dosage is limited by off-target effects (Rubin, JD, et al., Mol Diagn Ther 24, 375–396, 2020). Therefore, specific therapies are needed to improve transplant organ function and / or outcomes, and / or to increase the number of donor organs by repairing dysfunction in organs considered suboptimal or poor transplant candidates, in order to address the growing demand for donor organs and expand the donor organ pool for transplantation. Summary of the Invention

[0010] This disclosure provides an organ perfusion solution comprising a first polynucleotide encoding at least one regenerative factor and at least one vasodilator. In some aspects, the first polynucleotide encoding the at least one regenerative factor is operatively linked to a promoter. In some aspects, the organ perfusion solution further comprises at least one tonic agent. In some aspects, the organ perfusion solution further comprises at least one of a buffer, inorganic salt, amino acid, metabolic substrate, hormone, antioxidant, anti-inflammatory agent, anticoagulant, or antimicrobial agent. In some aspects, the organ perfusion solution further comprises a composition of human serum albumin, dextran, and extracellular electrolytes.

[0011] In some aspects, the tension agent is selected from dextran, glycerol, mannitol, potassium chloride, sodium chloride, and combinations thereof. In some aspects, the vasodilator is selected from carbon monoxide, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, calcium channel blockers, prostacyclin, hydralazine, minoxidil, nitroglycerin, and combinations thereof.

[0012] In some aspects, the organ perfusion fluid also contains an oxygenating agent. In some aspects, the oxygenating agent is selected from erythrocytes, hemoglobin, pyridine-oxygenated hemoglobin, synthetic hemoglobin-based oxygen carriers, and combinations thereof. In some aspects, the synthetic hemoglobin-based oxygen carrier is an oxygen carrier based on polymerized hemoglobin, Lifor... TM Aquix RS-I Or perfluorocarbon.

[0013] In some respects, the organ perfusion fluid also contains a second polynucleotide operably linked to a promoter that encodes at least one regeneration factor.

[0014] In some respects, the promoters of the first and second polynucleotides are inducible promoters.

[0015] In some aspects, the at least one regeneration factor encoded by the first polynucleotide is selected from Oct family transcription factors, Sox family transcription factors, Klf family transcription factors, and Myc family transcription factors. In some aspects, the at least one regeneration factor encoded by the second polynucleotide is selected from Oct family transcription factors, Sox family transcription factors, Klf family transcription factors, and Myc family transcription factors. In some aspects, the at least one regeneration factor encoded by the second polynucleotide is different from or not the same regeneration factor encoded by the first polynucleotide. In some aspects, the Oct family transcription factor is selected from Oct1, Oct3, Oct4, Oct6, and variants thereof. In some aspects, the Sox family transcription factor is selected from Sox1, Sox2, Sox3, Sox7, Sox15, Sox17, Sox18, and variants thereof. In some aspects, the Klf family transcription factor is selected from Kfl1, Klf4, Klf5, and variants thereof. In some aspects, the Myc family transcription factor is selected from c-Myc, L-Myc, N-Myc, and variants thereof.

[0016] In some aspects, the polynucleotide, the second polynucleotide, or both are encapsulated in nanoparticles. In some aspects, the nanoparticles are lipid nanoparticles, polymer nanoparticles, ligand-conjugated lipid nanoparticles, or ligand-conjugated polymer nanoparticles.

[0017] In some aspects, the polynucleotide, the second polynucleotide, or both are present in the viral genome, plasmid, microcircular vector, or transposon. In some aspects, the viral genome is selected from AAV genome, adenovirus genome, retrovirus genome, or lentivirus genome.

[0018] In some respects, organ perfusion fluids also contain regeneration factor-protein transfer domain fusion proteins.

[0019] In some respects, organ perfusion fluids also contain regenerative factor peptides.

[0020] In some aspects, the organ perfusion fluid also contains Oct family transcription factors, Sox family transcription factors, Klf family transcription factors, and / or Myc family transcription factors. In some aspects, the Oct family transcription factors are selected from Oct1, Oct3, Oct4, Oct6, and their variants. In some aspects, the Sox family transcription factors are selected from Sox1, Sox2, Sox3, Sox7, Sox15, Sox17, Sox18, and their variants. In some aspects, the Klf family transcription factors are selected from Kfl1, Klf4, Klf5, and their variants. In some aspects, the Myc family transcription factors are selected from c-Myc, L-Myc, N-Myc, and their variants.

[0021] In some respects, the organ perfusion fluid also contains enhancers selected from soluble Wnt, Wnt conditioned medium, BIX-01294 (G9a histone methyltransferase), PD0325901 (MEK inhibitor), DNA methyltransferase inhibitor, histone deacetylase (HDAC) inhibitor, valproic acid, 5'-azacytidine, dexamethasone, succinyl aniline isohydroxamic acid (SAHA), vitamin C, and trichosuccinimide (TSA), and combinations thereof.

[0022] This disclosure also provides a method for ex vivo organ regeneration, the method comprising: providing an organ; contacting the ex vivo organ with a composition comprising a polynucleotide encoding at least one regeneration factor, the polynucleotide being operatively linked to an inducible promoter; and adding a compound to the composition that induces the inducible promoter to promote the expression of the at least one regeneration factor, thereby expressing the at least one regeneration factor and regenerating the organ.

[0023] In some aspects of the methods described herein, the compound is added intermittently. In some aspects, the intermittent addition comprises adding the compound once a day for two consecutive days, and then not adding the compound for the next five consecutive days. In some aspects, the intermittent addition is performed 2-10 times. In some aspects, the intermittent addition is performed 2-10 times over a period of about 1 week to about 6 weeks.

[0024] In some aspects of the methods described herein, the composition further comprises a vasodilator. In some aspects, the composition further comprises a tension agent. In some aspects, the composition further comprises at least one of a buffer, an inorganic salt, an amino acid, a metabolic substrate, a hormone, an antioxidant, an anti-inflammatory agent, an anticoagulant, or an antimicrobial agent.

[0025] In some aspects of the methods described herein, the organ is provided in a perfusion system selected from the Hugo-Sachs system, organ assist system, OrganOX system, Radnoti system, ARK kidney system, and Aferetica system. system.

[0026] In some aspects of the methods described herein, the organ is the kidney or liver.

[0027] This disclosure also provides a method for transplanting an organ into a subject in need, the method comprising: providing an organ; contacting the isolated organ with a composition comprising a polynucleotide encoding at least one regeneration factor, the polynucleotide being operatively linked to an inducible promoter; adding a compound that induces the inducible promoter to the composition; and transplanting the organ into the subject.

[0028] In some aspects of the methods described herein, the compound is added to the composition over a period of about 1 minute to about 24 hours. In some aspects, the compound is added to the composition intermittently. In some aspects, the intermittent addition of the compound comprises adding the compound once a day for two consecutive days, and then not adding the compound for the next five consecutive days. In some aspects, the intermittent addition of the compound is repeated 2 to 10 times. In some aspects, the intermittent addition is performed 2 to 10 times over a period of about 1 week to about 6 weeks.

[0029] This disclosure also provides a method for transplanting an organ into a subject in need, the method comprising: providing an organ; contacting the isolated organ with a composition comprising a polynucleotide encoding at least one regeneration factor, the polynucleotide being operatively linked to an inducible promoter; transplanting the organ into the subject; and administering to the subject a compound that induces the inducible promoter.

[0030] In some aspects of the methods described herein, the compound is administered to the subject over approximately 1 minute to approximately 24 hours. In some aspects, the compound is administered to the subject intermittently. In some aspects, intermittent administration of the compound comprises administering the compound once daily for two consecutive days, followed by no administration of the compound for the next five consecutive days. In some aspects, intermittent administration of the compound is repeated approximately 2-10 times. In some aspects, intermittent administration of the compound is performed approximately 2-10 times every 3 months. In some aspects, intermittent administration of the compound is performed approximately 2-10 times every 6 months. In some aspects, the composition further comprises a vasodilator.

[0031] In some aspects of the methods described herein, the composition further comprises a tension agent. In some aspects, the composition further comprises at least one of a buffer, an inorganic salt, an amino acid, a metabolic substrate, a hormone, an antioxidant, an anti-inflammatory agent, an anticoagulant, or an antimicrobial agent.

[0032] In some aspects of the methods described herein, the removed organ is provided in a perfusion system selected from the Hugo-Sachs system, organ assist system, OrganOX system, Radnoti system, ARK kidney system, and Aferetica system. system.

[0033] In some aspects of the methods described herein, the organ mentioned is the kidney or liver.

[0034] In some aspects of the method described herein, the method further includes obtaining a biopsy from the organ prior to transplantation.

[0035] In some aspects of the method described herein, the method further includes obtaining a biopsy of the organ after transplantation.

[0036] In some aspects of the method described herein, the method further includes monitoring organ function in a subject who has received a transplanted organ. In some aspects, the monitoring includes measuring one or more of the following: blood urea level, serum creatinine level, bilirubin level, blood pH, blood bicarbonate level, blood sodium level, blood potassium level, or blood lactate level.

[0037] In some aspects of the method described herein, the method further includes administering an immunosuppressive drug to the subject.

[0038] This disclosure also provides an ex vivo organ for transplantation to a subject in need, the ex vivo organ comprising: (i) a perfusion fluid; and (ii) a polynucleotide encoding at least one regeneration factor. In some aspects, the ex vivo organ is damaged due to at least one of the following: aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, virus-induced hepatitis, alcoholism, or fibrosis unrelated to any known cause. In some aspects, the polynucleotide further comprises an inducible promoter operatively linked to the polynucleotide encoding the at least one regeneration factor. In some aspects, the perfusion fluid intermittently contains a compound that induces the inducible promoter. Attached Figure Description

[0039] Figure 1A The study demonstrates the direct injection of AAV vectors into rat kidneys via a clamped renal vein, and the expression of fluorescein in the injected and uninjected (contralateral) kidneys 7 days after in situ injection. Figure 1BThe in vivo luciferin levels are shown in rats with kidneys perfused with the GFP-Luc AAV vector (left animal), control rats (middle animal), and rats that received a systemic injection (via penile vein) of the GFP-Luc AAV vector (right animal).

[0040] Figure 2A A schematic diagram is shown of the allogeneic transplantation of aged Dark Agouti kidneys perfused with AAV OSKM vector into juvenile Lewis recipient rats, which were treated with doxycycline for 2 days a week starting from day 7 post-transplantation to induce OSKM expression. Figure 2B The image shows H&E staining of an aging kidney perfused with GFP-AAV after allogeneic transplantation, demonstrating immune and ischemic damage. Figure 2C The percentage survival rate of rats undergoing allogeneic transplantation with aging kidneys perfused with GFP and OSKM AAV vectors is shown up to 110 days post-transplantation, with doxycycline treatment discontinued on day 88. Figure 2D The BUN levels in rats were shown 2 weeks after transplantation of aging kidneys perfused with GFP and OSKM AAV vectors. Figure 2E Serum creatinine (SCr) levels in rats up to 7 weeks after transplantation of aging kidneys perfused with GFP AAV and OSKM AAV vectors (right panel) are shown compared to those in rats with kidneys perfused with GFP AAV vectors (left panel). Figure 2F Serum pH, HCO3, sodium, potassium, lactate, BUN, and serum creatinine levels in rats 2 weeks after transplantation of aged kidneys perfused with GFP AAV and OSKM AAV vectors are shown. Figure 2G Serum pH, HCO3, sodium, potassium, lactate, BUN, and serum creatinine levels in rats 3 weeks after transplantation of aging kidneys perfused with GFP AAV and OSKM AAV vectors are shown. Figure 2H Serum pH, HCO3, sodium, potassium, lactate, BUN, and serum creatinine levels in rats 4 weeks after transplantation of aged kidneys perfused with GFP AAV and OSKM AAV vectors are shown. Figure 2I Serum pH, HCO3, sodium, potassium, lactate, BUN, and serum creatinine levels in rats 2–4 weeks after transplantation of aged kidneys perfused with GFP AAV and OSKM AAV vectors (1–3 weeks after nephrectomy) are shown. Figure 2J The BUN levels in rats were shown 2–4 weeks after transplantation of aged kidneys perfused with GFP AAV and OSKM AAV vectors (1–3 weeks after nephrectomy). Figure 2KSerum creatinine (Cr) levels in rats that survived up to 15 weeks after transplantation of aging kidneys perfused with GFP AAV and OSKM AAV vectors are shown. Figure 2L The BUN levels in rats up to 14 weeks after transplantation of aging kidneys perfused with GFP AAV and OSKM AAV vectors are shown. Figure 2M Potassium levels in rats up to 12 weeks after transplantation of aging kidneys perfused with GFP AAV and OSKM AAV vectors are shown. Figure 2N Representative images of the transplanted kidney (right) from the OSKM group, collected 100 days post-transplantation, and the normal kidney (left) used for comparison are shown.

[0041] Figure 3A A schematic diagram of syngeneic transplantation in an ischemic kidney transplantation model is shown, in which a kidney from a juvenile Lewis rat perfused with the AAV OSKM vector is transplanted into a juvenile Lewis recipient rat. The juvenile Lewis recipient rat is treated with doxycycline for four days a week immediately after transplantation. Figure 3B The survival rates of rats with kidneys transplanted via AAV-free control and OSKM AAV vector infusion are shown. Figure 3C Serum creatinine levels in control rats that received a kidney transplanted with a control without AAV are shown (the number of surviving animals (n) is shown below the graph). Figure 3D Serum creatinine levels in rats with kidneys perfused with OSKM AAV are shown (the number of surviving animals (n) is shown below the graph). Figure 3E Serum creatinine levels in rats with kidneys perfused with GFP AAV (square graph on curve) and OSKM AAV (circular graph on curve) are shown. Figure 3F BUN levels in rats that received a kidney transplanted with a control without AAV are shown (the number of surviving animals (n) is shown below the graph). Figure 3G The BUN levels of rats with kidneys perfused with the OSKM AAV vector are shown (the number of surviving animals (n) is shown below the graph). Figure 3H The BUN levels of rats with kidneys transplanted via perfusion with AAV-free control (large circle) and OSKM AAV (small circle) are shown.

[0042] Figure 4 A schematic diagram of syngeneic transplantation in a cardiac arrest-induced kidney disease model is shown, in which juvenile Lewis rats are perfused with either control (without AAV) or treatment (perfusion in the presence of AAV / DJ-OSK+AAV / DJ cMyc-rtTA).

[0043] Figure 5AThe AST levels of rats were shown one week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 5B The ALT levels in rats were shown one week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 5C The caspase-3 levels in rats were shown one week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 5D The bilirubin levels in rats were shown one week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 5E The MPO levels in rats were shown one week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 5F The MDA levels in rats were shown one week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 5G The Ki67 levels in rats were shown one week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 5H The PCNA levels in rats were shown one week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 5I The HGF levels in rats were shown one week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 5J The TGF-β levels in rats were shown one week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 5K Representative H&E images of the liver are shown 1 week after intravenous injection of PBS (control). Figure 5L The image shows the liver H&E image one week after intravenous injection of the AAV-GFP / Luc control vector (the medium). Figure 5MThe image shows the liver H&E images one week after intravenous injection of AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment).

[0044] Figure 6A The AST levels of rats were shown 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 6B The ALT levels in rats were shown 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 6C The caspase 3 levels in rats were shown 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 6D The bilirubin levels in rats were shown 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 6E The MPO levels in rats were shown 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 6F The MDA levels in rats were shown 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 6G The Ki67 levels in rats were shown 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 6H The PCNA levels in rats were shown 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 6I The HGF levels in rats were shown 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 6J The TGF-β levels in rats were shown 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (mediator), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). Figure 6K Representative H&E images of the liver are shown 12 weeks after intravenous injection of PBS (control). Figure 6L The image shows the liver H&E images 12 weeks after intravenous injection of AAV-GFP / Luc control vector (the medium). Figure 6M The image shows the liver H&E images 12 weeks after intravenous injection of AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment).

[0045] Figure 7 A 10X view of a representative section of a kidney treated with OSKM and stained with hematoxylin and eosin is shown.

[0046] Figure 8 A 10X view of a representative section of a kidney treated with AAV-GFP / luc control and stained with hematoxylin and eosin is shown.

[0047] Figure 9A The results show the BUN levels in rats two weeks after kidney perfusion via OSKM-AAV or control without AAV in an ischemic injury model. Figure 9B The results show the BUN levels in rats three weeks after kidney perfusion via OSKM-AAV or control without AAV in an ischemic injury model. Figure 9C Serum creatine levels in rats two weeks after kidney perfusion via OSKM-AAV or control without AAV were shown in an ischemic injury model. Figure 9D Serum creatine levels were shown in rats three weeks after kidney perfusion via OSKM-AAV or control without AAV in an ischemic injury model.

[0048] Figure 10A The BUN levels of rats with kidneys perfused with AAV-free control (AAV-free vector) are shown (n = number of surviving animals). Figure 10B The BUN levels in rats with kidneys perfused with the OSKM AAV vector are shown (n = number of surviving animals). Figure 10C Serum creatine levels in rats with kidneys perfused with AAV-free control (solid circles) and OSKM AAV (hollow circles) are shown.

[0049] Figure 11 A rat model of segmental (70%) liver ischemia was shown.

[0050] Figure 12A The AST levels are shown one week after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 12BThe ALT levels are shown one week after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 12C The bilirubin levels are shown one week after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 12D The caspase 3 levels were shown one week after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 12E The MPO levels are shown one week after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 12F The MDA levels are shown one week after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 12G The PCNA levels are shown one week after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 12H The ki67 levels are shown one week after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA).

[0051] Figure 13A The AST levels are shown four days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 13B The ALT levels are shown four days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 13C The caspase 3 levels were shown four days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 13DThe MPO levels are shown four days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 13E The MDA levels are shown four days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 13F The PCNA levels are shown four days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 13G The ki67 levels are shown four days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 13H The HGF levels are shown four days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 13I The TGF-β levels are shown four days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA).

[0052] Figure 14A The AST levels are shown two days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 14B The ALT levels are shown two days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 14C The caspase 3 levels were shown two days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 14D The MPO levels are shown two days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 14EThe MDA levels are shown two days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 14F PCNA levels were shown two days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 14G The ki67 levels were shown two days after administration of the PBS control, the mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 14H The HGF levels are shown two days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). Figure 14I The TGF-β levels are shown two days after administration of PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA).

[0053] Figure 15A The changes in mean serum creatinine (SCr) levels in rats in the control and treatment groups were shown 8 days after transplantation. Figure 15B The mean change in BUN levels in rats 8 days after transplantation is shown. Figure 15C The left image shows urinary protein (UTP) and right image show urinary albumin (UAP) in rats from the control and treatment groups after transplantation. Figure 15D The flow and resistance records during normothermic renal perfusion are shown.

[0054] Figure 16A The expression profiles of neutrophil gelatinase-associated lipotransferase (NGAL) in control and OSKM-treated kidneys are shown. Figure 16B The expression profiles of hypoxia-inducible factor (HIF) in the kidneys of control and OSKM-treated patients are shown. Figure 16C The expression profiles of endothelin receptor (ETR) in the kidneys of control and OSKM-treated patients are shown. Figure 16D The expression profiles of transforming growth factor-β (TGFβ) in the kidneys of control and OSKM-treated patients are shown. Figure 16E The expression profiles of tumor necrosis factor (TNF) in the kidneys of control and OSKM-treated patients are shown. Figure 16F The expression profiles of monocyte chemoattractant protein 1 (MCP1) in control and OSKM-treated kidneys are shown. Figure 16GThe expression profiles of CXC motif chemokine ligand 10 (CXCL10) in control and OSKM-treated kidneys are shown. Figure 16H The expression profiles of the evaluated genes in the kidneys of control and OSKM-treated patients are shown.

[0055] Figure 17A Hematoxylin and eosin kidney staining showed four-fold glomerular compartments, revealing four-fold renal tubules and glomerular compartments without AAV administration. Figure 17B Hematoxylin and eosin kidney staining showed four-fold glomerular compartments, and four-fold renal tubules and glomerular compartments were observed after AAV-OSKM administration. Figure 17C Hematoxylin and eosin kidney staining showed 4x glomerular compartments and 10x renal tubules and glomerular compartments without AAV administration. Figure 17D Hematoxylin and eosin kidney staining showed four-fold glomerular compartments, and four-fold renal tubules and glomerular compartments were observed after AAV-OSKM administration. Figure 17E Masson's trichrome kidney staining, showing four-fold renal tubular and glomerular compartments, was performed without AAV administration. Figure 17F Masson's trichrome kidney staining, applied with AAV-OSKM, shows four-fold renal tubular and glomerular compartments.

[0056] Figure 18 The modified Remuzzi score analysis of OSKM treatment samples and control treatment samples is shown. Detailed Implementation

[0057] Materials and methods for organ restoration are provided to improve organ function and / or organ transplant outcomes, thereby increasing the number of donor organs by making organs considered suboptimal or poor transplant candidates suitable for transplantation. Materials and methods for organ restoration are also provided to improve organ function and / or organ transplant outcomes in autologous transplantation (i.e., restoration of the recipient's organ). The materials described herein include, but are not limited to, polynucleotides, carriers, and compositions introduced into ex vivo donor organs, for example, in organ perfusion fluids. The materials and methods are capable of expressing regenerative factors in the perfused organ, thereby potentially modifying age-related changes and treating aging or damaged organs to improve transplant suitability.

[0058] The methods and materials described herein can be used for ex vivo perfusion of human kidneys to reverse changes in the kidney associated with at least one of the following before transplantation to a human recipient: aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, virus-induced hepatitis, alcohol, or fibrosis unrelated to any known cause. The methods and materials described herein can also be used for ex vivo perfusion of human livers to reverse changes in the liver associated with at least one of the following before transplantation to a human recipient: aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, virus-induced hepatitis, alcohol, or fibrosis unrelated to any known cause. Advantageously, the methods and materials provided herein allow for the treatment of a kidney or liver by ex vivo perfusion with a perfusion solution containing the materials described herein, followed by measurement of the effect of the materials on the kidney or liver, for example by biopsy and functional assessment of the kidney or liver, and, if necessary, one or more further treatments of the kidney or liver with the methods and materials described herein before transplantation into a recipient patient in need.

[0059] Furthermore, the methods and materials used herein allow for the treatment of a kidney or liver in vivo following ex vivo perfusion with a perfusion solution containing the materials described herein, to modulate the effects of the materials on the kidney or liver in vivo. Measurements, such as biopsies or functional assessments, can be obtained from the transplant patient, and the kidney or liver can be treated based on the results of these measurements to enhance and / or maintain the effects of the materials supplied to the kidney or liver during pre-transplant ex vivo perfusion.

[0060] I. Definition

[0061] To make this disclosure more easily understood, certain terms are defined first. Further definitions are set forth throughout the detailed disclosure.

[0062] It should be noted that the term “a / an” refers to one or more of the same entity; for example, unless otherwise stated, “a polynucleotide sequence” should be understood to mean one or more polynucleotide sequences. Therefore, the terms “a” (or “a type”), “one or more”, and “at least one” are used interchangeably herein.

[0063] Furthermore, when used herein, “and / or” will be considered as a specific disclosure of each of the two specified features or components in the presence or absence of the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to include each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0064] It should be understood that whenever the language “contains” is used to describe an aspect in this document, other similar aspects described by “consisting of” and / or “substantially consisting of” are also provided.

[0065] The term “about” is used herein to mean approximately, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical value. Generally, the term “about” can modify values ​​above and below the stated value by, for example, a variation of 10% above or below (higher or lower).

[0066] The term "at least" preceding a number or series of numbers should be understood to include the number adjacent to the term "at least," as well as all subsequent numbers or integers that can logically be included, as is clearly apparent from the context. For example, the number of nucleotides in a polynucleotide molecule must be an integer. For instance, "at least 18 nucleotides in a polynucleotide molecule of 21 nucleotides" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" appears before a series of numbers or a range, it should be understood that "at least" can modify every single number in the series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18%, regardless of the number of significant figures).

[0067] As used herein, “not more than” or “less than” should be understood as the value and logical lower bound or integer adjacent to the phrase, such as from the context logical to zero. When “not more than” appears before a series of numbers or a range, it should be understood that “not more than” can modify each number in the series or range.

[0068] Furthermore, any reference referred to as “incorporated into this paper” should be interpreted as being incorporated in its entirety.

[0069] The terms “polynucleotide,” “polynucleotide,” and “oligonucleotide” are used interchangeably in this application. These terms refer only to the primary structure of a molecule. Thus, these terms include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. As used herein, the terms “polynucleotide,” “polynucleotide,” and “oligonucleotide” are defined as molecules that are generally understood by those skilled in the art to contain two or more covalently linked nucleosides. Oligonucleotides may also be referred to as oligomers. Oligonucleotides are typically prepared synthetically in the laboratory by solid-phase chemical synthesis followed by purification. Polynucleotides can be prepared by recombinant, enzymatic, or synthetic methods, for example, by solid-phase chemical synthesis followed by purification. When referring to an oligonucleotide, polynucleotide, or sequence of a polynucleotide, the sequence or order of the nucleobase portion of the covalently linked nucleotide or nucleoside or its modifications thereof is referred to.

[0070] As used herein, “nucleotide” refers to a monomeric unit of an oligonucleotide or polynucleotide containing a nucleoside and an inter-nucleoside bond. “G,” “C,” “A,” “T,” and “U” typically denote naturally occurring nucleotides containing guanine, cytosine, adenine, thymine, and uracil as nucleobases, respectively. However, it should be understood that the term “nucleotide” may refer to alternative nucleotides, or substitutes, as further detailed below. Oligonucleotides or polynucleotides are not limited to naturally occurring nucleosides but may contain non-natural nucleosides and bonds as disclosed herein, for example, to produce oligonucleotides modified to enhance their stability or cell permeability. Those skilled in the art will readily recognize that guanine, cytosine, adenine, and uracil can be substituted with other portions without substantially impairing the base-pairing properties of oligonucleotides or polynucleotides containing nucleotides with such substitute portions. For example, but not limited to, nucleotides containing inosine as a base may pair with nucleotides containing adenine, cytosine, or uracil. Therefore, in the nucleotide sequence of an oligonucleotide or polynucleotide, a nucleotide containing uracil, guanine, or adenine can be replaced by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide or polynucleotide can be replaced by guanine and uracil, respectively, to form a G-UWobble base pairing with the target RNA. Sequences containing such substitutions are suitable for the compositions and methods described herein.

[0071] The terms “nucleobase” and “base” are used interchangeably herein and include purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) portions present in nucleosides and nucleotides that form hydrogen bonds during polynucleotide hybridization. The term nucleobase also includes alternative nucleobases that may differ from naturally occurring nucleobases but function during polynucleotide hybridization. In this document, “nucleobase” refers to naturally occurring nucleobases such as adenine, guanine, cytosine, thymine, uracil, xanthine, and hypoxanthine, as well as alternative nucleobases. Such variants are described, for example, in Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Polynucleotide Chemistry Suppl. 37 1.4.1. Alternative nucleobases include modified purines or pyrimidines. For example, adenine and guanine can be replaced by other naturally occurring purines, including but not limited to N. 6 -Methyladenine, N 2 -Methylguanine, hypoxanthine, and 7-methylguanine. Cytosine, uracil, and thymine may be substituted with other naturally occurring pyrimidines, including but not limited to 5-methylcytosine, 5-hydroxymethylcytosine, pseudouracil, and 4-thiouracil. In some aspects, the oligomer may contain a thymine base instead of uracil. Other substituted nucleobases include isocytosine, pseudoisocytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uridine, 5-bromouridine, 5-thiazo-uridine, 2-thiouridine, pseudouridine, 1-methylpseudouridine, 5-methoxyuridine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, 2-chloro-6-aminopurine, agmatidine, lysidine, and 2-thiopyrimidines (e.g., 2-thiouracil, 2-thiothyronine). Adenosine), G-clamp and its derivatives, 5-substituted pyrimidines (e.g., 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, 5-hydroxybutynyl-2'-deoxyuridine), 7-deazoguanine, 7-deazoadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazoguanine, 8-aza-7-deazoadenine, 8-aza-7-deazo-2,6-diaminopurine, 8-aza-7-deazoguanosine and N4-ethylcytosine or its derivatives; N 2 -Cyclopentylguanine (cPent-G), N 2-Cyclopentyl-2-aminopurine (cPent-AP) and N 2 -propyl-2-aminopurine (Pr-AP), pseudouracil or its derivatives; and degenerate or universal bases, such as 2,6-difluorotoluene or baseless sites such as debasement sites (e.g., 1-deoxyribose, 1,2-dideoxyribose, l-deoxy-2-O-methylribose; or pyrrolidine derivatives in which the epoxide has been replaced by nitrogen (azaribose)).

[0072] Certain modified or substituted nucleobases are particularly useful for increasing the binding affinity of antisense oligonucleotides. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, and 5-methylcytosine.

[0073] The term "nucleoside" refers to a monomeric unit of an oligonucleotide or polynucleotide having a nucleobase and a sugar moiety. Nucleosides can include naturally occurring ones as well as alternative nucleosides, such as those described herein. The nucleobase of a nucleoside can be a naturally occurring nucleobase or an alternative nucleobase. Similarly, the sugar moiety of a nucleoside can be a naturally occurring sugar or an alternative sugar.

[0074] The term "alternative nucleoside" refers to a nucleoside that has a substitute sugar or a substitute nucleobase, such as those described herein.

[0075] "Sugar" or "sugar moiety" includes naturally occurring sugars having a furanose ring. Sugar also includes "alternative sugars," which are defined as structures capable of replacing the furanose ring of a nucleoside. In some respects, alternative sugars are non-furanose (or 4′-substituted furanose) rings or ring systems or open systems. Alternative sugars may include sugar substitutes in which the furanose ring has been replaced by another ring system (e.g., a morpholino or hexitol ring system). The sugar moiety that can be used to prepare oligonucleotides with motifs includes, but is not limited to, β-D-ribose, β-D-2′-deoxyribose, substituted sugars (such as 2′, 5′, and disubstituted sugars), 4′-S-sugars (such as 4′-S-ribose, 4′-S-2′-deoxyribose, and 4′-S-2′-substituted ribose), bicyclic substituted sugars (such as 2′-O—CH2-4′ or 2′-O—(CH2)2-4′ bridging ribose-derived bicyclic sugars), and sugar substitutes (such as when the ribose ring has been replaced by a morpholino or hexitol ring system). The type of heterocyclic base and nucleoside linkage used at each position is variable and not a determining factor in the motif. In most nucleosides with substituted sugar moieties, the heterocyclic nucleobase is generally maintained to allow hybridization. In some aspects, oligonucleotides or polynucleotides contain nucleosides with, for example, 2′-FANA modifications and nucleosides without 2′-FANA modifications, which may contain substituted sugars as described herein.

[0076] "Nucleoside linkage" refers to the linkage between nucleosides in oligonucleotides or polynucleotides, which may include phosphate linkages or alternative nucleoside linkages. Many "alternative nucleoside linkages" or "non-natural linkages" are known in the art, including but not limited to phosphate, thiophosphate, and borophosphate linkages. Alternative nucleosides include bicyclic nucleosides (BNAs) (e.g., locked nucleosides (LNAs) and restricted ethyl (cEt) nucleosides), peptide nucleosides (PNAs), phosphate triesters, thiophosphate esters, phosphoramides, and other variants of the phosphate backbone of natural nucleosides, including those described herein.

[0077] As used herein, “alternative nucleotide” refers to a nucleotide having an alternative nucleoside or an alternative bond between a sugar and a nucleoside, which may include an alternative nucleoside bond.

[0078] Oligonucleotides or polynucleotides may include (i) compounds having one or more 2'FANA-modified nucleosides, (ii) compounds in which one or more furanose moieties are replaced by furanose derivatives or by any cyclic or acyclic structure that can serve as a covalent attachment site for the base moieties, (iii) compounds in which one or more phosphodiester bonds are modified (e.g., in the case of aminophosphate or thiophosphate bonds), or are completely replaced by suitable linker moieties (e.g., in the case of methyl acetal or ribose acetal bonds), and / or (iv) compounds in which one or more linked furanose-phosphodiester bond moieties are replaced by any cyclic or acyclic structure that can serve as a covalent attachment site for the base moieties. Oligonucleotides or polynucleotides may contain one or more substituted nucleosides or nucleotides (e.g., including those described herein). It should also be understood that oligonucleotides include compositions lacking a sugar moiety or nucleobase but still capable of pairing or hybridizing with a target sequence. Oligonucleotides include short polynucleotides (e.g., nucleosides with 100 or fewer links). As used herein, a “chimeric” oligonucleotide or “chimera” is an oligonucleotide containing two or more chemically distinct regions, each region consisting of at least one monomeric unit, i.e., in the case of an oligonucleotide, consisting of either a nucleotide or a nucleoside. Chimeric oligonucleotides also include “gapmers.” For example, a chimeric oligonucleotide may contain an unmodified nucleoside and a 2'FANA-modified nucleoside. In some aspects, the 2'FANA-modified nucleoside is located at the 5' or 3' portion of the oligonucleotide, or at both portions. In some aspects, the 2'FANA-modified nucleoside is located throughout the oligonucleotide. In some aspects, a chimeric oligonucleotide comprises a 2'FANA-modified nucleoside located at the 5' and / or 3' portion and at least one unmodified nucleoside located at the center of the oligonucleotide.

[0079] As used herein and unless otherwise stated, the term “complementary” is used to describe a first nucleotide or nucleoside sequence relative to a second nucleotide or nucleoside sequence and refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide or nucleoside sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions and form a double-stranded structure, as will be understood by those skilled in the art. Such conditions can be, for example, stringent conditions, which may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12–16 hours, followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual,” Sambrook et al. (1989), Cold Spring Harbor Laboratory). (Press). Other conditions may be used, such as physiologically relevant conditions that may be encountered in vivo. Depending on the final application of the hybrid nucleotide or nucleoside, the technician will be able to determine a set of conditions best suited for testing the complementarity of the two sequences. Complementary sequences may include non-Watson-Crick base pairs and / or base pairs formed from non-naturally substituted nucleotides or nucleosides, or base pairs formed entirely from non-Watson-Crick base pairs and / or base pairs formed from non-naturally substituted nucleotides or nucleosides, provided that the above requirements for their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogstein base pairings. Complementary sequences between oligonucleotides and target sequences as described herein comprise oligonucleotides or polynucleotides containing the first nucleotide or nucleoside sequence along the full length of one or two nucleotide or nucleoside sequences and containing the second nucleotide or nucleoside sequence. Base pairing of nucleotides. Such sequences may be referred to herein as “perfectly complementary” relative to each other. Complementary sequences between oligonucleotides or polynucleotides and target sequences, as described herein, include base pairing of the oligonucleotide or polynucleotide containing the first nucleotide or nucleoside sequence with the oligonucleotide or polynucleotide containing the second nucleotide or nucleoside sequence over its full length of less than one or two nucleotide or nucleoside sequences. Such sequences may be referred to herein as “partially complementary” relative to each other. In some aspects, the oligonucleotide or polynucleotide comprises a continuous nucleotide region and may contain additional nucleotides or nucleosides. For example, nucleotide linker regions may be used to attach functional groups to the continuous nucleotide sequence. Nucleotide linker regions may be complementary to the target polynucleotide. In some aspects, the inter-nucleoside linkages between the nucleotides in the continuous nucleotide region are all phosphate-thionucleotide linkages. In some aspects, the continuous nucleotide region contains one or more sugar-modified nucleosides.

[0080] As used herein, the term "connector" or "linking group" refers to a link between two atoms that connect one or more chemical groups or segments of interest via one or more covalent bonds. The conjugate moiety may be attached to an oligonucleotide directly or via a linking portion (e.g., a connector or ligand). Connectors are used to covalently link a third region, such as connecting the conjugate moiety to an oligonucleotide or polynucleotide. In some aspects, conjugates, oligonucleotide conjugates, or polynucleotide conjugates may include a linker region located between the oligonucleotide or polynucleotide and the conjugate moiety. In some aspects, the linker between the conjugate and the oligonucleotide or polynucleotide is biocleavable. Biocleavable linkers containing phosphodiester are described in more detail in WO 2014 / 076195 (incorporated herein by reference).

[0081] As used herein, the term "promoter" refers to a DNA sequence recognized by the synthetic machinery introduced into the cell for the specific transcription of a gene. The term "promoter" is also intended to encompass those polynucleotide elements sufficient for promoter-dependent gene expression, which can be controlled by external signals or reagents to achieve cell-type-specific, tissue-specific, or inducible expression; such elements may be located in the 5' or 3' region of a natural gene. In some aspects, a promoter can be a constitutively active promoter, a cell-type-specific promoter, or an inducible promoter.

[0082] As used herein, the terms “operationally linked,” “operationally inserted,” “operationally positioned,” “operationally linked,” “under control,” or “under transcriptional control” mean that the promoter is in the correct position and orientation relative to the polynucleotide to control the initiation of RNA polymerase and gene expression. The term “operationally linked” or “operationally linked” means that the polynucleotide sequence and the regulatory sequence are linked in such a way that RNA expression is permitted when an appropriate molecule (e.g., a transcription activating protein) binds to the regulatory sequence. The term “operationally inserted” or “operationally inserted” means that the polynucleotide of interest introduced into the cell is positioned near the polynucleotide sequence that guides the transcription and translation of the introduced polynucleotide (i.e., promotes, for example, the production of a polypeptide encoded by the DNA of interest).

[0083] As used herein, the term "inducible promoter" refers to a promoter that is operatively or operably linked to a polynucleotide sequence in such a manner that RNA expression is permitted when a suitable molecule (e.g., a transcriptional activator) binds to the promoter sequence. In some respects, transcriptional activators are activated upon contact with an activator. Examples of inducible promoters include, but are not limited to, tetracycline-inducible promoters, hormone-inducible promoters (e.g., tamoxifen-inducible promoters) (promoter-binding domains fused to estrogen receptors), steroid-inducible promoters (promoter-binding domains fused to mutant progesterone receptors that bind RU486 but not endogenous progesterone), or coumarin-inducible promoters. Inducible promoters may contain tetracycline response elements (TREs) (e.g., TRE3G promoters, TRE2 promoters, or P-tight promoters). Inducible promoters may also contain mifepristone-inducible promoters (e.g., containing GAL4 upstream activating sequences), or coumarin / neomycin-inducible / repressive promoters. For example, the TRE (e.g., TRE3G) promoter may contain at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) of the same nucleic acid (e.g., engineered nucleic acid) sequence as SEQ ID NO:7.

[0084] As used herein, the term "tissue-specific promoter" refers to any promoter that activates higher transcription of polynucleotides in a particular tissue compared to other tissues. For example, tissue-specific promoters used in the polynucleotides described herein can specifically induce transcription in the following cells: endothelial cells (e.g., the Tie-2 promoter); smooth muscle cells (e.g., α-smooth muscle actin promoter, smooth muscle myosin heavy chain promoter, calponin promoter, transgelin promoter, h-caldesmon promoter, smooth protein promoter); pericytes (e.g., the p75 promoter, endosialin promoter, desmin promoter); and renal tubular epithelial cells (e.g., the sodium-dependent phosphate cotransporter gene NPT2a promoter, sodium-potassium-2-chloride cotransporter promoter, or aquaporin 2 promoter).

[0085] As used herein, the term "senescent cell-specific promoter" refers to a promoter that is expressed in senescent cells but not expressed or expressed in significantly reduced form in non-senescent cells. Examples of senescent cell-specific promoters include, but are not limited to, the p16 promoter, p21 promoter, mir146a promoter, ATF3 promoter, GADD45b promoter, MMP13 promoter, or BTG2 promoter.

[0086] As used in this article, the term "WPRE" refers to the post-translational regulatory element of marmot hepatitis virus, which is a tertiary DNA sequence that, when transcribed, enhances the expression of viral vector genes.

[0087] As used herein, an “IRES” is an element that facilitates direct entry of the internal ribosome into the cistron (protein-coding region) of the gene (such as ATG), thereby leading to cap-independent gene translation. See, for example, Jackson RJ et al., Trends Biochem Sci 15(12):477-83(199); Jackson RJ and Kaminski, A. RNA 1(10):985-1000(1995). Under the translational control of an IRES, translation proceeds in a cap-independent manner.

[0088] As used herein, the term "termination signal sequence" can refer to any genetic element that causes RNA polymerase to terminate transcription, such as a polyadenylation signal sequence. A polyadenylation signal sequence is a recognition region essential for endonuclease cleavage of RNA transcripts, followed by the polyadenylation concordance sequence AATAAA. The polyadenylation signal sequence provides a "polyA site," the site at which adenine residues are added to the RNA transcript via post-transcriptional polyadenylation.

[0089] As used herein, the term "protein hydrolysis cleavage site" refers to a polynucleotide that encodes an amino acid sequence that can be cleaved by protein hydrolysis, and includes, but is not limited to, self-processing cleavage sites and furin cleavage sites.

[0090] As used herein, the term "self-processing cleavage site" refers to a post-translational or co-translational processing cleavage site or sequence, which can be a DNA sequence or an amino acid sequence. This article uses 2A sites, sequences, or domains, or 2A-like sites, sequences, and domains as examples. A self-processing peptide is a peptide expression product of a DNA sequence encoding a self-processing cleavage site or sequence that mediates rapid intramolecular (cis) cleavage of a protein or polypeptide containing the self-processing cleavage site post-translation to produce discrete mature protein or polypeptide products.

[0091] As used herein, the term "furin cleavage site" refers to a polynucleotide encoding an amino acid sequence that can be cleaved by endogenous subtilisin-like proteases, such as furin in the protein secretion pathway and other serine proteases. In some aspects, furin cleavage sites include the concordant sequence RXK(R)R of SEQ ID NO:17.

[0092] As used herein, the term "vector" refers to any medium used to clone and / or transfer polynucleotides into host cells, such as plasmids, bacteriophages, transposons, microcircular vectors, kinases, chromosomes, artificial chromosomes, viruses, viral particles, etc. A vector can be a replicon to which another polynucleotide fragment can be attached to induce replication of the attachment segment. A "replicon" refers to an autonomous unit that acts as a replicator in vivo, i.e., any genetic element capable of replicating under its own control (e.g., plasmids, bacteriophages, kinases, chromosomes, viruses). The term "vector" includes viral and nonviral mediators used to introduce polynucleotides into cells in vitro, ex vivo, or in vivo. Vectors that can be used in the aspects described herein include, but are not limited to, plasmids, modified eukaryotic viruses, or modified bacterial viruses. In some aspects, insertion of a polynucleotide into a suitable vector can be accomplished by ligating an appropriate polynucleotide fragment into a selected vector having complementary sticky ends. Vectors can be engineered to encode selection markers or reporter genes that provide selection or identification of cells incorporating the vector. The expression of selective markers or reporter genes allows for the identification and / or selection of host cells incorporating and expressing additional coding regions contained on the vector. Examples of selective marker genes known and used in the art include genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, diammonium phosphate herbicide, sulfonamides, etc.; and genes used as phenotypic markers, such as anthocyanin regulatory genes, isopentenyltransferase genes, etc. Examples of reporter genes known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selective markers can also be considered reporter genes. In some aspects, the delivery vector is selected from viral vectors (e.g., AAV vectors), plasmids, lipids, cationic polymers, protein particles, bacterial vectors, and lysosomes. Some aspects of this disclosure relate to biological vectors, which may include viruses, particularly attenuated viruses and / or replication-defective viruses. In some aspects, the vector may include a microRNA targeting sequence to increase the specificity of vector-mediated transgene expression. In some respects, the delivery vectors disclosed herein are viral vectors selected from adeno-associated virus (AAV) vectors, adenovirus vectors, lentivirus vectors, retrovirus vectors, poxvirus vectors, baculovirus vectors, herpesvirus vectors, simian virus 40 (SV40), cytomegalovirus (CMV), mouse mammary tumor virus (MMTV) and Moloney mouse leukemia virus.

[0093] As used herein, the term "retroviral vector" refers to any vector containing or derived from retroviral vector components and suitable for infecting mammalian cells, preferably human cells. The term "retroviral vector" generally refers to a retroviral particle or viral particle or virus containing a payload. The terms "retroviral genome" and "retroviral vector" are used interchangeably and refer to retroviral nucleic acids into which the nucleic acid of interest has been inserted to replace certain viral sequences to produce a replication-defective virus. For packaging retroviral genomes or retroviral vectors into retroviral particles, packaging cell lines expressing the gag, pol, and env genes but without retroviral long terminal repeats (LTRs) can be provided. Retroviral vectors can integrate their genes into the host genome, thereby transferring large amounts of exogenous genetic material into the host genome.

[0094] As used herein, the term "retroviral particle" refers to a retrovirus comprising a capsid and a retroviral vector having at least one payload region (e.g., a polynucleotide encoding at least one regeneration factor) and at least one LTR. In some aspects, retroviral particles are pseudogenotyped by combining the retroviral vector with a heteroviral envelope protein. In some aspects, the heteroviral envelope protein is a naturally occurring envelope protein, such as that derived from vesicular stomatitits virus. In some aspects, the heteroviral envelope protein is an engineered viral envelope protein, for example, targeting a specific cell type. In some aspects, the retroviral vector contains a polynucleotide encoding a regeneration factor. In some aspects, the retroviral vector contains polynucleotides encoding Oct4 protein, Sox2 protein, Klf4 protein, and / or c-Myc protein. In some aspects, the retroviral vector contains polynucleotides containing a polycistronic cassette encoding Oct4 protein, Sox2 protein, Klf4 protein, and c-Myc protein. In some respects, retroviral vectors contain polynucleotides encoding polycistronic cassettes of Oct4, Sox2, and Klf4 proteins. In other respects, retroviral vectors contain polynucleotides encoding transcription activators and one or more of the following proteins: Oct4, Sox2, Klf4, c-Myc, and / or dnNFκBIA.

[0095] As used herein, the term "gag gene" refers to a nucleic acid that encodes a group-specific antigen protein, which is a major component of the capsid and specifically recognizes, binds to, and packages the retroviral genomic RNA into assembled viral particles.

[0096] As used in this article, the term "pol gene" refers to the gene that encodes pol enzymes required for viral replication, such as reverse transcriptase, protease, and integrase.

[0097] As used herein, the term "env gene" refers to a gene that encodes an envelope protein that enables retroviruses to bind to receptors on the surface of target cells and enter target cells via membrane fusion.

[0098] As used herein, the term "lentiviral vector" refers to any vector containing or derived from lentiviral vector components and suitable for infecting mammalian cells, preferably human cells. The term lentiviral vector generally refers to lentiviral particles or viral particles or viruses containing a payload. The terms "lentiviral genome" and "lentiviral vector" are used interchangeably and refer to lentiviral nucleic acids into which nucleic acids of interest have been inserted to replace certain viral sequences to produce replication-defective viruses. For packaging lentiviral genomes or lentiviral vectors into lentiviral particles, packaging cell lines containing the gag, pol, and env genes, and optionally the tat and rev genes, but without long terminal repeats (LTRs) are available. In some respects, the packaging cell lines contain the rev gene but not the tat gene. Lentiviral vectors are capable of integrating their genes into the host genome, thereby transferring large amounts of exogenous genetic material into the host genome. However, in some respects, lentiviral vectors are non-integrating. In some respects, lentiviral vectors are self-inactivating vectors. In some respects, the 3' end of the U3 region in the lentiviral 3'LTR is altered, causing the vector RNA to be produced from the intact 5'LTR in packaging cell lines but not regenerated in target cells. In some respects, the U3 region in the 5'LTR of the lentiviral vector is partially or completely replaced by a heterologous promoter and / or enhancer. In some respects, the lentiviral vector is a lentiviral loop without a lentiviral capsid and is introduced into cells as lentiviral DNA, for example, via electroporation or via nanoparticles. In some respects, the lentiviral loop does not contain integrase and remains free in the cell.

[0099] As used herein, the term "lentiviral particle" refers to a lentivirus comprising a capsid and a lentiviral vector having at least one payload region (e.g., a polynucleotide encoding at least one regeneration factor) and at least one LTR. In some aspects, lentiviral particles are pseudogenotyped by combining the lentiviral vector with a heteroviral envelope glycoprotein. In some aspects, the heteroviral envelope glycoprotein is a naturally occurring envelope glycoprotein, such as that derived from vesicular stomatitis virus. In some aspects, the heteroviral envelope glycoprotein is an engineered viral envelope glycoprotein, for example, targeting a specific cell type. In some aspects, the lentiviral vector contains a polynucleotide encoding a regeneration factor. In some aspects, the lentiviral vector contains a polynucleotide encoding Oct4 protein, Sox2 protein, Klf4 protein, and / or c-Myc protein. In some aspects, the lentiviral vector contains a polycistronic cassette encoding Oct4 protein, Sox2 protein, Klf4 protein, and c-Myc protein. In some aspects, the lentiviral vector contains a polycistronic cassette encoding Oct4 protein, Sox2 protein, and Klf4 protein. In some respects, lentiviral vectors contain one or more polynucleotides encoding transcription activators and one or more of the following proteins: Oct4, Sox2, Klf4, c-Myc, and / or dnNFκBIA.

[0100] As used herein, the term "adenoviral vector" refers to any vector containing or derived from adenoviral vector components and suitable for infecting mammalian cells, preferably human cells. The term adenoviral vector generally refers to adenoviral particles or viral particles containing a payload. The terms "adenoviral genome" and "adenoviral vector" are used interchangeably. Adenoviral vectors can be derived from various serotypes, such as serotype Ad2 or Ad5, or can contain elements from more than one serotype. In some respects, adenoviral vectors are helper virus-dependent, replication-defective adenoviral vectors.

[0101] As used herein, the term "adenovirus particle" refers to an adenovirus comprising an adenovirus capsid and an adenovirus vector having at least one payload region (e.g., a polynucleotide encoding a regeneration factor) and at least one inverted terminal repeat (ITR). In some aspects, adenovirus particles are prepared using a helper virus-dependent adenovirus vector comprising two ITRs, a payload, and optionally non-coding filler sequences, and a helper virus comprising genes for adenovirus replication, packaging, and capsid formation. In some aspects, the adenovirus vector contains a polynucleotide encoding a transcription activator. In some aspects, the adenovirus vector contains a polynucleotide encoding Oct4, Sox2, Klf4, and / or c-Myc proteins. In some aspects, the adenovirus vector contains a polynucleotide containing a polycistronic cassette encoding Oct4, Sox2, Klf4, and c-Myc proteins. In some aspects, the adenovirus vector contains a polycistronic cassette encoding Oct4, Sox2, and Klf4 proteins. In some respects, the adenovirus vector contains one or more polynucleotides encoding transcription activators and one or more of the following proteins: Oct4, Sox2, Klf4, c-Myc, and / or dnNFκBIA.

[0102] As used herein, the term "adeno-associated virus vector" or "AAV vector" refers to any vector containing or derived from adeno-associated vector components and suitable for infecting mammalian cells, preferably human cells. The term AAV vector generally refers to AAV-type viral particles or viral particles or viruses containing a payload. The terms "AAV genome" and "AAV vector" are used interchangeably. AAV vectors can be derived from various serotypes, including combinations of serotypes (i.e., "pseudotyped" AAV) or from various genomes (e.g., single-stranded or self-complementary). Furthermore, AAV vectors can be replication-defective and / or targeted. As used herein, the term “adeno-associated virus” (AAV) includes, but is not limited to, AAV 1, AAV 2, AAV 3 (including 3A and 3B), AAV 4, AAV 5, AAV 6, AAV 7, AAV 8, AAV 9, AAV 10, AAV 11, AAV 12, AAV 13, AAV-DJ, AAVrh8, AAVrh10, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, horse AAV, sheep AAV, goat AAV, shrimp AAV, those AAV serotypes and clades disclosed by Gao et al. (J.Virol.78:6381(2004)) and Moris et al. (Virol.33:375(2004)), as well as any other AAV now known or subsequently discovered. See, for example, FIELDS et al., VIROLOGY, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers). In some respects, “AAV” vectors include derivatives of known AAV vectors. In some respects, “AAV” vectors include modified or artificial AAV vectors. In some respects, the AAV vector is modified relative to the wild-type AAV serotype sequence.

[0103] As used herein, the term "AAV particle" refers to an AAV virus comprising an AAV capsid and an AAV vector having at least one payload region (e.g., a polynucleotide encoding a therapeutic protein or polypeptide) and at least one inverted terminal repeat (ITR). In some aspects, the AAV vector contains a polynucleotide encoding a transcription activator. In some aspects, the AAV vector contains a polynucleotide encoding Oct4 protein, Sox2 protein, Klf4 protein, and / or c-Myc protein. In some aspects, the AAV vector contains a polycistronic cassette encoding Oct4 protein, Sox2 protein, Klf4 protein, and c-Myc protein. In some aspects, the AAV vector contains a polycistronic cassette encoding Oct4 protein, Sox2 protein, and Klf4 protein. In some aspects, the AAV vector contains a polynucleotide encoding dnNFκBIA protein. In some aspects, the AAV vector contains a polynucleotide encoding a transcription activator and one or more of the following proteins: Oct4 protein, Sox2 protein, Klf4 protein, c-Myc, and / or dnNFκBIA protein.

[0104] As used herein, the term “AAV rep gene” refers to the large open reading frame (ORF) of the AAV genome, known as the AAV replication (rep) region. This ORF encodes the replication gene products Rep78, Rep68, Rep52, and Rep40, which are named according to their epigenetic molecular weight and allow for the replication, assembly, and packaging of complete AAV viruses.

[0105] As used herein, the term “AAV cap gene” refers to the large open reading frame (ORF) of the AAV genome, known as the AAV capsid region. This ORF encodes at least three capsid proteins: VP1, VP2, and VP3, which allow the assembly of the AAV capsid. The AAV genome is packaged into the capsid by means of the AAV Rep protein to produce the AAV virus.

[0106] As used herein, the phrase "AAV helper function for generating productive AAV infection" refers to the AAV rep and AAV cap genes provided from a source other than the polynucleotides containing the payload region and at least one ITR, enabling AAV capsid generation, AAV payload / ITR polynucleotide replication, and AAV payload / ITR insertion into the assembled AAV capsid. The AAV helper function can be provided by co-infecting AAV-producing cells with wild-type AAV virus particles, by providing AAV-producing cells with one or more plasmids containing the AAV rep and AAV cap genes, or by infecting AAV-producing cells with a non-AAV virus carrying the AAV rep and AAV cap genes. AAV particles generated according to the methods described herein lack the AAV rep and AAV cap genes and contain the AAV payload / ITR polynucleotides. When administered to a subject's cells, in the absence of the AAV rep and AAV cap genes, AAV virus particles cannot replicate or form more AAV virus particles in the subject's cells. Conversely, AAV viral particles release their payload / ITR polynucleotides upon entering the subject's cells, and the payload gene is transcribed in the subject's cells to produce the payload protein.

[0107] As used herein, the term “inhibition” is used interchangeably with “reduction,” “silencing,” “downregulation,” “blocking,” and other similar terms, and includes any level of inhibition.

[0108] As used herein, the term “in vitro” refers to an event that occurs in an artificial environment, such as in a test tube or reaction vessel, cell culture, culture dish, etc., where such event has not previously occurred in a living organism.

[0109] As used herein, the term "ex vivo" refers to an event that occurs outside a living organism, such as in a test tube, reaction vessel, cell culture, perfusion device, etc., rather than inside a living organism, where such event is preceded and / or followed by an event that occurred inside a living organism. For example, "ex vivo" perfusion of an organ refers to the perfusion of an organ that has been removed from a subject and is perfused outside the subject.

[0110] As used herein, the term "in vivo" refers to events that occur within an organism (e.g., an animal, a human, or its cells or tissues).

[0111] As used herein, the term "transfection" refers to a method of introducing exogenous polynucleotides into cells. Transfection methods include, but are not limited to, chemical methods, physical treatments, and cationic lipids or mixtures. The list of reagents that can be transfected into cells is large and includes, for example, siRNA, shRNA, sense sequences and / or antisense sequences, and DNA encoding one or more genes and organized into expression plasmids (e.g., vectors).

[0112] As used herein, the phrases “contacting cells with polynucleotides,” “contacting cells with oligonucleotides,” “contacting tissues with polynucleotides,” “contacting tissues with oligonucleotides,” “contacting organs with polynucleotides,” or “contacting organs with oligonucleotides,” “contacting cells with peptides,” “contacting tissues with peptides,” “contacting organs with peptides,” “contacting cells with compounds,” “contacting tissues with compounds,” or “contacting organs with compounds,” “contacting cells with nanoparticles,” or “contacting tissues with nanoparticles,” or “contacting organs with nanoparticles” include contacting cells or organs by any possible means. Contacting cells, tissues, or organs with polynucleotides, oligonucleotides, or compounds includes contacting cells, structures, or organs in vitro, in vivo, or ex vivo with said polynucleotides, oligonucleotides, peptides, nanoparticles, or compounds. Such contact can be direct or indirect. Therefore, for example, the polynucleotide, oligonucleotide, polypeptide, nanoparticle, or compound may be placed in physical contact with the cell, tissue, or organ by an individual performing the method, or alternatively, the polynucleotide, oligonucleotide, polypeptide, nanoparticle, or compound may be placed in a manner that would allow or result in its subsequent contact with the cell, tissue, or organ.

[0113] Contact with cells, tissues, or organs outside the body or in vitro can be achieved, for example, by incubating the cells, tissues, or organs with polynucleotides, oligonucleotides, peptides, nanoparticles, or compounds. Contact with cells, tissues, or organs in vivo can be achieved, for example, by injecting the polynucleotide, oligonucleotide, peptide, nanoparticle, or compound into or near the cells, tissues, or organs; by injecting the polynucleotide, oligonucleotide, peptide, nanoparticle, or compound into the bloodstream; or by injecting the polynucleotide, oligonucleotide, peptide, nanoparticle, or compound into another area, such as the bloodstream or subcutaneous space, such that the polynucleotide, oligonucleotide, peptide, nanoparticle, or compound subsequently reaches the tissue, cell, or organ to be contacted. For example, the polynucleotide, oligonucleotide, peptide, nanoparticle, or compound may contain and / or be coupled to a ligand that guides the polynucleotide, oligonucleotide, peptide, nanoparticle, or compound to the site of interest, such as the blood vessels of an organ. Combinations of in vitro and in vivo contact methods are also possible. For example, cells or tissues can be exposed to substances in vitro, or organs can be exposed to polynucleotides, oligonucleotides, peptides, nanoparticles, or compounds outside the body before being transplanted into the subject.

[0114] In some aspects, contacting cells, tissues, or organs with polynucleotides, oligonucleotides, peptides, nanoparticles, or compounds includes introducing or delivering the polynucleotides, oligonucleotides, peptides, nanoparticles, or compounds into the cells, tissues, or organs by promoting or achieving uptake or absorption into said cells, tissues, or organs. The uptake or absorption of polynucleotides, oligonucleotides, peptides, nanoparticles, or compounds can occur through unassisted diffusion or active cellular processes, or through adjuvants or devices. For example, in vitro introduction into cells, tissues, or organs includes methods known in the art, such as electroporation and lipid transfection. In vitro introduction into organs also includes methods such as perfusion. In some aspects, cells or organs are contacted in vitro or in vivo with polynucleotides, oligonucleotides, peptides, nanoparticles, compounds, AAV vectors, AAV capsids, retroviral vectors, lentiviral vectors, adenoviral vectors, or compositions thereof. For example, polynucleotides, oligonucleotides, polypeptides, nanoparticles, compounds, AAV vectors, AAV capsids, retroviral vectors, lentiviral vectors, adenoviral vectors, or compositions can be placed in physical contact with the cells, tissues, or organs, or in a manner that will allow or result in subsequent contact with the cells, tissues, or organs. In some aspects, contact with in vitro cells or tissues, or isolated tissues or organs, can be carried out, for example, by incubating the cells, tissues, or organs together with the polynucleotides, oligonucleotides, polypeptides, nanoparticles, compounds, AAV vectors, AAV capsids, retroviral vectors, lentiviral vectors, adenoviral vectors, or compositions. In some aspects, contact with isolated organs can be carried out, for example, by perfusing the organ with the polynucleotides, oligonucleotides, polypeptides, nanoparticles, compounds, AAV vectors, AAV capsids, retroviral vectors, lentiviral vectors, adenoviral vectors, or compositions. In some respects, contact with cells, tissues, or organs in the body can be achieved, for example, by injecting the polynucleotides, oligonucleotides, polypeptides, nanoparticles, compounds, AAV carriers, AAV capsids, retroviral vectors, lentiviral vectors, adenoviral vectors, or compositions of the present disclosure into or near the tissue containing the target cells, into the tissue, or into the organ; or by injecting the polynucleotides, oligonucleotides, polypeptides, nanoparticles, compounds, AAV carriers, AAV capsids, retroviral vectors, lentiviral vectors, adenoviral vectors, or compositions into an area, such as the bloodstream or subcutaneous space, such that the polynucleotides, oligonucleotides, polypeptides, nanoparticles, compounds, AAV carriers, AAV capsids, retroviral vectors, lentiviral vectors, adenoviral vectors, or compositions subsequently reach the tissue containing the cells to be contacted, the tissue containing the tissue, or the organ.Furthermore, polynucleotides, oligonucleotides, peptides, nanoparticles, compounds, AAV vectors, AAV viruses, retroviral vectors, lentiviral vectors, or adenoviral vectors can be encapsulated and / or conjugated to ligands that guide the polynucleotides, oligonucleotides, peptides, nanoparticles, compounds, AAV vectors, AAV viruses, retroviral vectors, lentiviral vectors, or adenoviral vectors to sites of interest, such as organs. Combinations of in vitro and in vivo contact methods are also possible. For example, cells or organs can be contacted in vitro with polynucleotides, oligonucleotides, peptides, nanoparticles, compounds, AAV vectors, AAV capsids, retroviral vectors, lentiviral vectors, adenoviral vectors, or combinations thereof and subsequently transplanted into a subject.

[0115] In some aspects, contacting cells or organs with polynucleotides, oligonucleotides, peptides, nanoparticles, compounds, AAV vectors, AAV capsids, retroviral vectors, lentiviral vectors, adenoviral vectors, or compositions as described herein includes “introducing” or “delivering” (directly or indirectly) the polynucleotides, oligonucleotides, peptides, nanoparticles, compounds, AAV vectors, AAV capsids, retroviral vectors, lentiviral vectors, adenoviral vectors, or compositions into the cells, tissues, or organs by promoting or achieving uptake or absorption into the cells, tissues, or organs. The introduction of polynucleotides, oligonucleotides, peptides, nanoparticles, compounds, AAV vectors, AAV capsids, retroviral vectors, lentiviral vectors, adenoviral vectors, or compositions into cells or organs can be ex vivo and / or in vivo.

[0116] As used herein, the term "lipid nanoparticle" refers to a vesicle containing a lipid layer encapsulating a pharmaceutically active molecule, such as a polynucleotide molecule, for example, a polynucleotide or oligonucleotide. Lipid nanoparticles can have an average diameter of 10 nanometers to 1000 nanometers and comprise a solid lipid core matrix and a surfactant. Lipid nanoparticles typically contain cationic lipids, non-cationic lipids, and lipids that prevent aggregation of the particles (e.g., PEG-lipid conjugates). Lipid nanoparticles are described, for example, in U.S. Patents 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated by reference. The lipids in the nanoparticles can be triglycerides, diglycerides, monoglycerides, fatty acids, steroids, or waxes, or mixtures thereof. Lipid nanoparticles can be prepared using high-shear homogenization, sonication, solvent emulsification / evaporation, or microemulsification. In some aspects, lipid nanoparticles comprise ionizable cationic lipids.

[0117] As used herein, the term "cationic lipid" refers to lipids that have a cationic or positive charge at physiological pH. Cationic lipids can take many forms, including but not limited to liposomes or micelles. Cationic lipids that can be used in certain aspects of this disclosure are known in the art and generally contain polar and nonpolar domains, bind polyanions such as nucleic acid molecules or negatively charged proteins, and are generally known to facilitate the delivery of nucleic acids into cells. Examples of useful cationic lipids include polyethyleneimine, polyamide amine (PAMAM) star-shaped dendritic polymers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, etc. (For example, 2000, LIPOF 3000 RNAiMAX, LTX), SAINT-RED (Synvolux Therapeutics, Groningen Netherlands), DOPE, Cytofectin (GileadSciences, Foster City, Calif.) and Eufectins (JBL, San Luis Obispo, Calif.). Exemplary cationic liposomes may be made of N-[l-(2,3-dioleoxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[l-(2,3-dioleoxy)-propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), 3-β[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3,-dioleoxy-N-[2(sperminecarbamoyl)ethyl]-N,N-dimethyl-1-propanetrifluoroacetate (DOSP A), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide; and dimethyl dioctadecylammonium bromide (DDAB).

[0118] As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer (e.g., one or more bilayers). Liposomes include monolayer and multilayer vesicles having a membrane formed of a lipophilic material and an aqueous interior. The aqueous portion contains an oligonucleotide composition. The lipophilic material isolates the aqueous interior from the aqueous exterior, which typically does not contain the oligonucleotide composition, but in some instances, it may contain the oligonucleotide composition. Liposomes also include "sterically stable" liposomes, as used herein; this term refers to liposomes containing one or more specialized lipids that, when incorporated into liposomes, result in a longer cycle life compared to liposomes lacking such specialized lipids.

[0119] As used in this article, the term "micelle" refers to a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules face inward, leaving the hydrophilic portions in contact with the surrounding water. If the environment is hydrophobic, the opposite arrangement exists.

[0120] As used herein, the term “organ” means any organ that can be processed with the materials and methods described herein and used for transplantation into a subject in need.

[0121] As used in this article, the term "organoid" refers to a miniaturized and simplified form of an organ produced from tissues, embryonic stem cells, or induced pluripotent stem cells in three-dimensional culture outside the body.

[0122] As used herein, the term "organ transplantation" refers to the procedure of removing an organ from a organism (called a donor) and transferring it into a recipient organism. In some respects, the donor and recipient are the same organism. Examples of organ transplantation include, but are not limited to, kidney transplantation, liver transplantation, lung transplantation, heart transplantation, pancreas transplantation, or corneal transplantation. Prior to transplantation, the organ may be processed or modified according to the methods described herein. In some respects, an organ is obtained from a organism (donor), processed with the materials and methods described herein, and introduced into a second organism (recipient). In some respects, an organ is obtained from a organism, processed with the materials and methods described herein, and introduced back into the same organism (autologous transplantation). In some respects, two organs, such as two kidneys, are obtained from a organism, processed with the materials and methods described herein, and both organs are introduced into a recipient, or one of the two organs is introduced into a recipient and the other into a different recipient. In some respects, two organs, such as two kidneys, are obtained from an organism, treated with the materials and methods described herein, and introduced into the same organism; or one kidney is introduced into the same organism while the other kidney is introduced into a different organism; or two kidneys are introduced into different organisms. In some respects, organ portions are obtained from an organism, treated with the materials and methods described herein, and introduced into different recipient organisms. In some respects, organ portions are obtained from an organism, treated with the materials and methods described herein, and introduced into the same organism (autologous transplantation). In some respects, one organ, such as a liver, is obtained from an organism, treated with the materials and methods described herein, then divided into two or more organ portions and each organ portion is introduced into a different recipient; or one organ portion is introduced into the organism from which the organ originated and one or more organ portions are introduced into one or more different recipients.

[0123] As used herein, the term "perfusion" refers to the delivery of fluid to tissues and / or organs via the circulatory or lymphatic system. In some aspects, perfusion includes fluid passing through blood vessels. In some aspects, perfusion includes fluid passing through lymphatic vessels. In some aspects, perfusion includes cold perfusion. In some aspects, perfusion includes normothermic perfusion. In some aspects, perfusion includes normothermic mechanical perfusion using a perfusion machine. In some aspects, normothermic mechanical perfusion is at about 38°C. In some aspects, normothermic mechanical perfusion is at about 22°C to about 38°C; or at about 23°C to about 37°C; at about 24°C to about 36°C; at about 25°C to about 35°C; at about 26°C to about 34°C; at about 27°C to about 33°C; at about 28°C to about 32°C; or at about 33°C; about 34°C; about 35°C; about 36°C; or about 37°C. In some aspects, perfusion includes cold perfusion followed by normothermic mechanical perfusion. In some aspects, the cold infusion is at about 2°C to about 21°C; or at about 3°C ​​to about 20°C; at about 4°C to about 19°C; at about 5°C to about 18°C; at about 6°C to about 17°C; at about 7°C to about 16°C; at about 8°C to about 15°C; at about 9°C to about 14°C; at about 10°C to about 13°C; or at about 2°C; about 3°C; about 4°C; about 5°C; or about 6°C; about 7°C; about 8°C; about 9°C; about 10°C; about 11°C; about 12°C; about 13°C; about 14°C; about 15°C; or about 16°C.

[0124] As used herein, the terms "perfusion machine," "perfusion system," or "perfusion apparatus" are interchangeable and refer to a machine, system, or apparatus that includes a pump, a reservoir containing an organ or organoid, a secondary reservoir containing perfusion fluid, and conduit connecting the organ-containing reservoir and the perfusion fluid reservoir. In some aspects, a perfusion system also includes conduit connecting the organ's blood vessels or lymphatic vessels to the perfusion fluid reservoir. For example, a perfusion system may be a Hugo Sachs / Harvard device, a Kidney Assist device, etc. TM System, OrganOX system, Radnoti system, ARK Kidney system and Aferetica system.

[0125] As used herein, the term "perfusion fluid" refers to a fluid composition that flows through the blood vessels or lymphatic vessels of an isolated organ or otherwise permeates an isolated organ. Perfusion fluid may contain at least one of the following: buffers, inorganic salts, amino acids, metabolic substrates, hormones, vasodilators, tonics, oxygenators, antioxidants, anti-inflammatory agents, anticoagulants, or antimicrobial agents; and at least one of the following: polynucleotides, oligonucleotides, compounds, AAV vectors, lentiviral vectors, retroviral vectors, or adenoviral vectors.

[0126] In the context of damaged tissue, the term "tissue repair" refers to the restoration of tissue structure, function, or a combination thereof following tissue damage. Tissue repair includes, but is not limited to, tissue regeneration, cell growth, and / or tissue replacement (reprogramming) of existing tissue.

[0127] The term "regeneration" refers to the generation of new tissue or cells within an existing tissue. In some implementations, the methods described herein facilitate organ regeneration.

[0128] As used herein, the terms "tissue regeneration" or "organ regeneration" refer to the at least partial regeneration, replacement, restoration, or regrowth of tissue, organ, or other body structure or part thereof, following loss, damage, or degeneration, wherein such tissue regeneration would not occur without the methods described in this disclosure. Organ regeneration is associated with, for example, an increase in the size and / or number of cells in a damaged or diseased organ.

[0129] The term "reprogramming" refers to the process of altering cells using reprogramming factors (e.g., reversing (all or part) or preventing (all or part) cellular changes that are causes of dysfunction, degeneration, cell death, or aging). Reprogramming can be complete, causing differentiated cells (e.g., somatic cells) to be reprogrammed into pluripotent stem cells. Cell reprogramming can be incomplete, causing differentiated cells (e.g., somatic cells) to retain their cellular characteristics (e.g., lineage-specific stem cells). Cell reprogramming can also be incomplete, for example, causing cells to revert to their original state or exhibit more youthful properties (e.g., increased survival, reduced inflammation, or the ability to divide).

[0130] As used herein, the terms “dedifferentiation,” “increased cellular potential,” or “increased developmental potential” refer to the process of altering or reversing the differentiation state of differentiated cells (e.g., somatic cells) by driving cells to differentiate backward into more undifferentiated or more primitive cell types. For example, cells with increased cellular potential have greater developmental plasticity (i.e., they can differentiate into more cell types).

[0131] As used herein, the term "enhancer" refers to a reagent or combination of reagents that enhances the efficiency or rate of reprogramming and / or restoration and / or regeneration, and may include soluble Wnt, Wnt conditioned medium, BIX-01294 (G9a histone methyltransferase), PD0325901 (MEK inhibitor), DNA methyltransferase inhibitor, histone deacetylase (HDAC) inhibitor, valproic acid, 5'-azacytidine, dexamethasone, and succinyl aniline isohydroxamic acid (SAHA). For example, MK0683, vorinostat or other isohydroxyxamic acids), vitamin C, trichosmin (TSA), BML-210, depudecin (e.g., (-)-depudecin), HC toxins, nullscript (4-(1,3-dioxo-1H,3H-benzo[de]isoquinoline-2-yl)-N-hydroxybutyramide), phenyl butyrate (e.g., sodium phenylbutyrate) and other short-chain fatty acids, Scriptaid, Suramin Sodium, APHA compound 8, Apidin, Sodium Butyrate, Pivanex (AN-9), Trapoxin B, Chlamydocin, Phenylephrine Peptide (also known as FR901228 or FK228), Benzamide (e.g., CI-994 (e.g., N-acetyldinarin) and MS-27-275), MGCD0103, NVPLAQ-824, CBHA (m-carboxycinnamic acid dihydroxamic acid), JNJ16241199, Tubacin, A-161906, proxamide, oxamflatin, 3-Cl-UCHA (e.g., 6-(3-chlorophenylurea)hexanoic acid isohydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP 50. Other reprogramming enhancers include, for example, dominant-negative forms of HDAC (e.g., non-catalytically active forms), siRNA inhibitors of HDAC, and antibodies that specifically bind to HDAC. Such inhibitors are available, for example, from BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Aton Pharma, Titan Pharmaceuticals, Schering AG, Pharmion, MethylGene, and SigmaAldrich.

[0132] As used herein, the term “potential” refers to the sum of all developmental options available to a cell (i.e., developmental potential). Cellular potential is a continuum, ranging from the most plastic cells (totipotent stem cells with the strongest developmental potential) to the least plastic cells (terminally differentiated cells with the weakest developmental potential). The continuum of cellular potential includes, but is not limited to, totipotent cells, pluripotent cells, pluripotent cells, oligopotent cells, unipotent cells, and terminally differentiated cells. As used herein, the term “pluripotency” refers to the ability of a cell to form all lineages of the body or somatic cells (i.e., the embryo itself). For example, embryonic stem cells are a type of pluripotent stem cell capable of forming cells from each of the three germ layers—ectoderm, mesoderm, and endoderm. Pluripotency can be partially determined by assessing the pluripotency characteristics of a cell. Pluripotency characteristics include, but are not limited to: (i) pluripotent stem cell morphology; (ii) unlimited self-renewal potential; (iii) expression of pluripotent stem cell markers, including but not limited to SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, Oct4, Nanog, Sox2, CD30 and / or CD50; (iv) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm and endoderm); (v) teratoma formation composed of the three somatic cell lineages; and (vi) embryoid formation composed of cells from the three somatic cell lineages.

[0133] As used herein, the term "regeneration factor" or "reprogramming factor" refers to an agent that can revert terminally differentiated cells to a low-differentiation state (such as a pluripotent or multipotent state). Examples of regeneration factors include, but are not limited to, Oct family genes, Sox family genes, Klf family genes, Myc family genes, SALL4, NANOG, LIN28, STELLA, NOBOX, ESRRB, NR5A2, CEBPA, or STAT family genes, including but not limited to STAT1, STAT2, STAT3, STAT4, STAT5 (STAT5A and STAT5B), and STAT6; and dominant-negative nuclear factor κB IA (dnNFκBIA). In some respects, the regeneration factors are Oct4, Sox2, Klf4 and c-Myc; Oct4, Sox2, Nanog and Lin28; Oct4, Sox2 and Klf4; or various combinations of Oct4, Sox2, klf4, nanog, ESRRB, NR5A2, CEBPA, Myc, Lin28A and Lin28B.

[0134] As used herein, the terms "preventing complete dedifferentiation" and "blocking complete dedifferentiation" refer to methods that revert cells to a low-differentiation state without allowing them to revert to a stem cell state. These methods may include exposing cells to at least one regenerative factor for a specific time period. The methods may also include exposing cells to at least one regenerative factor that does not induce a reversion to a stem cell state.

[0135] As used in this article, the term “restorative cell” is intended to include cellular causes that prevent or reverse (all or part) aging without inducing a pluripotent state.

[0136] As used herein, the term “Oct family genes” refers to the family of octamer transcription factors and includes, but is not limited to, Oct1, Oct3, Oct4, Oct6 and their variants.

[0137] As used herein, the term “Sox family genes” refers to the Syr-associated HMG box transcription factor family and includes, but is not limited to, Sox1, Sox2, Sox3, Sox7, Sox15, Sox17, Sox18 and their variants.

[0138] As used herein, the term "Klf family genes" refers to the Kruppel-like transcription factor family and includes, but is not limited to, Kfl1, Klf2, Klf4, Klf5 and their variants.

[0139] As used herein, the term "Myc family genes" refers to the Myc proto-oncogene transcription factor family and includes, but is not limited to, c-Myc, L-Myc, N-Myc, and their variants. For example, Myc variants may have amino acid deletions at positions 1 to 41 of the amino acid sequence of human c-Myc in SEQ ID NO:4. In some aspects, Myc variants have amino acid deletions at positions 1 to 64 of the amino acid sequence of human c-Myc. In some aspects, Myc variants have amino acid deletions at positions 1 to 107 of the amino acid sequence of human c-Myc. In some aspects, Myc variants have amino acid deletions at positions 1 to 13 of the amino acid sequence of human c-Myc and a mutation at position 135, wherein the mutation at position 135 is a deletion or substitution. In some aspects, Trp at position 135 of human c-Myc is replaced by Glu or Gly. In some aspects, L-Myc variants have at least the amino acid sequence from position 70 onwards in the amino acid sequence of human L-Myc as shown in SEQ ID NO:5. In some aspects, the L-Myc variant has at least position 45 and subsequent amino acid sequences in the human L-Myc amino acid sequence as shown in SEQ ID NO:5. In some aspects, the L-Myc variant has at least position 22 and subsequent amino acid sequences in the human L-Myc amino acid sequence as shown in SEQ ID NO:5. In some aspects, the L-Myc variant has at least position 321 and subsequent amino acid sequences in the human L-Myc amino acid sequence as shown in SEQ ID NO:5. In some aspects, the mutation at position 321 in SEQ ID NO:5 is a substitution or deletion. In some aspects, Val at position 321 in SEQ ID NO:5 is substituted with Asp. In some aspects, Myc is the N-Myc of SEQ ID NO:6.

[0140] As used herein, the term "peptide variant" generally exhibits at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity along its length with the peptide sequence shown herein. In one particular aspect, the variant or combination of variants employed retains the ability to induce pluripotency as described herein. Peptide variants may differ from naturally occurring peptides in that they may involve one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or can be generated synthetically, for example by modifying one or more of the peptide sequences described above and evaluating their effects.

[0141] As used herein, the term "percentage (%) sequence identity" relative to a reference polynucleotide or polypeptide sequence is defined as the percentage of polynucleotides or amino acids in a candidate sequence that are identical to those in a reference polynucleotide or polypeptide sequence after sequence alignment and the introduction of vacancies (if necessary) to achieve maximum percentage sequence identity. Alignment for determining the percentage of polynucleotide or amino acid sequence identity can be performed in a variety of ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences. For example, the percentage sequence identity value can be generated using the sequence comparison computer program BLAST. For example, the formula for calculating the percentage sequence identity between a given polynucleotide or amino acid sequence A and a given polynucleotide or amino acid sequence B (which can also be expressed as a certain percentage sequence identity between a given polynucleotide or amino acid sequence A and a given polynucleotide or amino acid sequence B) is as follows:

[0142] 100 × (fraction X / Y)

[0143] Where X is the number of nucleotides or amino acids that are scored as identical matches in the alignment of A and B by a sequence alignment program (e.g., BLAST), and Y is the total number of polynucleotides in B. It should be understood that when the length of polynucleotide or amino acid sequence A is not equal to the length of polynucleotide or amino acid sequence B, the percentage sequence identity between A and B will not be equal to the percentage sequence identity between B and A.

[0144] As used herein, the term "amino acid substitution" in variant terms refers to the result of replacing one amino acid with another, having similar structure and / or chemical properties; i.e., a conserved amino acid substitution. "Conserved" amino acid substitution can be based on similarity to any of a variety of properties, such as the side chain size, polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan, and methionine. Polar (hydrophilic) neutral amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In certain groups, certain substitutions may be of particular interest, for example, leucine replaced by isoleucine (or vice versa), serine replaced by threonine (or vice versa), or alanine replaced by glycine (or vice versa). Of course, non-conservative substitutions are generally also suitable for preserving function. In some aspects, substitution or deletion does not alter or remove amino acids that are important for activity. The size of insertions or deletions can range from about 1 to 20 amino acids, for example, 1 to 10 amino acids. In some cases, larger domains can be removed without substantially affecting function. In some embodiments of this disclosure, the sequence of a variant can be obtained by adding, deleting, or substituting a total of no more than 5, 10, 15, or 20 amino acids from the sequence of a naturally occurring enzyme. In some aspects, no more than 1%, 5%, 10%, or 20% of the amino acids in the polypeptide are insertions, deletions, or substitutions relative to the original polypeptide. By comparing the sequence of a specific polypeptide with that of a homologous polypeptide (e.g., from other organisms) and minimizing the number of amino acid sequence variations produced in highly homologous regions (conserved regions), or by substituting amino acids with those found in the homologous sequence, guidance can be obtained to determine which amino acid residues can be substituted, added, or deleted without eliminating or significantly reducing the activity of interest, since conserved amino acid residues are more likely to be important for activity than non-conserved amino acids across different species. In some aspects, polypeptide variants contain heterologous polypeptide moieties. Heterologous moieties typically have sequences not present in the original polypeptide or not homologous to the original polypeptide. Heterologous moieties can be, for example, 5 to about 5,000 amino acids long, or longer. Typically, their length is between 5 and about 1,000 amino acids. In some aspects, heterologous moieties contain sequences found in different polypeptides, such as functional domains. In some aspects, heterologous moieties contain sequences that can be used for the purification, expression, dissolution, and / or detection of the polypeptide. In some aspects, heterologous moieties contain polypeptide “tags,” such as affinity tags or epitope tags.For example, tags can be affinity tags (e.g., HA, TAP, Myc, 6xHis, Flag, GST), fluorescent or luminescent proteins (e.g., EGFP, ECFP, EYFP, Cerulean, DsRed, mCherry), or tags that enhance solubility (e.g., SUMO tags, NUS A tags, SNUT tags, or monomeric mutants of the Ocr protein of phage T7). See, for example, Esposito D and Chatterjee D K. Curr Opin Biotechnol.; 17(4):353-8 (2006). In some respects, tags can provide multiple functions. Tags are generally relatively small, for example, ranging from a few amino acids to about 100 amino acids in length. In some respects, tags are longer than 100 amino acids, for example, up to about 500 amino acids or more in length. In some respects, peptides have tags located at the N-terminus or C-terminus, for example, as N-terminal or C-terminal fusions. Peptides can contain multiple tags. In some aspects, the 6xHis tag and NUS tag are present, for example, at the N-terminus. In some aspects, the tag is cleavable, allowing it to be removed from the polypeptide, for example, by a protease. In some aspects, this is achieved by including a sequence encoding a protease cleavage site between the sequence encoding the portion homologous to the original polypeptide and the tag. Exemplary proteases include, for example, thrombin, TEV protease, factor Xa, PreScission protease, etc. In some aspects, a "self-cleaving" tag is used. The sequence encoding the tag may be located at the 5' or 3' (or both) of the polynucleotide encoding the polypeptide. In some aspects, the tag or other heterologous sequence is separated from the rest of the polypeptide by a polypeptide linker. For example, the linker may be a short polypeptide (e.g., 15-25 amino acids). Typically, the linker consists of small amino acid residues, such as serine, glycine, and / or alanine. The heterologous domain may include a transmembrane domain, a secretion signaling domain, etc.

[0145] As used herein, the term “level” refers to the level or activity of a protein or mRNA encoding one or more proteins (e.g., a regeneration factor), optionally compared to a reference. As defined herein, a reference can be any useful reference. A “decreased level” or “increased level” means a decrease or increase in protein level compared to a reference (e.g., a decrease or increase of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more; a decrease or increase of more than [amount missing] compared to a reference). Approximately 10%, approximately 15%, approximately 20%, approximately 50%, approximately 75%, approximately 100%, or approximately 200%; a decrease or increase of less than approximately 0.01, approximately 0.02, approximately 0.1, approximately 0.3, approximately 0.5, approximately 0.8, or less; or an increase of more than approximately 1.2, approximately 1.4, approximately 1.5, approximately 1.8, approximately 2.0, approximately 3.0, approximately 3.5, approximately 4.5, approximately 5.0, approximately 10, approximately 15, approximately 20, approximately 30, approximately 40, approximately 50, approximately 100, approximately 1000, or more. Protein levels can be expressed as mass / volume (e.g., g / dL, mg / mL, μg / mL, or ng / mL) or as a percentage of total protein or mRNA in the sample.

[0146] As used herein, the phrase “determining protein levels” means the direct or indirect detection of a protein or the mRNA encoding said protein by methods known in the art. “Direct determination” means performing a process, such as performing an assay or test on a sample, or analyzing a sample to obtain a physical entity or value. “Indirect determination” means receiving a physical entity or value from another party or source (e.g., a third-party laboratory that directly obtains the physical entity or value). Methods for measuring protein levels generally include, but are not limited to, Western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescence polarization, phosphorescence, immunohistochemistry, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, liquid chromatography (LC)-mass spectrometry, microcytometry, microscopy, fluorescence-activated cell sorting (FACS), and flow cytometry, as well as assays based on protein characteristics, including but not limited to enzymatic activity or interactions with other protein chaperones. Methods for measuring RNA levels include, but are not limited to, transcriptome sequencing, batch RNA sequencing, and single-cell RNA sequencing.

[0147] As used herein, the term "transcriptome profile" refers to the collection of all RNA molecules in a cell or population of cells. It is sometimes used to refer to all RNA or only mRNA, depending on the specific experimental setup. It differs from the exome in that it includes only those RNA molecules found in a particular population of cells and, in addition to molecular characteristics, usually includes the amount or concentration of each RNA molecule. Methods for obtaining transcriptome profiles include DNA microarrays and next-generation sequencing technologies such as RNA-Seq. Transcription can also be studied at the single-cell level through single-cell transcriptomics. One approach to inferring transcriptome sequences is to map sequence reads onto a reference genome, which is either that of the organism itself (whose transcriptome is being studied) or that of a closely related species. Another approach, de novo transcriptome assembly, uses software to directly infer transcripts from short sequence reads.

[0148] As used herein, the terms “tissue damage,” “cell damage,” and “organ damage” refer to any type of damage or injury to cells, tissues, or organs. In each respect, the term encompasses damage caused by aging, damage caused by disease, damage caused by physical trauma or surgery, damage caused by exposure to harmful substances, and other disruptions to the structure and / or function of cells, tissues, or organs.

[0149] As used herein, the term "subject" means any organism to which the materials or methods described herein may be applied, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes (and may be used interchangeably with "subject" herein). Exemplary organisms include mammals such as humans, non-human primates, rodents (e.g., mice, rats, rabbits), ungulates (e.g., sheep, cattle, horses, goat species), canines, and felines. Typically, a subject is an individual to whom a compound will be delivered, for example, for experimental, diagnostic, and / or therapeutic purposes, or to whom a sample will be obtained or to whom a diagnostic procedure will be performed (e.g., a sample or procedure to be used to assess tissue damage and / or to assess the effects of the compounds described in this disclosure).

[0150] As used herein, the term “donor” refers to a subject from whom an organ is obtained surgically and includes mammals such as humans, non-human primates, rodents (e.g., mice, rats, rabbits), ungulates (e.g., sheep, cattle, horses, goat species), canines, and felines.

[0151] As used herein, the term “recipient” refers to a subject to whom an organ is surgically transferred and includes mammals such as humans, non-human primates, rodents (e.g., mice, rats, rabbits), ungulates (e.g., sheep, cattle, horses, goat species), canines, and felines.

[0152] The terms “treat,” “treating,” “therapy,” “therapeutic,” and similar terms refer to therapeutic treatments and prophylactic or preventative measures aimed at preventing or alleviating (reducing) undesirable physical (e.g., age-related) symptoms, conditions, or diseases, or at achieving beneficial or desired clinical outcomes. In some respects, age-related symptoms include signs and / or symptoms associated with natural aging. In some respects, treatment reduces or alleviates symptoms associated with, for example, age-related diseases or conditions. In some respects, treatment produces beneficial or desired clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, reduction of symptoms; decrease in the severity of symptoms, conditions, or diseases; stabilization of the state of symptoms, conditions, or diseases (i.e., no worsening); delay or slowing of the onset of symptoms, conditions, or disease progression; improvement or relief of symptoms, conditions, or disease states (whether partial or complete), whether detectable or undetectable; improvement in at least one measurable physical parameter, not necessarily perceptible to the patient; or enhancement or improvement of symptoms, conditions, or diseases. In some respects, treatment includes evoking a clinically significant response without excessive side effects. In other respects, treatment includes prolonging survival compared to expected survival without treatment. As used herein, the term “improvement” refers to a reduction in the severity of at least one indicator of a symptom or disease. As used herein, the term “prevention” refers to delaying or preventing the onset, development, or progression of a symptom or disease over a period of time, including weeks, months, or years. Improving a disease or condition includes slowing its course or reducing the severity of age-related diseases or conditions that may develop later. An “effective preventative dose” can vary depending on the characteristics and composition of the agent, the method of administration, the degree of disease risk, and medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated. In some respects, treatments are provided to removed organs to regenerate and / or restore aging organs, to regenerate and / or restore damaged organs, and / or reverse (all or part) physical changes associated with at least one of the following: aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, fatty degeneration, virus-induced hepatitis, alcoholism, or fibrosis not associated with any known cause.

[0153] For example, the terms “effective amount,” “therapeutic effective amount,” and “sufficient amount,” as used herein with respect to polynucleotides, oligonucleotides, peptides, carriers, reagents, nanoparticles, or compositions, refer to an amount sufficient to achieve a beneficial or desired outcome (including clinical outcomes) when applied to a subject, tissue, or organ. Therefore, “effective amount” or its synonyms depend on the context of its application. In some aspects, a therapeutically effective amount of a reagent (e.g., a polynucleotide, carrier, nanoparticle, or composition described herein) is an amount that produces a beneficial or desired outcome in a subject or ex vivo tissue or organ, compared to a control. The amount of a given reagent (e.g., a polynucleotide, carrier, nanoparticle, or composition described herein) will vary depending on various factors, such as the given reagent, the pharmaceutical formulation, the route of administration, the type of disease or condition, the organ, and the characteristics of the subject, organ donor, and the organ recipient being treated (e.g., age, sex, and / or weight). For example, in the context of treating an ex vivo tissue or organ, an amount of reagent sufficient to achieve a desired response in said tissue or organ compared to a response obtained without the application of said reagent. The desired response could be, for example, the restoration of aging organs or the reversal of organ changes caused by at least one of the following: aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, fatty degeneration, virus-induced hepatitis, alcohol, or fibrosis unrelated to any known cause.

[0154] As used herein, the term "preventative effective amount" refers to an amount of polynucleotide, carrier, nanoparticle, or composition as described herein, when applied to a subject, tissue, or organ, sufficient to prevent or improve a disease or injury in that subject or organ. Improvement of disease or injury includes slowing the progression of a disease or injury-inducing process or reducing the severity of a disease or injury-inducing disease that may develop later. The preventative effective amount can vary depending on the therapeutic agent used and the manner of administration, the degree of risk of the disease or injury, the subject's (including organ donors and recipients') medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the subject, organ donor, and organ recipient. A preventive effective dose may refer to, for example, an amount of agent that reduces the level and / or activity of ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, virus-induced hepatitis, alcohol, or fibrosis unrelated to any known cause in an organ, and may refer to an amount that, when administered to a subject (including a human), tissue, or organ, is sufficient to delay the onset of one or more signs and symptoms of ischemia or injury described herein by at least 120 days, compared to the predicted onset, such as at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years, or longer.

[0155] As used herein, the terms “intermittent addition” or “intermittent administration” refer to the repeated addition of a polynucleotide, peptide, carrier, or nanoparticle to a composition, with each addition followed by a period of non-addition; or to the repeated administration of a polynucleotide, peptide, carrier, or nanoparticle to a subject, with each administration followed by a period of non-administration.

[0156] As used herein, the term "gene therapy" refers to the insertion of a polynucleotide sequence (e.g., a polynucleotide comprising a promoter operatively linked to a polynucleotide encoding a therapeutic molecule as disclosed herein) into the cells, tissues, and / or organs of an individual to treat a disease or age-related symptom, alleviate the symptoms of a disease or age-related symptom, or reduce the likelihood of a disease or age-related symptom. Gene therapy also includes transgenes whose insertion is inherently repressive, i.e., inhibiting, reducing, or diminishing the expression, activity, or function of endogenous genes or proteins, such as undesirable (e.g., damaging) or abnormal (e.g., pathogenic) genes or proteins. Such transgenes may be exogenous. Exogenous molecules or sequences should be understood as molecules or sequences that are abnormally present in the cells, tissues, organs, and / or individuals to be treated.

[0157] As used herein, the term "biopsy" refers to, for example, material obtained from a subject's tissue or organ by aspiration using a fine needle, or the procedure for obtaining such material. Biopsies typically contain multiple cell types from the tissue or organ and can provide information about the tissue's or organ's morphology, histopathology, and function.

[0158] As used herein, the term "pharmaceutical composition" means a composition comprising a compound or carrier described herein, such as a polynucleotide, oligonucleotide, AAV carrier, lentiviral carrier, retroviral carrier, or adenoviral carrier, formulated with pharmaceutically acceptable excipients, which may be manufactured or marketed as part of a treatment regimen for the treatment of a disease or organ prior to transplantation, subject to approval by a government regulatory agency.

[0159] As used herein, the term “pharmaceuticalally acceptable excipient” means any component other than the compounds or carriers described herein (e.g., a medium capable of suspending or dissolving an active compound or carrier) and having substantially non-toxic and non-inflammatory properties in a patient or organ.

[0160] II. Polynucleotides

[0161] A polynucleotide for expressing a regeneration factor is provided. In some aspects, the polynucleotide comprises a polynucleotide sequence encoding at least one regeneration factor. In some aspects, the polynucleotide comprises a polynucleotide sequence encoding at least one transcription activator. In some aspects, the polynucleotide comprises a polynucleotide sequence encoding at least one regeneration factor and at least one transcription activator.

[0162] In some aspects, the polynucleotide is non-integrating RNA. In some aspects, the polynucleotide is non-integrating DNA. In some aspects, the polynucleotide is integrated RNA. In some aspects, the polynucleotide is integrated DNA.

[0163] In some aspects, the polynucleotide is present in a vector. In some aspects, the polynucleotide is present in a plasmid. In some aspects, the polynucleotide is present in a viral vector. In some aspects, the polynucleotide is present in an AAV vector, a lentiviral vector, a retroviral vector, or an adenoviral vector.

[0164] In some aspects, the polynucleotide includes at least one inducible promoter. In some aspects, the polynucleotide includes at least one constitutive promoter. In some aspects, the polynucleotide includes at least one cell type-specific promoter.

[0165] In some aspects, non-integrating RNA encodes at least one regeneration factor that restores cells while maintaining their differentiated state. In some aspects, non-integrating DNA encodes at least one regeneration factor that restores cells while maintaining their differentiated state. In some aspects, integrating RNA encodes at least one regeneration factor operably linked to an inducible promoter, and contacting the cell with an inducing compound restores the cell while maintaining its differentiated state. In some aspects, integrating DNA encodes at least one regeneration factor operably linked to an inducible promoter, and contacting the cell with an inducing compound restores the cell while maintaining its differentiated state.

[0166] In some respects, the polynucleotide contains transposon elements.

[0167] In some respects, the polynucleotide comprises a polynucleotide sequence encoding at least one transcriptional transactivator.

[0168] In some aspects, the polynucleotide comprises an inducible promoter operatively linked to a polynucleotide sequence encoding at least one regeneration factor and a polynucleotide sequence encoding at least one transcriptional transactivator.

[0169] In some aspects, the inducible promoter is a tetracycline-inducible promoter; a hormone-inducible promoter; a steroid-inducible promoter; a cumate-inducible promoter; or a coumarin-inducible promoter. In some aspects, the hormone-inducible promoter is a tamoxifen-inducible promoter containing a transactivator domain fused to an estrogen receptor. In some aspects, the steroid-inducible promoter is a progesterone promoter containing a transactivator domain fused to a mutant progesterone receptor that binds RU486 but not endogenous progesterone. In some aspects, the coumarin-inducible promoter is a heterozygous promoter containing a transactivator domain that binds to the heterozygous promoter after homodimerization with coumarin. In some respects, cumulate-inducible promoters contain an operon site (CuO) downstream of the constitutive promoter and a repressor protein (CymR) that binds to the CuO site, wherein the addition of cumulate alleviates repression; or a chimeric transactivator formed by the fusion of CymR with an activation domain, which binds to CuO upstream of the constitutive promoter, and cumulate eliminates the DNA binding of the chimeric transactivator, thereby terminating transcriptional activation (see, for example, Mullick et al., BMC Biotechnology 6:43, 2006).

[0170] In some respects, tetracycline-inducible promoters contain multiple copies of the tet operon sequence that can bind to the inverse tetracycline-controlled transactivator protein (rtTA). In other respects, tetracycline-inducible promoters contain multiple copies of the tet operon sequence that can bind to the tTA-M2, rtTA3, or rtTA4 transactivator proteins.

[0171] In some aspects, in the presence of an inducible compound, the inducible promoter of the polynucleotide is bound by a transactivating protein. In some aspects, the tetracycline-inducible promoter of the polynucleotide is bound by an rTA protein in the presence of tetracycline. In some aspects, the tetracycline-inducible promoter is operatively linked to at least one regeneration factor. In some aspects, the tetracycline-inducible promoter is operatively linked to a polycistronic cassette containing a polynucleotide encoding at least two regeneration factors. In some aspects, the tetracycline-inducible promoter is operatively linked to a polycistronic cassette containing polynucleotides encoding Oct4 and Sox2. In some aspects, the polycistronic cassette further contains a polynucleotide encoding Klf4. In some aspects, the polycistronic cassette further contains a polynucleotide encoding c-Myc. In some aspects, the polynucleotide further contains a polynucleotide encoding a transcription transactivating factor. In some aspects, the polynucleotide further contains a polynucleotide encoding rtTA. In some aspects, the polynucleotide contains a polynucleotide encoding rtTA operatively linked to an inducible promoter. In some aspects, the polynucleotide contains a polynucleotide encoding rtTA operatively linked to a constitutive promoter. In some respects, polynucleotides contain polynucleotides encoding rtTA that are operatively linked to cell type-specific promoters.

[0172] In some respects, polynucleotides contain at least one constitutive promoter, such as CPI, CMV, EF1-α, SV40, PGK1, Ubc, human β-actin, CAG, CamKIIa, TEF1, GDS, CaMV35S, Grp78, Grp94, Hsp70, EGFR, H1, and U6 promoters.

[0173] In some aspects, the polynucleotide comprises a polynucleotide sequence encoding at least one regeneration factor operatively linked to a constitutive promoter, and a loxP sequence that frames the polynucleotide sequence encoding said at least one regeneration factor. In some aspects, the polynucleotide comprises more than one constitutive promoter and more than one expression cassette, said expression cassette optionally being framed by the loxP sequence.

[0174] In some aspects, the polynucleotide comprises an EF1-α promoter operably linked to a polynucleotide encoding a mutated inverse tetracycline-controlled transactivator rTA-M2. In some aspects, the polynucleotide comprises an EF1-α promoter operably linked to a polynucleotide encoding a mutated inverse tetracycline-controlled transactivator rTA-M2 and an inducible promoter operably linked to the c-Myc gene. In some aspects, the EF1-α promoter comprises the polynucleotide sequence of SEQ ID NO:12.

[0175] In some aspects, the polynucleotide comprises a TetO7 tetracycline operon promoter operatively linked to a polycistronic cassette, the polycistronic cassette comprising a polynucleotide sequence encoding the Oct4 gene, the Sox2 gene, and / or the Klf4 gene. In some aspects, the polynucleotide further comprises a TetO7 tetracycline operon promoter operatively linked to the c-Myc gene.

[0176] In some aspects, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:26. In some aspects, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:27. In some aspects, the polynucleotide comprises a binding site operatively linked to an estrogen receptor-binding transcription factor element of the c-Myc gene. In some aspects, the polynucleotide comprises a binding site operatively linked to an estrogen receptor-binding transcription factor element of a polycistronic cassette, the polycistronic cassette comprising a polynucleotide sequence encoding the Oct4 gene, the Sox2 gene, and / or the Klf4 gene. In some aspects, the polynucleotide comprises a binding site operatively linked to a chimeric regulatory protein of the c-Myc gene, the binding site being responsive to RU486 but not to endogenous progesterone. In some aspects, the polynucleotide comprises a binding site operatively linked to a chimeric regulatory protein of a polycistronic cassette, the binding site being responsive to RU486 but not to endogenous progesterone, the polycistronic cassette comprising a polynucleotide sequence encoding the Oct4 gene, the Sox2 gene, and / or the Klf4 gene.

[0177] In some aspects, the polynucleotide comprises an active promoter in aging cells. In some aspects, the promoter is the promoter of the cyclin-dependent kinase inhibitor 2A (CDKN2A) / p16 gene. In some aspects, the promoter is the p21 promoter. In some aspects, the promoter is the mir146A promoter. In some aspects, the promoter comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical polynucleotide sequence to the polynucleotide sequence of SEQ ID NO:19. In some aspects, the promoter comprises the polynucleotide sequence of SEQ ID NO:19. In some aspects, the promoter comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same polynucleotide sequence as SEQ ID NO:20. In some aspects, the promoter comprises the polynucleotide sequence of SEQ ID NO:20.

[0178] In some aspects, the promoter comprises a polynucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:7. In some aspects, the promoter comprises the polynucleotide sequence of SEQ ID NO:7. In some aspects, the polynucleotide comprises a polynucleotide encoding a transactivator that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:12. In some aspects, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:12.

[0179] In some aspects, the polynucleotide comprises a polynucleotide sequence encoding the Oct4 protein. In some aspects, the Oct4 protein is the human Oct4 protein. In some aspects, the polynucleotide comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical polynucleotide sequences to the human Oct4 sequence of SEQ ID NO:1. In some aspects, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:1. In some aspects, the Oct4 protein is the mouse Oct4 protein. In some aspects, the polynucleotide comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical polynucleotide sequences to the human Oct4 sequence of SEQ ID NO:8. In some respects, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:8.

[0180] In some aspects, the polynucleotide comprises a polynucleotide sequence encoding the Sox2 protein. In some aspects, the Sox2 protein is the human Sox2 protein. In some aspects, the polynucleotide comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical polynucleotide sequences to the human Sox2 sequence of SEQ ID NO:2. In some aspects, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:2. In some aspects, the Sox2 protein is the mouse Sox2 protein. In some aspects, the polynucleotide comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical polynucleotide sequences to the human Sox2 sequence of SEQ ID NO:9. In some respects, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:9.

[0181] In some aspects, the polynucleotide comprises a polynucleotide sequence encoding the Klf4 protein. In some aspects, the Klf4 protein is the human Klf4 protein. In some aspects, the polynucleotide comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical polynucleotide sequences to the human Klf4 sequence of SEQ ID NO:3. In some aspects, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:3. In some aspects, the Klf4 protein is the mouse Klf4 protein. In some aspects, the polynucleotide comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical polynucleotide sequences to the human Klf4 sequence of SEQ ID NO:10. In some respects, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:10.

[0182] In some aspects, the polynucleotide comprises a polynucleotide sequence encoding a c-Myc protein. In some aspects, the c-Myc protein is a human c-Myc protein. In some aspects, the polynucleotide comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical polynucleotide sequence to the human c-Myc sequence of SEQ ID NO:4. In some aspects, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:4. In some aspects, the c-Myc protein is a mouse c-Myc protein. In some aspects, the polynucleotide comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical polynucleotide sequence to the human c-Myc sequence of SEQ ID NO:11. In some respects, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:11.

[0183] In some aspects, the polynucleotide comprises a polynucleotide sequence encoding the human L-Myc protein of SEQ ID NO:5. In some aspects, the polynucleotide comprises a polynucleotide sequence encoding the rodent L-Myc protein. In some aspects, the polynucleotide comprises a polynucleotide sequence encoding the human N-Myc protein of SEQ ID NO:6. In some aspects, the polynucleotide comprises a polynucleotide sequence encoding the rodent N-Myc protein.

[0184] In some aspects, the polynucleotide comprises a polynucleotide sequence encoding the Oct4 protein and / or a polynucleotide sequence encoding the Sox2 protein and / or a polynucleotide sequence encoding the Klf4 protein, all of which are operatively linked to an inducible promoter. In some aspects, the polynucleotide comprises a polynucleotide sequence encoding the Oct4 protein and / or a polynucleotide sequence encoding the Sox2 protein and / or a polynucleotide sequence encoding the Klf4 protein and / or a c-Myc protein, all of which are operatively linked to an inducible promoter. In some aspects, the polynucleotide comprises a polycistronic cassette containing polynucleotide sequences encoding the Oct4, Sox2, and Klf4 proteins. In some aspects, the polynucleotide comprises a polycistronic cassette containing polynucleotide sequences encoding the Oct4, Sox2, Klf4, and c-Myc proteins. In some respects, one polynucleotide comprises a polynucleotide sequence encoding the Oct4 protein and / or a polynucleotide sequence encoding the Sox2 protein and / or a polynucleotide sequence encoding the Klf4 protein, all of which are operatively linked to an inducible promoter, and a second polynucleotide comprises a polynucleotide operatively linked to an inducible promoter encoding the c-Myc protein.

[0185] In some aspects, the polynucleotide further comprises a proteolytic cleavage site. In some aspects, the proteolytic cleavage site is a self-processing cleavage site or a furin protease cleavage site. In some aspects, the self-processing cleavage site is a P2A, E2A, F2A, or T2A peptide. In some aspects, the furin protease cleavage site comprises the concordant sequence RXK(R)R of SEQ ID NO:17. Therefore, the polypeptide produced when the polynucleotide is transcribed in the cell can be cleaved by proteases in the cell to release the Oct4, Sox2, and Klf4 proteins.

[0186] In some aspects, the polynucleotide comprises a polynucleotide sequence encoding the Oct4 protein, a polynucleotide sequence encoding the Sox2 protein, and a polynucleotide sequence encoding the Klf4 protein, which are arranged in a 5'-3' orientation on the polynucleotide such that a desired amount of each of Oct4, Sox2, and Klf4 is produced in cells transduced with the polynucleotide. The amounts of Oct4, Sox2, and Klf4 proteins required to induce partial reprogramming in a particular cell type can vary, and polynucleotides that provide the optimal ratio of Oct4, Sox2, and Klf4 protein levels for partial reprogramming of the corresponding cell type can be selected. In some aspects, the closer the polynucleotide sequence encoding the protein is to the promoter sequence, the higher the level of protein produced by the polynucleotide. In some aspects, Oct4, Sox2, and Klf4 are positioned in a 5'-3' order relative to the promoter sequence. In some aspects, Oct4, Sox2, Klf4, and c-Myc are positioned in a 5'-3' order relative to the promoter sequence.

[0187] In some aspects, the polynucleotide comprises an inducible promoter, a polynucleotide sequence encoding the Oct4 protein, a proteolytic cleavage site, and a polynucleotide sequence encoding the Sox2 protein.

[0188] In some aspects, the polynucleotide comprises an inducible promoter, a polynucleotide sequence encoding the Oct4 protein, a proteolytic cleavage site, and a polynucleotide sequence encoding the Klf4 protein.

[0189] In some aspects, the polynucleotide comprises an inducible promoter, a polynucleotide sequence encoding the Sox2 protein, a proteolytic cleavage site, and a polynucleotide sequence encoding the Klf4 protein.

[0190] In some aspects, the polynucleotide comprises an inducible promoter, a polynucleotide sequence encoding the Oct4 protein, a first proteolytic cleavage site, a polynucleotide sequence encoding the Sox2 protein, a second proteolytic cleavage site, and a polynucleotide sequence encoding the Klf4 protein.

[0191] In some aspects, the polynucleotide comprises, in the 5'-3' direction, an inducible promoter, a polynucleotide sequence encoding the Oct4 protein, a first proteolytic cleavage site, a polynucleotide sequence encoding the Klf4 protein, a second proteolytic cleavage site, and a polynucleotide sequence encoding the Sox2 protein.

[0192] In some aspects, the polynucleotide comprises, in the 5'-3' direction, an inducible promoter, a polynucleotide sequence encoding the Sox2 protein, a first proteolytic cleavage site, a polynucleotide sequence encoding the Oct4 protein, a second proteolytic cleavage site, and a polynucleotide sequence encoding the Klf4 protein.

[0193] In some aspects, the polynucleotide comprises, in the 5'-3' direction, an inducible promoter, a polynucleotide sequence encoding the Sox2 protein, a first proteolytic cleavage site, a polynucleotide sequence encoding the Ktlf4 protein, a second proteolytic cleavage site, and a polynucleotide sequence encoding the Oct4 protein.

[0194] In some aspects, the polynucleotide comprises, in the 5'-3' direction, an inducible promoter, a polynucleotide sequence encoding the Klf4 protein, a first proteolytic cleavage site, a polynucleotide sequence encoding the Oct4 protein, a second proteolytic cleavage site, and a polynucleotide sequence encoding the Sox2 protein.

[0195] In some aspects, the polynucleotide comprises, in the 5'-3' direction, an inducible promoter, a polynucleotide sequence encoding the Klf4 protein, a first proteolytic cleavage site, a polynucleotide sequence encoding the Sox2 protein, a second proteolytic cleavage site, and a polynucleotide sequence encoding the Oct4 protein.

[0196] In some respects, the inducible promoter is the tetracycline-inducible promoter or the RU486-inducible promoter.

[0197] In some respects, the first protein hydrolysis site is the concordant sequence of P2A peptide, E2A peptide, F2A peptide, T2A peptide, or RXK(R)R of SEQ ID NO:17.

[0198] In some respects, the second proteolytic site is the concordant sequence of P2A peptide, E2A peptide, F2A peptide, T2A peptide, or RXK(R)R of SEQ ID NO:17.

[0199] In some respects, the polynucleotide contains a second promoter.

[0200] In some aspects, the polynucleotide comprises, in the 5'-3' direction, a first inducible promoter, a polynucleotide sequence encoding the Oct4 protein, a second inducible or non-inducible promoter, a polynucleotide sequence encoding the Sox2 protein, a proteolytic cleavage site, and a polynucleotide sequence encoding the Klf4 protein.

[0201] In some aspects, the polynucleotide comprises, in the 5'-3' direction, a first inducible promoter, a polynucleotide sequence encoding the Oct4 protein, a second inducible or non-inducible promoter, a polynucleotide sequence encoding the Klf4 protein, a proteolytic cleavage site, and a polynucleotide sequence encoding the Sox2 protein.

[0202] In some aspects, the polynucleotide comprises, in the 5'-3' direction, a first inducible promoter, a polynucleotide sequence encoding the Oct4 protein, a proteolytic cleavage site, a polynucleotide sequence encoding the Sox2 protein, a second inducible or non-inducible promoter, and a polynucleotide sequence encoding the Klf4 protein.

[0203] In some aspects, the polynucleotide comprises, in the 5'-3' direction, a first inducible promoter, a polynucleotide sequence encoding the Sox2 protein, a proteolytic cleavage site, a polynucleotide sequence encoding the Oct4 protein, a second inducible or non-inducible promoter, and a polynucleotide sequence encoding the Klf4 protein.

[0204] In some aspects, the polynucleotide comprises, in the 5'-3' direction, a first inducible promoter, a polynucleotide sequence encoding the Oct4 protein, a proteolytic cleavage site, a polynucleotide sequence encoding the Klf4 protein, a second inducible or non-inducible promoter, and a polynucleotide sequence encoding the Sox2 protein.

[0205] In some aspects, the polynucleotide comprises, in the 5'-3' direction, a first inducible promoter, a polynucleotide sequence encoding the Klf4 protein, a proteolytic cleavage site, a polynucleotide sequence encoding the Oct4 protein, a second inducible or non-inducible promoter, and a polynucleotide sequence encoding the Sox2 protein.

[0206] In some respects, the first promoter is either inducible or non-inducible and the second promoter is inducible.

[0207] In some aspects, the first and second inducible promoters are induced by the same inducing compound. In some aspects, the first and second inducible promoters are induced by different inducing compounds. For example, the first inducible promoter may be a tetracycline-induced promoter, while the second inducible promoter may be a coumarin-induced promoter. In some aspects, the polynucleotide comprises a first inducible promoter operatively linked to a polynucleotide encoding at least one of Oct4, Sox2, and / or Klf4. In some aspects, the polynucleotide comprises a first inducible promoter that is a tetracycline-induced promoter and operatively linked to a polynucleotide encoding at least one of Oct4, Sox2, and / or Klf4. In some aspects, the polynucleotide further comprises a second inducible promoter operatively linked to c-Myc. In some aspects, the polynucleotide further comprises a second inducible promoter that is a coumarin-induced promoter operatively linked to c-Myc. In some respects, the first and second inducible promoters are located on different polynucleotides, the first inducible promoter being a tetracycline inducible promoter operatively linked to a polynucleotide encoding at least one of Oct4, Sox2, and / or Klf4, and the second inducible promoter being a coumarin inducible promoter operatively linked to c-Myc.

[0208] In some respects, the first and second promoters initiate transcription in the same direction. In other respects, the first and second promoters initiate transcription in different directions.

[0209] In some aspects, the polynucleotide further comprises a polyadenylation signal sequence. In some aspects, the polyadenylation signal sequence is the SV40 polyadenylation signal sequence, the human growth hormone polyadenylation signal sequence, or the bovine growth hormone polyadenylation signal sequence.

[0210] In some respects, the polynucleotide also contains a WPRE sequence.

[0211] In some respects, the polynucleotide also contains IRES.

[0212] In some aspects, the polynucleotide comprises, in the 5'-3' direction, an inducible promoter, a polynucleotide sequence encoding the Oct4 protein, a first IRES, a polynucleotide sequence encoding the Sox2 protein, a second IRES, and a polynucleotide sequence encoding the Klf4 protein.

[0213] In some aspects, the polynucleotide comprises, in the 5'-3' direction, an inducible promoter, a polynucleotide sequence encoding the Oct4 protein, a first IRES, a polynucleotide sequence encoding the Klf4 protein, a second IRES, and a polynucleotide sequence encoding the Sox2 protein.

[0214] In some aspects, the polynucleotide comprises, in the 5'-3' direction, an inducible promoter, a polynucleotide sequence encoding the Sox2 protein, a first IRES, a polynucleotide sequence encoding the Oct4 protein, a second IRES, and a polynucleotide sequence encoding the Klf4 protein.

[0215] In some aspects, the polynucleotide comprises, in the 5'-3' direction, an inducible promoter, a polynucleotide sequence encoding the Sox2 protein, a first IRES, a polynucleotide sequence encoding the Klf4 protein, a second IRES, and a polynucleotide sequence encoding the Oct4 protein.

[0216] In some aspects, the polynucleotide comprises, in the 5'-3' direction, an inducible promoter, a polynucleotide sequence encoding the Klf4 protein, a first IRES, a polynucleotide sequence encoding the Oct4 protein, a second IRES, and a polynucleotide sequence encoding the Sox2 protein.

[0217] In some aspects, the polynucleotide comprises, in the 5'-3' direction, an inducible promoter, a polynucleotide sequence encoding the Klf4 protein, a first IRES, a polynucleotide sequence encoding the Sox2 protein, a second IRES, and a polynucleotide sequence encoding the Oct4 protein.

[0218] In some aspects, the polynucleotide comprises, in the 5'-3' direction, an inducible promoter, a polynucleotide sequence encoding the Oct4 protein, a second inducible or non-inducible promoter, a polynucleotide sequence encoding the Sox2 protein, IRES, and a polynucleotide sequence encoding the Klf4 protein.

[0219] In some aspects, the polynucleotide comprises, in the 5'-3' direction, an inducible promoter, a polynucleotide sequence encoding the Oct4 protein, a second inducible or non-inducible promoter, a polynucleotide sequence encoding the Klf4 protein, IRES, and a polynucleotide sequence encoding the Sox2 protein.

[0220] In some aspects, the polynucleotide comprises, in the 5'-3' direction, a first inducible promoter, a polynucleotide sequence encoding the Oct4 protein, IRES, a polynucleotide sequence encoding the Sox2 protein, a second inducible or non-inducible promoter, and a polynucleotide sequence encoding the Klf4 protein.

[0221] In some aspects, the polynucleotide comprises, in the 5'-3' direction, a first inducible promoter, a polynucleotide sequence encoding the Sox2 protein, IRES, a polynucleotide sequence encoding the Oct4 protein, a second inducible or non-inducible promoter, and a polynucleotide sequence encoding the Klf4 protein.

[0222] In some aspects, the polynucleotide comprises, in the 5'-3' direction, a first inducible promoter, a polynucleotide sequence encoding the Oct4 protein, IRES, a polynucleotide sequence encoding the Klf4 protein, a second inducible or non-inducible promoter, and a polynucleotide sequence encoding the Sox2 protein.

[0223] In some aspects, the polynucleotide comprises, in the 5'-3' direction, a first inducible promoter, a polynucleotide sequence encoding the Klf4 protein, IRES, a polynucleotide sequence encoding the Oct4 protein, a second inducible or non-inducible promoter, and a polynucleotide sequence encoding the Sox2 protein.

[0224] In some aspects, the polynucleotide comprises an inducible promoter operably linked to a polynucleotide encoding a c-Myc protein. In some aspects, the polynucleotide comprises a non-inducible promoter operably linked to a polynucleotide encoding a trans-activator. In some aspects, the polynucleotide comprises an inducible promoter operably linked to a polynucleotide encoding a c-Myc protein and a non-inducible promoter operably linked to a polynucleotide encoding a trans-activator.

[0225] In some aspects, the polynucleotide also comprises a polynucleotide sequence encoding a marker protein. In some aspects, the marker protein is β-galactosidase protein, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, or blue fluorescent protein, tdTomato protein, or mCherry protein.

[0226] In some aspects, the polynucleotide also comprises intron sequences. In some aspects, the intron sequences comprise CMV intron sequences, β-actin intron sequences, SV40 enhancer sequences, or combinations thereof.

[0227] In some aspects, a polynucleotide is provided encoding a Cas9 fusion protein (CRISPR activator) and a guide RNA sequence, said guide RNA sequence targeting the promoter or enhancer of an endogenous locus of at least one of Oct4, Sox2, and Klf4. In some aspects, said polynucleotide encodes a Cas9 fusion protein and a guide RNA sequence, said guide RNA sequence targeting the promoter or enhancer of an endogenous locus of Oct4, Sox2, Klf4, and optionally c-Myc.

[0228] In some respects, the polynucleotide described herein is RNA. In some respects, the polynucleotide described herein is DNA. In some respects, the polynucleotide is present within a vector. In some respects, the polynucleotide is present within an integrated vector. In some respects, the polynucleotide is present within a non-integrating vector. In some respects, the polynucleotide is present within an AAV vector, an adenovirus vector, a lentiviral vector, or a retroviral vector. In some respects, the polynucleotide is non-integrating RNA.

[0229] III. Carriers and Cells

[0230] Vectors containing polynucleotides as described herein are also provided.

[0231] In some aspects, the vector is a viral vector. In some aspects, the vector is a non-viral vector. In some aspects, the vector is a lipid. In some aspects, the vector is a polymer.

[0232] In some aspects, the viral vector is an adeno-associated virus (AAV) vector, an adenovirus vector, a lentiviral vector, or a retroviral vector. In some aspects, the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAVrh9, AAV9, AAVrh10, AAV10, AAVRH10, AAV11, AAV12, or an AAV-DJ vector.

[0233] In some respects, AAV vectors with a broad target spectrum are selected to transduce various cell types. In some respects, the AAV vector is the AAV-DJ vector.

[0234] In some respects, AAV vectors with one or more selected target cells are selected. The target specificity of different AAV vectors is known in the art (see, for example, AAV Production Protocol, Genemedi Biotech, Inc. 2018.).

[0235] In some respects, AAV vectors are modified to target selected cell types. For example, the AAV cap sequence can be modified to remove cell-targeting epitopes from the capsid and introduce alternative cell-targeting sequences into the capsid. AAV cap sequences modified in this way are known in the art.

[0236] In some aspects, the viral vector is a retroviral vector and contains polynucleotides as described herein. In some aspects, the retroviral vector is a self-inactivating retroviral vector. In some aspects, the retroviral vector is a pseudotyped retroviral vector. In some aspects, the retroviral vector is modified to target one or more specific cell types. In some aspects, the retroviral vector contains polynucleotides encoding Oct4, Sox2, Klf4, and / or c-Myc proteins. In some aspects, the retroviral vector contains polynucleotides encoding Oct4, Sox2, Klf4, and c-Myc proteins. In some aspects, the retroviral vector contains polynucleotides containing polycistronic cassettes encoding Oct4, Sox2, Klf4, and / or c-Myc proteins. In some aspects, the retroviral vector contains polynucleotides encoding Oct4, Sox2, and Klf4 proteins. In some aspects, the retroviral vector contains polynucleotides containing polycistronic cassettes encoding Oct4, Sox2, and Klf4 proteins. In some respects, retroviral vectors contain one or more polynucleotides encoding transcription activators and one or more of the following proteins: Oct4, Sox2, Klf4, c-Myc, and / or dnNFκBIA.

[0237] In some aspects, the viral vector is a lentiviral vector and contains polynucleotides as described herein. In some aspects, the lentiviral vector is a self-inactivating lentiviral vector and contains at least one deletion in the enhancer / promoter unit of the U3 region of the 3'LTR. In some aspects, the lentiviral vector is a pseudotyped lentiviral vector. In some aspects, the lentiviral vector is pseudotyped using vesicular stomatitis virus glycoprotein (VSV G). In some aspects, the lentiviral vector is pseudotyped using glycoproteins from the families retroviridae, rhabdoviridae, arenaviridae, flaviviridae, paramyxoviridae, baculoviridae, filoviridae, or combinations thereof. In some aspects, the lentiviral vector is modified with a target cell-specific binding portion to target specific cells or cell types. In some aspects, the lentiviral vector contains a tat-independent heteropromoter that replaces a portion of the 5'LTR. In some respects, lentiviral vectors contain inactivating mutations in the integrase gene, allowing the lentiviral vector to remain free in the transduced cells.

[0238] In some aspects, the lentiviral vector contains polynucleotides encoding Oct4, Sox2, Klf4, and / or c-Myc proteins. In some aspects, the lentiviral vector contains polynucleotides encoding Oct4, Sox2, Klf4, and c-Myc proteins. In some aspects, the lentiviral vector contains polynucleotides encoding polycistronic cassettes of Oct4, Sox2, Klf4, and / or c-Myc proteins. In some aspects, the lentiviral vector contains polynucleotides encoding Oct4, Sox2, and Klf4 proteins. In some aspects, the lentiviral vector contains polycistronic cassettes encoding Oct4, Sox2, and Klf4 proteins. In some aspects, the lentiviral vector contains polynucleotides encoding transcription activators and one or more of the following proteins: Oct4, Sox2, Klf4, c-Myc, and / or dnNFκBIA. In some aspects, the non-viral vector is plasmid DNA, RNA, cationic polymers, lipids, lipid polymers, or chemical derivatives thereof.

[0239] In some respects, the amount of cationic polymer present is sufficient to make the ratio of amine nitrogen in the cationic polymer to phosphate in the DNA plasmid vector or RNA vector about 0.1:1 to about 100:1.

[0240] In some respects, the amount of cationic polymer present is sufficient to make the ratio of amine nitrogen in the cationic polymer to phosphate in the DNA plasmid vector or RNA vector about 0.1:1 to about 10:1.

[0241] In some respects, the amount of cationic polymer present is sufficient to make the ratio of amine nitrogen in the cationic polymer to phosphate in the DNA plasmid vector or RNA vector about 0.1:1 to about 5:1.

[0242] In some respects, the nonviral vector contains a polynucleotide complexed with a cationic polymer at a concentration of approximately 0.5 mg / ml to approximately 5.0 mg / ml. In some respects, the cationic polymer is a poly(ethyleneimine) (PEI) polymer, poly-L-lysine, poly(L-arginine) (PLA), polyallylamine (PAH), polyamide amine, diethylaminoethyl dextran, chitosan, poly(dimethylaminoethyl methacrylate) polyethyleneamine homopolymer or copolymer, poly(vinylbenzyl-tri-Cl-C4-alkylammonium salt), aliphatic or aryliphatic dihalides and polymers of aliphatic N,N,N',N'-tetra-Cl-C4-alkyl-alkylene diamines, poly(vinylpyridine) or poly(vinylpyridine salt), poly(N,N-diallyl-N,N-di-Cl-C4-alkyl-ammonium halide), quaternized di-C4-alkyl-aminoethyl acrylate or methacrylate, POLYQUAD™, polyaminoamide, etc.; or derivatives thereof.

[0243] Cells comprising polynucleotides or vectors as described herein are also provided. In some aspects, the cells may be bacterial cells, yeast cells, fungal cells, insect cells, or mammalian cells.

[0244] In some aspects, cells containing polynucleotides as described herein can be used to manufacture viral vectors. In some aspects, the cells are insect cells containing polynucleotides as described herein and other viral vector production components and are used to prepare viral vectors as described herein. In some aspects, the cells are mammalian cells containing polynucleotides as described herein and other viral vector production components and are used to prepare viral vectors as described herein.

[0245] In some respects, the cells are insect cells containing polynucleotides and AAV viral vector production components as described herein and are used to prepare AAV vectors.

[0246] In some aspects, the cells are mammalian cells containing polynucleotides and AAV viral vector production components as described herein and are used to prepare AAV vectors. In some aspects, the AAV vector production components contain polynucleotides encoding Rep and Cap proteins, and an AAV vector containing a polynucleotide encoding, for example, at least one regeneration factor and containing at least one ITR. In some aspects, the mammalian cells, after expressing Rep and Cap proteins, are capable of packaging the AAV vector encoding said at least one regeneration factor into an AAV capsid.

[0247] In some aspects, the cells are mammalian cells containing polynucleotides and retroviral vector production components as described herein and are used to prepare retroviral vectors. In some aspects, the retroviral vector production components contain polynucleotides encoding gag, pol, and env proteins, and a retroviral vector containing polynucleotides encoding, for example, at least one regeneration factor and containing an LTR. In some aspects, mammalian cells, after expressing gag, pol, and env proteins, are capable of packaging a retroviral vector encoding at least one regeneration factor into a retroviral capsid.

[0248] In some aspects, the cells are mammalian cells containing polynucleotides and lentiviral vector production components as described herein and are used to prepare lentiviral vectors. In some aspects, the lentiviral vector production components contain polynucleotides encoding gag, pol, and env proteins and optionally tat and rev proteins, and a retroviral vector containing polynucleotides encoding, for example, at least one regeneration factor and containing an LTR. In some aspects, mammalian cells, after expressing gag, pol, and env proteins and optionally tat and rev proteins, are capable of packaging a lentiviral vector encoding at least one regeneration factor into a lentiviral capsid.

[0249] In some aspects, cells comprising polynucleotides or carriers or nanoparticles as described herein are provided, said cells being present in organs. In some aspects, cells comprising polynucleotides as described herein are present in organs that have been exposed to ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, virus-induced hepatitis, alcohol, or fibrosis unrelated to any known cause.

[0250] In some aspects, cells containing polynucleotides as described herein can be used to treat subjects. In some aspects, cells containing polynucleotides as described herein can be administered to subjects requiring reversal of aging, wherein the cells express proteins encoded by the polynucleotides as described herein after administration, and the expressed proteins reverse cellular processes associated with aging phenotypes. In some aspects, cells containing polynucleotides as described herein replicate after administration to a subject. In some aspects, cells containing polynucleotides as described herein do not replicate after administration to a subject. In some aspects, cells containing polynucleotides as described herein can be hematopoietic progenitor cells. In some aspects, cells containing polynucleotides as described herein can be hematopoietic stem cells. In some aspects, cells containing polynucleotides as described herein can be muscle stem cells. In some aspects, cells containing polynucleotides as described herein can be adipose-derived stem cells.

[0251] IV. Nanoparticles

[0252] Lipid particles, such as lipid nanoparticles comprising polynucleotides as described herein, are also provided. In some aspects, the lipid nanoparticles have a size of about 10 nm to about 1000 nm. In some aspects, the lipid nanoparticles comprise a solid lipid core matrix and a surfactant. In some aspects, the lipid nanoparticles comprise cationic lipids. In some aspects, the lipid nanoparticles comprise cationic lipids, non-cationic lipids, and optionally lipids that prevent aggregation.

[0253] In some aspects, the lipid nanoparticles comprise amphiphilic lipids arranged in at least one bilayer having an aqueous interior containing a polynucleotide encoding a regeneration factor. In some aspects, the polynucleotide of the lipid nanoparticles is DNA, RNA, or a mixture of both. In some aspects, the lipid nanoparticles comprise more than one polynucleotide, and each polynucleotide encodes a regeneration factor. In some aspects, the lipid nanoparticles comprise RNA encoding Oct4. In some aspects, the lipid nanoparticles comprise RNA encoding Sox2. In some aspects, the lipid nanoparticles comprise RNA encoding Klf4. In some aspects, the lipid nanoparticles comprise RNA encoding c-Myc. In some aspects, the lipid nanoparticles comprise RNA encoding Oct4, RNA encoding Sox2, RNA encoding Klf4, and RNA encoding c-Myc. In some aspects, the lipid nanoparticles comprise RNA encoding Oct4, RNA encoding Sox2, and RNA encoding Klf4. In some aspects, the lipid nanoparticles comprise RNA encoding Oct4, Sox2, Klf4, and c-Myc. In some aspects, lipid nanoparticles contain RNA encoding Oct4, Sox2, and Klf4. In other aspects, lipid nanoparticles contain RNA encoding polycistronic cassettes of Oct4, Sox2, Klf4, and c-Myc. In other aspects, lipid nanoparticles contain RNA encoding polycistronic cassettes of Oct4, Sox2, and Klf4.

[0254] In some respects, lipid nanoparticles contain additional reagents. In other respects, lipid nanoparticles contain enhancers as described herein.

[0255] V. Compositions, perfusion solutions, and kits

[0256] Compositions comprising polynucleotides, carriers, and / or nanoparticles as described herein are also provided. In some aspects, perfusion solutions comprising polynucleotides, carriers, and / or nanoparticles as described herein are provided. In some aspects, the perfusion solution or composition is a liquid solution comprising polynucleotides, carriers, and / or nanoparticles as described herein, and inorganic salts.

[0257] In some respects, the perfusion fluid or composition contains a phosphate-bicarbonate buffer solution.

[0258] In some aspects, the irrigation solution or composition comprises at least one of sodium chloride, calcium chloride, potassium chloride, magnesium chloride, sodium bicarbonate, monopotassium phosphate, or disodium hydrogen phosphate. In some aspects, the irrigation solution or composition comprises sodium chloride, calcium chloride, potassium chloride, magnesium chloride, sodium bicarbonate, monopotassium phosphate, and disodium hydrogen phosphate.

[0259] In some aspects, the perfusion fluid or composition comprises a dextran / albumin solution. In some aspects, the perfusion fluid or composition comprises calcium gluconate.

[0260] In some aspects, the perfusion solution or composition comprises at least one vasodilator. In some aspects, said vasodilator is carbon monoxide, angiotensin-converting enzyme (ACE) inhibitor, angiotensin receptor blocker, calcium channel blocker, prostacyclin, hydralazine, minoxidil, and nitroglycerin. In some aspects, the perfusion solution or composition comprises a non-carbon monoxide vasodilator.

[0261] In some aspects, the infusion fluid or composition contains at least one tensioning agent. In some aspects, the tensioning agent is dextran, glycerol, mannitol, potassium chloride, or sodium chloride.

[0262] In some aspects, the perfusion fluid or composition comprises at least one of a buffer, an inorganic salt, a metabolic substrate, a growth factor, a hormone, an antioxidant, an anti-inflammatory agent, an immunosuppressant, an anticoagulant, or an antimicrobial agent. In some aspects, the metabolic substrate is an amino acid or glucose. In some aspects, the perfusion fluid or composition comprises insulin.

[0263] In some aspects, the growth factor is at least one of fibroblast growth factor (FGF), insulin-like growth factor (IGF), transforming growth factor β (TGF-P), epiregulin, epidermal growth factor (“EGF”), endothelial growth factor (“ECGF”), nerve growth factor (“NGF”), leukemia inhibitory factor (“LIF”), bone morphogenetic protein-4 (“BMP-4”), hepatocyte growth factor (“HGF”), vascular endothelial growth factor-A (“VEGF-A”), and octapeptide cholecystokinin.

[0264] In some aspects, the immunosuppressants include, but are not limited to, steroidal (e.g., prednisone) or nonsteroidal (e.g., sirolimus (Rapamune, Wyeth-Ayerst Canada), tacrolimus (Prograf, Fujisawa Canada), and anti-IL2R dalizumab (Zenapax, Roche Canada). In some aspects, the immunosuppressants are 15-deoxyguanidine, cyclosporine, methotrexate, rapamycin, rapamune (sirolimus / rapamycin), FK506, lisolophrenialine (LSF), mycophenolate mofetil, anti-thymocyte globulin, belacept, or everolimus.

[0265] In some respects, antimicrobial agents include benzalkonium chloride, benzyl chloride, benzyl alcohol, cetylpyridine chloride, chlorobutanol, phenol, phenylethanol, phenylmercuric nitrate, and thimersol; antibiotics, including but not limited to amoxicillin, penicillin, sulfonamides, cephalosporins, erythromycin, streptomycin, gentamicin, tetracycline, clarithromycin, ciprofloxacin, and azithromycin; antifungal agents, including but not limited to miconazole and terconazole; and combinations thereof.

[0266] In some respects, antioxidants include, but are not limited to, molecules with thiol groups, such as reduced glutathione (GSH) or its precursors, glutathione or glutathione analogs, glutathione monoesters and N-acetylcysteine; superoxide dismutase, catalase, vitamin E, Trolox, lipoic acid, lazaroid, butylated hydroxyanisole (BHA), vitamin K, etc.; and combinations thereof.

[0267] In some aspects, the perfusion fluid or composition contains an oxygenating agent. In some aspects, the oxygenating agent is plasma-free, leukocyte-depleted erythrocytes, hemoglobin, pyridoxine-oxygenated hemoglobin, or a synthetic hemoglobin-based oxygen carrier (including, but not limited to, polymerized hemoglobin-based oxygen carriers); or a cell-free oxygen-carrying medium, including but not limited to Lifor. TM , RS-I or STEEN solution TM In some respects, the oxygenating agent is Hemopure Or perfluorocarbon.

[0268] In some aspects, the perfusion fluid or composition comprises at least one of human serum albumin, dextran, and an extracellular electrolyte composition.

[0269] In some respects, the perfusion fluid or composition contains dextran / albumin solution, packed red blood cell solution, calcium gluconate, heparin, cefazolin, verapamil, amino acids, glucose, aninsluin, and lactated Ringer's solution.

[0270] In some respects, the perfusion fluid or composition contains thick red blood cell solutions and colloidal solutions (e.g., gelafuncin, BBraun), cefuroxime, heparin, sodium bicarbonate, calcium gluconate, insulin, sodium taurocholate, prostacyclin, amino acids, and glucose.

[0271] In some respects, the perfusion fluid or composition comprises polynucleotides as described herein and delivery agents or carriers as described herein.

[0272] In some respects, the delivery agent is a viral vector. In some respects, the delivery agent is a retroviral vector. In some respects, the delivery agent is a lentiviral vector. In some respects, the delivery agent is an AAV vector. In some respects, the delivery agent is an AAV-DJ vector. In some respects, the delivery agent is a nanoparticle. In some respects, the delivery agent is a lipid nanoparticle. In some respects, the delivery agent is a polymer nanoparticle. In some respects, the lipid or polymer nanoparticle is partially conjugated with a ligand to target the lipid or polymer nanoparticle to specific cells, tissues, or organs. In some respects, the delivery agent is a cationic polymer. In some respects, the delivery agent is a lipid, a lipid polymer, or a chemical derivative thereof.

[0273] In some respects, the amount of the cationic polymer present in the composition is sufficient to make the ratio of amine nitrogen in the cationic polymer to phosphate in the DNA plasmid or RNA about 0.1:1 to about 100:1.

[0274] In some respects, the cationic polymer of the composition is present in an amount sufficient to make the ratio of amine nitrogen in the cationic polymer to phosphate in the DNA plasmid or RNA about 0.1:1 to about 10:1.

[0275] In some respects, the cationic polymer of the composition is present in an amount sufficient to make the ratio of amine nitrogen in the cationic polymer to phosphate in the DNA plasmid or RNA about 0.1:1 to about 5:1.

[0276] In some aspects, the polynucleotides described herein are co-present with the cationic polymer of the composition at a concentration of about 0.5 mg / ml to about 5.0 mg / ml. In some aspects, the cationic polymer of the composition is a poly(ethyleneimine) (PEI) polymer, poly-L-lysine, polyamide amine, diethylaminoethyl dextran, chitosan, poly(dimethylaminoethyl methacrylate), or derivatives thereof.

[0277] Pharmaceutical compositions comprising polynucleotides, carriers, and / or nanoparticles as described herein are also provided. In some aspects, the pharmaceutical compositions comprise pharmaceutically acceptable carriers or excipients.

[0278] Some examples of materials that can be used as pharmaceutically acceptable carriers include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) astragalus powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) Polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) Esters, such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, such as magnesium hydroxide and aluminum hydroxide; (15) Alginate; (16) Atherless water; (17) Isotonic saline; (18) Ringer's solution; (19) Ethanol; (20) pH buffer solution; (21) Polyesters, polycarbonates and / or polyanhydrides; and (22) Other non-toxic and compatible substances used in pharmaceutical preparations.

[0279] Other non-limiting examples of reagents suitable for the compositions described herein include: PEG-conjugated polynucleotides, phospholipid-conjugated polynucleotides, polynucleotides containing a lipophilic moiety, thiophosphates, P-glycoprotein inhibitors (such as Pluronic P85), which can enhance drug delivery to various tissues; and biodegradable polymers, such as poly(DL-lactide-co-glycolic acid) microspheres for sustained release delivery after transplantation.

[0280] In some respects, the composition also includes wetting agents, emulsifiers and lubricants, release agents, coating agents, preservatives and antioxidants.

[0281] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0282] In some aspects, the compositions disclosed herein comprise an excipient selected from cyclodextrins, cellulose, liposomes, micelle forming agents (e.g., bile acids), and polymer carriers (e.g., polyesters and polyanhydrides); and the polynucleotides described herein. In some aspects, the compositions impart oral bioavailability to the polynucleotides described herein.

[0283] Methods for preparing these compositions, perfusion solutions, or pharmaceutical compositions include the step of associating a polynucleotide, as described herein, with a carrier and optionally one or more auxiliary components. Generally, perfusion solution compositions or pharmaceutical compositions are prepared by homogeneously and tightly associating a polynucleotide with a liquid carrier or a subdivided solid carrier, or both.

[0284] Liquid dosage forms of the compositions, infusion solutions, or pharmaceutical compositions described herein include pharmaceutically acceptable emulsions, microemulsions, solutions, and suspensions. In addition to the active ingredient, liquid dosage forms may contain inert diluents (such as water or other solvents), solubilizers, and emulsifiers commonly used in the art, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofuranol, polyethylene glycol, and sorbitol fatty acid esters, and mixtures thereof.

[0285] In addition to polynucleotides or carriers, suspensions may also contain suspending agents such as, for example, isostearyl alcohol ethoxylate, polyoxyethylene sorbitol and sorbitol esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth gum and mixtures thereof.

[0286] Pharmaceutical compositions suitable for parenteral or infusion administration may comprise one or more polynucleotides, carriers, or nanoparticles in combination with one or more pharmaceutically acceptable reagents, including sterile isotonic or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions just prior to use. These may contain sugars, alcohols, antioxidants, buffers, antibacterial agents, solutes that make the composition isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of this disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Appropriate flowability may be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0287] These pharmaceutical compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial action on the polynucleotides, carriers, or nanoparticles described herein can be ensured by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, sorbitol, etc.).

[0288] Depot forms can be prepared by forming microcapsule matrixes of polynucleotides or carriers in biodegradable polymers such as polylactic-co-glycolic acid. The release rate of the polynucleotides or carriers can be controlled depending on the ratio of the polynucleotides or carriers to the polymer and the properties of the specific polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Formulations can also be prepared by trapping the polynucleotides or carriers in organ-tissue compatible liposomes or microemulsions.

[0289] Regardless of the chosen route of administration, the polynucleotides, carriers, or nanoparticles described herein and / or the pharmaceutical compositions disclosed herein can be formulated into pharmaceutically acceptable dosage forms using conventional methods known to those skilled in the art. The actual dose level of the polynucleotide or carrier in the pharmaceutical composition can be varied to obtain an amount of polynucleotide or carrier that effectively achieves the desired therapeutic response for a particular organ or patient, composition, and administration mode, without causing unacceptable toxicity to the organ or patient.

[0290] For example, the dosage level of polynucleotides, carriers, or nanoparticles in the perfusion fluid will depend on a variety of factors, including the activity of the specific polynucleotide, carrier, or nanoparticle used, the type of organ being perfused, the perfusion method, the perfusion time, and / or other drugs, compounds, and / or materials used in combination with the specific polynucleotide, carrier, or nanoparticle used, as well as the age, sex, weight, condition, general health status, and medical history of the subject from whom the organ is obtained, and the age, sex, weight, condition, general health status, and medical history of the subject who receives the organ.

[0291] In some respects, the polynucleotides or carriers described herein are applied to cells and / or organs through a variety of methods, including but not limited to iontophoresis therapy, incorporation media such as liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. In some respects, microemulsion techniques can be used to improve the bioavailability of polynucleotides or carriers as described herein, and to enhance bioavailability by directing absorption into the lymphatic system rather than the circulatory system of, for example, transplanted organs.

[0292] In some aspects, the formulation comprises micelles formed from the polynucleotide or carrier described herein and at least one amphiphilic carrier, wherein the average diameter of the micelles is less than about 100 nm. In some aspects, micelles with an average diameter of less than about 50 nm are used in conjunction with the polynucleotide or carrier described herein. In some aspects, micelles with an average diameter of less than about 30 nm, or even less than about 20 nm, are used.

[0293] While all suitable amphiphilic carriers have been considered, those currently preferred are typically those with a Generally Recognized As Safe (GRAS) status. Examples of amphiphilic carriers include saturated and monounsaturated polyethylene glycol-modified fatty acid glycerides, such as those obtained from fully or partially hydrogenated vegetable oils. These oils can advantageously consist of triglycerides, diglycerides, and monoglycerides, as well as polyethylene glycol esters and monoglycerides of the corresponding fatty acids. Particularly preferred fatty acid compositions include 4-10% decanoic acid, 3-9% decanoic acid, 40-50% lauric acid, 14-24% myristic acid, 4-14% palmitic acid, and 5-15% stearic acid. Another class of useful amphiphilic carriers includes dehydrated sorbitol and / or sorbitol partially esterified with saturated or monounsaturated fatty acids (SPAN series) or corresponding ethoxylated analogs (TWEEN series).

[0294] Commercially available amphiphilic carriers may be useful, including the Gelucire series, Labrafil, Labrasol or Lauroglycol (all manufactured and distributed by Gattefosse Corporation, Saint Priest, France), PEG-monooleate, PEG-dioleate, PEG-monolaurate and dilaurate, lecithin, polysorbate 80, etc.

[0295] Suitable hydrophilic polymers for use with the polynucleotides or carriers described herein are those that are readily soluble in water, can be covalently attached to lipids forming vesicles, and are tolerated in vivo without toxicity (i.e., biocompatibility). Suitable polymers include polyethylene glycol (PEG), polylactic acid (also known as polylactide), polyglycolic acid (also known as polyglycolic acid), polylactic acid-polyglycolic acid copolymers, and polyvinyl alcohol. In some aspects, the polymer has a molecular weight of about 100 or 120 Daltons to about 5,000 or 10,000 Daltons, or about 300 Daltons to about 5,000 Daltons. In some aspects, the polymer is polyethylene glycol with a molecular weight of about 100 to about 5,000 Daltons, or about 300 to about 5,000 Daltons. In some aspects, the polymer is polyethylene glycol of 750 Daltons (PEG(750)). Polymers can also be defined by the number of monomers in them; in some respects, polymers with at least about three monomers are used, such as PEG polymers consisting of three monomers (about 150 Daltons).

[0296] Other hydrophilic polymers suitable for use in this disclosure include polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derived celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0297] In some aspects, the compositions disclosed herein comprise biocompatible polymers selected from: polyamides, polycarbonates, polyalkylene compounds, polymers of acrylates and methacrylates, polyethylene polymers, polyglycolic acid, polysiloxanes, polyurethanes and copolymers thereof, cellulose, polypropylene, polyethylene, polystyrene, polymers of lactic acid and glycolic acid, polyanhydrides, poly(orthocyanidates), poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acid, polycyanoacrylates and blends, mixtures or copolymers thereof.

[0298] In some aspects, the compositions disclosed herein comprise a cationic polymer. In some aspects, the cationic polymer comprises a polyethyleneimine (PEI) backbone. In some aspects, the PEI backbone is linked to a lipid or polyethylene glycol. In some aspects, the cationic polymer comprises cationic dextran, cationic chitosan, cationic gelatin, cationic cellulose, or cationic cyclodextrin.

[0299] In some aspects, the compositions disclosed herein comprise liposomes. Liposomes consist of at least one lipid bilayer membrane enclosing an aqueous internal compartment. Liposomes can be characterized by the type and size of their membranes. Small monolayer vesicles (SUVs) have a single membrane and are typically between 0.02 μm and 0.05 μm in diameter; large monolayer vesicles (LUVs) are typically greater than 0.05 μm. Oligolayer macrovesicles and multilayer vesicles have multiple typically concentric membrane layers and are typically greater than 0.1 μm. Liposomes having several non-concentric membranes, i.e., several smaller vesicles contained within a larger vesicle, are referred to as multivesicles.

[0300] In some respects, the lipid bilayer of the liposome contains lipids derived from polyethylene glycol (PEG), such that PEG chains extend from the inner surface of the lipid bilayer into the encapsulated internal space of the liposome and from the outer surface of the lipid bilayer into the surrounding environment. Polymer-derived lipids, such as PEG lipids, can inhibit micelle / membrane fusion. The active agents contained in the liposomes described herein, such as polynucleotides or carriers, are in dissolved form. Aggregates of surfactants and active agents (such as emulsions or micelles containing polynucleotides or carriers) can be trapped within the internal space of the liposomes according to this disclosure.

[0301] Liposomes according to this disclosure can be prepared by any of a variety of techniques known in the art. See, for example, U.S. Patent 4,235,871; published PCT application WO 96 / 14057; New RRC, Liposomes: A Practical Approach, IRL Press, Oxford (1990), pp. 33-104; Lasic DD, Liposomes from Physics to Applications, Elsevier Science Publishers BV, Amsterdam, 1993. For example, the liposomes described herein can be prepared by diffusing lipids derived from a hydrophilic polymer into pre-formed liposomes, such as by exposing the pre-formed liposomes to micelles composed of lipid-grafted polymers, the lipid concentration corresponding to the final molar percentage of the desired derived lipids in the liposomes. Liposomes containing hydrophilic polymers can also be formed by homogenization, lipid field hydration, or extrusion techniques known in the art.

[0302] In some aspects, the surfactant is first dispersed in lysophosphatidylcholine or other low-CMC surfactants (including polymer-grafted lipids) by sonication. The resulting micellar suspension of the surfactant is then used to rehydrate a dried lipid sample containing a suitable molar percentage of polymer-grafted lipids or cholesterol. The lipid and surfactant suspension is then formed into liposomes using extrusion techniques known in the art, and the resulting liposomes are separated from the unencapsulated solution by standard column separation.

[0303] In some aspects, liposomes are prepared to have a substantially uniform size within a selected size range. An efficient sieving method involves extruding an aqueous suspension of liposomes through a series of polycarbonate membranes having selected uniform pore sizes; the pore sizes of the membranes will approximately correspond to the maximum size of the liposomes produced by extrusion through the membrane. In some aspects, reagents such as and It can be used to introduce polynucleotides or vectors into cells.

[0304] The release characteristics of the formulations disclosed herein depend on the encapsulating material, the concentration of the encapsulated polynucleotide or carrier, and the presence of a release modifier. For example, release can be manipulated to be pH-dependent, for instance, by using a pH-sensitive coating that releases only at low or high pH levels. The coating can be used to prevent release from occurring until after the organ containing the formulation has been transplanted into a subject. For example, a variety of coatings or mixtures of coatings can be used to encapsulate polynucleotides or carriers as described herein for use in perfusion solutions or compositions, such that the encapsulated polynucleotides or carriers are absorbed by the cells of the perfused organ, but the polynucleotides or carriers are released only in the presence of low pH levels, such as metabolic acidosis as the function of the transplanted kidney deteriorates.

[0305] Release can also be manipulated by including salts or pore-forming agents, which can increase the water absorption or release of the composition by diffusing from the encapsulation. Excipients that alter the solubility of the composition can also be used to control the release rate. Agents that enhance matrix degradation or release from the encapsulating matrix can also be incorporated. These agents can be added to the composition as a separate phase (i.e., as microparticles) or can be co-soluble in the polymer phase, depending on the composition. In some aspects, the amount is between 0.1% and 30% (w / w polymer). Types of degradation enhancers include inorganic salts such as ammonium sulfate and ammonium chloride, organic acids such as citric acid, benzoic acid, and ascorbic acid, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide, and organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine, as well as surfactants such as and

[0306] In some respects, the encapsulated particles are coated with cell adhesion polymers. Examples include polymers with free carboxyl groups, such as chitosan, cellulose, and especially polyacrylates (as used herein, polyacrylates refer to polymers that include acrylate groups and modified acrylate groups, such as cyanoacrylates and methacrylates).

[0307] In some aspects, polynucleotides or carriers can be formulated to be contained in, or adapted to be released by, a surgical or medical device or implant. Examples of medical devices include vascular stents implanted prior to organ transplantation, such as stents implanted in an organ's artery or ureter to keep it open. In some aspects, implants can be coated or otherwise treated with polynucleotides or carriers. For example, hydrogels or other polymers, such as biocompatible and / or biodegradable polymers, can be used to coat implants with compositions as described herein (e.g., by using hydrogels or other polymers, the compositions can be adapted for use with medical devices). Polymers and copolymers for coating medical devices with reagents are well known in the art.

[0308] VI. Organs and perfusion system

[0309] This provides organs for transplantation and methods for preparing organs for transplantation. In some aspects, the organ is removed from a donor. In some aspects, an organ is removed from a subject (donor), treated with materials and methods as described herein, and transplanted into another subject (recipient). In some aspects, an organ is removed from a subject (donor), treated with materials and methods as described herein, and transplanted into the same subject (donor and recipient are the same subject). In some aspects, the donor is a human subject. In some aspects, the recipient is a human subject requiring organ transplantation. In some aspects, the organ is an in vitro generated organoid. In some aspects, the organoid is reconstituted using the methods described herein before transplantation into the recipient. In some aspects, the organoid includes a vascular system, including blood vessels and lymphatic vessels, and is perfused with perfusion fluid as described herein through conduits connected to blood vessels and / or lymphatic vessels.

[0310] In some aspects, the organ is perfused before, during, and / or after treatment with the materials and methods described herein. In some aspects, the organ is cold-perfused before, during, and / or after treatment with the materials and methods described herein. In some aspects, the organ is perfused at room temperature before, during, and / or after treatment with the materials and methods described herein. In some aspects, the organ is cold-perfused before treatment with the materials and methods described herein and perfused at room temperature during and / or after treatment with the materials and methods described herein. In some aspects, the organ is cold-perfused before and during treatment with the materials and methods described herein and perfused at room temperature after treatment with the materials and methods described herein. In some aspects, cold perfusion and / or room temperature perfusion are performed using a perfusion system.

[0311] In some aspects, the infusion system is an ambient temperature mechanical infusion system. In other aspects, the infusion system is a cryogenic (cold) mechanical infusion system.

[0312] In some aspects, an infusion system is used in the methods described herein. In some aspects, the infusion system is a Hugo Sachs / Harvard device, Kidney Assist... TM System, OrganOX system, Radnoti system, ARKKidney system or Aferetica system.

[0313] In some aspects, the organ is operatively connected to the perfusion system such that perfusion fluid moved by the perfusion system enters the organ through blood vessels. In some aspects, the organ is operatively connected to the perfusion system such that perfusion fluid moved by the perfusion system enters the organ through arteries. In some aspects, the organ is operatively connected to the perfusion system such that perfusion fluid moved by the perfusion system enters the organ through veins.

[0314] In some respects, the organ is a kidney and is operatively connected to the perfusion system such that perfusion fluid moved by the perfusion system enters the organ through the ureter of the organ.

[0315] In some aspects, the organ is operatively connected to the perfusion system via a perfusion bath, whereby the organ is contained within the perfusion bath, and the perfusion system moves the perfusion fluid such that the perfusion fluid enters or permeates the organ via a blood vessel, ureter, or lymphatic vessel.

[0316] In some aspects, the organ is perfused with a perfusion solution comprising at least one of a composition of albumin, dextran, and extracellular electrolytes. In some aspects, the perfusion solution comprises a composition of human serum albumin, dextran, and extracellular electrolytes.

[0317] In some aspects, the organ is perfused with a perfusion solution comprising the polynucleotides, carriers and / or nanoparticles described herein, and at least one inorganic salt. In other aspects, the organ is perfused with a perfusion solution or composition comprising a phosphate-bicarbonate buffer solution.

[0318] In some aspects, the organ is perfused with an perfusion solution containing at least one of sodium chloride, calcium chloride, potassium chloride, magnesium chloride, sodium bicarbonate, monopotassium phosphate, or disodium hydrogen phosphate. In other aspects, the organ is perfused with an perfusion solution containing sodium chloride, calcium chloride, potassium chloride, magnesium chloride, sodium bicarbonate, monopotassium phosphate, and disodium hydrogen phosphate.

[0319] In some aspects, the organ is perfused with a perfusion solution containing at least one vasodilator. In some aspects, the organ is perfused with a perfusion solution containing at least one tension agent. In some aspects, the organ is perfused with a perfusion solution containing at least one of a buffer, inorganic salt, amino acid, nutrient, cytokine, growth factor, hormone, antioxidant, anti-inflammatory agent, immunosuppressant, anticoagulant, or antimicrobial agent.

[0320] In some aspects, the organ is perfused with an oxygenating fluid containing an oxygenating agent, said oxygenating agent being plasma-free, leukocyte-depleted red blood cells, hemoglobin, synthetic hemoglobin-based oxygen carriers, cell-free oxygen-carrying mediators, polymerized hemoglobin-based oxygen carriers, or pyridinoxylated hemoglobin. In some aspects, the organ is perfused with an oxygenating fluid containing a cell-free oxygen-carrying medium selected from Lifor. TM , RS-I, STEEN Solution TM , And perfluorocarbons.

[0321] In some respects, the organ is perfused with an infusion containing a tension agent, the tension agent comprising dextran, glycerol, mannitol, potassium chloride, or sodium chloride.

[0322] In some aspects, the organ is perfused with a perfusion solution containing a vasodilator, said vasodilator being carbon monoxide, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin receptor blocker, a calcium channel blocker, prostacyclin, hydralazine, minoxidil, or nitroglycerin. In some aspects, the organ is perfused with a perfusion solution containing carbon monoxide-releasing molecules (e.g., CORM-3, CORM-A1, see, for example, Csongradi et al., Curr Pharm Biotechnol. 13:819-26, 2012). In some aspects, the organ is perfused with a perfusion solution to which carbon monoxide has already been added. In some aspects, the organ is perfused with a perfusion solution containing approximately 20 ppm to 250 ppm of carbon monoxide. In some aspects, the perfusion solution does not contain carbon monoxide as a vasodilator.

[0323] In some aspects, the organ is perfused with a perfusion solution comprising polynucleotides, peptides, lipid nanoparticles, polymer nanoparticles, or ligand-conjugated lipid or polymer nanoparticles as described herein. In some aspects, the organ is perfused with a perfusion solution comprising a regeneration factor-protein transfer domain fusion protein. In some aspects, the organ is perfused with a perfusion solution comprising a viral vector containing a polynucleotide encoding at least one regeneration factor as described herein. In some aspects, the organ is perfused with a perfusion solution comprising an AAV vector, an adenovirus vector, a retroviral vector, or a lentiviral vector. In some aspects, the organ is perfused with a perfusion solution comprising a regeneration factor peptide.

[0324] In some aspects, the organ is the kidney and is perfused with a perfusion solution comprising at least one of a composition of human serum albumin, dextran, and extracellular electrolytes. In some aspects, the kidney is perfused with a perfusion solution comprising at least one of a composition of human serum albumin, dextran, and extracellular electrolytes.

[0325] In some aspects, the organ is the kidney and the perfusion system for perfusing the kidney is an ambient temperature perfusion system. In some aspects, the organ is the kidney and the perfusion system is a Hugo Sachs / Harvard device, Kidney Assist... TM System, OrganOX system, Radnoti system, ARK Kidney system or Aferetica The system. In some aspects, the kidneys are perfused using a normothermic perfusion system with a perfusion solution containing dextran / albumin solution, thick red blood cell solution, calcium gluconate, heparin, antibiotics, vasodilators, amino acids, glucose, insulin, and lactated Ringer's solution.

[0326] In some aspects, the organ is the liver and the perfusion system used for perfusing the liver is an ambient temperature perfusion system. In some aspects, the organ is the liver and the perfusion system is a Hugo Sachs / Harvard device or a Kidney Assist device. TM System, OrganOX system, Radnoti system, ARK Kidney system or Aferetica The system. In some respects, the liver is perfused using a normothermic perfusion system with a perfusion solution containing thick red blood cells, a colloidal solution, sodium bicarbonate, calcium gluconate, heparin, antibiotics, vasodilators, amino acids, glucose and insulin, and sodium taurocholate in an isotonic saline solution.

[0327] In some aspects, the organ is a kidney and is perfused with a perfusion solution comprising the polynucleotides, peptides, carriers, and / or nanoparticles described herein. In some aspects, prior to exposure to the perfusion solution described herein, the kidney is characterized by at least one of the following: aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, virus-induced hepatitis, alcoholism, or fibrosis unrelated to any known cause. In some aspects, the aging or damaged kidney is exposed to the perfusion solution for different time periods and / or to perfusion solutions containing different components, depending on age and / or the type and extent of injury. In some aspects, the aging or damaged kidney may be exposed to the perfusion solution for several days or weeks prior to transplantation to a recipient. In some aspects, the aging or damaged kidney may be evaluated by diagnostic tests and / or biopsy before and / or after exposure to the perfusion solution described herein to determine whether signs of aging or injury, including but not limited to, inflammatory cell infiltration, renal tubular injury, interstitial edema, or fibrotic changes, are reduced after exposure to the perfusion solution compared to before exposure. In some respects, aging or damaged kidneys may be exposed to the perfusion fluid for one or more additional time periods. In some respects, the composition of the perfusion fluid may be adjusted based on the age or damage present in the kidney before perfusion. In some respects, the composition of the perfusion fluid may be adjusted based on the remaining damage in the kidney after the first perfusion cycle to perfuse the kidney in a second or more perfusion cycles.

[0328] In some respects, the kidneys are perfused with the perfusion fluid described herein at temperatures ranging from about 2°C to about 38°C; or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22°C; or about 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38°C.

[0329] In some aspects, the organ is the liver and is perfused with a perfusion fluid comprising the polynucleotides, peptides, carriers, and / or nanoparticles described herein. In some aspects, prior to exposure to the perfusion fluid described herein, the liver is characterized by aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, virus-induced hepatitis, alcoholism, or fibrosis unrelated to any known cause. In some aspects, the aged or damaged liver is exposed to the perfusion fluid for different time periods and / or to perfusion fluids containing different components, depending on age and / or the type and / or extent of injury. In some aspects, the aged or damaged liver may be exposed to the perfusion fluid for several days or weeks prior to transplantation to the recipient. In some aspects, the aged or damaged liver may be assessed by diagnostic tests and / or biopsy before and / or after exposure to the perfusion fluid described herein to determine signs of age or injury, including but not limited to, inflammatory cell infiltration, renal tubular injury, interstitial edema, or fibrotic changes, whether these signs are reduced after exposure to the perfusion fluid compared to before exposure. In some respects, an aging or damaged liver may be exposed to the perfusion fluid for one or more additional time periods. In some respects, the composition of the perfusion fluid may be adjusted based on the age or damage present in the liver prior to perfusion. In some respects, the composition of the perfusion fluid may be adjusted based on the remaining damage in the liver after the first perfusion cycle to perfuse the liver in a second or more perfusion cycles.

[0330] In some respects, the liver is perfused with the perfusion fluid described herein at temperatures ranging from about 2°C to about 38°C; or about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C; or about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C.

[0331] VII. Methods

[0332] Methods are provided for regulating (e.g., inducing) partial cell reprogramming, cell restoration, cell regeneration, tissue repair, tissue regeneration, tissue restoration, partial tissue reprogramming, organ regeneration, organ restoration, partial organ reprogramming, or any combination thereof, said methods comprising contacting cells, tissues, or organs with polynucleotides, oligonucleotides, peptides, compounds, carriers, or nanoparticles as described herein. In some aspects, said methods include contacting isolated cells, tissues, or organs. In some aspects, said methods include contacting cells, tissues, or organs with polynucleotides, such as engineered polynucleotides encoding at least one regeneration factor.

[0333] In some aspects, the method includes organ regeneration. In some aspects, the method includes organ restoration, comprising contacting an isolated organ with a polynucleotide, oligonucleotide, polypeptide, compound, carrier, or nanoparticle as described herein, wherein the polynucleotide, oligonucleotide, polypeptide, compound, carrier, or nanoparticle contains or encodes at least one regeneration factor, wherein the at least one regeneration factor is expressed in or present in the organ, and the organ is restored. In some aspects, the method includes organ restoration, the organ being characterized by at least one of the following: aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, fatty degeneration, virus-induced hepatitis, alcoholism, or fibrosis unrelated to any known cause.

[0334] In some aspects, the method includes transplanting an organ comprising a polynucleotide, oligonucleotide, polypeptide, compound, carrier, or nanoparticle as described herein into a subject in need (the recipient).

[0335] In some aspects, the method includes contacting an isolated organ with a polynucleotide, the polynucleotide comprising a polynucleotide encoding Oct4. In some aspects, the method includes contacting an isolated organ with a polynucleotide, the polynucleotide comprising a polynucleotide encoding Sox2. In some aspects, the method includes contacting an isolated organ with a polynucleotide, the polynucleotide comprising a polynucleotide encoding Klf4. In some aspects, the method includes contacting an isolated organ with a polynucleotide, the polynucleotide comprising a polynucleotide encoding c-Myc.

[0336] In some aspects, the method includes contacting the organ with a first polynucleotide encoding Oct4. In some aspects, the method further includes contacting the organ with a second polynucleotide encoding Sox2. In some aspects, the method further includes contacting the organ with a third polynucleotide encoding Klf4. In some aspects, the method further includes contacting the organ with a fourth polynucleotide encoding c-Myc.

[0337] In some aspects, the method includes contacting an organ with a polynucleotide encoding Oct4, Sox2, and Klf4. In some aspects, the polynucleotide also encodes c-Myc. In some aspects, the method includes contacting the organ with a polynucleotide comprising a polycistronic cassette encoding Oct4, Sox2, and Klf4. In some aspects, the method further includes contacting the organ with a polynucleotide encoding c-Myc.

[0338] In some aspects, the method includes contacting an organ with a polynucleotide comprising a first inducible promoter operably linked to a polynucleotide encoding at least one of Oct4, Sox2, and / or Klf4. In some aspects, the method includes contacting an organ with a polynucleotide comprising a first inducible promoter, which is a tetracycline-inducible promoter and operably linked to a polynucleotide encoding at least one of Oct4, Sox2, and / or Klf4. In some aspects, the method includes contacting an organ with a polynucleotide comprising a second inducible promoter operably linked to c-Myc. In some aspects, the method further includes contacting the organ with an inducible compound such that the inducible promoter promotes the expression of at least one of Oct4, Sox2, Kl4, and / or c-Myc.

[0339] In some aspects, the method includes contacting an organ with a polynucleotide comprising a first inducible promoter, which is a tetracycline-inducible promoter and operably linked to a polynucleotide encoding at least one of Oct4, Sox2, and / or Klf4. In some aspects, the method further includes contacting the organ with a second polynucleotide comprising a second inducible promoter, which is a coumarin-inducible promoter operably linked to c-Myc. In some aspects, the method includes contacting an organ with a polynucleotide comprising a first inducible promoter, which is a tetracycline-inducible promoter and operably linked to a polynucleotide encoding at least one of Oct4, Sox2, and / or Klf4, and the polynucleotide further comprising a second inducible promoter, which is a coumarin-inducible promoter and operably linked to c-Myc.

[0340] In some aspects, the organ is contacted with a polynucleotide encoding at least one regenerative factor for about 1 minute to about 24 hours; about 1 minute; about 2 minutes; about 5 minutes; about 10 minutes; about 15 minutes; about 20 minutes; about 25 minutes; about 30 minutes; about 35 minutes; about 40 minutes; about 45 minutes; about 50 minutes; about 55 minutes; about 60 minutes; about 70 minutes; about 80 minutes; about 90 minutes; about 2 hours; about 3 hours; about 4 hours; about 5 hours; Approximately 6 hours; approximately 7 hours; approximately 8 hours; approximately 9 hours; approximately 10 hours; approximately 12 hours; approximately 14 hours; approximately 16 hours; approximately 18 hours; approximately 20 hours; approximately 22 hours; approximately 24 hours; approximately 36 hours; approximately 48 hours; approximately 60 hours; approximately 72 hours; approximately 84 hours; approximately 96 hours; approximately 108 hours; approximately 120 hours; approximately 132 hours; approximately 144 hours; approximately 156 hours; or approximately 168 hours, or any time period between these. In some aspects, the organ is contacted with a polynucleotide encoding at least one regenerative factor for approximately 1 minute to approximately 72 hours, followed by a period of no contact and another period of contact with the polynucleotide for approximately 1 minute to approximately 72 hours. In some aspects, the organ is repeatedly contacted with a polynucleotide as described herein, with periods of no contact in between. For example, in some aspects, the organ is contacted with a polynucleotide encoding at least one regenerative factor for about 1 minute to about 12 hours, followed by a 12-hour non-contact period, followed by another period of contact with the polynucleotide for about 1 minute to about 12 hours, followed by a 12-hour non-contact period, followed by another contact period. In some aspects, the periods of contact with the polynucleotide and the non-contact periods have different lengths. In some aspects, the periods of contact with the polynucleotide have different lengths, while the non-contact periods have the same length. In some aspects, the periods of contact with the polynucleotide have different lengths, and the non-contact periods have different lengths. In some aspects, organ function is measured after contact with the polynucleotide, and the non-contact period and the subsequent contact period are based on the results of the organ function measurement.

[0341] In some respects, contact takes place immediately after organ donation; or approximately 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, or more after organ donation. The procedure can be performed at approximately 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after organ donation; or approximately 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or 9 weeks after organ donation.

[0342] In some aspects, the interval between the step of contacting the organ with a polynucleotide containing at least one regenerative factor operatively linked to an inducible promoter and the step of contacting the organ with a compound that activates the inducible promoter can be 0 minutes or about 10 minutes to about 9 weeks or longer; for example, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, or about 17 hours. Approximately 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, approximately 14 days; or approximately 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or 9 weeks.

[0343] In some cases, the organ is exposed to the compound intermittently about 2-10 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, or about 10 times.

[0344] In some respects, the organ intermittent contact with the compound occurs approximately 2-10 times in a cycle of approximately 1 week to approximately 6 weeks, approximately 2-10 times in a cycle of approximately 1 week to approximately 5 weeks, approximately 2-10 times in a cycle of approximately 1 week to approximately 4 weeks, approximately 2-10 times in a cycle of approximately 1 week to approximately 3 weeks, approximately 2-10 times in a cycle of approximately 1 week to approximately 2 weeks, approximately 2-10 times in a cycle of approximately 2 weeks to approximately 6 weeks, approximately 2-10 times in a cycle of approximately 3 weeks to approximately 6 weeks, approximately 2-10 times in a cycle of approximately 4 weeks to approximately 6 weeks, approximately 2-10 times in a cycle of approximately 5 weeks to approximately 6 weeks, or any combination thereof. Or approximately 2 times in a period of approximately 1 to 6 weeks, approximately 3 times in a period of approximately 1 to 6 weeks, approximately 4 times in a period of approximately 1 to 6 weeks, approximately 5 times in a period of approximately 1 to 6 weeks, approximately 6 times in a period of approximately 1 to 6 weeks, approximately 7 times in a period of approximately 1 to 6 weeks, approximately 8 times in a period of approximately 1 to 6 weeks, approximately 9 times in a period of approximately 1 to 6 weeks, approximately 10 times in a period of approximately 1 to 6 weeks, or approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 times in any period of approximately 1 to 6 weeks.

[0345] In some respects, organs are contacted with perfusion fluids containing no polynucleotides, peptides, carriers, or nanoparticles for a certain period of time, followed by contact with perfusion fluids containing polynucleotides, peptides, carriers, or nanoparticles as described herein.

[0346] In some aspects, the method further includes contacting an organ containing a polynucleotide, a nanoparticle containing a polynucleotide, or a carrier containing at least one regenerative factor polynucleotide operatively linked to an inducible promoter, with an inducible compound.

[0347] In some respects, as described herein, the step of contacting an organ with a compound that induces the inducible promoter present in the organ includes administering the compound that induces the inducible promoter to a subject who has already received the organ.

[0348] In some aspects, the method includes contacting an organ containing a polynucleotide, a nanoparticle containing a polynucleotide, or a carrier as described herein with an inducing compound prior to transplanting the organ into a recipient. In some aspects, the method includes contacting an organ containing a polynucleotide, a nanoparticle containing a polynucleotide, or a carrier as described herein with an inducing compound after transplanting the organ into a recipient, wherein the compound is applied to the recipient.

[0349] In some aspects, the method includes exposing the organ to an inducing compound for about 1 minute to about 24 hours after transplantation into the recipient; about 1 minute; about 2 minutes; about 5 minutes; about 10 minutes; about 15 minutes; about 20 minutes; about 25 minutes; about 30 minutes; about 35 minutes; about 40 minutes; about 45 minutes; about 50 minutes; about 55 minutes; about 60 minutes; about 70 minutes; about 80 minutes; about 90 minutes; about 2 hours; about 3 hours; about 4 hours; about 5 hours The time period may be approximately 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 132 hours, 144 hours, 156 hours, or 168 hours, or any time period between these intervals. In some aspects, the method includes exposing the organ to the inducing compound more than once at the time periods described above after transplantation into the recipient.

[0350] In some aspects, the method includes exposing the organ to the time period described above after transplantation to the recipient, followed by non-existence. In some aspects, the method includes exposing the organ to the time period described above after transplantation to the recipient, followed by organ biopsy and organ function measurements, optionally, exposing the organ to another time period based on the results of the biopsy and organ function measurements. In some aspects, organ biopsies and function measurements are repeated periodically, for example, monthly, every 2 months, every 3 months, every 4 months, every 5 months, or every 6 months, and the organ is exposed to or not exposed to the polynucleotides described herein within the time period described herein, based on the results of each biopsy and function measurement.

[0351] In some aspects, the organ is exposed to the compound that activates the inducible promoter a specified number of times on one day of the week, and the organ is no longer exposed to the inducible compound on the sixth day of the week. In some aspects, the organ is exposed to the inducible compound a specified number of times on two days of the week, and the organ is no longer exposed to the inducible compound on the fifth day of the week. In some aspects, the organ is exposed to the inducible compound a specified number of times on three days of the week, and the organ is no longer exposed to the inducible compound on the fourth day of the week. In some aspects, the organ is exposed to the inducible compound a specified number of times on four days of the week, and the organ is no longer exposed to the inducible compound on the third day of the week. In some aspects, the organ is exposed to the inducible compound a specified number of times on five days of the week, and the organ is no longer exposed to the inducible compound on the second day of the week. In some aspects, the organ is exposed to the inducible compound a specified number of times on six days of the week, and the organ is no longer exposed to the inducible compound on the first day of the week.

[0352] In some respects, the number of days in a week during which the organ is exposed to the inducing compound can be continuous or discontinuous. For example, the organ may be exposed to the inducing compound the number of times described above on days 1, 3, and 5 of the week, while not exposed to the inducing compound on days 2, 4, 6, and 7 of the week.

[0353] In some aspects, the method further includes contacting the organ with a first inducible compound that activates a first inducible promoter. In some aspects, the first inducible promoter is a tetracycline-inducible promoter and the first inducible compound is tetracycline. In some aspects, the method further includes contacting the organ with a second inducible compound that activates a second inducible promoter. In some aspects, the second inducible promoter is a coumarin-inducible promoter and the second inducible compound is coumarin. In some aspects, the method further includes contacting the organ with the first inducible compound for a different time period than contacting the organ with the second inducible compound. For example, in some aspects, the method includes contacting the organ with tetracycline for two days per week for about 1-10 weeks and contacting the organ with coumarin for one day per week for about 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-5 weeks, 1-6 weeks, 1-7 weeks, 1-8 weeks, or 1-9 weeks. In some aspects, the method includes exposing the organ to tetracycline for two days per week for 1-10 weeks and to coumarin for two days per week for about 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-5 weeks, 1-6 weeks, 1-7 weeks, 1-8 weeks, or 1-9 weeks. In some aspects, the method includes exposing the organ to tetracycline for two days per week and to coumarin for one day per week, wherein on the days the organ is exposed to tetracycline, the organ is also exposed to coumarin. In some aspects, the method includes exposing the organ to tetracycline for two days per week and to coumarin for one day per week, wherein on the days the organ is not exposed to tetracycline, the organ is exposed to coumarin.

[0354] In some aspects, the method includes exposing the organ to tetracycline for 4 days per week for about 1-10 weeks and to coumarin for 1-4 days per week for about 1-10 weeks. In some aspects, the method includes exposing the organ to tetracycline for 4 days per week and to coumarin for 1-4 days per week, wherein on the days the organ is exposed to tetracycline, the organ is also exposed to coumarin. In some aspects, the method includes exposing the organ to tetracycline for 4 days per week and to coumarin for 1-3 days per week, wherein on the days the organ is not exposed to tetracycline, the organ is exposed to coumarin.

[0355] In some aspects, the period of contact between the organ and the first inducing compound is longer than the period of contact with the second inducing compound at each contact. In some aspects, the organ is contacted with the first and second inducing compounds for the same amount of time. In some aspects, the organ is contacted with the first inducing compound more than once, and the organ is contacted with the second inducing compound once. In some aspects, the organ is contacted with the first inducing compound once, and the organ is contacted with the second inducing compound more than once.

[0356] In some aspects, the organ is contacted with a polynucleotide operably linked to a first inducible promoter encoding at least one of Oct4, Sox2, and / or Klf4, and a second polynucleotide operably linked to a second inducible promoter encoding c-Myc. In some aspects, the method includes contacting the organ with a composition containing a first inducible compound for a first time period, and adding a second inducible compound to the composition for a second time period. In some aspects, the first and second time periods have the same length. In some aspects, the first and second inducible compounds are added to the composition at the same time. In some aspects, the first and second inducible compounds are added at different times. In some aspects, the first and / or second inducible compounds are added to the composition, for example, 2, 3, or 4 days per week for 2 months. In some aspects, the first and / or second inducible compounds are added to the composition, for example, 2, 3, or 4 days per week for 1 month. In some aspects, the first and / or second inducible compounds are added to the composition, for example, every other week for 2, 3, or 4 days per week for 2 months. In some respects, a first inducing compound and / or a second inducing compound are added to the composition, for example, every other week for 2, 3 or 4 days per week for 1 month.

[0357] In some aspects, the method includes administering a compound that induces an inducible promoter present in the polynucleotide or carrier to a subject who has received an organ containing a polynucleotide, carrier, or nanoparticle as described herein, and further includes administering an additional agent, such as an immunosuppressant, to the subject. In some aspects, immunosuppressants include, but are not limited to, steroidal (e.g., prednisone) or nonsteroidal (e.g., sirolimus (Rapamune, Wyeth-Ayerst Canada), tacrolimus (Prograf, Fujisawa Canada), anti-IL2R antibodies (e.g., dalizumab) (Zenapax, Roche Canada), 15-deoxyguanidine, cyclosporine, methotrexate, rapamycin, rapamune (sirolimus / rapamycin), FK506, lisolophrenialine (LSF) or mycophenolate mofetil, anti-thymocyte globulin, beraceptide, or everolimus.

[0358] In some aspects, the method includes administering an immunosuppressant, such as a steroid (e.g., prednisone), a nonsteroidal anti-steroid (e.g., sirolimus (Rapamune, Wyeth-Ayerst Canada), tacrolimus (Prograf, Fujisawa Canada), an anti-IL2R antibody (e.g., dalizumab) (Zenapax, Roche Canada), 15-deoxyguanidine, cyclosporine, methotrexate, rapamycin, rapamune (sirolimus / rapamycin), FK506, lisolophrenialine (LSF), or mycophenolate mofetil, an anti-thymocyte globulin, beraceptide, or everolimus, to a subject who has received an organ containing a polypeptide as described herein.

[0359] In some aspects, the method includes contacting an organ that has previously been contacted with a polynucleotide encoding at least one regeneration factor with an inducible compound, the polynucleotide being operatively linked to an inducible promoter.

[0360] In some aspects, the method includes administering an inducing compound to a recipient who has received an organ previously in vitro contact with a polynucleotide encoding at least one regeneration factor, the polynucleotide being operatively linked to an inducible promoter. In some aspects, the method includes administering an inducing compound to a recipient who has received an organ containing a foreign polynucleotide encoding at least one regeneration factor, the foreign polynucleotide being operatively linked to an inducible promoter. In some aspects, the recipient has received an organ that has previously been contacted with a polynucleotide operatively linked to a first inducible promoter encoding at least one of Oct4, Sox2, and / or Klf4 and a second polynucleotide operatively linked to a second inducible promoter encoding c-Myc. In some aspects, the method includes administering a first inducing compound to the recipient at a first time period and administering a second inducing compound to the recipient at a second time period. In some aspects, the first and second time periods have the same length. In some aspects, the first and second inducing compounds are administered at the same time. In some aspects, the first and second inducing compounds are administered at different times. In some aspects, the first inducing compound and / or the second inducing compound are applied, for example, 2, 3, or 4 days per week, for 1-10 weeks every 3 months. In some aspects, the first inducing compound and / or the second inducing compound are applied, for example, 2, 3, or 4 days per week, for 1-10 weeks every 4 months. In some aspects, the first inducing compound and / or the second inducing compound are applied, for example, 2, 3, or 4 days per week, for 1-10 weeks every 6 months. In some aspects, the first inducing compound and / or the second inducing compound are applied, for example, 2, 3, or 4 days per week, for 1-10 weeks every 8 months. In some aspects, the first inducing compound and / or the second inducing compound are applied, for example, 2, 3, or 4 days per week, for 1-10 weeks every 10 months. In some aspects, the first inducing compound and / or the second inducing compound are applied, for example, 2, 3, or 4 days per week, for 1-10 weeks every 12 months.

[0361] In some respects, for each application of the first and second compounds to the recipient, the first inducing compound is applied over a longer period than the second inducing compound. In some respects, the first and second inducing compounds are applied to the recipient in the same amount over the same period. In some respects, the first inducing compound is applied over a shorter period than the second inducing compound. In some respects, the first inducing compound is applied to the recipient more than once, while the second inducing compound is applied once. In some respects, the first inducing compound is applied to the recipient once, while the second inducing compound is applied more than once.

[0362] In some aspects, the method includes (i) contacting an organ with a polynucleotide comprising a first inducible promoter operatively linked to a polynucleotide encoding at least one of Oct4, Sox2, or Klf4; (ii) contacting the organ with an inducer that induces activity of the first promoter and causes expression of at least one of Oct4, Sox2, or Klf4; (iii) contacting the organ with a polynucleotide comprising a second inducible promoter operatively linked to a polynucleotide encoding c-Myc; and (iv) contacting the organ with a second inducer that induces activity of a second promoter and causes expression of c-Myc. In some aspects, step (ii) of the described method is performed more than once before step (iii). In some aspects, step (ii) of the described method is performed once before step (iii). In some aspects, step (ii) is performed more than once, while step (iv) is performed only once. In some aspects, step (ii) is performed 2 to 100 times, while step (iv) is performed only 1 to 10 times. In some respects, step (iv) is performed every 2-5 times of step (ii). In other respects, step (iv) is performed every 1-5 times, 6-10 times, 11-15 times, 16-20 times, 21-25 times, 26-30 times, 31-35 times, 36-40 times, 41-45 times, or 46-50 times of step (ii).

[0363] In some respects, steps (ii) and (iv) are performed multiple times within a one-month cycle, but not within the next three consecutive months. In some respects, steps (ii) and (iv) are performed multiple times within a one-month cycle, but not within the next six consecutive months. In some respects, steps (ii) and (iv) are performed multiple times within a one-month cycle, but not within the next nine consecutive months. In some respects, steps (ii) and (iv) are performed multiple times within a one-month cycle, but not within the next twelve consecutive months.

[0364] In some aspects, the method includes (i) contacting an organ with a polynucleotide comprising a first inducible promoter operatively linked to a polynucleotide encoding at least one of Oct4, Sox2, or Klf4, and further contacting the organ with a polynucleotide comprising a second inducible promoter operatively linked to a polynucleotide encoding c-Myc; (ii) contacting the organ with an inducible compound inducing activity of the first promoter; and (iii) contacting the organ with a second inducible compound inducing activity of the second promoter. In some aspects, step (ii) of the method is performed more than once, while step (iii) is performed once. In some aspects, step (ii) is performed 2 to 100 times, while step (iii) is performed 1 to 10 times. In some aspects, step (iii) is performed every 2 to 5 times of step (ii). In some respects, step (iv) is performed every 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, or 46-50 times of step (ii).

[0365] In some respects, steps (ii) and (iii) are performed multiple times within a one-month cycle, but not within the next three consecutive months. In some respects, steps (ii) and (iii) are performed multiple times within a one-month cycle, but not within the next six consecutive months. In some respects, steps (ii) and (iii) are performed multiple times within a one-month cycle, but not within the next nine consecutive months. In some respects, steps (ii) and (iii) are performed multiple times within a one-month cycle, but not within the next twelve consecutive months.

[0366] In some respects, organs in contact with the compositions described herein in vitro are characterized by at least one of the following: aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, fatty degeneration, virus-induced hepatitis, alcohol, or fibrosis unrelated to any known cause.

[0367] In some respects, the organ is an aging organ. In some respects, the organ is 70 years of age or older (i.e., the organ was taken from a subject 70 years of age or older). In some respects, the organ is over 60 years of age. In some respects, the organ is 70-79 years of age. In some respects, the organ is 60-69 years of age. In some respects, the organ is 50-59 years of age. In some respects, the organ is 40-49 years of age. In some respects, the organ is under 60 years of age. In some respects, the organ is 50 years of age or younger. In some respects, the organ is between 6 and 50 years of age.

[0368] In some respects, the organ in question is the kidney or the liver.

[0369] In some aspects, the kidney is 70 years of age or older. In some aspects, the kidney is 60 years of age or older; 70-79 years of age; 60-69 years of age; 50-59 years of age; or 40-49 years of age. In some aspects, the kidney is under 60 years of age. In some aspects, the kidney is 50 years of age or younger. In some aspects, the kidney is between 6 and 50 years of age.

[0370] In some aspects, the kidney is from an elderly donor. In some aspects, the kidney is from a donor aged 70-79; 60-69; 50-59; or 40-49. In some aspects, the kidney is from a donor with an age-related disease or condition. In some aspects, the age-related disease or condition is a history of hypertension. In some aspects, the age-related disease or condition is a history of atherosclerosis. In some aspects, the age-related disease or condition is a history of toxic kidney injury. In some aspects, the age-related disease or condition is a history of renal immune injury. In some aspects, the age-related disease or condition is a history of renal perfusion injury. In some aspects, the age-related disease or condition is fibrosis. In some aspects, the kidney is from a donor with a serum creatinine level greater than or equal to 1.5 mg / dL. In some aspects, the kidney is from a donor with any two of the following: a history of hypertension, a creatinine level greater than or equal to 1.5 mg / dL, or death due to stroke. In some aspects, the kidney is from a donor under 50 years of age. In some aspects, the kidney is from a donor under 50 years of age who is brain dead (e.g., due to trauma or other causes such as stroke). In some aspects, the donor is from a donor without a heartbeat (donated after cardiac death, DCD). In some aspects, the donor is from a brain-dead donor (donated after brain death, DBD). In some aspects, the kidney is from a DCD donor who died from uncontrolled circulatory death, also known as Maastricht Class II, cardiac arrest with unsuccessful resuscitation. In some aspects, the kidney is from a donor with controlled circulatory death (also known as Maastricht Class III, cardiac arrest occurring in a controlled manner in a hospital).

[0371] In some respects, the kidney is from a living donor. In other respects, the kidney is from a deceased donor.

[0372] In some aspects, the liver is 70 years of age or older. In some aspects, the liver is 60 years of age or older; 70-79 years of age; 60-69 years of age; 50-59 years of age; or 40-49 years of age. In some aspects, the kidney is from a donor suffering from an age-related disease or condition. In some aspects, the age-related disease or condition is steatosis. In some aspects, the age-related disease or condition is liver fibrosis.

[0373] In some respects, the liver is characterized by steatosis. In some respects, the liver is from a heartless donor (post-cardiac death donation, DCD). In some respects, the liver is from a brain-dead donor (post-brain-dead donation, DBD). In some respects, the liver is a segmented liver.

[0374] In some respects, the liver is from a living donor. In other respects, the liver is from a deceased donor.

[0375] Organ function can be characterized before and after transplantation using assays and tests known in the art. In some aspects, the organ is characterized by at least one of the following: aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, virus-induced hepatitis, alcoholism, or fibrosis unrelated to any known cause. In some aspects, aging or damaged organs are assessed by diagnostic tests and / or biopsies before and / or after exposure to the perfusion fluid as described herein to determine signs of age or damage, including but not limited to, inflammatory cell infiltration, renal tubular injury, interstitial edema, or fibrotic changes, whether these signs are reduced after exposure to the perfusion fluid compared to before exposure. In some aspects, aging or damaged organs may be exposed to the perfusion fluid for one or more additional time periods. In some aspects, the composition of the perfusion fluid may be adjusted based on the age or damage present in the organ before perfusion. In some aspects, the composition of the perfusion fluid may be adjusted based on the remaining damage in the organ after the first perfusion cycle to perfuse the organ in a second or more perfusion cycles.

[0376] In some respects, renal function is characterized by testing one or more of the following: BUN, pH, bicarbonate, sodium, potassium, serum creatinine (e.g., eGFR at 1 week / 1 month / 3 months post-transplantation), protein-to-creatinine ratio (urine), serum KIM1, serum Klotho, CXCL10 / 9 (e.g., measured in urine), and / or blood pressure.

[0377] In some respects, the renal function of a recipient is characterized by detecting and / or measuring urine production.

[0378] In some respects, renal histology can be characterized by testing one or more of the following: Banff score (Masson tricolor), tubular necrosis score, proliferation (e.g., by measuring Ki67, PCNA), apoptosis (e.g., by TUNEL assay) and / or immune infiltrates (e.g., by measuring CD68, CD3 and / or CD19).

[0379] In some aspects, liver damage, inflammation, regeneration, and / or tissue repair are evaluated. In some aspects, liver function is evaluated / characterized by one or more of the following: AST, ALT, bilirubin, MPO levels, and / or MDA levels. In some aspects, liver histology is characterized, for example, by testing one or more of the following: Ki67, PCNA, HGF, and / or TGFβ; by TUNEL and / or caspase 3 assays. H&E staining can be performed to evaluate liver damage.

[0380] In some respects, the liver is characterized by one or more of the following (evaluating one or more of the following for the liver): steatosis score (e.g., steatosis, lobular inflammation, and hepatocellular ballooning degeneration) grade 0 (healthy, <5%), grade 1 (mild, 5%–33%), grade 2 (moderate, 34%–66%), and grade 3 (severe, >66%), necrosis score, and / or the presence of immune infiltrates (e.g., determination of CD68, CD3, and / or CD19).

[0381] In some respects, liver function is evaluated by measuring alkaline phosphatase (ALP), albumin and total protein, bilirubin, gamma-glutamyl transferase (GGT), L-lactate dehydrogenase (LD), and / or prothrombin time (PT).

[0382] In some aspects, epigenetic clocks are used to characterize organs (e.g., organ biopsies). In a further embodiment, the Horvath epigenetic clock is used to determine the DNA methylation age of an organ. The Horvath epigenetic clock can be used as an age estimation method based on DNA methylation at CpG dinucleotide motifs in DNA.

[0383] In some respects, renal function in recipients is characterized by testing one or more of the following: BUN, serum creatinine, serum pH, bicarbonate, sodium, potassium, or lactate levels. In some respects, kidneys exposed to the polynucleotides described herein exhibit improved BUN, serum creatinine, serum pH, bicarbonate, sodium, potassium, and / or lactate levels compared to before exposure to the polynucleotides.

[0384] In some aspects, liver damage, inflammation, and regeneration / tissue repair of the transplanted liver are evaluated. In others, recipient liver function is characterized by testing one or more of the following: broadly standardized liver disease parameters (AST, ALT, and bilirubin), apoptosis parameters (cysteine ​​3), hepatocyte proliferation (Ki67 / PCNA, HGF, and TGF-β), liver inflammation (MPO activity due to neutrophil accumulation, MDA levels due to oxidative stress), and / or liver histology (H&E).

[0385] In some respects, livers exposed to the polynucleotides described herein exhibited reduced levels of AST, ALT, bilirubin, and caspase-3 compared to pre-existing exposure, increased levels of hepatocyte proliferation markers Ki67 / PCNA, HGF, and TGF-β, decreased MPO activity, reduced MDA levels, and fewer histological signs of liver damage.

[0386] In some respects, the methods for restoring aging cells, tissues or organs described herein restore or improve at least one function of aging cells, tissues or organs, such as mitochondrial function, proteolytic activity, heterochromatin levels, histone methylation, nuclear lamina polypeptides, and cytokine levels.

[0387] In some respects, contact between a cell, tissue, or organ and a polynucleotide as described herein results in a change in the aggregated methylation state of an individual set of methylation sites within the cell, tissue, or organ, which measures actual age (DNA methylation clock).

[0388] In some respects, contact of cells, tissues, or organs with polynucleotides as described herein leads to an increase in one or more nuclear markers and / or epigenetic markers compared to reference values. In some respects, contact of cells, tissues, or organs with polynucleotides as described herein leads to increased expression of HP1γ. In some respects, contact of cells, tissues, or organs with polynucleotides as described herein leads to an increase in H3K9me3. In some respects, contact of cells, tissues, or organs with polynucleotides as described herein leads to increased expression of the laminar support protein LAP2α. In some respects, contact of cells, tissues, or organs with polynucleotides as described herein leads to increased gene expression of the SIRT1 protein.

[0389] In some respects, cells, tissues, or organs exposed to the polynucleotides described herein exhibit improved mitochondrial health and function compared to those not exposed. In some respects, this improved mitochondrial health and function is measured as changes in mitochondrial membrane potential, reactive oxygen species (ROS), or combinations thereof. For example, mitochondrial membrane potential can be measured using tetramethylrhodamine, methyl ester, perchlorate (Thermo), as described in PCT Publication WO2019 / 178296, which is a dye chelated by mitochondria based on their membrane potential. Mitosoidal ROS can be measured using MitoSOX, as described in PCT Publication WO2019 / 178296, which is a fluorescent dye oxidized by superoxide in mitochondria.

[0390] In some aspects, methods for preparing organs for transplantation are provided. In some aspects, the method includes contacting the organ with a composition comprising the polynucleotides, carriers, or nanoparticles described herein prior to transplantation. In some aspects, the composition is a perfusion fluid as described herein. In some aspects, the method includes contacting the organ with a perfusion fluid comprising the polynucleotides, carriers, or nanoparticles described herein prior to transplantation, wherein the organ is rejuvenated. In some aspects, the method includes contacting an organ characterized by at least one of aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, virus-induced hepatitis, alcoholism, or fibrosis unrelated to any known cause with a perfusion fluid comprising the polynucleotides, carriers, or nanoparticles described herein prior to transplantation, wherein at least one of aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, virus-induced hepatitis, alcoholism, or fibrosis unrelated to any known cause is treated, improved, prevented, and / or reversed. In some aspects, the method includes contacting an organ with a perfusion fluid containing the polynucleotides, carriers, or nanoparticles described herein prior to transplantation, in which at least one of aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, fatty degeneration, virus-induced hepatitis, alcoholism, or fibrosis unrelated to any known cause is improved, prevented, and / or reversed.

[0391] In some aspects, the method includes contacting the organ with the polynucleotides, carriers, or nanoparticles described herein. In some aspects, contact is achieved by immersing the organ in a perfusion solution containing the polynucleotides, carriers, or nanoparticles described herein. In some aspects, contact is achieved by perfusing the organ with a perfusion solution containing the polynucleotides, carriers, or nanoparticles described herein. In some aspects, perfusion is achieved by connecting a conduit to a blood vessel of the organ. In some aspects, perfusion is achieved by connecting a conduit to a lymphatic vessel of the organ.

[0392] In some aspects, methods for preparing organoids for transplantation are provided. In some aspects, the organoids are generated in vitro from pluripotent stem cells. In some aspects, the organoids are contacted with the polynucleotides described herein. In some aspects, the organoids contain a vascular system. In some aspects, contact is achieved by immersing the organoids in a perfusion solution containing polynucleotides, carriers, or nanoparticles as described herein. In some aspects, contact is achieved by connecting the vascular bundles of the organoids to conduits and perfusing the organoids with a perfusion solution containing polynucleotides, carriers, or nanoparticles as described herein. In some aspects, perfusion is achieved by connecting conduits to the blood vessels of the organoids. In some aspects, perfusion is achieved by connecting conduits to the lymphatic vessels of the organoids.

[0393] In some cases, organs or organoids come into contact with transfection agents. In some cases, transfection agents include lipofectamine, LT-1, dextran, calcium phosphate, and lectin. In some cases, electroporation, microinjection, or combinations thereof are used to transfect organs or organoids.

[0394] In some respects, organs or organoids come into contact with compositions containing combination therapies.

[0395] In some respects, the composition comprises polynucleotides, peptides, carriers or nanoparticles as described herein, and enhancers. In some aspects, the composition comprises polynucleotides, peptides, carriers, or nanoparticles as described herein, and one or more of the following: soluble Wnt, Wnt conditioned medium, BIX-01294, PD0325901, DNA methyltransferase inhibitors, histone deacetylase inhibitors, valproic acid, 5'-azacytidine, dexamethasone, succinyl aniline isohydroxamic acid (SAHA (e.g., MK0683, vorinostat, or other isohydroxamic acids), vitamin C, trichomoniasisin (TSA), BML-210, depudecin (e.g., (-)-depudecin), HC toxin, Nullscript (4-(1,3-dioxo-1H,3H-benzo[de]isoquinoline-2-yl)-N-hydroxybutyramide), phenyl butyrate (e.g., sodium phenylbutyrate) or other short-chain fatty acids, Scriptaid, suramin sodium. Sodium), APHA compound 8, apicidin, sodium butyrate, pivanex (AN-9), tramoxin B, chlamydocin, phenyl condensate (also known as FR901228 or FK228), benzamide (e.g., CI-994 (e.g., N-acetyldinarin) and MS-27-275), MGCD0103, NVPLAQ-824, CBHA (m-carboxycinnamic acid dihydroxamic acid), JNJ16241199, Tubacin, A-161906, proxamide, oxamflatin, 3-Cl-UCHA (e.g., 6-(3-chlorophenylurea)hexanoic acid isohydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 or CHAP 50. In some aspects, the composition comprises polynucleotides, peptides, carriers or nanoparticles as described herein, and a dominant-negative form of HDAC (e.g., a non-catalytically active form), an HDAC siRNA inhibitor, or an antibody that specifically binds to HDAC.

[0396] VIII. Reagent Kit

[0397] In some aspects, kits are provided comprising compositions or perfusion solutions as described herein, said compositions or perfusion solutions comprising polynucleotides, carriers and / or nanoparticles, and one or more pharmaceutically acceptable carriers, excipients, and / or mediators. In some aspects, kits are provided comprising a first composition or perfusion solution and optionally a second composition or perfusion solution, said first composition or perfusion solution comprising polynucleotides, carriers and / or nanoparticles as described herein, and one or more pharmaceutically acceptable carriers, excipients, and / or mediators, said second composition or perfusion solution comprising a second polynucleotide, carrier and / or nanoparticles as described herein, and one or more pharmaceutically acceptable carriers, excipients, and / or mediators. In some aspects, said kits further comprise inducing compounds. In some aspects, said kits comprise a first inducing compound and a second inducing compound.

[0398] In some respects, the kit comprises a composition or perfusion solution as described herein, the composition or perfusion solution being packaged in a manner that facilitates its use in practicing the methods of this disclosure.

[0399] In some aspects, the kit also includes instructions on how to administer the composition or perfusion solution, as well as tools for preparing and administering the pharmaceutical composition or perfusion solution in a perfusion system as described herein.

[0400] Example

[0401] Example 1. Methods and Materials

[0402] animal

[0403] Allogeneic and allogeneic kidney transplantation models were established. For the allogeneic kidney transplantation model, 3-month-old male Lewis (LEW, Janvier Labs, Le Genest-Saint-Isle, France) rats were recipients of 3-month-old male LEW transplants (Lewin, E. et al., Scand J Urol vol. 27, 1993). For allogeneic kidney transplantation, male LEW rats were recipients of 9-month-old male Dark Agouti (DA, Janvier Labs, Le Genest-Saint-Isle, France) transplants. The DA and LEW strains differ partially in the major histocompatibility complex and various non-MHC sites, conferring a weaker histocompatibility combination and driving transplant rejection. Systolic blood pressure, proteinuria, and diuresis were monitored to assess the progression of hypertensive nephropathy.

[0404] Renal normothermic perfusion and AAV administration

[0405] For normothermic renal perfusion, the Hugo Sachs / Harvard Apparatus device was used. In short, the system consists of a water-jacketed annular chamber with a glass cap assembly. Two cannulas for organ perfusion are connected to the kidney via the vena cava and aorta. The system includes various support components attached to the cannulas to maintain the organ under maximally physiological conditions: a membrane oxygenator, reservoir, cannula clamps, perfusion fluid, gas and water lines, a bubble trap, and a manifold for water, gas, and perfusion fluid control. The perfusion fluid is preheated at 37°C and oxygenated for 30 minutes prior to organ connection. The perfusion fluid consists of Gey's solution (Sigma) and 1% penicillin / streptomycin (Sigma). After organ connection, pressure-controlled perfusion is performed, with pressure gradually increased over 5 minutes to reach an average pressure of 70 mmHg. When the target pressure is reached, AAV is administered to the perfusion fluid, and perfusion continues for 1 hour after AAV administration.

[0406] Human kidney perfusion

[0407] Using a perfusion system, human kidneys are ex vivo perfused with polynucleotides, carriers, and / or nanoparticles as described herein, for kidneys that would not be used for transplantation due to, for example, ischemic injury, toxic injury, perfusion injury, or other injuries, or due to age. The perfusion solution for human kidney perfusion contains:

[0408] 215 mL dextran / albumin solution (Steen Solution, XVIVO Perfusion AB, Goteborg, Sweden); 400 mL concentrated red blood cell solution; 2 mL 10% calcium gluconate; 1300 U / L heparin; 400 mg cefazolin; verapamil (vasodilator); amino acids, glucose, and insulin; and

[0409] Lactated Ringer's solution to compensate for urine loss

[0410] Before adding the polynucleotides, carriers, and / or nanoparticles as described herein, the human kidney will be connected to a perfusion system and perfused for at least one hour.

[0411] Human renal function in renal biopsies will then be assessed before and after perfusion by determining the Banff score (Masson tricolor); renal tubular necrosis score; measuring diffusion markers (Ki67, PCNA); apoptosis markers (TUNEL); immune infiltrates (CD68, CD3 and / or CD19); and by single-nuclear RNA sequencing (snRNAseq) and methylome analysis.

[0412] Human liver perfusion

[0413] The human liver will be perfused using the Organox Metra protocol. The perfusion system will be started using 3 units of concentrated red blood cells and colloid solution (Gelafundin, B Braun 500 mL).

[0414] Prior to liver connection, the blood-based perfusion is supplemented with: cefuroxime 750 mg (antibiotic); heparin 10,000 IU (anticoagulant to prevent thrombosis in the circuit); sodium bicarbonate (buffer to adjust the pH of the infusion before placing the liver in the device); calcium gluconate 10% 10 ml (to correct citrate binding with calcium); and after connection to the human liver and during liver perfusion, the following components are added to the perfusion fluid: insulin (200 IU) (to control the level of perfusion glucose); heparin (25,000 IU) (to maintain anticoagulation); and 2% sodium taurocholate solution in isotonic saline solution (to compensate for bile salt loss). 0.5 mg prostacyclin (a vasodilator optimized for microperfusion); and parenteral nutrition solution containing amino acids and glucose for liver maintenance.

[0415] Once the circuit is started and the infusion is connected, the perfusion fluid will automatically circulate, oxygenate, and heat to 37°C, and the human liver will be connected to the perfusion system.

[0416] The infusion rate of glucose and amino acids (parenteral nutrition) is variable and the glucose level is manually infused every 4 hours.

[0417] As an alternative, human liver perfusion systems can use non-RBC oxygen carriers, including Hemopure Or perfluorocarbon.

[0418] Human liver is monitored by measuring AST / ALT, ALP, albumin, bilirubin, prothrombin time, L-lactate dehydrogenase, bilirubin, and gamma-glutamyl transferase in the perfusion fluid. Human liver biopsies obtained before and at the end of perfusion can be examined using SnRNASeq. Methylome and histological analyses will be performed to assess proliferation markers (Ki67, PCNA), apoptosis markers (e.g., TUNEL), steatosis scores (e.g., steatosis, lobular inflammation, and hepatocyte ballooning) at grades 0 (healthy, <5%), 1 (mild, 5%–33%), 2 (moderate, 34%–66%), and 3 (severe, >66%), necrosis scores, and immune infiltrates (CD68, CD3, and / or CD19).

[0419] According to the supplier's plan, the tissue is dissolved and homogenized and then used. RSC instrument (Promega), using tissue... Total RNA was extracted using RSC miRNA (Promega). RNA purity and concentration were assessed using a NanoDrop™ spectrophotometer (ThermoFisher). cDNA was synthesized from the RNA template using an Invitrogen cDNA synthesis kit, following the manufacturer's instructions. The resulting cDNA was diluted and used to determine the expression levels of GFP, NGAL, MCP1, CXCL10, HIF, ETR, TFGβ, and TNF. GADPH was used as a housekeeping gene. Primers used in the cDNA analysis are shown in Table 2.

[0420] Real-time qPCR was performed on a QuantStudio 7 device (ThermoFisher) using SYBR Green PCR Master Mix reagent (Appliedbiosystems) in 384-well plates with the corresponding primers for the genes of interest. Samples were run in triplicate in 10 μl reaction, and the mRNA expression of the target gene was normalized relative to GADPH mRNA and expressed as relative gene expression relative to the control group.

[0421] Adeno-associated virus (AAV)

[0422] DNA encoding key reprogrammed genes under a regulated promoter was delivered using adeno-associated virus (AAV) prepared by Vector Biolabs. This allows for efficient delivery and regulated expression of the reprogrammed gene products. Reprogramming factors (Oct4, Sox2, Klf4, and cMyc) were cloned under the Tet-ON promoter, which was activated by the transactivator rtTA in the presence of doxycycline (SEQ ID NO:26 AAV TRE3G SK short WPRE SV40 polyA; SEQ ID NO:27 AAV TRE3G cMyc short WPRE SV40 polyA+EF1a TetON3G; SEQ ID NO:28 AAV-TRE3G-hMyc-EF1a-rTta; SEQ ID NO:29 plasmid AAV-CAG-CBRLuc-GFP). Sequences of the polynucleotides and their components are provided in Table 2.

[0423] To take advantage of its broad cell tropism, AAV with a DJ serum capsid was used (Grimm, D. et al., J Virol 82, 5887–5911, 2008). Gene expression was activated by adding commercially available doxycycline (Dox) after the programming factor was delivered to the organ via AAV-mediated delivery. From 2 weeks post-transplantation until the end of the study, rats were weighed and placed in metabolic cages, with urine and tail vein blood collected weekly for 24 hours. Blood urea nitrogen (BUN), urine, and serum creatinine (Crea) were determined.

[0424] Luc fluorescence analysis.

[0425] Seven days after graft perfusion and kidney transplantation, rats were anesthetized and injected intraperitoneally with 1 ml of fluorescein (15 mg / mL). Thirty minutes later, luminescence images were captured using the IVIS bioluminescence imaging system according to standard methods.

[0426] Statistical analysis

[0427] Statistical analysis was performed using GraphPad Prism 5 statistical software (GraphPad Software Inc.). Univariate analysis was performed using the log-rank test (Kaplan–Meier curves) to assess rat survival (time from kidney transplantation to death). The Man Whitney test was used to compare the two groups. A value <0.05 was considered significant. Values ​​are given as mean ± standard deviation.

[0428] We chose to use the effective score (Table Z) used in kidney transplantation to assess kidneys from human donors to analyze chronic kidney disease 6 weeks after transplantation (Remuzzi et al. J Am Soc Nephrol 10:2591–2598, 1999), modified as described below. This overall score evaluates glomerular sclerosis, arterial stenosis, tubular necrosis, tubular atrophy, and interstitial fibrosis.

[0429] Improved Remuzzi rating analysis

[0430] The modified Remuzzi score was used to analyze chronic kidney disease 6 weeks after transplantation from human donors (Remuzzi et al. J Am Soc Nephrol 10:2591–2598, 1999), modified as described below. This overall score evaluates glomerular sclerosis, arterial stenosis, tubular necrosis, tubular atrophy, and interstitial fibrosis.

[0431] Remuzzi rating (Rs) (Remuzzi et al., see above)

[0432] Global glomerular sclerosis (G) score is as follows:

[0433] 0: No overall hardening

[0434] 1: <20% of the total glomeruli are sclerotic

[0435] 2: 20% to 50% of the total glomeruli are sclerotic.

[0436] 3: >50% of the total glomeruli are sclerotic

[0437] The renal tubular atrophy (T) score is as follows:

[0438] 0: Does not exist

[0439] 1: <20% of renal tubules are affected

[0440] 2: 20% to 50% of the renal tubules are affected.

[0441] 3. More than 50% of the renal tubules are affected.

[0442] The interstitial fibrosis (I) score is as follows:

[0443] 0: Does not exist

[0444] 1: <20% of kidney tissue was replaced by fibrous connective tissue.

[0445] 2: 20% to 50% of kidney tissue is replaced by fibrous connective tissue.

[0446] 3: >50% of kidney tissue is replaced by fibrous connective tissue.

[0447] The arterial and arteriole stenosis (A) scores are as follows:

[0448] 0: Does not exist

[0449] 1: The wall thickness increases, but the degree is less than that of the lumen diameter.

[0450] 2: The wall thickness is equal to or slightly greater than the lumen diameter.

[0451] 3: The wall thickness far exceeds the diameter of the lumen, resulting in an extremely narrow or blocked lumen.

[0452] The modified Remuzzi score excludes glomerulosclerosis factor (ischemic injury does not affect the glomerular compartments, therefore all glomeruli in all samples were normal) and arteriolar / arteriole stenosis (arteries were not observed in some slides). Furthermore, acute tubular necrosis (ATN) was calculated on four distinct grades: none (0), low (1), moderate (2), and high (4).

[0453] Example 2. AAV targeting kidney GFP / luc

[0454] AAV vectors were injected into clamped rat renal veins, and weak luciferin expression was detected in both the injected kidney and the uninjected contralateral kidney 7 days after in situ injection (see [link to original text]). Figure 1A The weak signal observed in the kidneys indicates systemic leakage of AAV, which could lead to adverse effects. This is attributed to the low transduction efficiency of AAV carriers in the kidneys.

[0455] Then, two room-temperature mechanical perfusion (NMP) time periods were tested using the AAV-GFP / Luc vector: 45 minutes and 90 minutes. The injected viral titer was approximately 1 x 10⁻⁶. 12 The total distribution volume in the perfusion system was estimated to be approximately 60–70 ml. Rat transplanted kidneys were perfused in vitro with GFP / luc AAV for 45 min via normothermic mechanical perfusion (NMP) as described in the Methods and Materials section above. Luciferin was detected only in the perfused kidneys (see [link to Methods and Materials section]). Figure 1B (Left animal). In contrast, in rats injected intravenously (iv) (via penile vein), fluorescein localized to the liver, while no fluorescein was observed in the kidneys (see [link]). Figure 1B (Rats on the right). Therefore, isolated normothermic mechanical perfusion (NMP) of rat kidneys with GFP / luc AAV showed specific delivery to the perfused kidneys and no significant "leakage" to other organs and tissues.

[0456] Ninety minutes after perfusion (flow rate set to approximately 4 ml / min), the perfused left kidney showed in vivo fluorescein expression, while no fluorescein expression was observed in the lung or the contralateral kidney (right kidney, i.e., unperfused). The lowest fluorescein levels were observed in the liver.

[0457] Example 3. OSKM treatment improves renal function and rat survival in aging and allogeneic transplantation models.

[0458] In the kidneys, aging is associated with decreased glomerular filtration rate, increased vascular and interstitial lesions, and increased susceptibility to acute injury (e.g., ischemia-reperfusion injury) (Stenvinkel, P. and Larsson, TE Am. J. Kidney Dis. 62, 339–351, 2013; Anderson, S. and Brenner, BMA Am. J. Med. 80, 435–442, 1986; and Baylis, C. and Corman, B., J. Am. Soc. Nephrol. JASN 9, 699–709, 1998). Clinically relevant models of kidney allogeneic transplantation from aging donors were used to test the effects of ex vivo AAV-delivered Yamanaka factors (Oct-3 / 4, Sox2, Klf4, and c-Myc [OSKM]) on kidney transplant function and kidney function-dependent rat survival (see [link to study]). Figure 2A In this model, ischemic injury occurs due to organ donation and normothermic perfusion, while immune injury is caused by the different genetic backgrounds of the donor (Dark agouti strain) and the recipient (Lewis strain).

[0459] Donor kidneys from aged (9-month-old) Dark agouti rats (DA rats) were perfused with NMP at room temperature for approximately 1 hour, using inducible AAV-OSK and AAV c-Myc / rtTA (approximately 1 x 10⁻⁶ each vector). 12 One; or a total titer of 2 x 10-1 12 Then, tacrolimus, an anti-rejection drug, was administered for 5 days post-kidney transplant to prevent early transplant rejection. One week post-transplant, the contralateral kidney was removed. Starting one week post-transplant, OSKM expression was driven by rtTA activation via doxycycline (Dox) supplementation in drinking water twice weekly. AAV GFP / luc (1x10⁻¹) at the same titer was used. 12) Perfusion media control. In the AAV OSKM group, Dox administration was discontinued 87 days post-transplantation (80 days after Dox treatment).

[0460] Histological analysis revealed inflammatory infiltration, tubular injury, and interstitial edema in the kidneys of aged donors who had received AAV-GFP perfusion (see [link to original text]). Figure 2B Previous studies have shown that inflammatory infiltration is primarily caused by alloimmune injury, but it can also be caused by ischemic injury. Renal tubular injury is more prominent in ischemic injury, but it can also be caused by alloimmune injury. Interstitial edema can be observed in both alloimmune and ischemic injuries.

[0461] The survival rate in the OSKM treatment group was significantly higher than that in the control group (see...). Figure 2C All animals in the control group died by day 70 or earlier. Dox treatment was discontinued on day 87 post-transplantation. One rat died one week after discontinuation (renal function was observed to decline in this rat), while three other rats survived for another three weeks, at which point the animals were analyzed (see [link to study]). Figure 2C In the three surviving rats, renal function was normal, as evidenced by stable serum creatinine levels three weeks after discontinuation of dox (see [link to original text]). Figure 2K Notably, the OSKM-treated group had lower serum creatinine levels compared to the AAV-GFP / luc-treated control group, and these levels remained consistent throughout the study. In rats receiving OSKM-perfused kidneys, renal function was significantly enhanced compared to the AAV-GFP / luc-treated control group, as evidenced by lower blood urea nitrogen (BUN) levels at 2 weeks post-transplantation (i.e., better indicator function) (see [link to study report]). Figure 2D and Figure 2FBUN levels, pH, bicarbonate, sodium, potassium, and lactate levels remained stable from 3 to 12 weeks post-transplantation (see [link]). Figure 2G , Figure 2H , Figure 2I , Figure 2J , Figure 2L and Figure 2M BUN levels were particularly stable in rats that survived for 3 weeks after dox cessation (see [link to dox cessation]). Figure 2L Note that the highest BUN level was 120 mg / dL; while the normal BUN level range is 15-20 mg / dL. Transplanted kidneys were collected from surviving OSKM-treated rats 100 days post-transplantation (no control rats were surviving at this time). No evidence of tumors was found in the kidneys (see [link to original text]). Figure 2N Macroscopically, the kidneys of these OSKM-treated animals were enlarged and had irregular surfaces; these features are typically observed in allogeneic transplantation models due to alloimmune injury. Overall, the results indicate that partial reprogramming with OSKM can improve renal function following ischemic and immune injury.

[0462] Then, the kidney sections treated with OSKM were stained with hematoxylin and eosin (see [link]). Figure 7 Immune infiltration, vascular damage, and renal tubular damage were observed in the above sections, consistent with alloimmune injury (transplant rejection) (see [link]). Figure 7 For most of the study period, OSKM-treated mice did not receive anti-rejection drugs. The treated kidneys did not show signs of tumors or malignancy (see [link to study]). Figure 7 ).

[0463] To evaluate the level of immune infiltration in the kidneys of the AAV-GFP / luc control group, kidneys were collected from two pre-mortem rats (i.e., mice showing signs of impending death) and studied in the AAV-GFP / luc group at 15 and 16 days post-transplantation. Hematoxylin-eosin (H&E) stained sections showed allogeneic rejection features with extensive immune infiltration, as expected given that the mice were not treated with anti-rejection drugs (except for those noted in the early stages of the protocol) (see [link to relevant documentation]). Figure 8 These results demonstrate that the level of immune rejection (immune infiltration) was lower than expected in OSKM-treated mice. Because OSKM-treated mice also survived longer and exhibited improved renal function, the results indicate that OSKM treatment has a beneficial effect in preventing allogeneic transplant rejection.

[0464] Example 4. OSKM treatment improves ischemic injury in an endogenic kidney transplant model

[0465] Ischemia-reperfusion injury (IRI) is a leading cause of acute kidney injury and a constant feature of kidney transplantation. IRI is tissue damage that occurs when blood supply to an organ is interrupted and then restored. IRI is typically associated with robust inflammatory and oxidative stress responses to hypoxia and reperfusion (e.g., in kidney transplantation), which disrupt organ function. Organ ischemia can cause tissue damage by reducing the level of the energy storage molecule ATP. This alteration leads to the accumulation of harmful reactive oxygen species, damage to mitochondrial organelles, and triggers an inflammatory response when oxygenated blood flow is restored to the transplanted organ. These symptoms are particularly severe in high-risk donor organs, which constitute an increasingly large proportion of current organ grafts.

[0466] Organ retrieval, transport, and transplantation in kidney transplantation can lead to organ resorption and ischemia-reperfusion injury (IRI). Cold ischemia causes IRI and can occur during organ transplantation, after organ retrieval and subsequent transport when the organ is cooled with cold perfusion fluid. Warm ischemia also contributes to IRI and can occur, for example, in cases of donor death according to cardiovascular criteria (DCD), where donor hypoxia can persist between the time of DCD and the time of organ retrieval.

[0467] To understand whether OSKM can overcome ischemic injury, kidney perfusion with OSKM was performed in an allogeneic transplantation model (LEW to LEW, lack of allogeneic immune injury) (see [link to study]). Figure 3A ).like Figure 3A As shown, 3-month-old donor kidneys tolerated NMP for approximately 1 hour, during which they were treated with inducible AAV-OSK and AAV c-Myc / rtTA (1x10⁻¹ of each vector). 12 Total titer 2x10 12 One week post-transplantation, the contralateral (right) kidney was then removed. Starting one week post-transplantation, OSKM expression was driven by rtTA activation through doxycycline (Dox) added to drinking water approximately four days per week. The control kidney was then perfused, but AAV was not administered. In the AAVOSKM group, Dox administration was discontinued on day 105 post-transplantation (56 days after Dox treatment). Organs were collected on day 105 post-transplantation for analysis.

[0468] In this ischemic model, organ donation surgery and normothermic perfusion (1 hour) were associated with ischemic injury and ischemia-reperfusion lesions in the isolated perfused kidney, despite continuous oxygenation of the perfusion fluid. The perfusion fluid in this model, a Grignard equilibrium solution, lacked oxygen carriers, which is believed to lead to reduced oxygen transport to cells. Ten of the eleven animals in the control group died by day 63 or earlier. Compared to the OSKM treatment group, three animals died within the first 15 days; however, six animals survived to the end of the study (approximately 125 days). Renal function was stable in the six surviving rats, evident from the stable serum creatinine and blood urea nitrogen (BUN) levels observed at the end of the study.

[0469] Ex vivo delivery of OSKM significantly improved rat survival in an syngeneic kidney injury model, as the survival rate of OSKM-treated animals was >60% at 100 days post-kidney transplantation (see [link to original text]). Figure 3B Importantly, compared to the control (see...). Figure 3C , Figure 9C-9D -D and Figure 10C Compared to the OSKM treatment group, the serum creatinine level was lower (see...). Figure 3D , Figure 9C-9D and Figure 10C This difference is statistically significant (see [link to relevant documentation]). Figure 3E , Figure 9C-9D and Figure 10C ). Compared with the control group (see Figure 3F , Figures 9A-9B , Figure 10B Compared to the OSKM treatment group, Figure 3G , Figures 9A-9B , Figure 10A The BUN levels in the samples were also lower (the expected effect). This difference was statistically significant (see [link to relevant documentation]). Figure 3H , Figures 9A-9B , Figure 10A and Figure 10B Macroscopic examination of the kidneys harvested from the OSKM group revealed no tumors or teratomas in the OSKM-treated kidneys.

[0470] Overall, the results indicate that partial reprogramming with OSKM improves ischemic injury in transplanted kidneys. The results also demonstrate that transplanted kidney function was preserved after syngeneic transplantation in an ischemic ex vivo transplantation model.

[0471] Example 5. Short-term safety study of OSKM treatment in rat liver

[0472] In Wistar rats (220–250 g) with healthy livers, the infection and safety of AAV-OSKM were evaluated for up to one week via administration of the AAV-OSKM vector (intravenous administration via the penis). In the control group, rats were administered PBS via the penis, and on day 6 after PBS administration, doxycycline (diluted to 1 mg / mL in drinking water) was administered. On day 7, rats were sacrificed, and liver, blood, and kidneys were collected (kidneys served as controls in this model). In the vector control group, the vector (AAV-GFP / Luc, 1 x 10⁻⁶ per rat) was administered via the penis. 12 On day 6 following the administration of the medium, doxycycline (diluted to 1 mg / mL) was administered to the drinking water. On day 7, the rats were sacrificed and their livers, blood, and kidneys were collected (as controls).

[0473] In the treatment group, AAV-OSK+AAV-cMyc-rtTA vector (1x10⁻¹ per rat) was administered via the penile route. 12 Each carrier was administered individually, and on day 6 post-carrier administration, doxycycline (diluted to 1 mg / mL) was administered to drinking water. On day 7, rats were sacrificed and liver, blood, and kidneys were collected (kidneys served as controls). Blood and tissue samples were then collected for examination. Liver damage, inflammation, and regeneration / tissue repair were evaluated. Broadly standardized liver pathogenesis parameters (e.g., AST, ALT, and bilirubin), apoptosis parameters (e.g., caspase 3), and hepatocyte proliferation (e.g., Ki67 / PCNA, HGF, and TGF-β) were analyzed. Regarding liver inflammation: neutrophil accumulation was assessed by myeloperoxidase (MPO) activity, and oxidative stress was assessed by malondialdehyde (MDA) levels. Liver damage was evaluated using histological analysis (H&E) (10X). Data are presented as mean ± standard error and statistically analyzed by the Kruskal-Wallis test, followed by post-hoc Dunn multiple comparisons test if necessary. p < 0.05 was considered significant.

[0474] Results of biochemical parameters (AST, ALT, bilirubin) indicating liver damage and liver function showed that the levels of AST, ALT, and bilirubin were similar across all study groups (see [link to study]). Figure 5A , Figure 5B and Figure 5D Compared with the control group, a decrease in caspase-3 levels was observed in the AAV-OSKM treatment group (see [link to treatment group]). Figure 5C ).

[0475] Liver inflammatory parameters (neutrophil accumulation and oxidative stress) determined by MPO and MDA levels were similar across all groups (see [link]). Figure 5E and Figure 5F This indicates the absence / absence of liver inflammation when AAV-OSKM is administered (intravenously). Furthermore, parameters for liver proliferation / tissue repair, determined by Ki67, PCNA, and HGF levels, were similar across all groups (see [link to relevant documentation]). Figure 5G , Figure 5H and Figure 5I Compared to the control group, a decrease in TGFβ (a potent pro-proliferative and fibrotic molecule) was observed in the AAV-OSKM treatment group (see [link to relevant documentation]). Figure 5J ).

[0476] No obvious necrotic lesions were observed in any of the evaluated groups (see [link]). Figure 5K , Figure 5L and Figure 5M This is consistent with the results of biochemical measurements.

[0477] These results demonstrate that short-term in vivo treatment is safe, as determined by measuring parameters of liver damage and function, liver inflammation, and liver repair / regeneration.

[0478] Example 6. Long-term safety study of OSKM treatment in rat liver

[0479] The safety of AAV-OSKM infection was evaluated for up to 12 weeks following intravenous administration of AAV-OSKM to Wistar rats (220–250 g) with healthy livers. In the control group, rats were administered PBS via the penile route (interchangeably referred to as penic) and doxycycline (diluted to 1 mg / mL in drinking water) for 24 hours starting on day 6 after PBS administration. Oral doxycycline was administered once weekly for 12 weeks. Rats were then sacrificed after 12 weeks (treated with Dox), and liver, blood, pancreas, kidney, adipose tissue, skin, spleen, heart, lungs, intestines, and muscle were collected. In the vector control group, the vector (AAV-GFP / Luc, 1 x 10⁻⁶ per rat) was administered via the penile route. 12 (1) Starting from day 6 after administration of the medium, doxycycline was administered to drinking water for 24 hours (diluted to 1 mg / mL in drinking water) and continued orally for 12 weeks for 1 day / week. At this time, the rats were sacrificed and liver, blood, pancreas, kidney, adipose tissue, skin, spleen, heart, lung, intestine and muscle were obtained.

[0480] In the treatment group, AAV-OSK+AAV-cMyc vector (1x10⁻¹ per rat) was administered via the penile route. 12 Each carrier was administered individually, and starting on day 6 after carrier administration, doxycycline was administered to drinking water for 24 hours (diluted to 1 mg / mL in drinking water). Oral doxycycline was continued for 12 weeks, once daily. After 12 weeks, rats were sacrificed, and liver, blood, pancreas, kidneys, adipose tissue, skin, spleen, heart, lungs, intestines, and muscle were collected.

[0481] Biochemical parameters of liver damage and liver function (AST, ALT, bilirubin) showed similar levels of AST, ALT, and bilirubin across all groups (see [link to relevant documentation]). Figure 6A , Figure 6B and Figure 6D The same applies to the level of caspase-3 (see...). Figure 6C ). Parameters of liver inflammation (neutrophil accumulation - measured by MPO and oxidative stress - measured by MPO, (see...) Figure 6E and Figure 6F ) and parameters of liver proliferation / liver tissue repair (levels of Ki67, PCNA, HGF, and TGFβ, (see...) Figure 6G, 6H No differences were observed between the groups for 6I and 6J. Liver histology in control and AAV-OSKM-treated rats showed no liver damage at 12 weeks (see [link to relevant documentation]). Figure 6K , Figure 6L and Figure 6M Data demonstrate that intravenous administration of AAV-OSKM and weekly cyclic Dox activation for 12 weeks are safe.

[0482] Example 7. The role of AAV-delivered pre-injury liver reprogramming in rats in a model of liver ischemia-reperfusion injury (an acute clinically relevant liver injury).

[0483] The role of pre-injury OSKM reprogramming was evaluated in a rat model of segmental (70%) liver ischemia (also known as hot liver ischemia-reperfusion injury). This model is generally described in Peralta et al., J Hepatology 59, 1094-1106 (2013).

[0484] Acute warm hepatic ischemia-reperfusion injury was induced by clamping major blood vessels to the liver for one hour and then releasing the clamps for varying time periods. This generated warm hepatic ischemia-reperfusion (I / R) injury, which was modeled in relation to I / R injuries observed during hepatectomy and liver transplantation. Warm hepatic ischemia-reperfusion injury occurs with surgical resection of the liver because major blood vessels connected to the liver are clamped during the procedure to prevent blood loss during hepatectomy (i.e., liver resection), the removal of part or all of the liver. Acute warm ischemia-reperfusion injury is also associated with the anhepatic period (the time from the physical removal of the liver from the recipient to graft recirculation) during liver transplantation. The remaining liver after surgical resection is then able to regenerate to replace the removed portion; however, warm ischemia-reperfusion can negatively impact the regeneration of the remaining liver after hepatectomy, leading to serious postoperative complications. Warm ischemia / reperfusion injury associated with liver transplantation can result in adverse postoperative outcomes.

[0485] PBS (control) and mediator control were administered to rats via the penile route (5x10). 11 5 x 10 AAV-GFP / Luc+ 11 AAV-rtTA) and OSKM AAV (AAV-OSK+AAV-cMyc vector (5x10) 11 One AAV-OSK + 5x10 11 Animals were treated with doxycycline after delivering AAV (cMyc / rtTA) or control to the liver and then treated with doxycycline at different time intervals (1 day, 3 days, or 5 days) to induce OSKM expression, as described below.

[0486] To induce warm hepatic ischemia-reperfusion injury, animals were anesthetized with isoflurane. After anesthesia, a midline laparotomy was performed, and the hepatic artery and portal vein of the left and middle lobes of the liver were occluded for 60 minutes. Reperfusion was initiated by removing the occlusion clamps. Four hours after reperfusion, the animals were sacrificed, and organs / tissues were collected for analysis.

[0487] Liver pathological parameters (AST, ALT, and bilirubin), apoptosis parameters (cysteine ​​3), and hepatocyte proliferation (Ki67 / PCNA, HGF, and TGF-β) were analyzed. Regarding liver inflammation: neutrophil accumulation was assessed by MPO activity, and oxidative stress was assessed by MDA levels. Data are expressed as mean ± standard error and were statistically analyzed using the Kruskal-Wallis test, followed by a post-hoc Dunn multiple comparison test if necessary. p < 0.05 was considered statistically significant.

[0488] Test the 1-week, 4-day, and 2-day protocols.

[0489] During the one-week protocol, PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA) were administered. On day 6 post-administration, doxycycline was added to the rats' drinking water (diluted to 1 mg / mL). On day 7 post-administration, liver infusion / reperfusion was performed as described above. Four hours after reperfusion, the rats were sacrificed and organs / tissues were harvested.

[0490] Compared with the liver I / R effect of the control mediator group + OSKM, intravenous administration of OSKM (AAV-OSK + AAV-cMyc + AAV-rtTa) one week prior to induced partial hepatic ischemia-reperfusion induced statistically significant reductions in the levels of transaminases (i.e., AST and ALT), biochemical markers of liver injury and function. However, no difference in bilirubin levels was observed between the two groups. More time may be needed post-injury to detect changes in BUN measurements, as such differences are typically observed at longer reperfusion times. Apoptosis parameters (cysteine ​​3), hepatocyte proliferation (Ki67 / PCNA, HGF), neutrophil accumulation (MPO activity), and oxidative stress (MDA levels) were also assessed. No significant differences were observed for any of these measurements, indicating that the observed beneficial effects were not due to changes in oxidative stress, neutrophil accumulation, and / or cell proliferation parameters (see [link to relevant documentation]). Figures 12A-12H ).

[0491] In the four-day regimen, PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA) were administered as described above. On day 3 post-administration, doxycycline was added to the rats' drinking water (diluted to 1 mg / mL). On day 4 post-administration, liver infusion / reperfusion was performed as described above. Four hours after reperfusion, the rats were sacrificed and organs / tissues were harvested. Treatment with AAV-OSK+AAV-cMyc+AAV-rtTA in the livers subjected to liver infusion / reperfusion induced transaminases (assessed by ALT and AST, see [reference]). Figures 13A-13B )), oxidative stress (measured by MDA (see Figure 13E The value of )) decreased. No effect was observed on the levels of caspase 3, MPO, PCNA, Ki67, HGF, and TGFB (see [references] for details). Figure 13C-13D and Figure 13F-13I ).

[0492] In the two-day protocol, PBS control, mediator control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA) were administered as described above. On day 1 post-administration, doxycycline was added to drinking water (diluted to 1 mg / mL). On day 2 post-administration, liver infusion / reperfusion was performed as described above. Four hours after reperfusion, rats were sacrificed and organs / tissues were harvested.

[0493] For example, in 4 days (see Figure 13A and Figure 13B ) and the 1-week plan (see Figure 12A and Figure 12B As observed in [reference needed], AST and ALT levels were not decreased in the OSKM-AAV treatment group compared to the control group (PBS and mediator). ALT levels were observed in rats with warm ischemia compared to the control group without warm ischemia (see [reference needed]). Figure 14A ) and AST level (see Figure 14B The levels of caspase 3, MPO, MDA, PCNA, Ki67, HGF, and TGFb were significantly elevated between the control and treatment arms (see [references]). Figure 14C-14I ).

[0494] Therefore, in the warm ischemia-induced liver injury model, at least 3 days were required between AAV injection and doxycycline activation of OSKM to observe the protective effect of liver injury markers. In contrast, no beneficial effect was observed when OSKM was activated only 2 days after AAV injection.

[0495] Therefore, treatment with OSKM prior to acute liver injury due to warm hepatic ischemia-reperfusion produced beneficial effects, especially when AAV was injected 4 days or 1 week before injury, when assessed by liver injury markers 4 hours after reperfusion. In contrast, treatment with OSKM only when AAV was injected 2 days before injury did not show remarkable effects (because OSKM expression from the AAV vector takes more than two days).

[0496] Example 8. OSKM treatment improves ischemic injury in a kidney transplant model following cardiac arrest death.

[0497] Ischemia-reperfusion injury is a major cause of acute kidney injury during in vivo kidney and organ transplantation. Depending on the severity of ischemic injury, tubular lesions and renal function impairment may resolve within a few days (usually 3 weeks in humans) or lead to chronic tubulointerstitial lesions and fibrosis (Chawla et al., “Acute kidney injury and chronic kidney disease as interconnected syndromes”, NEJM 371(1):58-66(2014)). To closely simulate ischemia induced by cardiac death, an animal (rat) model of kidney transplantation following warm kidney ischemia induced by cardiac arrest (45 minutes) was studied (see [link to study]). Figure 4 ).

[0498] First, rats were subjected to cardiac arrest, which caused immediate cessation of renal artery blood flow. Cardiac arrest was induced by clamping the donor's ascending aorta for 45 minutes prior to kidney harvesting. Kidney transplant perfusion was performed for 1 hour using either control perfusion (without AAV) or treatment (perfusion in the presence of AAV / DJ-OSK + AAV / DJ cMyc-rtTA).

[0499] Kidney transplantation was performed using 9-0 continuous nylon sutures, with end-to-side anastomoses between the aortic root of the donor kidney and the recipient aorta, as well as between the recipient's inferior vena cava and the donor renal vein. The mean anastomosis time was 24.8 ± 3.3 minutes. Ureteroureterostomy was performed using 11-0 nylon sutures with an end-to-end interrupted suture technique. The ipsilateral native kidney was removed at implantation (kidney transplantation). After systemic administration of heparin sodium (1000 UI, Rovi), the donor kidney was flushed with sterile saline at 4°C. Non-perfused nephrectomy of the kidney was performed 7 days post-transplantation to prevent early death due to acute kidney injury from ischemic kidney disease.

[0500] Then, starting from the day of transplantation, 1 mg / mL doxycycline (Sigma-Aldrich) was administered to the recipient rats' drinking water for 4 days during the first week post-surgery. From the second week post-transplantation until sacrifice, doxycycline (DOX) was administered weekly, cycling between 2 days of DOX administration per week, followed by 5 days without DOX administration. Following contralateral nephrectomy (i.e., 1 week post-transplantation), blood was analyzed every other day using the EPOC blood analysis system. Blood tests were performed to measure blood urea nitrogen (BUN) and serum creatinine (SCr) until day 14 post-transplantation. Thereafter, blood and urine analyses (proteinuria and microalbuminuria) were performed weekly until the animal was euthanized.

[0501] Eight days post-transplantation (i.e., one day after contralateral nephrectomy), serum creatine (SCr) levels were similar in the control and treatment groups (0.59 ± 0.16 mg / dL in the control group and 0.52 ± 0.03 mg / dL in the treatment group, P = 0.12) (see [link to relevant documentation]). Figure 15A ).

[0502] However, compared to the control group who underwent their first kidney transplant, the SCr in the OSKM group began to decline at day 10 post-transplant, with improvement not observed until day 12. The worst renal function was achieved at day 10 (see [link to original text]). Figure 15A Notably, despite improvements in the transplanted kidney, the SCr value remained consistently higher during follow-up compared to the OSKM group. Figure 1A These results indicate that OSKM treatment improves renal repair following ischemic injury, reduces ischemia-induced chronic lesions, and increases long-term renal function.

[0503] Increased BUN levels were observed in both groups at 8 and 10 days post-transplantation (90.56±33.01 mg / dL and 106.73±33.85 mg / dL for the control group and 102.50±19.79 mg / dL and 112.91±15.89 mg / dL for the treatment group at 8 and 10 days post-transplantation, respectively, P=0.26) (see [link to relevant documentation]). Figure 15B Following this time point, BUN levels decreased in both groups. This decrease occurred earlier in the treated animals (109.10 ± 36.57 mg / dL for the control group and 74.72 ± 30.10 mg / dL for the treatment group, P = 0.01). This accelerated improvement observed in the treatment group throughout the experiment resulted in better renal function compared to the control group (see Table 1).

[0504] Table 1. Scores and total Remuzzi score for the improved Remuzzi scoring criterion.

[0505] Comparison mOSKM Renal tubular atrophy 1.70+0.67 1.14+0.38 Interstitial fibrosis** 2.1+0.32 1.43+0.53 ATN rating 2.4+0.84 1.29+0.49 Overall score 6.2+.140 3.86+1.21

[0506] Similar kinetics were observed for total urinary protein and albumin: urinary protein levels were comparable in both groups until 28 days post-transplantation, with an increase observed in the control group. Significantly, from 35 days post-transplantation, both total urinary protein (57.89±41.68 mg / 24h in the control group and 30.76±9.86 mg / 24h in the treatment group, P=0.02) and urinary albumin (7.22±10.72 mg / 24h in the control group and 0.63±0.52 mg / 24h in the treatment group, P=0.01) increased significantly. These differences persisted during follow-up (see [link to follow-up]). Figure 15C These results demonstrate that OSKM treatment improves renal repair following ischemic injury, reduces ischemia-induced chronic lesions, and increases long-term renal function.

[0507] Regarding hemodynamic monitoring of the perfused kidneys during normothermic perfusion, we observed an initial increase in vascular resistance in both groups, which gradually decreased during perfusion. However, in the OSKM-treated kidneys, this resistance remained higher t...

Claims

1. An organ perfusion solution comprising a first polynucleotide encoding at least one regeneration factor and at least one vasodilator.

2. The organ perfusion fluid of claim 1, wherein the first polynucleotide encoding the at least one regeneration factor is operatively linked to a promoter.

3. The organ perfusion solution as described in claim 1 or 2, further comprising at least one tensioning agent.

4. The organ perfusion solution according to any one of claims 1-3, further comprising at least one of a buffer, inorganic salt, amino acid, metabolic substrate, hormone, antioxidant, anti-inflammatory agent, anticoagulant or antimicrobial agent.

5. The organ perfusion solution according to any one of claims 1-3, further comprising a composition of human serum albumin, dextran, and extracellular electrolytes.

6. The organ perfusion solution according to any one of claims 3-5, wherein the tensioning agent is selected from dextran, glycerol, mannitol, potassium chloride, sodium chloride, and combinations thereof.

7. The organ perfusion solution according to any one of claims 1-6, wherein the vasodilator is selected from carbon monoxide, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, calcium channel blockers, prostacyclin, hydralazine, minoxidil, nitroglycerin, and combinations thereof.

8. The organ perfusion fluid according to any one of claims 1-7, further comprising an oxygenating agent.

9. The organ perfusion solution of claim 8, wherein the oxygenating agent is selected from erythrocytes, hemoglobin, pyridine-oxygenated hemoglobin, oxygen carriers based on synthetic hemoglobin, and combinations thereof.

10. The organ perfusion fluid of claim 9, wherein the synthetic hemoglobin-based oxygen carrier is a polymerized hemoglobin-based oxygen carrier, Lifor TM Aquix RS-I Or perfluorocarbon.

11. The organ perfusion fluid according to any one of claims 1-10, further comprising a second polynucleotide operably linked to a promoter encoding at least one regeneration factor.

12. The organ perfusion fluid according to any one of claims 1-11, wherein the promoters of the first polynucleotide and the second polynucleotide are inducible promoters.

13. The organ perfusion fluid according to any one of claims 1-12, wherein the at least one regeneration factor encoded by the first polynucleotide is selected from Oct family transcription factors, Sox family transcription factors, Klf family transcription factors, and Myc family transcription factors.

14. The organ perfusion fluid of any one of claims 11-13, wherein the at least one regeneration factor encoded by the second polynucleotide is selected from Oct family transcription factors, Sox family transcription factors, Klf family transcription factors, and Myc family transcription factors.

15. The organ perfusion fluid of claim 13 or 14, wherein the Oct family transcription factors are selected from Oct1, Oct3, Oct4, Oct6 and their variants.

16. The organ perfusion fluid of claim 13 or 14, wherein the Sox family transcription factor is selected from Sox1, Sox2, Sox3, Sox7, Sox15, Sox17, Sox18 and variants thereof.

17. The organ perfusion fluid of claim 13 or 14, wherein the Klf family transcription factors are selected from Kfl1, Klf4, Klf5 and their variants.

18. The organ perfusion fluid of claim 13 or 14, wherein the Myc family transcription factor is selected from c-Myc, L-Myc, N-Myc and variants thereof.

19. The organ perfusion fluid according to any one of claims 1-18, wherein the first polynucleotide, the second polynucleotide, or both are encapsulated in nanoparticles.

20. The organ perfusion solution of claim 19, wherein the nanoparticles are lipid nanoparticles, polymer nanoparticles, ligand-conjugated lipid nanoparticles, or ligand-conjugated polymer nanoparticles.

21. The organ perfusion fluid according to any one of claims 1-20, wherein the first polynucleotide, the second polynucleotide, or both are present in a viral genome, plasmid, microcircular vector, or transposon.

22. The organ perfusion fluid of claim 21, wherein the viral genome is selected from AAV genome, adenovirus genome, retrovirus genome, or lentivirus genome.

23. The organ perfusion fluid according to any one of claims 1-22, wherein the perfusion fluid further comprises a regeneration factor-protein transfer domain fusion protein.

24. The organ perfusion solution according to any one of claims 1-23, wherein the perfusion solution further comprises a regeneration factor polypeptide.

25. The organ perfusion fluid according to any one of claims 1-24, wherein the perfusion fluid further comprises Oct family transcription factors, Sox family transcription factors, Klf family transcription factors and / or Myc family transcription factors.

26. The organ perfusion fluid of claim 25, wherein the Oct family transcription factors are selected from Oct1, Oct3, Oct4, Oct6 and their variants.

27. The organ perfusion fluid of claim 25 or 26, wherein the Sox family transcription factor is selected from Sox1, Sox2, Sox3, Sox7, Sox15, Sox17, Sox18 and variants thereof.

28. The organ perfusion fluid according to any one of claims 25-27, wherein the Klf family transcription factors are selected from Kfl1, Klf4, Klf5 and their variants.

29. The organ perfusion fluid according to any one of claims 25-28, wherein the Myc family transcription factor is selected from c-Myc, L-Myc, N-Myc and variants thereof.

30. The organ perfusion fluid according to any one of claims 1-29, further comprising a enhancer selected from soluble Wnt, Wnt conditioned medium, BIX-01294 (G9a histone methyltransferase), PD0325901 (MEK inhibitor), DNA methyltransferase inhibitor, histone deacetylase (HDAC) inhibitor, valproic acid, 5'-azacytidine, dexamethasone, succinylanilide hydroxamic acid (SAHA), vitamin C, and trichosuccinimide (TSA), and combinations thereof.

31. A method for the reconstructive function of an ex vivo organ, the method comprising: (a) To provide organs; (b) Contact the isolated organ with a composition comprising a polynucleotide encoding at least one regeneration factor, the polynucleotide being operatively linked to an inducible promoter; as well as (c) Adding a compound that induces the inducible promoter to the composition to promote the expression of the at least one regenerative factor, thereby expressing the at least one regenerative factor and restoring the organ.

32. The method of claim 31, wherein the compound is added intermittently.

33. The method of claim 32, wherein the intermittent addition comprises adding the compound once a day for two consecutive days, and not adding the reagent for the next five consecutive days.

34. The method of claim 31 or 32, wherein the intermittent addition is performed 2-10 times.

35. The method of claim 34, wherein the intermittent addition is performed 2-10 times over a period of about 1 week to about 6 weeks.

36. The method according to any one of claims 31-35, wherein the composition further comprises a vasodilator.

37. The method according to any one of claims 31-36, wherein the composition further comprises a tension agent.

38. The method of any one of claims 31-37, wherein the composition further comprises at least one of a buffer, an inorganic salt, an amino acid, a metabolic substrate, a hormone, an antioxidant, an anti-inflammatory agent, an anticoagulant, or an antimicrobial agent.

39. The method of any one of claims 31-38, wherein the organ is provided in a perfusion system selected from the Hugo-Sachs system, organ assist system, OrganOX system, Radnoti system, ARK kidney system, and Aferetica system. system.

40. The method of any one of claims 31-39, wherein the organ is the kidney or the liver.

41. A method for transplanting an organ into a subject in need, the method comprising: (a) To provide organs; (b) Contact the isolated organ with a composition comprising a polynucleotide encoding at least one regeneration factor, the polynucleotide being operatively linked to an inducible promoter; (c) adding a compound that induces the inducible promoter to the composition; and (d) Transplanting the organ into the subject.

42. The method of claim 41, wherein the compound is added to the composition within about 1 minute to about 24 hours.

43. The method of claim 41 or 42, wherein the compound is added to the composition intermittently.

44. The method of claim 43, wherein the intermittent addition of the composition comprises adding the compound once a day for two consecutive days, and not adding the compound for the next five consecutive days.

45. The method of claim 44, wherein the intermittent addition of the compound is repeated 2-10 times.

46. ​​The method of claim 45, wherein the intermittent addition is performed 2-10 times over a period of about 1 week to about 6 weeks.

47. A method for transplanting an organ into a subject in need, the method comprising: (a) To provide organs; (b) Contact the isolated organ with a composition comprising a polynucleotide encoding at least one regeneration factor, the polynucleotide being operatively linked to an inducible promoter; (c) Transplanting the organ into the subject; and (d) Administering the subject a compound that induces the inducible promoter.

48. The method of claim 47, wherein the compound is administered to the subject within about 1 minute to about 24 hours.

49. The method of claim 47 or 48, wherein the compound is administered intermittently to the subject.

50. The method of claim 49, wherein the intermittent application of the composition comprises applying the compound once a day for two consecutive days, and not applying the compound for the next five consecutive days.

51. The method of claim 49 or 50, wherein the intermittent application of the compound is repeated about 2 to 10 times.

52. The method of claim 51, wherein the intermittent application of the compound is performed approximately 2-10 times every 3 months.

53. The method of claim 52, wherein the intermittent application of the compound is performed approximately 2-10 times every 6 months.

54. The method according to any one of claims 31-53, wherein the composition further comprises a vasodilator.

55. The method according to any one of claims 31-54, wherein the composition further comprises a tensioning agent.

56. The method according to any one of claims 31-55, further comprising at least one of a buffer, an inorganic salt, an amino acid, a metabolic substrate, a hormone, an antioxidant, an anti-inflammatory agent, an anticoagulant, or an antimicrobial agent.

57. The method of any one of claims 31-56, wherein the transplanted organ is provided in a perfusion system selected from the Hugo-Sachs system, organ assist system, OrganOX system, Radnoti system, ARK kidney system, and Aferetica system. system.

58. The method of any one of claims 31-57, wherein the organ is the kidney or liver.

59. The method of any one of claims 31-58, wherein the method further comprises obtaining a biopsy from the organ prior to transplantation.

60. The method of any one of claims 31-58, wherein the method further comprises obtaining a biopsy of the organ after transplantation.

61. The method of any one of claims 31-60, further comprising monitoring organ function in a subject who has received said organ transplant.

62. The method of claim 61, wherein the monitoring comprises measuring one or more of the following: blood urea level, serum creatinine level, bilirubin level, blood pH, blood bicarbonate level, blood sodium level, blood potassium level, or blood lactate level.

63. The method of any one of claims 31-62, further comprising administering an immunosuppressive drug to the subject.

64. An ex vivo organ for transplantation to a subject in need, the ex vivo organ comprising: (i) a perfusion fluid; and (ii) a polynucleotide encoding at least one regeneration factor.

65. The ex vivo organ of claim 64, wherein the ex vivo organ is damaged by at least one of the following: aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, fatty degeneration, virus-induced hepatitis, alcohol, or fibrosis unrelated to any known cause.

66. The ex vivo organ of claim 64 or 65, wherein the polynucleotide further comprises an inducible promoter operatively linked to the polynucleotide encoding the at least one regeneration factor.

67. The ex vivo organ of any one of claims 64-66, wherein the perfusion fluid intermittently contains a compound that induces the inducible promoter.

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