Use of regenerative factors in organ transplantation

Abstract

Materials and methods are provided for ex vivo organ rejuvenation prior to transplantation into a subject in need thereof. The methods and materials can be used to treat, improve, rejuvenate, and / or regenerate organs, such as kidneys and livers, that are aged and / or characterized by organ damage resulting from ischemic injury, toxic injury, perfusion injury, hypertensive injury, immune injury, physical injury, steatosis, viral-induced hepatitis, alcohol, or fibrosis unrelated to any known cause. The provided materials and methods can increase the pool of available donor organs, such as kidneys and livers. Furthermore, the provided materials and methods can be used to treat organs that are then autologously transplanted.

Description

[Technical Field]

[0001] Reference to sequence listings submitted electronically via EFS-WEB The contents of the electronically submitted Sequence Listing (Name: 4967_023PC01_Seqlisting_ST26.XML, Size: 166,421 bytes, Created: January 19, 2024) submitted with this application are hereby incorporated by reference in their entirety.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from EP Application No. 23382042.2, filed January 19, 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0003] At any given time, tens of thousands of patients in countries around the world are on waiting lists for organ transplants and are unable to receive treatment due to a shortage of suitable organs. In aging populations, the potential number of available aging organs increases, but insufficient organ quality often prevents their use in transplants. This need is particularly acute in common chronic diseases that affect organs such as the kidney and liver.

[0004] Chronic kidney disease (CKD) represents a global health burden due to the aging population and the high and increasing prevalence of diabetes and hypertension worldwide. Renal aging is associated with a decline in glomerular filtration rate, increased vascular and interstitial lesions, and increased susceptibility to acute kidney injury (e.g., ischemia-reperfusion injury (Weinstein, JR & Anderson, S. Advances in Chronic Kidney Disease vol. 17 302-307, 2010)). Currently, transplantation is the best replacement therapy for end-stage renal disease, but many patients are unable to receive a transplant in a timely manner due to organ shortages. Furthermore, 20–30% of organs harvested from donors are discarded due to insufficient quality for transplantation, increasing shortages and delaying life-saving transplants. Donor organ rejection rates are extremely high in the United States, especially among marginal donors (Aubert et al., JAMA Int. Med. 179(10):1365-74, 2019). The need for kidneys and other organs is not being met, and one possibility is to expand the donor pool to include donor organs currently considered to be of insufficient quality, such as aging organs, which are often considered to be of insufficient quality due to a history of hypertension or other pathologies (Ojo, AO et al. American Journal of Transplantation vol. 4 27-37, 2004).

[0005] Chronic liver disease is also a global burden due to the aging population and the high and increasing prevalence of diseases worldwide, such as hepatitis, hepatocellular carcinoma (HCC), and cirrhosis. Currently, transplantation is the best replacement therapy for end-stage liver disease and HCC, but many patients cannot receive a transplant in a timely manner due to organ shortages. The current shortage of donor livers can be addressed by expanding the donor pool to include elderly donors by repairing dysfunction in donor livers currently considered to be of insufficient quality, such as aging livers.

[0006] Reprogramming of somatic cells 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 long and complex, and complete reprogramming can lead to tumorigenesis (Takahashi, K. et al. Cell 131, 861-872, 2007; Takahashi, K. & Yamanaka, S. Cell 126, 663-676, 2006; Polo, J. Met al. Cell 151, 1617-1632, 2012; Abad, M. et al. Nature 502, 340-345, 2013). Therefore, alternative strategies to reverse the consequences of donor organ aging are needed.

[0007] Partial reprogramming has emerged as an alternative to complete reprogramming and a potential treatment for the physical effects of aging. However, challenges in translating reprogramming from basic research to clinical application need to be addressed, both in general and in kidneys and organs specifically. For example, viral vectors do not deliver therapeutic agents to the kidney when injected systemically because infection is generally inefficient, with most vectors taken up by the liver, and / or vector doses are limited by off-target effects (Rubin, J. Det al. Mol Diagn Ther 24, 375-396, 2020). Therefore, specific therapies that improve transplanted organ function and / or outcomes and / or increase the number of donor organs by correcting dysfunction in organs considered suboptimal or poor candidates for transplantation are needed to address the increasing need for donor organs and expand the donor organ pool for transplantation. Summary of the Invention

[0008] The present disclosure provides an organ perfusion solution comprising a first polynucleotide encoding at least one regeneration factor and at least one vasodilator. In some embodiments, the first polynucleotide encoding the at least one regeneration factor is operably linked to a promoter. In some embodiments, the organ perfusion solution further comprises at least one tonicity agent. In some embodiments, the organ perfusion solution 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 antibacterial agent. In some embodiments, the organ perfusion solution further comprises human serum albumin, dextran, and an extracellular electrolyte composition.

[0009] In some embodiments, the tonicity agent is selected from the group consisting of dextrose, glycerin, mannitol, potassium chloride, sodium chloride, and combinations thereof. In some embodiments, the vasodilator is selected from the group consisting of carbon monoxide, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, calcium channel blockers, prostacyclin, hydralazine, minoxidil, nitroglycerin, and combinations thereof.

[0010] In some embodiments, the organ perfusion solution further comprises an oxygenating agent. In some embodiments, the oxygenating agent is selected from the group consisting of red blood cells, hemoglobin, pyridoxylated hemoglobin, a synthetic hemoglobin-based oxygen carrier, and combinations thereof. In some embodiments, the synthetic hemoglobin-based oxygen carrier is a polymerized hemoglobin-based oxygen carrier, Lifor™, Aquix RS-I, Hemarina®, or a perfluorocarbon.

[0011] In some embodiments, the organ perfusion solution further comprises a second polynucleotide encoding at least one regeneration factor operably linked to a promoter.

[0012] In some embodiments, the promoters of the first polynucleotide and the second polynucleotide are inducible promoters.

[0013] In some embodiments, the at least one regeneration factor encoded by the first polynucleotide is selected from the group consisting of an Oct family transcription factor, a Sox family transcription factor, a Klf family transcription factor, and a Myc family transcription factor. In some embodiments, the at least one regeneration factor encoded by the second polynucleotide is selected from the group consisting of an Oct family transcription factor, a Sox family transcription factor, a Klf family transcription factor, and a Myc family transcription factor. In some embodiments, the at least one regeneration factor encoded by the second polynucleotide is different from or is not the same regeneration factor encoded by the first polynucleotide. In some embodiments, the Oct family transcription factor is selected from the group consisting of Oct1, Oct3, Oct4, Oct6, and variants thereof. In some embodiments, the Sox family transcription factor is selected from the group consisting of Sox1, Sox2, Sox3, Sox7, Sox15, Sox17, Sox18, and variants thereof. In some embodiments, the Klf family transcription factor is selected from the group consisting of Kfl1, Klf4, Klf5, and variants thereof. In some embodiments, the Myc family transcription factor is selected from the group consisting of c-Myc, L-Myc, N-Myc, and variants thereof.

[0014] In some embodiments, the polynucleotide, the second polynucleotide, or both are encapsulated in a nanoparticle. In some embodiments, the nanoparticle is a lipid nanoparticle, a polymer nanoparticle, a ligand-conjugated lipid nanoparticle, or a ligand-conjugated polymer nanoparticle.

[0015] In some embodiments, the polynucleotide, the second polynucleotide, or both are present in a viral genome, a plasmid, a minicircle vector, or a transposon. In some embodiments, the viral genome is selected from an AAV genome, an adenovirus genome, a retrovirus genome, or a lentivirus genome.

[0016] In some embodiments, the organ perfusate further comprises a regenerative factor-protein transport domain fusion protein.

[0017] In some embodiments, the organ perfusion solution further comprises a regeneration factor polypeptide.

[0018] In some embodiments, the organ perfusion solution further comprises an Oct family transcription factor, a Sox family transcription factor, a Klf family transcription factor, and / or a Myc family transcription factor. In some embodiments, the Oct family transcription factor is selected from the group consisting of Oct1, Oct3, Oct4, Oct6, and variants thereof. In some embodiments, the Sox family transcription factor is selected from the group consisting of Sox1, Sox2, Sox3, Sox7, Sox15, Sox17, Sox18, and variants thereof. In some embodiments, the Klf family transcription factor is selected from the group consisting of Kfl1, Klf4, Klf5, and variants thereof. In some embodiments, the Myc family transcription factor is selected from the group consisting of c-Myc, L-Myc, N-Myc, and variants thereof.

[0019] In some embodiments, the organ perfusion solution further comprises an enhancer selected from the group consisting of 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, suberoylanilide, hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), and combinations thereof.

[0020] The present disclosure also provides a method for rejuvenating an organ ex vivo, comprising providing an organ; contacting the organ ex vivo with a composition comprising a polynucleotide encoding at least one regenerative factor operably linked to an inducible promoter; and adding to the composition a compound that induces the inducible promoter to promote expression of the at least one regenerative factor, wherein addition of the compound results in expression of the at least one regenerative factor and rejuvenates the organ.

[0021] In some embodiments of the methods described herein, the compound is administered intermittently. In some embodiments, the intermittent administration comprises administering the compound once daily for two consecutive days, followed by five consecutive days without administering the compound. In some embodiments, the intermittent administration is performed 2 to 10 times. In some embodiments, the intermittent administration is performed 2 to 10 times over a period of about one week to about six weeks.

[0022] In some embodiments of the methods described herein, the composition further comprises a vasodilator. In some embodiments, the composition further comprises a tonicity agent. In some embodiments, the composition further comprises at least one of a buffering agent, an inorganic salt, an amino acid, a metabolic substrate, a hormone, an antioxidant, an anti-inflammatory agent, an anticoagulant, or an antibacterial agent.

[0023] In some aspects of the methods described herein, the organ is provided in a perfusion system selected from the Hugo-Sachs system, the Organ Assist system, the OrganOX system, the Radnoti system, the ARK Kidney system, and the Aferetica PerLife® system.

[0024] In some aspects of the methods described herein, the organ is a kidney or a liver.

[0025] The present disclosure also provides a method of transplanting an organ in a subject in need thereof, comprising providing the organ, contacting the organ ex vivo with a composition comprising a polynucleotide encoding at least one regenerative factor operably linked to an inducible promoter, adding to the composition a compound that induces the inducible promoter, and transplanting the organ into the subject.

[0026] In some embodiments 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 embodiments, the compound is added intermittently to the composition. In some embodiments, the intermittent addition of the compound comprises adding the compound once daily for two consecutive days, followed by not adding the compound for five consecutive days. In some embodiments, the intermittent addition of the compound is repeated 2 to 10 times. In some embodiments, the intermittent addition is repeated 2 to 10 times over a period of about 1 week to about 6 weeks.

[0027] The present disclosure also provides a method of transplanting an organ in a subject in need thereof, comprising providing the organ, contacting the organ ex vivo with a composition comprising a polynucleotide encoding at least one regenerative factor operably linked to an inducible promoter, transplanting the organ into the subject, and administering to the subject a compound that induces the inducible promoter.

[0028] In some embodiments of the methods described herein, the compound is administered to the subject over a period of about 1 minute to about 24 hours. In some embodiments, the compound is administered intermittently to the subject. In some embodiments, the intermittent administration of the compound comprises administering the compound once daily for two consecutive days, followed by five consecutive days without administering the compound. In some embodiments, the intermittent administration of the compound is repeated about 2-10 times. In some embodiments, the intermittent administration of the compound is administered about 2-10 times every three months. In some embodiments, the intermittent administration of the compound is administered about 2-10 times every six months. In some embodiments, the composition further comprises a vasodilator.

[0029] In some embodiments of the methods described herein, the composition further comprises an isotonicity agent, hi some embodiments, the composition further comprises at least one of a buffering agent, an inorganic salt, an amino acid, a metabolic substrate, a hormone, an antioxidant, an anti-inflammatory agent, an anticoagulant, or an antibacterial agent.

[0030] In some aspects of the methods described herein, the explanted organ is provided in a perfusion system selected from the Hugo-Sachs system, the Organ Assist system, the OrganOX system, the Radnoti system, the ARK Kidney system, and the Aferetica PerLife® system.

[0031] In some aspects of the methods described herein, the organ is a kidney or a liver.

[0032] In some aspects of the methods described herein, the methods further comprise taking a biopsy from the organ prior to transplantation.

[0033] In some aspects of the methods described herein, the methods further comprise taking a biopsy of the organ after transplantation.

[0034] In some embodiments of the methods described herein, the method further comprises monitoring the organ transplanted subject for organ function. In some embodiments, the monitoring comprises measuring one or more of blood urea levels, serum creatinine levels, bilirubin levels, blood pH, blood bicarbonate levels, blood sodium levels, blood potassium levels, or blood lactate levels.

[0035] In some aspects of the methods described herein, the methods further comprise administering an immunosuppressant to the subject.

[0036] The present disclosure also provides an ex vivo organ for transplantation into a subject in need thereof, the ex vivo organ comprising (i) a perfusion solution and (ii) a polynucleotide encoding at least one regenerative factor. In some embodiments, the ex vivo organ has been damaged by at least one of aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, virally induced hepatitis, alcohol, or fibrosis unrelated to any known cause. In some embodiments, the polynucleotide further comprises an inducible promoter operably linked to the polynucleotide encoding the at least one regenerative factor. In some embodiments, the perfusion solution intermittently comprises a compound that induces the inducible promoter. [Brief explanation of the drawings]

[0037] [Figure 1A] Direct injection of AAV vector into rat kidney via clamped renal vein and luciferin expression in injected and uninjected (contralateral) kidney 7 days after in situ injection are shown. [Figure 1B] In vivo bolusciferin levels are shown in a rat transplanted with a kidney perfused with the GFP-Luc AAV vector (left animal), a control rat (middle animal), and a rat systemically injected (via the penile vein) with the GFP-Luc AAV vector (right animal). [Figure 2A]Schematic diagram of allotransplantation of aged Dark Agouti kidneys perfused with AAV OSKM vectors into young Lewis recipient rats. To induce OSKM expression, the recipient rats were treated with doxycycline 2 days per week starting on day 7 post-transplant. [Figure 2B] H&E stained images of aged kidneys perfused with GFP-AAV after allograft transplantation are shown, demonstrating immune and ischemic injury. [Figure 2C] The survival rates (%) of rats allografted with GFP vector-perfused senescent kidneys and OSKM AAV vector-perfused senescent kidneys up to 110 days after transplantation are shown. Doxycycline treatment was discontinued on day 88 in these rats. [Figure 2D] BUN levels in rats 2 weeks after transplantation of aged kidneys perfused with GFP vector and aged kidneys perfused with OSKM AAV vector are shown. [Figure 2E] Serum creatinine (SCr) levels in rats transplanted with aged kidneys perfused with GFP AAV vector and OSKM AAV vector (right panel) are compared with those in rats transplanted with kidneys perfused with GFP AAV vector (left panel) up to 7 weeks after transplantation. [Figure 2F-1] Serum pH, HCO3, sodium, potassium, lactate, BUN, and serum creatinine levels are shown in rats 2 weeks after transplantation of senescent kidneys perfused with GFP AAV vectors and senescent kidneys perfused with OSKM AAV vectors. [Figure 2F-2] Serum pH, HCO3, sodium, potassium, lactate, BUN, and serum creatinine levels are shown in rats 2 weeks after transplantation of senescent kidneys perfused with GFP AAV vectors and senescent kidneys perfused with OSKM AAV vectors. [Figure 2G-1] Serum pH, HCO3, sodium, potassium, lactate, BUN, and serum creatinine levels are shown in rats 3 weeks after transplantation of senescent kidneys perfused with GFP AAV vectors and senescent kidneys perfused with OSKM AAV vectors. [Figure 2G-2]Serum pH, HCO3, sodium, potassium, lactate, BUN, and serum creatinine levels are shown in rats 3 weeks after transplantation of senescent kidneys perfused with GFP AAV vectors and senescent kidneys perfused with OSKM AAV vectors. [Figure 2H-1] Serum pH, HCO3, sodium, potassium, lactate, BUN, and serum creatinine levels are shown in rats 4 weeks after transplantation of senescent kidneys perfused with GFP AAV vectors and senescent kidneys perfused with OSKM AAV vectors. [Figure 2H-2] Serum pH, HCO3, sodium, potassium, lactate, BUN, and serum creatinine levels are shown in rats 4 weeks after transplantation of senescent kidneys perfused with GFP AAV vectors and senescent kidneys perfused with OSKM AAV vectors. [Figure 2I-1] Serum pH, HCO3, sodium, potassium, lactate, BUN, and serum creatinine levels are shown in rats 2 to 4 weeks after transplantation (1 to 3 weeks after nephrectomy) of senescent kidneys perfused with GFP AAV vectors and senescent kidneys perfused with OSKM AAV vectors. [Figure 2I-2] Serum pH, HCO3, sodium, potassium, lactate, BUN, and serum creatinine levels are shown in rats 2 to 4 weeks after transplantation (1 to 3 weeks after nephrectomy) of senescent kidneys perfused with GFP AAV vectors and senescent kidneys perfused with OSKM AAV vectors. [Figure 2J] BUN levels in rats 2 to 4 weeks after transplantation (1 to 3 weeks after nephrectomy) of senescent kidneys perfused with GFP AAV vector and senescent kidneys perfused with OSKM AAV vector are shown. [Figure 2K] Serum creatinine (Cr) levels in surviving rats up to 15 weeks after transplantation of aged kidneys perfused with GFP AAV vectors and aged kidneys perfused with OSKM AAV vectors are shown. [Figure 2L] 1 shows BUN levels in rats up to 14 weeks after transplantation of aged kidneys perfused with GFP AAV vectors and aged kidneys perfused with OSKM AAV vectors. [Figure 2M] 1 shows potassium levels in rats up to 12 weeks after transplantation of aged kidneys perfused with GFP AAV vectors and aged kidneys perfused with OSKM AAV vectors. [Figure 2N] Representative photographs of transplanted kidneys from the OSKM group (right) and normal kidneys (left) for comparison, harvested 100 days after transplantation, are shown. [Figure 3A] 1 shows a schematic diagram of syngeneic transplantation in an ischemic renal transplantation model in which young Lewis rat kidneys perfused with AAV OSKM vectors are transplanted into young Lewis recipient rats that have been chronically treated with doxycycline 4 days per week immediately after transplantation. [Figure 3B] Shown are the survival rates of rats transplanted with kidneys perfused with a no-AAV control and with OSKM AAV vectors. [Figure 3C] Serum creatinine levels in control rats transplanted with kidneys perfused with control AAV are shown (number of surviving animals (n) is indicated below the graph). [Figure 3D] Serum creatinine levels in rats transplanted with kidneys perfused with OSKM AAV are shown (number of surviving animals (n) is indicated below the graph). [Figure 3E] Serum creatinine levels are shown in rats transplanted with kidneys perfused with GFP AAV (square plots on graph) and rats transplanted with kidneys perfused with OSKM AAV (circular plots on graph). [Figure 3F] BUN levels in rats transplanted with kidneys perfused with control AAV are shown (number of surviving animals (n) is indicated below the graph). [Figure 3G] BUN levels in rats transplanted with kidneys perfused with OSKM AAV vectors are shown (number of surviving animals (n) is indicated below the graph). [Figure 3H] BUN levels are shown in rats transplanted with kidneys perfused with no AAV control (large circles) and rats transplanted with kidneys perfused with OSKM AAV (small circles). [Figure 4]A schematic diagram of syngeneic transplantation in a kidney model of cardiac arrest-induced nephropathy (young Lewis rats perfused with control (no AAV) or treatment (perfusion in the presence of AAV / DJ-OSK+AAV / DJ cMyc-rtTA)) is shown. [Figure 5A] AST levels in rats 1 week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment) are shown. [Figure 5B] ALT levels in rats 1 week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment) are shown. [Figure 5C] Caspase 3 levels are shown in rats one week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). [Figure 5D] Bilirubin levels in rats 1 week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment) are shown. [Figure 5E] MPO levels are shown in rats one week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). [Figure 5F] MDA levels in rats 1 week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment) are shown. [Figure 5G] Ki67 levels are shown in rats one week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). [Figure 5H]PCNA levels are shown in rats one week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). [Figure 5I] Shown are HGF levels in rats one week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). [Figure 5J] Shown are TGF-β levels in rats one week after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). [Figure 5K] Representative H&E images of the liver 1 week after intravenous injection of PBS (control) are shown. [Figure 5L] H&E images of the liver 1 week after intravenous injection of AAV-GFP / Luc control vector (vehicle) are shown. [Figure 5M] Shown is an H&E image of the liver one week after intravenous injection of the AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). [Figure 6A] AST levels in rats 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment) are shown. [Figure 6B] ALT levels in rats 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment) are shown. [Figure 6C] Caspase 3 levels are shown in rats 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). [Figure 6D]Bilirubin levels in rats 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment) are shown. [Figure 6E] MPO levels are shown in rats 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). [Figure 6F] MDA levels in rats 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment) are shown. [Figure 6G] Ki67 levels in rats 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment) are shown. [Figure 6H] PCNA levels are shown in rats 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). [Figure 6I] Shown are HGF levels in rats 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). [Figure 6J] Shown are TGF-β levels in rats 12 weeks after intravenous injection of PBS (control), AAV-GFP / Luc control vector (vehicle), or AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). [Figure 6K] Representative H&E images of the liver 12 weeks after intravenous injection of PBS (control) are shown. [Figure 6L] H&E images of the liver 12 weeks after intravenous injection of AAV-GFP / Luc control vector (vehicle) are shown. [Figure 6M]Shown is an H&E image of the liver 12 weeks after intravenous injection of the AAV-OSK+AAV-cMyc-rtTA treatment vector (treatment). [Figure 7] A 10x magnification image of a representative section of a hemoxylin-eosin stained OSKM treated kidney is shown. [Figure 8] A 10x magnification image of a representative section of a hematoxylin-eosin stained kidney treated with AAV-GFP / luc control is shown. [Figure 9A] 1 shows BUN levels in rats 2 weeks after transplantation in an ischemic injury model involving kidneys perfused with OSKM-AAV or a no-AAV control. [Figure 9B] 1 shows BUN levels in rats 3 weeks after transplantation in an ischemic injury model involving kidneys perfused with OSKM-AAV or a no-AAV control. [Figure 9C] 1 shows serum creatine levels in rats 2 weeks after transplantation in an ischemic injury model involving kidneys perfused with OSKM-AAV or a no-AAV control. [Figure 9D] 1 shows serum creatine levels in rats 3 weeks after transplantation in an ischemic injury model involving kidneys perfused with OSKM-AAV or a no-AAV control. [Figure 10A] BUN levels in rats transplanted with kidneys perfused with no AAV control (no AAV vector) are shown (n=number of surviving animals). [Figure 10B] BUN levels in rats transplanted with kidneys perfused with OSKM AAV vectors are shown (n=number of surviving animals). [Figure 10C] Serum creatinine levels are shown in rats transplanted with kidneys perfused with no AAV control (filled circles) and rats transplanted with kidneys perfused with OSKM AAV (open circles). [Figure 11] A rat model of partial (70%) hepatic ischemia is shown. [Figure 12A]AST levels are shown one week after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 12B] ALT levels are shown one week after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 12C] Bilirubin levels are shown one week after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 12D] Caspase 3 levels are shown one week after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 12E] MPO levels are shown one week after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 12F] MDA levels are shown one week after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 12G] PCNA levels are shown one week after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 12H] Ki67 levels are shown one week after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 13A]AST levels are shown 4 days after administration of PBS control, vehicle control, (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 13B] ALT levels are shown 4 days after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 13C] Caspase 3 levels are shown 4 days after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 13D] MPO levels are shown 4 days after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 13E] MDA levels are shown 4 days after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 13F] PCNA levels are shown 4 days after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 13G] Ki67 levels are shown 4 days after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 13H] HGF levels are shown 4 days after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 13I]TGF-β levels are shown 4 days after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 14A] AST levels are shown 2 days after administration of PBS control, vehicle control, (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 14B] ALT levels are shown 2 days after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 14C] Caspase 3 levels are shown 2 days after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 14D] MPO levels are shown 2 days after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 14E] MDA levels are shown 2 days after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 14F] PCNA levels are shown 2 days after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 14G] Ki67 levels are shown 2 days after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 14H]HGF levels are shown 2 days after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 14I] TGF-β levels are shown 2 days after administration of PBS control, vehicle control (AAV-GFP / Luc+AAV-rtTA), or AAV-OSKM (AAV-OSK+AAV cMyc / rtTA). [Figure 15A] The levels of mean serum creatinine (SCr) change in both control and treated groups of rats 8 days after implantation are shown. [Figure 15B] The mean BUN change levels in rats 8 days after transplantation are shown. [Figure 15C] Individual urinary proteins (left panel) and urinary albumin (right panel) are shown in both control and treated groups of rats after transplantation. [Figure 15D] Flow and resistance registry during normothermic kidney perfusion. [Figure 16A] 1 shows the expression profile of neutrophil gelatinase-associated lipocalin (NGAL) in control and OSKM-treated kidneys. [Figure 16B] 1 shows the expression profile of hypoxia inducible factor (HIF) in control and OSKM-treated kidneys. [Figure 16C] 1 shows the expression profile of endothelin receptors (ETR) in control and OSKM-treated kidneys. [Figure 16D] Transforming growth factor beta (TGFβ) expression profile in control and OSKM-treated kidneys. [Figure 16E] 1 shows the expression profile of tumor necrosis factor (TNF) in control and OSKM-treated kidneys. [Figure 16F] 1 shows the expression profile of monocyte chemoattractant protein 1 (MCP1) in control and OSKM-treated kidneys. [Figure 16G] 1 shows the expression profile of C-X-C motif chemokine ligand 10 (CXCL10) in control and OSKM-treated kidneys. [Figure 16H] Expression profiles of the evaluated genes in control and OSKM-treated kidneys are shown. [Figure 17A] A 4x magnification image of the glomerular compartment of a non-AAV-treated kidney stained with hematoxylin and eosin is shown, showing the tubular and glomerular compartments at 4x magnification. [Figure 17B] A 4x magnification image of the glomerular compartment of an AAV-OSKM-treated kidney stained with hematoxylin and eosin is shown, showing the tubular and glomerular compartments at 4x magnification. [Figure 17C] A 4x magnification of the glomerular compartment of a non-AAV-treated kidney stained with hematoxylin and eosin is shown, with the tubular and glomerular compartments shown at 10x magnification. [Figure 17D] A 4x magnification image of the glomerular compartment of an AAV-OSKM-treated kidney stained with hematoxylin and eosin is shown, showing the tubular and glomerular compartments at 4x magnification. [Figure 17E] Masson's trichrome stained tubular and glomerular compartments of an AAV-untreated kidney are shown in 4x magnification. [Figure 17F] Masson's trichrome stained tubular and glomerular compartments of AAV-OSKM-treated kidneys are shown in 4x magnification. [Figure 18] 1 shows a modified Remuzzi score analysis of OSKM-treated and control-treated samples. DETAILED DESCRIPTION OF THE INVENTION

[0038] Materials and methods are provided for rejuvenating organs to improve organ function and / or organ transplant outcomes, and to increase the number of donor organs by making organs considered suboptimal or poor candidates suitable for transplantation. Materials and methods are also provided for rejuvenating organs for autologous transplantation (i.e., rejuvenating a target organ) to improve organ function and / or organ transplant outcomes. Materials described herein include, but are not limited to, polynucleotides, vectors, and compositions introduced into donor organs ex vivo, e.g., in organ perfusion fluid. The materials and methods enable the expression of regenerative factors in perfused organs, thereby ameliorating, for example, age-related changes and treating aged or damaged organs to improve their suitability for transplantation.

[0039] The methods and materials described herein can be used for ex vivo perfusion of a human kidney to reverse changes associated with at least one of aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, viral-induced hepatitis, alcohol, or fibrosis not associated with any known cause in the kidney prior to transplantation of the kidney into a human recipient. The methods and materials described herein can also be used for ex vivo perfusion of a human liver to reverse changes associated with at least one of aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, viral-induced hepatitis, alcohol, or fibrosis not associated with any known cause in the liver prior to transplantation of the liver into a human recipient. Advantageously, the methods and materials provided herein allow for the kidney or liver to be treated by ex vivo perfusion with a perfusion solution containing the materials described herein, followed by measuring the effect of the material on the kidney or liver, for example, by kidney or liver biopsy and functional assessment, and, if desired, subjecting the kidney or liver to one or more further treatments using the methods and materials described herein before transplanting the kidney or liver into a recipient patient in need thereof.

[0040] Furthermore, the methods and materials used herein allow for ex vivo perfusion of a kidney or liver with a perfusion solution containing a material described herein, followed by in vivo treatment of the kidney or liver to modulate the effect of the material on the kidney or liver in vivo. For example, measurements can be obtained from the transplanted patient by biopsy or functional assessment, and based on the results of these measurements, the kidney or liver can be treated to enhance and / or prolong the effect of the material provided to the kidney or liver during ex vivo perfusion prior to transplantation.

[0041] I. Definition In order that this disclosure may be more readily understood, certain terms are first defined. Further definitions are set forth throughout the detailed disclosure.

[0042] It should be noted that unless otherwise specified, an entity designated by the term "a" or "an" refers to one or more of that entity; for example, "a polynucleotide sequence" is understood to refer to one or more polynucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0043] Furthermore, as used herein, "and / or" should be understood as a specific disclosure of each of two particular features or components, with or without the other. Thus, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: 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).

[0044] Whenever an embodiment is described herein in conjunction with the word "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0045] The term "about" is used herein to mean approximately, roughly, around, or within a range thereof. 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 values. In general, the term "about" can modify a numerical value above and below the stated value by, for example, a variance of up to or below 10 percent (higher or lower).

[0046] The term "at least" preceding a number or series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that are logically included from the context. For example, the number of nucleotides in a polynucleotide molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide polynucleotide molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" precedes a series or range, it is understood that "at least" can modify each number in the series or range. "At least" is not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, and 5.18%, regardless of the number of significant digits).

[0047] As used herein, "less than" or "below" is understood to refer to the value adjacent to the term and to any logically lower value or integer up to zero where the context makes sense. When "less than" precedes a series of numbers or ranges, it is understood that the "less than" can modify each of the numbers in the series or range.

[0048] Additionally, any reference referred to as "incorporated herein" is understood to be incorporated in its entirety.

[0049] The terms "polynucleotide," "polynucleotide," and "oligonucleotide" are used interchangeably in this application. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA. As used herein, the terms "polynucleotide," "polynucleotide," and "oligonucleotide" are defined as a molecule containing two or more covalently linked nucleosides, as commonly understood by those skilled in the art. An oligonucleotide may also be referred to as an oligomer. Oligonucleotides are generally synthetically produced in the laboratory by solid-phase chemical synthesis followed by purification. Polynucleotides can be produced recombinantly, enzymatically, or synthetically, for example, by solid-phase chemical synthesis followed by purification. Reference to an oligonucleotide, polynucleotide, or sequence of a polynucleotide refers to the sequence or order of the nucleobase moieties or modifications thereof of the covalently linked nucleotides or nucleosides.

[0050] As used herein, "nucleotide" refers to a monomeric unit of an oligonucleotide or polynucleotide, including a nucleoside and an internucleoside linkage. "G," "C," "A," "T," and "U" each generally represent naturally occurring nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as nucleobases, respectively. However, it is understood that the term "nucleotide" can also refer to alternative nucleotides or alternative replacement moieties, as described in more detail below. Oligonucleotides or polynucleotides are not limited to naturally occurring nucleosides, but can contain non-natural nucleosides and linkages as disclosed herein, for example, to generate modified oligonucleotides for increased stability or cell permeability. Those skilled in the art will appreciate that guanine, cytosine, adenine, and uracil can be substituted by other moieties without substantially impairing the base-pairing properties of an oligonucleotide or polynucleotide containing a nucleotide with such a replacement moiety. For example, without limitation, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Therefore, the nucleotide containing uracil, guanine or adenine can be substituted with the nucleotide containing inosine in the nucleotide sequence of oligonucleotide or polynucleotide.In another example, the adenine and cytosine at any position in oligonucleotide or polynucleotide can be substituted with guanine and uracil, respectively, to form GU wobble base pair with target RNA.The sequence containing such a substituted portion is suitable for the composition and method described herein.

[0051] 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) moieties present in nucleosides and nucleotides that form hydrogen bonds during polynucleotide hybridization. The term nucleobase also encompasses alternative nucleobases that may differ from naturally occurring nucleobases but are functional during polynucleotide hybridization. In this context, "nucleobase" refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, 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, page 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 substituted with N. 6 -methyladenine, N 2Other naturally occurring purines may be substituted, including, but not limited to, 5-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 embodiments, the oligomer may contain a thymine base in place of uracil. Other alternative nucleobases include isocytosine, pseudoisocytosine, 5-thiazolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uridine, 5-bromouridine-5-thiazolo-uridine, 2-thio-uridine, pseudouridine, 1-methylpseudouridine, 5-methoxyuridine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, 2-chloro-6-aminopurine, agmatidine, lysidine, 2-thiopyrimidines (e.g., 2-thiouracil, 2-thiothymine), G-clamps and their 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-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, 8-aza-7-deazaguanosine, and N4-ethylcytosine, or derivatives thereof; 2 -Cyclopentylguanine (cPent-G), N 2 -cyclopentyl-2-aminopurine (cPent-AP), and N 2 -propyl-2-aminopurine (Pr-AP), pseudouracil or their derivatives; and degenerate or universal bases such as 2,6-difluorotoluene, or missing bases such as abasic sites (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives in which the ring oxygen is replaced with a nitrogen (azaribose)).

[0052] 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, such as 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, and 5-methylcytosine.

[0053] 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 and 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.

[0054] The term "alternative nucleoside" refers to a nucleoside having an alternative sugar or alternative nucleobase, such as those described herein.

[0055] "Sugar" or "sugar moiety" includes naturally occurring sugars having a furanose ring. Sugar also includes "sugar substitutes," which are defined as structures that can replace the furanose ring of a nucleoside. In some embodiments, the sugar substitute is a non-furanose (or 4'-substituted furanose) ring or ring system or an open system. The sugar substitute can include sugar substitutes in which the furanose ring is replaced with another ring system, for example, a morpholino or hexitol ring system. Sugar moieties useful for preparing oligonucleotides having a motif include, but are not limited to, β-D-ribose, β-D-2'-deoxyribose, substituted sugars (e.g., 2', 5', and bis-substituted sugars), 4'-S-sugars (e.g., 4'-S-ribose, 4'-S-2'-deoxyribose, and 4'-S-2'-substituted riboses), bicyclic sugar surrogates (e.g., bicyclic sugars derived from 2'-O-CH2-4' or 2'-O-(CH2)2-4' bridged ribose), and sugar surrogates (e.g., when the ribose ring is replaced with a morpholino or hexitol ring system). The type of heterocyclic base and internucleoside linkage used at each position is variable and is not a determining factor for the motif. In most nucleosides with surrogate sugar moieties, the heterocyclic nucleobase is generally maintained to allow hybridization. In some embodiments, an oligonucleotide or polynucleotide contains, for example, nucleosides with and without 2'FANA modifications, which nucleosides may contain sugar substitutes as described herein.

[0056] "Internucleoside linkage" refers to the linkage between nucleosides in an oligonucleotide or polynucleotide, which may include a phosphate linkage or an alternative internucleoside linkage. Many "alternative internucleoside linkages" or "non-natural linkages" are known in the art, including, but not limited to, phosphate linkages, phosphorothioate linkages, and boronophosphate linkages. Alternative nucleosides include bicyclic nucleosides (BNAs) (e.g., locked nucleosides (LNAs) and constrained ethyl (cEt) nucleosides), peptide nucleosides (PNAs), phosphotriesters, phosphorothioates, phosphoramidates, and other variants of the phosphate backbone of natural nucleosides, including those described herein.

[0057] As used herein, "alternative nucleotide" refers to a nucleotide having an alternative nucleoside or sugar and an internucleoside linkage that may include alternative nucleosides or sugars and alternative internucleoside linkages.

[0058] Oligonucleotides or polynucleotides may include (i) compounds with one or more 2'FANA-modified nucleosides, (ii) compounds with one or more furanose moieties replaced by furanose derivatives or any structure (cyclic or acyclic) that can be used as a covalent attachment point for the base moiety, (iii) compounds with one or more phosphodiester linkages that are modified, such as with phosphoramidate or phosphorothioate linkages, or completely replaced by suitable linkages, such as with formacetal or riboacetal linkages, and / or (iv) compounds with one or more furanose phosphodiester linkages that are replaced by any structure (cyclic or acyclic) that can be used as a covalent attachment point for the base moiety. Oligonucleotides or polynucleotides may include one or more alternative nucleosides or nucleotides (e.g., including those described herein). It is also understood that oligonucleotides include compositions that lack sugar moieties or nucleobases but are still capable of pairing or hybridizing with target sequences. Oligonucleotides include short polynucleotides (e.g., 100 or fewer linked nucleosides). As used herein, a "chimeric" oligonucleotide or "chimera" is an oligonucleotide that contains two or more chemically distinct regions, each of which is composed of at least one monomer unit (i.e., a nucleotide or nucleoside in the case of an oligonucleotide). Chimeric oligonucleotides also include "gapmers." For example, chimeric oligonucleotides can contain unmodified nucleosides and 2'FANA-modified nucleosides. In some embodiments, the 2'FANA-modified nucleosides are located at the 5' or 3' portion of the oligonucleotide, or both. In some embodiments, the 2'FANA-modified nucleosides are located throughout the oligonucleotide. In some embodiments, chimeric oligonucleotides contain 2'FANA-modified nucleosides located at the 5' and / or 3' portion, as well as at least one unmodified nucleoside in the center of the oligonucleotide.

[0059] As used herein, unless otherwise indicated, the term "complementary" is used to describe a first nucleotide or nucleoside sequence in relation to a second nucleotide or nucleoside sequence, and as understood by one of skill in the art, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide or nucleoside sequence to hybridize to an oligonucleotide or polynucleotide comprising a second nucleotide sequence to form a duplex structure under specified conditions. Such conditions may be, for example, stringent conditions, which may include 400 mM NaCl, 40 mM PIPES (pH 6.4), 1 mM EDTA, at 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, 1999). (See, e.g., Press). Other conditions, such as physiologically relevant conditions that may be encountered within an organism, can be used. One skilled in the art can determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides or nucleosides. Complementary sequences can contain, or be formed entirely from, non-Watson-Crick base pairs and / or base pairs formed from unnatural and alternative nucleotides or nucleosides, so long as the above requirements for hybridization are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogstein base pairs. Complementary sequences between oligonucleotides and target sequences described herein include base pairs between an oligonucleotide or polynucleotide containing a first nucleotide or nucleoside sequence and an oligonucleotide or polynucleotide containing a second nucleotide or nucleoside sequence across the entire length of one or both nucleotide or nucleoside sequences. Such sequences may be referred to herein as "fully complementary" with respect to each other.A complementary sequence between an oligonucleotide or polynucleotide and a target sequence described herein includes base pairs spanning less than the entire length of one or both nucleotide or nucleoside sequences of an oligonucleotide or polynucleotide containing a first nucleotide or nucleoside sequence and an oligonucleotide or polynucleotide containing a second nucleotide or nucleoside sequence. Such sequences may be referred to herein as "partially complementary" to each other. In some embodiments, an oligonucleotide or polynucleotide comprises a contiguous nucleotide region and may further comprise nucleotide(s) or nucleoside(s). For example, a nucleotide linker region may be used to attach a functional group to a contiguous nucleotide sequence. The nucleotide linker region may be complementary to a target polynucleotide. In some embodiments, all internucleoside linkages present between nucleotides in the contiguous nucleotide region are phosphorothioate internucleoside linkages. In some embodiments, the contiguous nucleotide region comprises one or more sugar-modified nucleosides.

[0060] As used herein, the term "linker" or "linking group" refers to a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. The conjugate moiety may be attached to the oligonucleotide directly or via a linking moiety (e.g., a linker or tether). The linker functions to covalently attach a third region, e.g., the conjugate moiety, to the oligonucleotide or polynucleotide. In some embodiments, the conjugate, oligonucleotide conjugate, or polynucleotide conjugate may include a linker region located between the oligonucleotide or polynucleotide and the conjugate moiety. In some embodiments, the linker between the conjugate and the oligonucleotide or polynucleotide is biocleavable. Phosphodiester-containing biocleavable linkers are described in more detail in WO2014 / 076195 (incorporated herein by reference).

[0061] As used herein, the term "promoter" refers to a DNA sequence recognized by cellular or introduced synthetic machinery required to initiate specific transcription of a gene. The term "promoter" is also meant to encompass polynucleotide elements sufficient for promoter-dependent gene expression that is controllable for cell-type-specific, tissue-specific, or inducible expression by external signals or agents; such elements may be located in the 5' or 3' regions of the native gene. In some embodiments, the promoter may be a constitutively active promoter, a cell-type-specific promoter, or an inducible promoter.

[0062] The terms "operably linked," "operably inserted," "operably positioned," "operably linked," "under control," or "under transcriptional control," as used herein, mean that a promoter is in the correct location and orientation with respect to a polynucleotide to control RNA polymerase initiation and expression of a gene. The terms "operably linked" or "operably linked" mean that a polynucleotide sequence and a regulatory sequence(s) are connected in a manner that allows for RNA expression when the appropriate molecule (e.g., a transcriptional activator protein) is bound to the regulatory sequence(s). The terms "operably inserted" or "operably inserted" mean that a polynucleotide of interest introduced into a cell is positioned adjacent to a polynucleotide sequence that directs the transcription and translation of the introduced polynucleotide (i.e., promotes the production of a polypeptide encoded by the DNA of interest, for example).

[0063] The term "inducible promoter," as used herein, refers to a promoter that is operably or operably linked to a polynucleotide sequence to enable RNA expression when an appropriate molecule (e.g., a transcriptional activator protein) binds to the promoter sequence(s). In some embodiments, the transcriptional activator protein is activated when contacted with an activating agent. Examples of inducible promoters include, but are not limited to, a tetracycline-inducible promoter, a hormone-inducible promoter, such as a tamoxifen-inducible promoter (a promoter-binding domain fused to an estrogen receptor), a steroid-inducible promoter (a promoter-binding domain fused to a mutant progesterone receptor that binds RU486 but not endogenous progesterone), or a coumermycine-inducible promoter. An inducible promoter may include a tetracycline-responsive element (TRE) (e.g., a TRE3G promoter, a TRE2 promoter, or a P tight promoter). Inducible promoters can also include mifepristone-responsive promoters (e.g., containing a GAL4 upstream activation sequence), or coumermycin / novobiocin-inducible / repressible promoters. By way of example, a TRE (e.g., TRE3G) promoter can include a nucleic acid (e.g., an engineered nucleic acid) sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO:7.

[0064] As used herein, the term "tissue-specific promoter" refers to any promoter that activates the transcription of a polynucleotide in a specific tissue more highly than in other tissues.For example, the tissue-specific promoter used in the polynucleotide described herein can specifically induce transcription in endothelial cells (e.g., 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, smoothelin promoter); pericytes (e.g., p75 promoter, endosialin promoter, desmin promoter); or renal tubular epithelial cells (e.g., sodium-dependent phosphate cotransporter gene NPT2a promoter, sodium-potassium-2-chloride cotransporter promoter, or aquaporin 2 promoter).

[0065] The term "senescent cell-specific promoter" as used herein refers to a promoter that is expressed in senescent cells but not expressed or significantly less expressed 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.

[0066] The term "WPRE," as used herein, refers to the woodchuck hepatitis virus post-translational regulatory element, a DNA sequence that, when transcribed, creates a tertiary structure that enhances expression of the viral vector's genes.

[0067] The term "IRES" as used herein refers to an element that promotes direct internal ribosome entry to an initiation codon, such as ATG, of a cistron (protein coding region), thereby resulting in cap-independent translation of the gene. See, for example, Jackson RJ et al., Trends Biochem Sci 15(12):477-83(199); Jackson RJ and Kaminski, A. RNA1(10):985-1000(1995). Under the translational control of an IRES, translation proceeds in a cap-independent manner.

[0068] The term "termination signal sequence," as used herein, may be any genetic element that causes RNA polymerase to terminate transcription, such as a polyadenylation signal sequence. A polyadenylation signal sequence is a recognition region required for endonuclease cleavage of an RNA transcript, followed by the polyadenylation consensus sequence AATAAA. A polyadenylation signal sequence provides a "poly A site," i.e., a site on an RNA transcript where adenine residues are added by post-transcriptional polyadenylation.

[0069] The term "proteolytic cleavable site," as used herein, refers to a polynucleotide that encodes an amino acid sequence that can be proteolytically cleaved, including, but not limited to, self-processing cleavage sites and furin cleavage sites.

[0070] The term "self-processing cleavage site," as used herein, refers to a post- or co-translational processing cleavage site or sequence, which may be a DNA or amino acid sequence, and is exemplified herein by a 2A site, sequence, or domain, or a 2A-like site, sequence, or domain. A self-processing peptide is a peptide expression product of a DNA sequence encoding the self-processing cleavage site or sequence, which, upon translation, mediates rapid intramolecular (cis) cleavage of a protein or polypeptide containing the self-processing cleavage site, resulting in a distinct mature protein or polypeptide product.

[0071] The term "furin cleavage site," as used herein, refers to a polynucleotide that encodes an amino acid sequence that can be cleaved by endogenous subtilisin-like proteases, such as furin and other serine proteases in the protein secretory pathway. In some embodiments, the furin cleavage site comprises the consensus sequence RXK(R)R of SEQ ID NO: 17.

[0072] The term "vector," as used herein, refers to any vehicle for cloning and / or introducing a polynucleotide into a host cell, e.g., a plasmid, phage, transposon, minicircle vector, cosmid, chromosome, artificial chromosome, virus, virion, etc. A vector may be a replicon to which another polynucleotide segment may be attached so as to bring about replication of the attached segment. A "replicon" refers to any genetic element (e.g., a plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous replication unit in vivo (i.e., capable of replication under its own control). The term "vector" includes both viral and non-viral vehicles for introducing polynucleotides into cells in vitro, ex vivo, or in vivo. Vectors useful in the embodiments described herein include, but are not limited to, plasmids, modified eukaryotic viruses, or modified bacterial viruses. In some embodiments, insertion of a polynucleotide into a suitable vector can be accomplished by ligating an appropriate polynucleotide fragment into a selected vector with complementary cohesive termini. Vectors can be engineered to encode a selectable marker or reporter that allows for the selection or identification of cells that have incorporated the vector. Expression of the selectable marker or reporter allows for the identification and / or selection of host cells that have incorporated and expressed other coding regions contained in the vector. Examples of selectable marker genes known and used in the art include genes that confer resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc., as well as genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc. Examples of reporters known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc.A selectable marker can also be considered a reporter. In some aspects, the delivery vector is selected from the group consisting of a viral vector (e.g., an AAV vector), a plasmid, a lipid, a cationic polymer, a protein particle, a bacterial vector, and a lysosome. Some aspects of the present disclosure are directed to biological vectors, which may include viruses, particularly attenuated 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 aspects, the delivery vector of the present disclosure is a viral vector selected from the group consisting of an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, a retroviral vector, a poxvirus vector, a baculovirus vector, a herpesvirus vector, simian virus 40 (SV40), cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), and Moloney murine leukemia virus.

[0073] The term "retroviral vector," as used herein, refers to any vector that contains or is derived from retroviral vector components and is suitable for infecting mammalian cells, preferably human cells. The term retroviral vector typically refers to a retroviral particle, virion, or virus containing a payload. The terms "retroviral genome" and "retroviral vector" can be used interchangeably and refer to a retroviral nucleic acid into which a nucleic acid of interest is inserted in place of a specific viral sequence to generate a replication-deficient virus. To package a retroviral genome or retroviral vector into retroviral particles, packaging cell lines expressing the gag, pol, and env genes but lacking the retroviral long terminal repeats (LTRs) can be provided. Retroviral vectors have the ability to integrate their genes into the host genome, thereby transferring large amounts of heterologous genetic material to the host genome.

[0074] 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 retrieval factor) and at least one LTR. In some embodiments, the retroviral particle is pseudotyped by combining the retroviral vector with a heterologous viral envelope protein. In some embodiments, the heterologous viral envelope protein is, for example, a naturally occurring envelope protein from vesicular stomatitis virus. In some embodiments, the heterologous viral envelope protein is, for example, a viral envelope protein engineered to target a specific cell type. In some embodiments, the retroviral vector comprises a polynucleotide encoding a retrieval factor. In some embodiments, the retroviral vector comprises a polynucleotide encoding an Oct4 protein, a Sox2 protein, a Klf4 protein, and / or a c-Myc protein. In some embodiments, the retroviral vector comprises a polynucleotide comprising a polycistronic cassette encoding an Oct4 protein, a Sox2 protein, a Klf4 protein, and a c-Myc protein. In some embodiments, the retroviral vector comprises a polynucleotide comprising a polycistronic cassette encoding an Oct4 protein, a Sox2 protein, and a Klf4 protein, hi some embodiments, the retroviral vector comprises a polynucleotide encoding a transcriptional activator and one or more of an Oct4 protein, a Sox2 protein, a Klf4 protein, a c-Myc protein, and / or a dnNFκBIA protein.

[0075] As used herein, the term "gag gene" refers to a nucleic acid that encodes a group-specific antigen protein that is a major component of the capsid and that specifically recognizes, binds to, and packages retroviral genomic RNA into assembled virions.

[0076] As used herein, the term "pol gene" refers to a gene that encodes the pol enzymes required for viral replication, such as reverse transcriptase, protease, and integrase.

[0077] As used herein, the term "env gene" refers to a gene encoding an envelope protein that enables a retrovirus to bind to a target cell surface receptor and enter the target cell by membrane fusion.

[0078] The term "lentiviral vector," as used herein, refers to any vector that contains or is derived from lentiviral vector components and is suitable for infecting mammalian cells, preferably human cells. The term lentiviral vector typically refers to a lentiviral particle, virion, or virus containing a payload. The terms "lentiviral genome" and "lentiviral vector" can be used interchangeably and refer to a lentiviral nucleic acid into which a nucleic acid of interest is inserted in place of a specific viral sequence to generate a replication-deficient virus. To package a lentiviral genome or lentiviral vector into a lentiviral particle, a packaging cell line containing the gag, pol, and env genes, and optionally the tat and rev genes, but lacking long terminal repeats (LTRs) can be provided. In some embodiments, the packaging cell line contains the rev gene but not the tat gene. Lentiviral vectors have the ability to integrate their genes into the host genome, thereby transferring large amounts of heterologous genetic material to the host genome. However, in some embodiments, lentiviral vectors are non-integrating. In some embodiments, lentiviral vectors are self-inactivating vectors. In some embodiments, the 3' end of the U3 region in the lentiviral 3'LTR is modified so that the vector RNA is produced from the intact 5'LTR in the packaging cell line but cannot be reproduced in the target cell. In some embodiments, the U3 region in the 5'LTR of the lentiviral vector is partially or completely replaced with a heterologous promoter and / or enhancer. In some embodiments, the lentiviral vector does not contain a lentiviral capsid and is a lentiviral circle that is introduced into cells as lentiviral DNA, for example, by electroporation or via nanoparticles. In some embodiments, the lentiviral circle does not contain integrase and still exists as an episome in the cell.

[0079] 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 retinal factor) and at least one long terminal repeat (LTR). In some embodiments, the lentiviral particle is pseudotyped by combining the lentiviral vector with a heterologous viral envelope glycoprotein. In some embodiments, the heterologous viral envelope glycoprotein is, for example, a naturally occurring envelope glycoprotein from vesicular stomatitis virus. In some embodiments, the heterologous viral envelope glycoprotein is, for example, a viral envelope glycoprotein engineered to target a specific cell type. In some embodiments, the lentiviral vector comprises a polynucleotide encoding a retinal factor. In some embodiments, the lentiviral vector comprises a polynucleotide encoding an Oct4 protein, a Sox2 protein, a Klf4 protein, and / or a c-Myc protein. In some embodiments, the lentiviral vector comprises a polynucleotide comprising a polycistronic cassette encoding an Oct4 protein, a Sox2 protein, a Klf4 protein, and a c-Myc protein. In some embodiments, the lentiviral vector comprises a polynucleotide comprising a polycistronic cassette encoding an Oct4 protein, a Sox2 protein, and a Klf4 protein, hi some embodiments, the lentiviral vector comprises a polynucleotide encoding a transcriptional activator and one or more of an Oct4 protein, a Sox2 protein, a Klf4 protein, a c-Myc protein, and / or a dnNFκBIA protein.

[0080] The term "adenoviral vector," as used herein, refers to any vector that contains or is derived from adenoviral vector components and is suitable for infecting mammalian cells, preferably human cells. The term adenoviral vector typically refers to an adenoviral particle or virion containing a payload. The terms "adenoviral genome" and "adenoviral vector" can be used interchangeably. Adenoviral vectors can be derived from various serotypes, such as serotype Ad2 or serotype Ad5, or can contain elements of multiple serotypes. In some embodiments, the adenoviral vector is a helper-dependent, replication-deficient adenoviral vector.

[0081] As used herein, the term "adenoviral particle" refers to an adenovirus comprising an adenoviral capsid and an adenoviral 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 embodiments, the adenoviral particle is prepared with a helper-dependent adenoviral vector comprising two ITRs, a payload, and optionally a non-coding stuffer sequence, and a helper virus comprising adenoviral replication, packaging, and capsid genes. In some embodiments, the adenoviral vector comprises a polynucleotide encoding a transcriptional activator. In some embodiments, the adenoviral vector comprises a polynucleotide encoding Oct4 protein, Sox2 protein, Klf4 protein, and / or c-Myc protein. In some embodiments, the adenoviral vector comprises a polynucleotide comprising a polycistronic cassette encoding Oct4 protein, Sox2 protein, Klf4 protein, and c-Myc protein. In some embodiments, the adenoviral vector comprises a polynucleotide comprising a polycistronic cassette encoding an Oct4 protein, a Sox2 protein, and a Klf4 protein, hi some embodiments, the adenoviral vector comprises a polynucleotide encoding a transcriptional activator and one or more of an Oct4 protein, a Sox2 protein, a Klf4 protein, a c-Myc protein, and / or a dnNFκBIA protein.

[0082] The term "adeno-associated viral vector" or "AAV vector," as used herein, refers to any vector that contains or is derived from adeno-associated vector components and is suitable for infecting mammalian cells, preferably human cells. The term AAV vector typically refers to an AAV-type viral particle, virion, or virus that contains a payload. The terms "AAV genome" and "AAV vector" may be used interchangeably. AAV vectors may be derived from various serotypes, including combinations of serotypes (i.e., "pseudotyped" AAV), or may be derived from various genomes (e.g., single-stranded or self-complementary). Furthermore, AAV vectors may be replication-deficient and / or targetable. As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAV-DJ, AAVrh8, AAVrh10, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, the AAV serotypes and clades disclosed by Gao et al. (J. Virol. 78:6381 (2004)) and Morris et al. (Virol. 33:375 (2004)), and any other AAV now known or later discovered. See, for example, FIELDS et al. VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). In some embodiments, the AAV vector includes a derivative of a known AAV vector. In some embodiments, "AAV vector" includes a modified or artificial AAV vector. In some embodiments, the AAV vector is modified compared to the wild-type AAV serotype sequence.

[0083] 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 peptide) and at least one inverted terminal repeat (ITR). In some embodiments, the AAV vector comprises a polynucleotide encoding a transcriptional activator. In some embodiments, the AAV vector comprises a polynucleotide encoding an Oct4 protein, a Sox2 protein, a Klf4 protein, and / or a c-Myc protein. In some embodiments, the AAV vector comprises a polynucleotide comprising a polycistronic cassette encoding an Oct4 protein, a Sox2 protein, a Klf4 protein, and a c-Myc protein. In some embodiments, the AAV vector comprises a polynucleotide comprising a polycistronic cassette encoding an Oct4 protein, a Sox2 protein, and a Klf4 protein. In some embodiments, the AAV vector comprises a polynucleotide encoding a dnNFκBIA protein. In some embodiments, the AAV vector comprises a polynucleotide encoding a transcriptional activator and one or more of an Oct4 protein, a Sox2 protein, a Klf4 protein, a c-Myc protein, and / or a dnNFκBIA protein.

[0084] The term "AAV rep gene," as used herein, refers to the large open reading frame (ORF) known as the AAV replication (rep) region of the AAV genome. This ORF encodes the replication gene products Rep78, Rep68, Rep52, and Rep40, named for their apparent molecular weight, and enables replication, assembly, and packaging of the complete AAV virus.

[0085] The term "AAV cap gene," as used herein, refers to the large open reading frame (ORF) known as the AAV capsid region of the AAV genome. This ORF encodes at least three capsid proteins: VP1, VP2, and VP3, which enable assembly of the AAV capsid in which the AAV genome is packaged by the AAV Rep proteins to produce the AAV virus.

[0086] The phrase "AAV helper functions for generating productive AAV infection" as used herein refers to AAV rep and AAV cap genes provided by a source other than a polynucleotide containing a payload region and at least one ITR, such that AAV capsid production, AAV payload / ITR polynucleotide replication, and AAV payload / ITR insertion into assembled AAV capsids can occur. AAV helper functions can be provided by co-infecting AAV producer cells with wild-type AAV virions, by providing AAV producer cells with one or more plasmids containing the AAV rep and AAV cap genes, or by infecting AAV producer cells with a non-AAV virus harboring the AAV rep and AAV cap genes. AAV particles produced according to the methods described herein lack the AAV rep and AAV cap genes and contain AAV payload / ITR polynucleotides. When administered to a subject's cells, AAV virions are unable to replicate or form more AAV virions within the subject's cells in the absence of the AAV rep and AAV cap genes. Instead, AAV virions release their payload / ITR polynucleotides upon entry into the cells of the subject, and the payload gene is transcribed in the cells of the subject to produce the payload protein.

[0087] As used herein, the term "inhibiting" is used interchangeably with "reduce," "silencing," "downregulate," "suppress," and other similar terms, and includes any level of inhibition.

[0088] The term "in vitro," as used herein, refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in a cell culture, in a Petri dish, etc., and which events are not preceded by events occurring within an organism.

[0089] The term "ex vivo," as used herein, refers to events that occur outside an organism, e.g., not within an organism, but in a test tube, reaction vessel, cell culture, perfusion device, etc., where the events are preceded and / or followed by events that occur within an organism, e.g., "ex vivo" organ perfusion refers to perfusion of an organ that has been removed from a subject and perfused outside the subject's body.

[0090] The term "in vivo," as used herein, refers to events that take place within an organism (e.g., an animal, a human, or a cell or tissue thereof).

[0091] The term "transfection" as used herein refers to a method for 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 agents that can be transfected into cells is large, including, for example, siRNA, shRNA, sense and / or antisense sequences, DNA encoding one or more genes and organized into expression plasmids, e.g., vectors.

[0092] The phrases "contacting a cell with a polynucleotide," "contacting a cell with an oligonucleotide," "contacting a tissue with a polynucleotide," "contacting a tissue with an oligonucleotide," "contacting an organ with a polynucleotide," or "contacting an organ with an oligonucleotide," "contacting a cell with a polypeptide," "contacting a tissue with a polypeptide," "contacting an organ with a polypeptide," "contacting a cell with a compound," "contacting a tissue with a compound," or "contacting an organ with a compound," "contacting a cell with nanoparticles," "contacting a tissue with nanoparticles," or "contacting an organ with nanoparticles," as used herein, include contacting a cell or organ by any conceivable means. Contacting a cell, tissue, or organ with a polynucleotide, oligonucleotide, or compound includes contacting a cell, tissue, or organ with a polynucleotide, oligonucleotide, polypeptide, nanoparticle, or compound in vitro, in vivo, or ex vivo. Contacting can be direct or indirect. Thus, for example, the polynucleotide, oligonucleotide, polypeptide, nanoparticle, or compound can be brought into physical contact with the cell, tissue, or organ by the individual performing the method, or alternatively, the polynucleotide, oligonucleotide, polypeptide, nanoparticle, or compound can be placed in a situation that allows or causes it to subsequently contact the cell, tissue, or organ.

[0093] Contacting cells, tissues, or organs in vitro or ex vivo can be achieved, for example, by incubating the cells, tissues, or organs with a polynucleotide, oligonucleotide, polypeptide, nanoparticle, or compound. Contacting cells, tissues, or organs in vivo can be achieved, for example, by injecting a polynucleotide, oligonucleotide, polypeptide, nanoparticle, or compound into or near the cell, tissue, or organ, by injecting a polynucleotide, oligonucleotide, polypeptide, nanoparticle, or compound into the bloodstream, or by injecting a polynucleotide, oligonucleotide, polypeptide, nanoparticle, or compound into another region, such as the bloodstream or subcutaneous space, so that the polynucleotide, oligonucleotide, polypeptide, nanoparticle, or compound subsequently reaches the tissue, cell, or organ to be contacted. For example, the polynucleotide, oligonucleotide, polypeptide, nanoparticle, or compound can contain and / or be bound to a ligand that directs the polynucleotide, oligonucleotide, polypeptide, nanoparticle, or compound to the desired site, such as the blood vessels of an organ. Combinations of in vitro and in vivo contacting methods are also contemplated. For example, cells or tissues can be contacted in vitro, or organs can be contacted with polynucleotides, oligonucleotides, polypeptides, nanoparticles, or compounds ex vivo and then transplanted into a subject.

[0094] In some embodiments, contacting a cell, tissue, or organ with a polynucleotide, oligonucleotide, polypeptide, nanoparticle, or compound includes introducing or delivering the polynucleotide, oligonucleotide, polypeptide, nanoparticle, or compound into the cell, tissue, or organ by promoting or effecting uptake or absorption into the cell, tissue, or organ. Absorption or uptake of the polynucleotide, oligonucleotide, polypeptide, nanoparticle, or compound can occur via unassisted diffusion or active cellular processes, or by an auxiliary agent or device. For example, introduction into a cell, tissue, or organ in vitro includes methods known in the art, such as electroporation and lipofection. Introduction into an organ ex vivo further includes methods such as perfusion. In some embodiments, a cell or organ is contacted with a polynucleotide, oligonucleotide, polypeptide, nanoparticle, compound, AAV vector, AAV capsid, retroviral vector, lentiviral vector, adenoviral vector, or composition in vitro or in vivo. For example, a polynucleotide, oligonucleotide, polypeptide, nanoparticle, compound, AAV vector, AAV capsid, retroviral vector, lentiviral vector, adenoviral vector, or composition can be brought into physical contact with a cell, tissue, or organ, or placed in a situation that allows or allows for subsequent contact with the cell, tissue, or organ. In some embodiments, contacting a cell or tissue in vitro or a tissue or organ ex vivo can be achieved, for example, by incubating the cell, tissue, or organ with the polynucleotide, oligonucleotide, polypeptide, nanoparticle, compound, AAV vector, AAV capsid, retroviral vector, lentiviral vector, adenoviral vector, or composition.In some embodiments, contacting the organ ex vivo can be accomplished by, for example, perfusing the organ with the polynucleotide, oligonucleotide, polypeptide, nanoparticle, compound, AAV vector, AAV capsid, retroviral vector, lentiviral vector, adenoviral vector, or composition. In some aspects, contacting a cell, tissue, or organ in vivo can be accomplished by, for example, injecting a polynucleotide, oligonucleotide, polypeptide, nanoparticle, compound, AAV vector, AAV capsid, retroviral vector, lentiviral vector, adenoviral vector, or composition of the present disclosure into or near the tissue, tissue, or organ where the target cell is located, or by injecting a polynucleotide, oligonucleotide, polypeptide, nanoparticle, compound, AAV vector, AAV capsid, retroviral vector, lentiviral vector, adenoviral vector, or composition into a region, e.g., the bloodstream or subcutaneous space, so that the polynucleotide, oligonucleotide, polypeptide, nanoparticle, compound, AAV vector, AAV capsid, retroviral vector, lentiviral vector, adenoviral vector, or composition will then reach the tissue where the cell to be contacted is located, tissue, or organ is located. Furthermore, the polynucleotide, oligonucleotide, polypeptide, nanoparticle, compound, AAV vector, AAV virus, retroviral vector, lentiviral vector, or adenoviral vector can be encapsulated and / or bound to a ligand that directs the polynucleotide, oligonucleotide, polypeptide, nanoparticle, compound, AAV vector, AAV virus, retroviral vector, lentiviral vector, or adenoviral vector to a desired site, for example, an organ. A combination of in vitro and in vivo contact methods is also contemplated. For example, cells or organs can be contacted with a polynucleotide, oligonucleotide, polypeptide, nanoparticle, compound, AAV vector, AAV capsid, retroviral vector, lentiviral vector, adenoviral vector, or composition ex vivo and then transplanted into a subject.

[0095] In some aspects, contacting a cell or organ with a polynucleotide, oligonucleotide, polypeptide, nanoparticle, compound, AAV vector, AAV capsid, retroviral vector, lentiviral vector, adenoviral vector, or composition as described herein comprises "introducing" or "delivering" (directly or indirectly) the polynucleotide, oligonucleotide, polypeptide, nanoparticle, compound, AAV vector, AAV capsid, retroviral vector, lentiviral vector, adenoviral vector, or composition into the cell or organ by promoting or effecting uptake or absorption into the cell, tissue, or organ. Introduction of the polynucleotide, oligonucleotide, polypeptide, nanoparticle, compound, AAV vector, AAV capsid, retroviral vector, lentiviral vector, adenoviral vector, or composition into the cell or organ can occur ex vivo and / or in vivo.

[0096] As used herein, the term "lipid nanoparticle" refers to a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a polynucleotide molecule, e.g., a polynucleotide or oligonucleotide. Lipid nanoparticles may have an average diameter of 10 to 1000 nanometers and may include a solid lipid core matrix and surfactant. Lipid nanoparticles typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). Lipid nanoparticles are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference. The lipid in the nanoparticles may be triglycerides, diglycerides, monoglycerides, fatty acids, steroids, or waxes, and mixtures thereof. Lipid nanoparticles can be prepared using high-shear homogenization, ultrasound, solvent emulsification / evaporation, or microemulsion. In some embodiments, the lipid nanoparticles comprise ionizable cationic lipids.

[0097] As used herein, the term "cationic lipid" refers to a lipid that is cationic or positively charged at physiological pH. Cationic lipids can take various forms, including, but not limited to, liposomes or micelles. Cationic lipids useful in certain embodiments of the present disclosure are known in the art and generally contain both polar and non-polar domains, bind to polyanions such as nucleic acid molecules or negatively charged proteins, and typically promote the delivery of nucleic acids into cells. Examples of useful cationic lipids include polyethyleneimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE® (e.g., LIPOFECTAMINE® 2000, LIPOFECTAMINE® 3000, LIPOFECTAMINE® RNAiMAX, LIPOFECTAMINE® LTX), SAINT-RED (Synvolux Therapeutics, Groningen, Netherlands), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.). Exemplary cationic liposomes can be made from N-[1-(2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 3-β[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide, and dimethyldioctadecylammonium bromide (DDAB).

[0098] 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 unilamellar and multilamellar vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the oligonucleotide composition. The lipophilic material separates the aqueous interior from the aqueous exterior. The aqueous exterior typically does not contain the oligonucleotide composition, but in some instances, it can. Liposomes also include "sterically stabilized" liposomes, which, as used herein, refer to liposomes containing one or more specialized lipids that, when incorporated into the liposome, result in an extended circulation life compared to liposomes lacking such specialized lipids.

[0099] As used herein, the term "micelle" refers to a specific type of molecular construct in which amphiphilic molecules are arranged in a spherical structure such that all hydrophobic portions of the molecules are oriented inward and the hydrophilic portions remain in contact with the surrounding aqueous phase. The reverse arrangement exists when the environment is hydrophobic.

[0100] As used herein, the term "organ" refers to any organ that can be subjected to the materials and methods described herein and used for transplantation into a subject in need thereof.

[0101] As used herein, the term "organoid" refers to miniaturized and simplified versions of organs generated in three-dimensional culture outside the body from tissues, embryonic stem cells, or induced pluripotent stem cells.

[0102] As used herein, the term "organ transplantation" refers to a procedure in which an organ is removed from an organism (called a donor) and transplanted into a recipient organism. In some embodiments, the donor and recipient are the same organism. Examples of organ transplants include, but are not limited to, kidney, liver, lung, heart, pancreas, or corneal transplants. The organ may be treated or modified according to the methods described herein prior to transplantation. In some embodiments, the organ is obtained from one organism (the donor), subjected to the materials and methods described herein, and introduced into a second organism (the recipient). In some embodiments, the organ is obtained from one organism, subjected to the materials and methods described herein, and reintroduced into the same organism (autologous transplant). In some embodiments, two organs, e.g., two kidneys, are obtained from one organism, subjected to the materials and methods described herein, and the two organs are introduced into one recipient, or one of the two organs is introduced into one recipient and the other into a different recipient. In some embodiments, two organs, e.g., two kidneys, are obtained from one organism and subjected to the materials and methods described herein, and the two kidneys are introduced into the same organism, or one kidney is introduced into the same organism and the other kidney is introduced into a different organism, or both kidneys are introduced into different organisms. In some embodiments, organ parts are obtained from an organism, subjected to the materials and methods described herein, and introduced into different recipient organisms. In some embodiments, organ parts are obtained from an organism, subjected to the materials and methods described herein, and introduced into the same organism (autologous transplant). In some embodiments, a single organ, e.g., a liver, is obtained from one organism, subjected to the materials and methods described herein, and then divided into two or more organ parts, and each organ part is introduced into a different recipient, or one organ part is introduced into the organism from which the organ originated and one or more organ parts are introduced into one or more different recipients.

[0103] The term "perfusion," as used herein, refers to the passage of a fluid through the circulatory or lymphatic system to a tissue and / or organ. In some embodiments, perfusion includes the passage of a fluid through blood vessels. In some embodiments, perfusion includes the passage of a fluid through lymphatic vessels. In some embodiments, perfusion includes cold perfusion. In some embodiments, perfusion includes normothermic perfusion. In some embodiments, perfusion includes normothermic mechanical perfusion using a perfusion machine. In some embodiments, normothermic mechanical perfusion is performed at about 38°C. In some embodiments, normothermic mechanical perfusion is performed at about 22°C to about 38°C; or about 23°C to about 37°C; about 24°C to about 36°C; about 25°C to about 35°C; about 26°C to about 34°C; about 27°C to about 33°C; about 28°C to about 32°C; or about 33°C; about 34°C; about 35°C; about 36°C; or about 37°C. In some embodiments, the perfusion comprises cold perfusion followed by normothermic mechanical perfusion. In some embodiments, the cold perfusion is performed at about 2°C to about 21°C; or about 3°C ​​to about 20°C; about 4°C to about 19°C; about 5°C to about 18°C; about 6°C to about 17°C; about 7°C to about 16°C; about 8°C to about 15°C; about 9°C to about 14°C; 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.

[0104] As used herein, the terms "perfusion machine," "perfusion system," or "perfusion device" are used interchangeably and refer to a machine, system, or device that includes a pump, a reservoir for holding an organ or organoid, a second reservoir for holding perfusate, and tubing for connecting the reservoir for holding the organ to the perfusate reservoir. In some embodiments, the perfusion system further includes tubing connecting the blood vessels or lymphatic vessels of the organ to the perfusate reservoir. For example, the perfusion system may be a Hugo Sachs / Harvard Apparatus, a Kidney Assist™ system, an OrganOX system, a Radnoti system, an ARK Kidney system, or an Aferetica PerLife® system.

[0105] As used herein, the term "perfusate" refers to a liquid composition that passes through the blood or lymphatic vessels of an organ ex vivo, or that otherwise permeates an organ ex vivo. The perfusate may include at least one of a buffer, inorganic salt, amino acid, metabolic substrate, hormone, vasodilator, isotonicity agent, oxygenator, antioxidant, anti-inflammatory agent, anticoagulant, or antimicrobial agent, and at least one of a polynucleotide, oligonucleotide, compound, AAV vector, lentiviral vector, retroviral vector, or adenoviral vector.

[0106] The term "tissue repair" in reference to damaged tissue refers to the restoration of tissue structure, function, or a combination thereof following tissue injury. Tissue repair includes, but is not limited to, tissue regeneration, cell proliferation, and / or tissue replacement (reprogramming) of existing tissue.

[0107] The term "regeneration" refers to the generation of new tissue or cells within a tissue. In some embodiments, the methods provided herein promote organ regeneration.

[0108] As used herein, the term "tissue regeneration" or "organ regeneration" refers to the at least partial regeneration, replacement, restoration, or regrowth of a tissue, organ, or other body structure, or part thereof, e.g., after loss, injury, or degeneration, which tissue regeneration would not occur without the methods described herein. Organ regeneration relates, for example, to an increase in size and / or cell number in a damaged or diseased organ.

[0109] The term "reprogramming" refers to the process of modifying cells using reprogramming factors (e.g., reversing (in whole or in part) or preventing (in whole or in part) cellular changes that lead to dysfunction, deterioration, cell death, or aging). Reprogramming can be complete, such that a differentiated cell (e.g., a somatic cell) is reprogrammed into a pluripotent stem cell. Cell reprogramming can be incomplete, such that a differentiated cell (e.g., a somatic cell) retains its cellular identity (e.g., a lineage-specific stem cell). Cell reprogramming can be incomplete, such that, for example, a cell rejuvenates or acquires more juvenile characteristics (e.g., increased survival, reduced inflammation, or the ability to divide).

[0110] As used herein, the terms "dedifferentiation," "increased cell potential," or "increased developmental potential" refer to a process that alters or reverses the differentiation state of a differentiated cell (e.g., a somatic cell) by reverting the differentiation of the cell toward a less differentiated or more primitive cell type. For example, a cell with increased cell potential has greater developmental plasticity (i.e., can differentiate into more cell types).

[0111] As used herein, the term "enhancing agent" refers to an agent or combination of agents that enhances the efficiency or rate of reprogramming and / or rejuvenation and / or regeneration, and includes, but is not limited to, soluble Wnt, Wnt-conditioned medium, BIX-01294 (G9a histone methyltransferase), PD0325901 (MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide, hydroxamic acids (SAHA (e.g., MK0683, vorinostat, and other hydroxamic acids)), vitamin C, trichostatin (TSA), BML-210, depudecin (e.g., (-)-depudecin), HC toxin, nullscript (N script (4-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), phenylbutyrates (e.g., sodium phenylbutyrate) and other short-chain fatty acids, scriptaid, suramin sodium, APHA compound 8, apicidin, sodium butyrate, pivaloyloxymethyl butyrate (Pivanex, AN-9), trapoxin B, chlamydocin, depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994 (e.g., N-acetyldinaline) and MS-27-275), MGCD0103, NVPLAQ-824, CBHA (m-carboxycinnaminic acid acid)bishydroxamic acid), JNJ16241199, tubacin, A-161906, proxamide, oxamflatin, 3-Cl-UCHA (e.g., 6-(3-chlorophenylureido)caproic acid hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31, and CHAP50. Other reprogramming enhancers include, for example, dominant-negative forms of HDACs (e.g., catalytically inactive forms), siRNA inhibitors of HDACs, and antibodies that specifically bind to HDACs.Such inhibitors are available from, for example, BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Aton Pharma, Titan Pharmaceuticals, Schering AG, Pharmion, MethylGene, and Sigma Aldrich.

[0112] As used herein, the term "potency" refers to the sum of all developmental options available to a cell (i.e., developmental ability). Cell potential is a continuum from the most developmentally potent, most plastic cell, i.e., totipotent stem cells, to the least developmentally potent, least plastic cell, i.e., terminally differentiated cells. The continuum of cell potential includes, but is not limited to, totipotent cells, pluripotent cells, multipotent 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 embryonic body). For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency can be determined, in part, by assessing the pluripotency characteristics of a cell. Pluripotency characteristics include, but are not limited to, (i) pluripotent stem cell morphology; (ii) the potential for unlimited self-renewal; (iii) the 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) the formation of teratomas composed of the three somatic cell lineages; and (vi) the formation of embryoid bodies composed of cells of the three somatic cell lineages.

[0113] As used herein, the term "regenerative factor" or "reprogramming factor" refers to an agent that can revert terminally differentiated cells to less differentiated cells, such as a multipotent or pluripotent state. Examples of regenerative 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 kappa B IA (dnNFκBIA). In some embodiments, the regenerative factors are various combinations of Oct4, Sox2, Klf4, and c-Myc; Oct4, Sox2, Nanog, and Lin28; Oct4, Sox2, and Klf4; or Oct4, Sox2, klf4, nanog, ESRRB, NR5A2, CEBPA, Myc, Lin28A, and Lin28B.

[0114] As used herein, the terms "preventing complete dedifferentiation" and "blocking complete dedifferentiation" refer to a method of reverting a cell to a less differentiated state without causing the cell to revert to a stem cell state. The method may include contacting the cell with at least one regenerative factor for a period of time. The method may include contacting the cell with at least one regenerative factor that does not induce the cell to revert to a stem cell state.

[0115] As used herein, the term "rejuvenating a cell" is meant to include preventing or reversing (in whole or in part) the causes of cellular senescence without inducing a pluripotent state.

[0116] As used herein, the term "Oct family gene" refers to a family of octameric transcription factors, including, but not limited to, Oct1, Oct3, Oct4, Oct6, and variants thereof.

[0117] As used herein, the term "Sox family gene" refers to a family of Syr-related HMG box transcription factors, including, but not limited to, Sox1, Sox2, Sox3, Sox7, Sox15, Sox17, Sox18, and variants thereof.

[0118] As used herein, the term "Klf family gene" refers to a family of Kruppel-like factor transcription factors, including, but not limited to, Kfl1, Klf2, Klf4, Klf5, and variants thereof.

[0119] As used herein, the term "Myc family gene" refers to the family of Myc proto-oncogene transcription factors, including, but not limited to, c-Myc, L-Myc, N-Myc, and variants thereof. For example, a Myc variant may have a deletion of amino acids 1-41 in the amino acid sequence of human c-Myc set forth in SEQ ID NO: 4. In some embodiments, a Myc variant has a deletion of amino acids 1-64 in the amino acid sequence of human c-Myc. In some embodiments, a Myc variant has a deletion of amino acids 1-107 in the amino acid sequence of human c-Myc. In some embodiments, a Myc variant has a deletion of amino acids 1-13 in the amino acid sequence of human c-Myc and a mutation at position 135. The mutation at position 135 is a deletion or substitution. In some embodiments, Trp at position 135 of human c-Myc is substituted with Glu or Gly. In some embodiments, the L-Myc variant has an amino acid sequence of at least position 70 or thereafter in the amino acid sequence of human L-Myc set forth in SEQ ID NO: 5. In some embodiments, the L-Myc variant has an amino acid sequence of at least position 45 or thereafter in the amino acid sequence of human L-Myc set forth in SEQ ID NO: 5. In some embodiments, the L-Myc variant has an amino acid sequence of at least position 22 or thereafter in the amino acid sequence of human L-Myc set forth in SEQ ID NO: 5. In some embodiments, the L-Myc variant has a mutation at position 321 in the amino acid sequence of human L-Myc set forth in SEQ ID NO: 5. In some embodiments, the mutation at position 321 in SEQ ID NO: 5 is a substitution or deletion. In some embodiments, Val at position 321 in SEQ ID NO: 5 is substituted with Asp. In some embodiments, the Myc is human N-Myc set forth in SEQ ID NO: 6.

[0120] As used herein, the term "polypeptide variant" typically exhibits at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity along its length to a polypeptide sequence described herein. In certain embodiments, the variant or combination of variants used retains the ability to induce pluripotency as described herein. Polypeptide variants may differ from naturally occurring polypeptides in one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences and assessing their effects.

[0121] The term "percent (%) sequence identity," as used herein with respect 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 the reference polynucleotide or polypeptide sequence after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percent sequence identity. Alignment for purposes of determining percent polynucleotide or amino acid sequence identity can be achieved 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 aligning sequences, including any algorithms needed to achieve maximum alignment over the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. As an example, the percent sequence identity of a given polynucleotide or amino acid sequence A to, or relative to, a given polynucleotide or amino acid sequence B (which can alternatively be expressed as a given polynucleotide or amino acid sequence A having a particular percent sequence identity to, or relative to, a given polynucleotide or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) (where X is the number of nucleotides or amino acids scored by a sequence alignment program (e.g., BLAST) as a perfect match in that program's alignment of A and B, and Y is the total number of polynucleotides in B.) It will be understood that if the length of polynucleotide or amino acid sequence A is not equal to the length of polynucleotide or amino acid sequence B, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0122] As used herein, the term "amino acid substitution" in a variant refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties, i.e., a conservative amino acid substitution. "Conservative" amino acid substitutions can be made based on similarity in either diversity or properties, such as the side chain size, polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity 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. Within a particular group, certain substitutions may be of particular interest, such as the substitution of leucine with isoleucine (or vice versa), serine with threonine (or vice versa), or alanine with glycine (or vice versa). Of course, non-conservative substitutions can often be compatible with retaining function. In some aspects, the substitution or deletion does not change or delete amino acids important for activity. Insertions or deletions can range in size from about 1 to 20 amino acids, e.g., 1 to 10 amino acids. In some cases, larger domains can be removed without substantially affecting function. In certain embodiments of the present disclosure, variant sequences can be obtained by adding, deleting, or substituting a total of 5, 10, 15, or 20 or fewer amino acids to the sequence of a naturally occurring enzyme. In some aspects, no more than 1%, 5%, 10%, or 20% of the amino acids in a polypeptide are inserted, deleted, or substituted relative to the original polypeptide.Guidance for determining which amino acid residues can be substituted, added, or deleted without eliminating or substantially reducing a desired activity can be obtained by comparing the sequence of a particular polypeptide to that of a homologous polypeptide (e.g., from another organism) and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by substituting amino acids with amino acids found in the homologous sequence, since amino acid residues that are conserved among various species are more likely to be important for activity than amino acids that are not conserved. In some embodiments, a variant of a polypeptide comprises a heterologous polypeptide portion. The heterologous portion often has a sequence that is not present in the original polypeptide or that is not homologous to the original polypeptide. The heterologous portion can be, for example, from 5 to about 5,000 amino acids in length, or longer. Often, it is from 5 to about 1,000 amino acids in length. In some embodiments, the heterologous portion comprises a sequence found in a different polypeptide, e.g., a functional domain. In some embodiments, the heterologous portion comprises a sequence useful for purifying, expressing, solubilizing, and / or detecting the polypeptide. In some embodiments, the heterologous moiety comprises a polypeptide "tag," e.g., an affinity tag or an epitope tag. For example, the tag can be an affinity tag (e.g., HA, TAP, Myc, 6xHis, Flag, GST), a fluorescent or luminescent protein (e.g., EGFP, ECFP, EYFP, Cerulean, DsRed, mCherry), or a solubility-enhancing tag (e.g., a SUMO tag, a NUS A tag, a SNUT tag, or a monomeric variant of the Ocr protein of bacteriophage T7). See, e.g., Esposito D and Chatterjee D K. Curr Opin Biotechnol.;17(4):353-8(2006). In some embodiments, a tag can serve multiple functions. Tags are often relatively small, e.g., ranging from a few amino acids to about 100 amino acids in length. In some embodiments, the tag is more than 100 amino acids in length, e.g., up to about 500 amino acids in length, or more.In some embodiments, a polypeptide has a tag located at the N- or C-terminus, e.g., as an N- or C-terminal fusion. A polypeptide can contain multiple tags. In some embodiments, a 6xHis tag and an NUS tag are present, e.g., at the N-terminus. In some embodiments, the tag is cleavable, such that it can be removed from the polypeptide, e.g., by a protease. In some embodiments, this is achieved by including a sequence encoding a protease cleavage site between the tag and the sequence encoding the portion homologous to the original polypeptide. Exemplary proteases include, e.g., thrombin, TEV protease, Factor Xa, PreScission protease, etc. In some embodiments, a "self-cleaving" tag is used. The sequence encoding the tag can be located 5' or 3' (or both) relative to the polynucleotide encoding the polypeptide. In some embodiments, the tag or other heterologous sequence is separated from the remainder of the polypeptide by a polypeptide linker. For example, the linker can be a short polypeptide (e.g., 15-25 amino acids). Often, the linker is composed of small amino acid residues such as serine, glycine, and / or alanine. The heterologous domain may include a transmembrane domain, a secretory signal domain, and the like.

[0123] 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 regenerative factor), optionally compared to a reference. The reference can be any useful reference as defined herein. A "decreased level" or "increased level" of a protein refers to a decrease or increase in protein level compared to a reference (e.g., 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 decrease or increase; a decrease or increase of about 10% compared to a reference). a decrease or increase of more than about 15%, about 20%, about 50%, about 75%, about 100%, or about 200%; a decrease or increase of less than about 0.01-fold, about 0.02-fold, about 0.1-fold, about 0.3-fold, about 0.5-fold, about 0.8-fold, or less; or an increase of more than about 1.2-fold, about 1.4-fold, about 1.5-fold, about 1.8-fold, about 2.0-fold, about 3.0-fold, about 3.5-fold, about 4.5-fold, about 5.0-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 1000-fold, or more). Protein levels can be expressed as mass / volume (e.g., g / dL, mg / mL, μg / mL, or ng / mL) or percentage of total protein or mRNA in the sample.

[0124] As used herein, the phrase "determining the level of a protein" refers to directly or indirectly detecting a protein or mRNA encoding the protein by methods known in the art. "Directly determining" means obtaining a physical entity or value by performing a process, such as running an assay or test on a sample or analyzing the sample. "Indirectly determining" refers to 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 properties (including, but not limited to, enzymatic activity or interactions with other protein partners).Methods for measuring RNA levels include, but are not limited to, transcriptome sequencing, bulk RNA sequencing, and single-cell RNA sequencing.

[0125] As used herein, the term "transcriptome profile" refers to the set of all RNA molecules in a cell or population of cells. Depending on the specific experimental setup, it may be used to refer to all RNA or simply mRNA. It differs from the exome in that it includes only the RNA molecules found in a particular cell population and typically includes the amount or concentration of each RNA molecule in addition to its molecular identity. Methods for obtaining transcriptome profiles include next-generation sequencing technologies such as DNA microarrays and RNA-Seq. Transcription can also be studied at the level of individual cells through single-cell transcriptomics. One approach to estimating transcriptome sequence is to map sequence reads onto a reference genome, either of the organism itself (whose transcriptome is being studied) or of a closely related species. Another approach, de novo transcriptome assembly, uses software to directly estimate transcripts from short sequence reads.

[0126] As used herein, the terms "tissue damage," "cell damage," and "organ damage" refer to any type of damage or injury to a cell, tissue, or organ. In various aspects, the terms encompass damage due to aging, damage due to disease, damage due to physical trauma or surgery, damage caused by exposure to harmful substance(s), and other disruptions of the structure and / or functionality of a cell, tissue, or organ.

[0127] As used herein, the term "subject" refers to any organism (which may be used interchangeably herein) to which the materials or methods described herein may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Exemplary organisms include mammals, e.g., humans, non-human primates, rodents (e.g., mice, rats, rabbits), ungulates (e.g., sheep, cattle, horses, caprine species), dogs, and cats. Often, a subject refers to an individual to whom a compound is to be delivered, or from whom a sample is obtained, or from whom a diagnostic procedure (e.g., a sample or procedure to be used to assess tissue damage and / or to evaluate the effect of a compound described in the present disclosure) is performed, for example, for experimental, diagnostic, and / or therapeutic purposes.

[0128] As used herein, the term "donor" refers to a subject from whom an organ is obtained by surgery, and includes mammals, such as humans, non-human primates, rodents (e.g., mice, rats, rabbits), ungulates (e.g., ovine, bovine, equine, caprine), dogs, and cats.

[0129] As used herein, the term "recipient" refers to a subject into which an organ is surgically transplanted, and includes mammals, such as humans, non-human primates, rodents (e.g., mice, rats, rabbits), ungulates (e.g., ovine, bovine, equine, caprine), dogs, and cats.

[0130] The terms "treat," "treating," "therapy," "therapeutic," and similar terms, as used herein with respect to a subject, refer to both therapeutic treatment and prophylactic or preventative measures aimed at preventing or slowing (alleviating) an undesirable physiological, e.g., age-related, condition, disorder, or disease, or achieving a beneficial or desired clinical result. In some embodiments, an age-related condition includes signs and / or symptoms associated with natural aging. In some embodiments, treatment reduces or alleviates symptoms associated, for example, with an age-related disease or disorder. In some embodiments, treatment results in a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; reduction in the severity of a condition, disorder, or disease; stabilization of a condition, disorder, or disease (i.e., not worsening); delay in the onset or slowing of progression of a condition, disorder, or disease; improvement or remission (whether partial or complete) of a condition, disorder, or disease state, whether detectable or undetectable; improvement in at least one measurable physical parameter, not necessarily discernible by the patient; or improvement or amelioration of a condition, disorder, or disease. In some embodiments, treatment involves eliciting a clinically significant response without excessive levels of side effects. In some embodiments, treatment involves prolonging survival compared to the expected survival in the absence of treatment. As used herein, the term "improvement" or "ameliorating" refers to a reduction in the severity of at least one indicator of a condition or disease. As used herein, the term "preventing" or "prevention" refers to delaying or forestalling the onset, development, or progression of a condition or disease for a period of time, e.g., weeks, months, or years. Amelioration of a disease or disorder includes slowing the course of the disease or disorder or reducing the severity of a later-onset age-related disease or disorder.A "prophylactically effective amount" may vary depending on the characteristics of the agent, the composition, the method of administration of the agent, the degree of risk of disease, and the medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if applicable), and other individual characteristics of the patient being treated. In some embodiments, treatment is provided to a harvested organ to, for example, regenerate and / or rejuvenate an aging organ, regenerate and / or rejuvenate a damaged organ, and / or reverse (in whole or in part) physical changes associated with at least one of aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, viral-induced hepatitis, alcohol, or fibrosis not associated with any known cause.

[0131] For example, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of a polynucleotide, oligonucleotide, polypeptide, vector, agent, nanoparticle, or composition described herein refer to an amount sufficient to produce a beneficial or desired result, e.g., a clinical outcome, when administered to a subject, tissue, or organ. Thus, "effective amount" or its synonyms depend on the context in which it is used. In some aspects, a therapeutically effective amount of an agent (e.g., a polynucleotide, vector, nanoparticle, or composition described herein) is an amount that produces a beneficial or desired result in a subject or ex vivo tissue or organ compared to a control. The amount of a given agent (e.g., a polynucleotide, vector, nanoparticle, or composition described herein) will vary depending on various factors, such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, organ, identity of the subject, organ donor, and organ recipient undergoing treatment (e.g., age, sex, and / or weight). For example, in the context of treating an ex vivo tissue or organ, it is the amount of agent sufficient to achieve a desired response in the tissue or organ compared to the response obtained without administration of the agent. The desired response may be, for example, rejuvenation of an aging organ or reversal of changes in the organ resulting from at least one of aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, virally induced hepatitis, alcohol, or fibrosis not associated with any known cause.

[0132] As used herein, the term "prophylactically effective amount" refers to an amount of a polynucleotide, vector, nanoparticle, or composition described herein that, when administered to a subject, tissue, or organ, is sufficient to prevent or ameliorate disease or injury in the subject or organ. Amelioration of disease or injury includes slowing the course of the disease- or injury-induced process or reducing the severity of any subsequent disease or injury-induced condition. A "prophylactically effective amount" may vary depending on the therapeutic agent used, the method of administration of the therapeutic agent, the degree of risk of disease or injury, and the medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any) of the subject (including the organ donor and organ recipient), and other individual characteristics of the subject. A prophylactically effective amount can 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, virally induced hepatitis, alcohol, or fibrosis not associated with any known cause in an organ, and can refer to an amount that, when administered to a subject, e.g., a human, tissue, or organ, is sufficient to delay the onset of one or more of the signs and symptoms of ischemia or injury described herein by at least 120 days, e.g., 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 more, relative to the expected onset.

[0133] As used herein, the term "intermittent addition" or "intermittent administration" refers to the repeated addition of a polynucleotide, polypeptide, vector, or nanoparticle to a composition (where each addition is followed by a period of no addition) or the repeated administration of a polynucleotide, polypeptide, vector, or nanoparticle to a subject (where each administration is followed by a period of no administration).

[0134] As used herein, the term "gene therapy" refers to the insertion of a polynucleotide sequence (e.g., a polynucleotide comprising a promoter operably linked to a polynucleotide encoding a therapeutic molecule disclosed herein) into the cells, tissues, and / or organs of an individual to treat, alleviate symptoms of, or reduce the likelihood of a disease or aging-related condition. Gene therapy also includes the insertion of a transgene that is inhibitory in nature, i.e., inhibits, reduces, or decreases the expression, activity, or function of an endogenous gene or protein, such as an undesirable (e.g., injury-induced) or aberrant (e.g., pathogenic) gene or protein. Such a transgene may be exogenous. An exogenous molecule or sequence is understood to be a molecule or sequence that does not normally occur in the cell, tissue, organ, and / or individual being treated.

[0135] As used herein, the term "biopsy" refers to a material or a procedure for obtaining such material from a tissue or organ of a subject, for example, by fine needle aspiration. A biopsy typically contains some cell types of the tissue or organ and can provide information about the morphology, histopathology, and functionality of the tissue or organ.

[0136] As used herein, the term "pharmaceutical composition" refers to a composition comprising a compound or vector described herein, e.g., a polynucleotide, oligonucleotide, AAV vector, lentiviral vector, retroviral vector, or adenoviral vector described herein, formulated with a pharmaceutically acceptable excipient, and capable of being manufactured or sold with the approval of a government regulatory agency as part of a therapeutic regimen for the treatment of disease or the treatment of an organ prior to transplantation.

[0137] As used herein, the term "pharmaceutically acceptable excipient" refers to any ingredient other than a compound or vector described herein (e.g., a vehicle capable of suspending or dissolving an active compound or vector) that has the properties of being substantially non-toxic and non-inflammatory in a patient or organ.

[0138] II. Polynucleotides Polynucleotides for expression of regeneration factors are provided. In some embodiments, the polynucleotides comprise polynucleotide sequences encoding at least one regeneration factor. In some embodiments, the polynucleotides comprise polynucleotide sequences encoding at least one transcriptional activator. In some embodiments, the polynucleotides comprise polynucleotide sequences encoding at least one regeneration factor and at least one transcriptional activator.

[0139] In some embodiments, the polynucleotide is non-integrated RNA. In some embodiments, the polynucleotide is non-integrated DNA. In some embodiments, the polynucleotide is integrated RNA. In some embodiments, the polynucleotide is integrated DNA.

[0140] In some embodiments, the polynucleotide is present in a vector. In some embodiments, the polynucleotide is present in a plasmid. In some embodiments, the polynucleotide is present in a viral vector. In some embodiments, the polynucleotide is present in an AAV vector, a lentiviral vector, a retroviral vector, or an adenoviral vector.

[0141] In some embodiments, the polynucleotide comprises at least one inducible promoter. In some embodiments, the polynucleotide comprises at least one constitutive promoter. In some embodiments, the polynucleotide comprises at least one cell type-specific promoter.

[0142] In some embodiments, the non-integrated RNA encodes at least one regenerative factor that rejuvenates the cell while maintaining the cell in a differentiated state. In some embodiments, the non-integrated DNA encodes at least one regenerative factor that rejuvenates the cell while maintaining the cell in a differentiated state. In some embodiments, the integrated RNA encodes at least one regenerative factor operably linked to an inducible promoter, and contacting the cell with an inducing compound rejuvenates the cell while maintaining the cell in a differentiated state. In some embodiments, the integrated DNA encodes at least one regenerative factor operably linked to an inducible promoter, and contacting the cell with an inducing compound rejuvenates the cell while maintaining the cell in a differentiated state.

[0143] In some aspects, the polynucleotide comprises a transposon element.

[0144] In some embodiments, the polynucleotide comprises a polynucleotide sequence encoding at least one transcriptional transactivator.

[0145] In some embodiments, the polynucleotide comprises an inducible promoter operably linked to a polynucleotide sequence encoding at least one regeneration factor and a polynucleotide sequence encoding at least one transcriptional transactivator.

[0146] In some embodiments, the inducible promoter is a tetracycline-inducible promoter, a hormone-inducible promoter, a steroid-inducible promoter, a cumate-inducible promoter, or a coumermycin-inducible promoter. In some embodiments, the hormone-inducible promoter is a tamoxifen-inducible promoter comprising a transactivator domain fused to an estrogen receptor. In some embodiments, the steroid-inducible promoter is a progesterone promoter comprising a transactivator domain fused to a mutant progesterone receptor that binds to RU486 but not endogenous progesterone. In some embodiments, the coumermycin-inducible promoter is a hybrid promoter comprising a transactivator domain that binds to the hybrid promoter after being homodimerized by coumermycin. In some embodiments, a cumate-inducible promoter comprises an operator site (CuO) downstream of a constitutive promoter and a repressor protein (CymR) bound to the CuO site (addition of cumate relieves repression); or a chimeric transactivator protein formed by fusing CymR with an activation domain (the chimeric transactivator protein binds to a CuO upstream of the constitutive promoter, and cumate inhibits DNA binding of the chimeric transactivator, thereby terminating transcriptional activation) (see, e.g., Mullick et al., BMC Biotechnology 6:43, 2006).

[0147] In some embodiments, the tetracycline-inducible promoter contains multiple copies of a tet operator sequence that can bind to the reverse tetracycline-controlled transactivator protein (rtTA). In some embodiments, the tetracycline-inducible promoter contains multiple copies of a tet operator sequence that can bind to the tTA-M2, rtTA3, or rtTA4 transactivator protein.

[0148] In some embodiments, the inducible promoter of the polynucleotide is bound by a transactivator protein in the presence of an inducing compound. In some embodiments, the tetracycline-inducible promoter of the polynucleotide is bound by an rTA protein in the presence of tetracycline. In some embodiments, the tetracycline-inducible promoter is operably linked to at least one regeneration factor. In some embodiments, the tetracycline-inducible promoter is operably linked to a polycistronic cassette comprising polynucleotides encoding at least two regeneration factors. In some embodiments, the tetracycline-inducible promoter is operably linked to a polycistronic cassette comprising polynucleotides encoding Oct4 and Sox2. In some embodiments, the polycistronic cassette further comprises a polynucleotide encoding Klf4. In some embodiments, the polycistronic cassette further comprises a polynucleotide encoding c-Myc. In some embodiments, the polynucleotide further comprises a polynucleotide encoding a transcriptional transactivator. In some embodiments, the polynucleotide further comprises a polynucleotide encoding rtTA. In some embodiments, the polynucleotide comprises a polynucleotide encoding rtTA operably linked to an inducible promoter. In some embodiments, the polynucleotide comprises a polynucleotide encoding rtTA operably linked to a constitutive promoter. In some embodiments, the polynucleotide comprises a polynucleotide encoding rtTA operably linked to a cell type-specific promoter.

[0149] In some embodiments, the polynucleotide comprises at least one constitutive promoter, such as CPI, CMV, EF1-alpha, SV40, PGK1, Ubc, human beta-actin, CAG, CamKIIa, TEF1, GDS, CaMV35S, Grp78, Grp94, Hsp70, EGFR, H1, and U6 promoters.

[0150] In some embodiments, the polynucleotide comprises a polynucleotide sequence encoding at least one regeneration factor operably linked to a constitutive promoter and a loxP sequence framing the polynucleotide sequence encoding the at least one regeneration factor, hi some embodiments, the polynucleotide comprises multiple constitutive promoters and multiple expression cassettes, optionally framed by loxP sequences.

[0151] In some embodiments, the polynucleotide comprises an EF1-alpha promoter operably linked to a polynucleotide encoding the mutant reverse tetracycline-controlled transactivator rTA-M2. In some embodiments, the polynucleotide comprises an EF1-alpha promoter operably linked to a polynucleotide encoding the mutant reverse tetracycline-controlled transactivator rTA-M2 and an inducible promoter operably linked to the c-Myc gene. In some embodiments, the EF1-alpha promoter comprises the polynucleotide sequence of SEQ ID NO: 12.

[0152] In some embodiments, the polynucleotide comprises a TetO7 tetracycline operator promoter operably linked to a polycistronic cassette comprising a polynucleotide sequence encoding an Oct4 gene, a Sox2 gene, and / or a Klf4 gene, hi some embodiments, the polynucleotide further comprises a TetO7 tetracycline operator promoter operably linked to a c-Myc gene.

[0153] In some embodiments, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 26. In some embodiments, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 27. In some embodiments, the polynucleotide comprises a binding site for an estrogen receptor binding transcription factor element operably linked to a c-Myc gene. In some embodiments, the polynucleotide comprises a binding site for an estrogen receptor binding transcription factor element operably linked to a polycistronic cassette comprising a polynucleotide sequence encoding an Oct4 gene, a Sox2 gene, and / or a Klf4 gene. In some embodiments, the polynucleotide comprises a binding site for a chimeric regulatory protein responsive to RU486 but not endogenous progestin operably linked to a c-Myc gene. In some embodiments, the polynucleotide comprises a binding site for a chimeric regulatory protein responsive to RU486 but not endogenous progestin operably linked to a polycistronic cassette comprising a polynucleotide sequence encoding an Oct4 gene, a Sox2 gene, and / or a Klf4 gene.

[0154] In some embodiments, the polynucleotide comprises a promoter that is active in senescent cells. In some embodiments, the promoter is the promoter of the cyclin-dependent kinase inhibitor 2A (CDKN2A) / p16 gene. In some embodiments, the promoter is the p21 promoter. In some embodiments, the promoter is the mir146A promoter. In some embodiments, 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: 19. In some embodiments, the promoter comprises the polynucleotide sequence of SEQ ID NO: 19. In some embodiments, 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: 20. In some embodiments, the promoter comprises the polynucleotide sequence of SEQ ID NO: 20.

[0155] In some embodiments, 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 embodiments, the promoter comprises the polynucleotide sequence of SEQ ID NO: 7. In some embodiments, 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 embodiments, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 12.

[0156] In some embodiments, the polynucleotide comprises a polynucleotide sequence encoding an Oct4 protein. In some embodiments, the Oct4 protein is a human Oct4 protein. In some embodiments, the polynucleotide 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 human Oct4 sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 1. In some embodiments, the Oct4 protein is a mouse Oct4 protein. In some embodiments, the polynucleotide 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 human Oct4 sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:8.

[0157] In some embodiments, the polynucleotide comprises a polynucleotide sequence encoding a Sox2 protein. In some embodiments, the Sox2 protein is a human Sox2 protein. In some embodiments, the polynucleotide 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 human Sox2 sequence of SEQ ID NO: 2. In some embodiments, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 2. In some embodiments, the Sox2 protein is a mouse Sox2 protein. In some embodiments, the polynucleotide 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 human Sox2 sequence of SEQ ID NO: 9. In some embodiments, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:9.

[0158] In some embodiments, the polynucleotide comprises a polynucleotide sequence encoding a Klf4 protein. In some embodiments, the Klf4 protein is a human Klf4 protein. In some embodiments, the polynucleotide 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 human Klf4 sequence of SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 3. In some embodiments, the Klf4 protein is a mouse Klf4 protein. In some embodiments, the polynucleotide 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 human Klf4 sequence of SEQ ID NO: 10. In some embodiments, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:10.

[0159] In some embodiments, the polynucleotide comprises a polynucleotide sequence encoding a c-Myc protein. In some embodiments, the c-Myc protein is a human c-Myc protein. In some embodiments, the polynucleotide 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 human c-Myc sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 4. In some embodiments, the c-Myc protein is a mouse c-Myc protein. In some embodiments, the polynucleotide 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 human c-Myc sequence of SEQ ID NO: 11. In some embodiments, the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:11.

[0160] In some embodiments, the polynucleotide comprises a polynucleotide sequence encoding a human L-Myc protein of SEQ ID NO: 5. In some embodiments, the polynucleotide comprises a polynucleotide sequence encoding a rodent L-Myc protein. In some embodiments, the polynucleotide comprises a polynucleotide sequence encoding a human N-Myc protein of SEQ ID NO: 6. In some embodiments, the polynucleotide comprises a polynucleotide sequence encoding a rodent N-Myc protein.

[0161] In some embodiments, the polynucleotide comprises a polynucleotide sequence encoding an Oct4 protein, a polynucleotide sequence encoding a Sox2 protein, and / or a polynucleotide sequence encoding a Klf4 protein, all operably linked to an inducible promoter. In some embodiments, the polynucleotide comprises a polynucleotide sequence encoding an Oct4 protein, a polynucleotide sequence encoding a Sox2 protein, a polynucleotide sequence encoding a Klf4 protein, and / or a polynucleotide sequence encoding a c-Myc protein, all operably linked to an inducible promoter. In some embodiments, the polynucleotide comprises a polycistronic cassette comprising polynucleotide sequences encoding an Oct4 protein, a Sox2 protein, and a Klf4 protein. In some embodiments, the polynucleotide comprises a polycistronic cassette comprising polynucleotide sequences encoding an Oct4 protein, a Sox2 protein, a Klf4 protein, and a c-Myc protein. In some embodiments, one polynucleotide comprises a polynucleotide sequence encoding an Oct4 protein and / or a polynucleotide sequence encoding a Sox2 protein and / or a polynucleotide sequence encoding a Klf4 protein, all of which are operably linked to an inducible promoter, and the second polynucleotide comprises a polynucleotide encoding a c-Myc protein operably linked to an inducible promoter.

[0162] In some embodiments, the polynucleotide further comprises a proteolytic cleavable site. In some embodiments, the proteolytic cleavable site is a self-processing cleavage site or a furin cleavage site. In some embodiments, the self-processing cleavage site is a P2A, E2A, F2A, or T2A peptide. In some embodiments, the furin cleavage site comprises the consensus sequence RXK(R)R of SEQ ID NO: 17. Thus, the polypeptide produced when the polynucleotide is transcribed in a cell can be cleaved by cellular proteases to release the Oct4, Sox2, and Klf4 proteins.

[0163] In some embodiments, the polynucleotide comprises a polynucleotide sequence encoding Oct4 protein, a polynucleotide sequence encoding Sox2 protein, and a polynucleotide sequence encoding Klf4 protein, arranged in 5' to 3' order on the polynucleotide so that desired amounts of Oct4, Sox2, and Klf4, respectively, are produced in cells transduced with the polynucleotide. The amounts of Oct4, Sox2, and Klf4 proteins required for a particular cell type to induce partial reprogramming may vary, and polynucleotides that provide Oct4, Sox2, and Klf4 protein levels in the optimal ratio for partial reprogramming of each cell type may be selected. In some embodiments, the level of protein produced from the polynucleotide is highest when the polynucleotide sequences encoding the proteins are located closest to the promoter sequence. In some embodiments, Oct4, Sox2, and Klf4 are arranged in 5' to 3' order from the promoter sequence. In some embodiments, Oct4, Sox2, Klf4, and c-Myc are arranged in 5' to 3' order from the promoter sequence.

[0164] In some embodiments, the polynucleotide comprises an inducible promoter, a polynucleotide sequence encoding an Oct4 protein, a proteolytic cleavable site, and a polynucleotide sequence encoding a Sox2 protein.

[0165] In some embodiments, the polynucleotide comprises an inducible promoter, a polynucleotide sequence encoding an Oct4 protein, a proteolytic cleavable site, and a polynucleotide sequence encoding a Klf4 protein.

[0166] In some embodiments, the polynucleotide comprises an inducible promoter, a polynucleotide sequence encoding a Sox2 protein, a proteolytic cleavable site, and a polynucleotide sequence encoding a Klf4 protein.

[0167] In some embodiments, the polynucleotide comprises an inducible promoter, a polynucleotide sequence encoding an Oct4 protein, a first proteolytic cleavable site, a polynucleotide sequence encoding a Sox2 protein, a second proteolytic cleavable site, and a polynucleotide sequence encoding a Klf4 protein.

[0168] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, an inducible promoter, a polynucleotide sequence encoding an Oct4 protein, a first proteolytic cleavable site, a polynucleotide sequence encoding a Klf4 protein, a second proteolytic cleavable site, and a polynucleotide sequence encoding a Sox2 protein.

[0169] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, an inducible promoter, a polynucleotide sequence encoding a Sox2 protein, a first proteolytic cleavable site, a polynucleotide sequence encoding an Oct4 protein, a second proteolytic cleavable site, and a polynucleotide sequence encoding a Klf4 protein.

[0170] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, an inducible promoter, a polynucleotide sequence encoding a Sox2 protein, a first proteolytic cleavable site, a polynucleotide sequence encoding a Ktlf4 protein, a second proteolytic cleavable site, and a polynucleotide sequence encoding an Oct4 protein.

[0171] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, an inducible promoter, a polynucleotide sequence encoding a Klf4 protein, a first proteolytic cleavable site, a polynucleotide sequence encoding an Oct4 protein, a second proteolytic cleavable site, and a polynucleotide sequence encoding a Sox2 protein.

[0172] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, an inducible promoter, a polynucleotide sequence encoding a Klf4 protein, a first proteolytic cleavable site, a polynucleotide sequence encoding a Sox2 protein, a second proteolytic cleavable site, and a polynucleotide sequence encoding an Oct4 protein.

[0173] In some embodiments, the inducible promoter is a tetracycline-inducible or RU486-inducible promoter.

[0174] In some embodiments, the first proteolytic site is a P2A peptide, an E2A peptide, an F2A peptide, a T2A peptide, or the RXK(R)R consensus sequence of SEQ ID NO:17.

[0175] In some embodiments, the second proteolytic site is a P2A peptide, an E2A peptide, an F2A peptide, a T2A peptide, or the RXK(R)R consensus sequence of SEQ ID NO:17.

[0176] In some embodiments, the polynucleotide comprises a second promoter.

[0177] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, a first inducible promoter, a polynucleotide sequence encoding an Oct4 protein, a second inducible or non-inducible promoter, a polynucleotide sequence encoding a Sox2 protein, a proteolytic cleavage site, and a polynucleotide sequence encoding a Klf4 protein.

[0178] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, a first inducible promoter, a polynucleotide sequence encoding an Oct4 protein, a second inducible or non-inducible promoter, a polynucleotide sequence encoding a Klf4 protein, a proteolytic cleavage site, and a polynucleotide sequence encoding a Sox2 protein.

[0179] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, a first inducible promoter, a polynucleotide sequence encoding an Oct4 protein, a proteolytic cleavable site, a polynucleotide sequence encoding a Sox2 protein, a second inducible or non-inducible promoter, and a polynucleotide sequence encoding a Klf4 protein.

[0180] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, a first inducible promoter, a polynucleotide sequence encoding a Sox2 protein, a proteolytic cleavable site, a polynucleotide sequence encoding an Oct4 protein, a second inducible or non-inducible promoter, and a polynucleotide sequence encoding a Klf4 protein.

[0181] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, a first inducible promoter, a polynucleotide sequence encoding an Oct4 protein, a proteolytic cleavable site, a polynucleotide sequence encoding a Klf4 protein, a second inducible or non-inducible promoter, and a polynucleotide sequence encoding a Sox2 protein.

[0182] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, a first inducible promoter, a polynucleotide sequence encoding a Klf4 protein, a proteolytic cleavable site, a polynucleotide sequence encoding an Oct4 protein, a second inducible or non-inducible promoter, and a polynucleotide sequence encoding a Sox2 protein.

[0183] In some embodiments, the first promoter is inducible or non-inducible and the second promoter is inducible.

[0184] In some embodiments, the first and second inducible promoters are induced by the same inducing compound. In some embodiments, the first and second inducible promoters are induced by different inducing compounds. For example, the first inducible promoter can be a tetracycline-inducible promoter and the second inducible promoter can be a coumermycin-inducible promoter. In some embodiments, the polynucleotide comprises a first inducible promoter operably linked to a polynucleotide encoding at least one of Oct4, Sox2, and / or Klf4. In some embodiments, the polynucleotide comprises a first inducible promoter that is a tetracycline-inducible promoter and operably linked to a polynucleotide encoding at least one of Oct4, Sox2, and / or Klf4. In some embodiments, the polynucleotide further comprises a second inducible promoter operably linked to c-Myc. In some embodiments, the polynucleotide further comprises a second inducible promoter that is a coumermycin-inducible promoter operably linked to c-Myc. In some embodiments, the first inducible promoter, which is a tetracycline-inducible promoter and is operably linked to a polynucleotide encoding at least one of Oct4, Sox2, and / or Klf4, and the second inducible promoter, which is a coumermycin-inducible promoter and is operably linked to c-Myc, are present on different polynucleotides.

[0185] In some embodiments, the first and second promoters initiate transcription in the same direction. In some embodiments, the first and second promoters initiate transcription in different directions.

[0186] In some embodiments, the polynucleotide further comprises a polyadenylation signal sequence, hi some embodiments, the polyadenylation signal sequence is an SV40 polyadenylation signal sequence, a human growth hormone polyadenylation signal sequence, or a bovine growth hormone polyadenylation signal sequence.

[0187] In some embodiments, the polynucleotide further comprises a WPRE sequence.

[0188] In some embodiments, the polynucleotide further comprises an IRES.

[0189] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, an inducible promoter, a polynucleotide sequence encoding an Oct4 protein, a first IRES, a polynucleotide sequence encoding a Sox2 protein, a second IRES, and a polynucleotide sequence encoding a Klf4 protein.

[0190] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, an inducible promoter, a polynucleotide sequence encoding an Oct4 protein, a first IRES, a polynucleotide sequence encoding a Klf4 protein, a second IRES, and a polynucleotide sequence encoding a Sox2 protein.

[0191] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, an inducible promoter, a polynucleotide sequence encoding a Sox2 protein, a first IRES, a polynucleotide sequence encoding an Oct4 protein, a second IRES, and a polynucleotide sequence encoding a Klf4 protein.

[0192] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, an inducible promoter, a polynucleotide sequence encoding a Sox2 protein, a first IRES, a polynucleotide sequence encoding a Klf4 protein, a second IRES, and a polynucleotide sequence encoding an Oct4 protein.

[0193] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, an inducible promoter, a polynucleotide sequence encoding a Klf4 protein, a first IRES, a polynucleotide sequence encoding an Oct4 protein, a second IRES, and a polynucleotide sequence encoding a Sox2 protein.

[0194] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, an inducible promoter, a polynucleotide sequence encoding a Klf4 protein, a first IRES, a polynucleotide sequence encoding a Sox2 protein, a second IRES, and a polynucleotide sequence encoding an Oct4 protein.

[0195] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, an inducible promoter, a polynucleotide sequence encoding an Oct4 protein, a second inducible or non-inducible promoter, a polynucleotide sequence encoding a Sox2 protein, an IRES, and a polynucleotide sequence encoding a Klf4 protein.

[0196] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, an inducible promoter, a polynucleotide sequence encoding an Oct4 protein, a second inducible or non-inducible promoter, a polynucleotide sequence encoding a Klf4 protein, an IRES, and a polynucleotide sequence encoding a Sox2 protein.

[0197] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, a first inducible promoter, a polynucleotide sequence encoding an Oct4 protein, an IRES, a polynucleotide sequence encoding a Sox2 protein, a second inducible or non-inducible promoter, and a polynucleotide sequence encoding a Klf4 protein.

[0198] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, a first inducible promoter, a polynucleotide sequence encoding a Sox2 protein, an IRES, a polynucleotide sequence encoding an Oct4 protein, a second inducible or non-inducible promoter, and a polynucleotide sequence encoding a Klf4 protein.

[0199] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, a first inducible promoter, a polynucleotide sequence encoding an Oct4 protein, an IRES, a polynucleotide sequence encoding a Klf4 protein, a second inducible or non-inducible promoter, and a polynucleotide sequence encoding a Sox2 protein.

[0200] In some embodiments, the polynucleotide comprises, in a 5' to 3' direction, a first inducible promoter, a polynucleotide sequence encoding a Klf4 protein, an IRES, a polynucleotide sequence encoding an Oct4 protein, a second inducible or non-inducible promoter, and a polynucleotide sequence encoding a Sox2 protein.

[0201] In some embodiments, the polynucleotide comprises an inducible promoter operably linked to a polynucleotide encoding a c-Myc protein. In some embodiments, the polynucleotide comprises a non-inducible promoter operably linked to a polynucleotide encoding a transactivator. In some embodiments, 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 transactivator.

[0202] In some embodiments, the polynucleotide further comprises a polynucleotide sequence encoding a marker protein, ie, β-galactosidase protein, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, or blue fluorescent protein, tdTomato protein, or mCherry protein.

[0203] In some embodiments, the polynucleotide further comprises an intron sequence, hi some embodiments, the intron sequence comprises a CMV intron sequence, a β-actin intron sequence, an SV40 enhancer sequence, or a combination thereof.

[0204] In some embodiments, polynucleotides are provided that encode a Cas9 fusion protein (CRISPR activator) and a guide RNA sequence that targets a promoter or enhancer of at least one of the endogenous loci of Oct4, Sox2, and Klf4. In some embodiments, the polynucleotides encode a Cas9 fusion protein and a guide RNA sequence that targets a promoter or enhancer of the endogenous locus of Oct4, Sox2, Klf4, and optionally c-Myc.

[0205] In some embodiments, the polynucleotides described herein are RNA. In some embodiments, the polynucleotides described herein are DNA. In some embodiments, the polynucleotides are present in a vector. In some embodiments, the polynucleotides are present in an integrating vector. In some embodiments, the polynucleotides are present in a non-integrating vector. In some embodiments, the polynucleotides are present in an AAV vector, an adenoviral vector, a lentiviral vector, or a retroviral vector. In some embodiments, the polynucleotides are non-integrating RNA.

[0206] III. Vectors and Cells Also provided are vectors comprising the polynucleotides described herein.

[0207] 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.

[0208] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAVrh9, AAV9, AAVrh10, AAV10, AAVRH10, AAV11, AAV12, or AAV-DJ vector.

[0209] In some embodiments, an AAV vector with a broad targeting spectrum is selected to transduce a variety of cell types. In some embodiments, the AAV vector is an AAV-DJ vector.

[0210] In some embodiments, an AAV vector is selected that has one or more selected target cells. The target specificity of various AAV vectors is known in the art (see, for example, AAV Production Protocol, Genemedi Biotech, Inc. 2018).

[0211] In some embodiments, AAV vectors are modified to target one or more selected cell types. For example, AAV cap sequence can be modified to remove cell targeting epitopes from capsid and introduce alternative cell targeting sequences into capsid. AAV cap sequence modified in this way is known in the art.

[0212] In some embodiments, the viral vector is a retroviral vector and comprises a polynucleotide described herein. In some embodiments, the retroviral vector is a self-inactivating retroviral vector. In some embodiments, the retroviral vector is a pseudotyped retroviral vector. In some embodiments, the retroviral vector is modified to target a specific cell type or cell types. In some embodiments, the retroviral vector comprises a polynucleotide encoding an Oct4 protein, a Sox2 protein, a Klf4 protein, and / or a c-Myc protein. In some embodiments, the retroviral vector comprises a polynucleotide encoding an Oct4 protein, a Sox2 protein, a Klf4 protein, and a c-Myc protein. In some embodiments, the retroviral vector comprises a polynucleotide comprising a polycistronic cassette encoding an Oct4 protein, a Sox2 protein, a Klf4 protein, and / or a c-Myc protein. In some embodiments, the retroviral vector comprises a polynucleotide encoding an Oct4 protein, a Sox2 protein, a Klf4 protein, and / or a c-Myc protein. In some embodiments, the retroviral vector comprises a polynucleotide comprising a polycistronic cassette encoding an Oct4 protein, a Sox2 protein, and a Klf4 protein, hi some embodiments, the retroviral vector comprises a polynucleotide encoding a transcriptional activator and one or more of an Oct4 protein, a Sox2 protein, a Klf4 protein, a c-Myc protein, and / or a dnNFκBIA protein.

[0213] In some embodiments, the viral vector is a lentiviral vector and comprises the polynucleotide described herein.In some embodiments, the lentiviral vector is a self-inactivating lentiviral vector and comprises at least one deletion in the enhancer / promoter unit in the U3 region of 3'LTR.In some embodiments, the lentiviral vector is a pseudotyped lentiviral vector.In some embodiments, the lentiviral vector is pseudotyped with vesicular stomatitis virus glycoprotein (VSV G).In some embodiments, the lentiviral vector is pseudotyped with glycoproteins from Retroviridae, Rhabdoviridae, Arenaviridae, Flaviviridae, Paramyxoviridae, Baculoviridae, Filoviridae, or a combination thereof.In some embodiments, the lentiviral vector is modified with a target cell-specific binding moiety to target specific cells or cell types.In some embodiments, the lentiviral vector comprises a tat-independent heterologous promoter that replaces a portion of 5'LTR. In some aspects, the lentiviral vector contains an inactivating mutation in the integrase gene so that the lentiviral vector remains episomal in the transduced cells.

[0214] In some embodiments, the lentiviral vector comprises a polynucleotide encoding an Oct4 protein, a Sox2 protein, a Klf4 protein, and / or a c-Myc protein. In some embodiments, the lentiviral vector comprises a polynucleotide encoding an Oct4 protein, a Sox2 protein, a Klf4 protein, and a c-Myc protein. In some embodiments, the lentiviral vector comprises a polynucleotide comprising a polycistronic cassette encoding an Oct4 protein, a Sox2 protein, a Klf4 protein, and / or a c-Myc protein. In some embodiments, the lentiviral vector comprises a polynucleotide encoding an Oct4 protein, a Sox2 protein, and a Klf4 protein. In some embodiments, the lentiviral vector comprises a polynucleotide comprising a polycistronic cassette encoding an Oct4 protein, a Sox2 protein, and a Klf4 protein. In some embodiments, the lentiviral vector comprises a polynucleotide encoding a transcriptional activator and one or more of an Oct4 protein, a Sox2 protein, a Klf4 protein, a c-Myc protein, and / or a dnNFκBIA protein. In some embodiments, the non-viral vector is a plasmid DNA, RNA, a cationic polymer, a lipid, a lipopolymer, or a chemical derivative thereof.

[0215] In some embodiments, the cationic polymer is present in an amount sufficient to produce a ratio of amine nitrogens in the cationic polymer to phosphates in the DNA plasmid vector or RNA vector of about 0.1:1 to about 100:1.

[0216] In some embodiments, the cationic polymer is present in an amount sufficient to produce a ratio of amine nitrogens in the cationic polymer to phosphates in the DNA plasmid vector or RNA vector of about 0.1:1 to about 10:1.

[0217] In some embodiments, the cationic polymer is present in an amount sufficient to produce a ratio of amine nitrogens in the cationic polymer to phosphates in the DNA plasmid vector or RNA vector of about 0.1:1 to about 5:1.

[0218] In some embodiments, the non-viral vector comprises about 0.5 mg / ml to about 5.0 mg / ml of polynucleotide complexed with a cationic polymer. In some embodiments, the cationic polymer is poly(ethyleneimine) (PEI) polymer, poly-L-lysine, poly(L-arginine) (PLA), polyallylamine (PAH), polyamidoamine, diethylaminoethyl dextran, chitosan, poly(dimethylaminoethyl methacrylate) polyvinylamine homo- or copolymer, poly(vinylbenzyl-tri-Cl-C4-alkylammonium salt), polymer of aliphatic or aralkylaliphatic dihalide with aliphatic N,N,N',N'-tetra-Cl-C4-alkyl-alkylenediamine, poly(vinylpyridine) or poly(vinylpyridinium salt), poly(N,N-diallyl-N,N-di-Cl-C4-alkyl-ammonium halide), homo- or copolymer of quaternized diethyl-C4-alkyl-aminoethyl acrylate or methacrylate, POLYQUAD™, polyaminoamide, etc., or derivatives thereof.

[0219] Additionally provided are cells comprising the polynucleotides described herein or the vectors described herein, hi some aspects, the cells may be bacterial cells, yeast cells, fungal cells, insect cells, or mammalian cells.

[0220] In some aspects, cells comprising the polynucleotides described herein can be used to manufacture viral vectors. In some aspects, the cells are insect cells comprising the polynucleotides described herein and additional viral vector production components and are used to prepare the viral vectors described herein. In some aspects, the cells are mammalian cells comprising the polynucleotides described herein and additional viral vector production components and are used to prepare the viral vectors described herein.

[0221] In some aspects, the cells are insect cells that contain the polynucleotides and AAV viral vector production components described herein and are used to prepare AAV vectors.

[0222] In some embodiments, the cell is a mammalian cell comprising the polynucleotides and AAV viral vector production components described herein and is used to prepare an AAV vector. In some embodiments, the AAV vector production components include a polynucleotide encoding a Rep protein and a Cap protein, and an AAV vector comprising, for example, a polynucleotide encoding at least one regeneration factor and comprising at least one ITR. In some embodiments, the mammalian cell, upon expression of the Rep and Cap proteins, allows the AAV vector encoding at least one regeneration factor to be packaged into an AAV capsid.

[0223] In some embodiments, the cell is a mammalian cell comprising the polynucleotides and retroviral vector production components described herein and is used to prepare a retroviral vector. In some embodiments, the retroviral vector production components include polynucleotides encoding gag, pol, and env proteins, and a retroviral vector comprising, for example, a polynucleotide encoding at least one regeneration factor and comprising an LTR. In some embodiments, the mammalian cell, upon expression of the gag, pol, and env proteins, allows for packaging of a retroviral vector encoding at least one regeneration factor into a retroviral capsid.

[0224] In some embodiments, the cell is a mammalian cell comprising the polynucleotides and lentiviral vector production components described herein and is used to prepare a lentiviral vector. In some embodiments, the lentiviral vector production components include polynucleotides encoding gag, pol, and env proteins, and optionally, tat and rev proteins, and a retroviral vector comprising, for example, a polynucleotide encoding at least one regeneration factor and including an LTR. In some embodiments, the mammalian cell, upon expression of the gag, pol, and env proteins, and optionally, the tat and rev proteins, allows the lentiviral vector encoding at least one regeneration factor to be packaged into a lentiviral capsid.

[0225] In some aspects, a cell comprising a polynucleotide, vector, or nanoparticle described herein and present in an organ is provided. In some aspects, the cell comprising a polynucleotide described herein is present in an organ that has been exposed to ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, viral-induced hepatitis, alcohol, or fibrosis not associated with any known cause.

[0226] In some aspects, cells comprising the polynucleotides described herein can be used to treat a subject. In some aspects, cells comprising the polynucleotides described herein can be administered to a subject in need of reversing senescence, whereupon the cells express a protein encoded by the polynucleotide described herein, and the expressed protein reverses cellular processes associated with the senescent phenotype. In some aspects, cells comprising the polynucleotides described herein replicate after administration to a subject. In some aspects, cells comprising the polynucleotides described herein do not replicate after administration to a subject. In some aspects, cells comprising the polynucleotides described herein can be hematopoietic progenitor cells. In some aspects, cells comprising the polynucleotides described herein can be hematopoietic stem cells. In some aspects, cells comprising the polynucleotides described herein can be muscle stem cells. In some aspects, cells comprising the polynucleotides described herein can be adipose stem cells.

[0227] IV. Nanoparticles Further provided are lipid particles, e.g., lipid nanoparticles, comprising the polynucleotides described herein. In some embodiments, the lipid nanoparticles are about 10 nm to about 1000 nm in size. In some embodiments, the lipid nanoparticles comprise a solid lipid core matrix and a surfactant. In some embodiments, the lipid nanoparticles comprise a cationic lipid. In some embodiments, the lipid nanoparticles comprise a cationic lipid, a non-cationic lipid, and, optionally, an aggregation-preventing lipid.

[0228] In some embodiments, the lipid nanoparticles comprise amphiphilic lipids arranged in at least one bilayer having an aqueous interior containing a polynucleotide encoding a regenerative factor. In some embodiments, the polynucleotide of the lipid nanoparticle is DNA, RNA, or a mixture of both. In some embodiments, the lipid nanoparticles comprise multiple polynucleotides, each polynucleotide encoding a regenerative factor. In some embodiments, the lipid nanoparticles comprise RNA encoding Oct4. In some embodiments, the lipid nanoparticles comprise RNA encoding Sox2. In some embodiments, the lipid nanoparticles comprise RNA encoding Klf4. In some embodiments, the lipid nanoparticles comprise RNA encoding c-Myc. In some embodiments, the lipid nanoparticles comprise RNA encoding Oct4, RNA encoding Sox2, RNA encoding Klf4, and RNA encoding c-Myc. In some embodiments, the lipid nanoparticles comprise RNA encoding Oct4, RNA encoding Sox2, and RNA encoding Klf4. In some embodiments, the lipid nanoparticles comprise RNA encoding Oct4, Sox2, Klf4, and c-Myc. In some embodiments, the lipid nanoparticles comprise RNA encoding Oct4, Sox2, and Klf4. In some embodiments, the lipid nanoparticles comprise RNA comprising a polycistronic cassette encoding Oct4, Sox2, Klf4, and c-Myc. In some embodiments, the lipid nanoparticles comprise RNA comprising a polycistronic cassette encoding Oct4, Sox2, and Klf4.

[0229] In some embodiments, the lipid nanoparticles comprise an additional agent, hi some embodiments, the lipid nanoparticles comprise an enhancer agent as described herein.

[0230] V. Compositions, Perfusion Solutions, and Kits Also provided are compositions comprising the polynucleotides, vectors, and / or nanoparticles described herein. In some embodiments, perfusion solutions are provided comprising the polynucleotides, vectors, and / or nanoparticles described herein. In some embodiments, the perfusion solution or composition is a liquid solution comprising the polynucleotides, vectors, and / or nanoparticles described herein and an inorganic salt.

[0231] In some embodiments, the perfusate or composition comprises a phosphate bicarbonate buffer.

[0232] In some embodiments, the perfusion solution or composition comprises at least one of sodium chloride, calcium chloride, potassium chloride, magnesium chloride, sodium bicarbonate, monopotassium phosphate, or dibasic sodium phosphate. In some embodiments, the perfusion solution or composition comprises sodium chloride, calcium chloride, potassium chloride, magnesium chloride, sodium bicarbonate, monopotassium phosphate, and dibasic sodium phosphate.

[0233] In some embodiments, the perfusion solution or composition comprises a dextran / albumin solution. In some embodiments, the perfusion solution or composition comprises calcium gluconate.

[0234] In some embodiments, the perfusion solution or composition includes at least one vasodilator. In some embodiments, the vasodilator is carbon monoxide, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, calcium channel blockers, prostacyclin, hydralazine, minoxidil, and nitroglycerin. In some embodiments, the perfusion solution or composition includes a vasodilator that is not carbon monoxide.

[0235] In some embodiments, the perfusate or composition comprises at least one tonicity agent, hi some embodiments, the tonicity agent is dextrose, glycerin, mannitol, potassium chloride, or sodium chloride.

[0236] In some embodiments, the perfusion solution 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 embodiments, the metabolic substrate is an amino acid or glucose. In some embodiments, the perfusion solution or composition comprises insulin.

[0237] In some embodiments, the growth factor is at least one of fibroblast growth factor (FGF), insulin-like growth factor (IGF), transforming growth factor beta (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 cholecystokinin octapeptide.

[0238] In some embodiments, immunosuppressants include, but are not limited to, steroidal (e.g., prednisone) or non-steroidal (e.g., sirolimus (Rapamune, Wyeth-Ayerst Canada), tacrolimus (Prograf, Fujisawa Canada), and anti-IL2R daclizumab (Zenapax, Roche Canada)). In some embodiments, the immunosuppressant is 15-deoxyspergualin, cyclosporine, methotrexate, rapamycin, rapamune (sirolimus / rapamycin), FK506, lisofylline (LSF), mycophenolate mofetil, antithymocyte globulin, belatacept, or everolimus.

[0239] In some embodiments, the antibacterial agent is an antibiotic, including but not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol; an antibiotic, including but not limited to, amoxicillin, penicillin, sulfonamides, cephalosporins, erythromycin, streptomycin, gentamicin, tetracycline, clarithromycin, ciproflozacin, and azithromycin; an antifungal, including but not limited to, myconazole and terconazole; and combinations thereof.

[0240] In some embodiments, antioxidants include, but are not limited to, molecules with a thiol group, 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, lazaroids, butylated hydroxyanisole (BHA), vitamin K, and the like; and combinations thereof.

[0241] In some embodiments, the perfusate or composition includes an oxygenating agent. In some embodiments, the oxygenating agent is plasma-free leukocyte-depleted red blood cells, hemoglobin, pyridoxylated hemoglobin, a synthetic hemoglobin-based oxygen carrier (including, but not limited to, a polymerized hemoglobin-based oxygen carrier); or a cell-free oxygen-carrying medium (including, but not limited to, Lifor™, Aqix™ RS-I, or STEEN solution™). In some embodiments, the oxygenating agent is HEMOXYCarrier™, Hemopure™, Hemarina™, or a perfluorocarbon.

[0242] In some embodiments, the perfusion solution or composition comprises at least one of human serum albumin, dextran, and an extracellular electrolyte composition. In some embodiments, the perfusion solution or composition comprises human serum albumin, dextran, and an extracellular electrolyte composition.

[0243] In some embodiments, the perfusion solution or composition comprises a dextran / albumin solution, packed red blood cells, calcium gluconate, heparin, cefazolin, verapamil, amino acids, glucose, aninsluin, and lactated Ringer's solution.

[0244] In some embodiments, the perfusion solution or composition comprises packed red blood cells and colloid solution (e.g., Gelafuncin, B Braun), cefuroxime, heparin, sodium bicarbonate, calcium gluconate, insulin, sodium taurocholate, prostacyclin, amino acids, and glucose.

[0245] In some aspects, the perfusate or composition comprises a polynucleotide described herein and a delivery agent or vector described herein.

[0246] In some embodiments, the delivery agent is a viral vector. In some embodiments, the delivery agent is a retroviral vector. In some embodiments, the delivery agent is a lentiviral vector. In some embodiments, the delivery agent is an AAV vector. In some embodiments, the delivery agent is an AAV-DJ vector. In some embodiments, the delivery agent is a nanoparticle. In some embodiments, the delivery agent is a lipid nanoparticle. In some embodiments, the delivery agent is a polymer nanoparticle. In some embodiments, the lipid or polymer nanoparticle is conjugated to a ligand moiety to target the lipid or polymer nanoparticle to a specific cell, tissue, or organ. In some embodiments, the delivery agent is a cationic polymer. In some embodiments, the delivery agent is a lipid, lipopolymer, or chemical derivative thereof.

[0247] In some embodiments, the cationic polymer of the composition is present in an amount sufficient to produce a ratio of amine nitrogens in the cationic polymer to phosphates in the DNA plasmid or RNA of about 0.1:1 to about 100:1.

[0248] In some embodiments, the cationic polymer of the composition is present in an amount sufficient to produce a ratio of amine nitrogens in the cationic polymer to phosphates in the DNA plasmid or RNA of about 0.1:1 to about 10:1.

[0249] In some embodiments, the cationic polymer of the composition is present in an amount sufficient to produce a ratio of amine nitrogens in the cationic polymer to phosphates in the DNA plasmid or RNA of about 0.1:1 to about 5:1.

[0250] In some embodiments, the polynucleotides described herein are present at about 0.5 mg / ml to about 5.0 mg / ml complexed with a cationic polymer of the composition, hi some embodiments, the cationic polymer of the composition is poly(ethyleneimine) (PEI) polymer, poly-L-lysine, polyamidoamine, diethylaminoethyl dextran, chitosan, poly(dimethylaminoethyl methacrylate), or derivatives thereof.

[0251] Also provided are pharmaceutical compositions comprising the polynucleotides, vectors, and / or nanoparticles described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient.

[0252] Some examples of materials that can function as pharmaceutically acceptable carriers include: (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 carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; and (10) glycols, such as cellulose acetate, ... , propylene glycol, (11) polyols, such as glycerin, 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) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffers, (21) polyesters, polycarbonates, and / or polyanhydrides, and (22) other non-toxic, compatible substances used in pharmaceutical formulations.

[0253] Additional non-limiting examples of suitable agents for the compositions described herein include PEG-conjugated polynucleotides, phospholipid-conjugated polynucleotides, polynucleotides containing lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (e.g., Pluronic P85) that may facilitate drug entry into various tissues, and biodegradable polymers (e.g., poly(DL-lactide-co-glycolide) microspheres) for sustained delivery after implantation.

[0254] In some embodiments, the composition further comprises wetting agents, emulsifiers and lubricants, release agents, coating agents, preservatives and antioxidants.

[0255] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0256] In some aspects, the compositions of the present disclosure comprise an excipient selected from cyclodextrins, celluloses, liposomes, micelle-forming agents such as bile acids, and polymeric carriers such as polyesters and polyanhydrides; and a polynucleotide described herein. In some aspects, the compositions render the polynucleotide described herein orally bioavailable.

[0257] Methods of preparing these compositions, perfusates, or pharmaceutical compositions include combining the polynucleotides described herein with a carrier and, optionally, one or more accessory ingredients. Generally, the compositions, perfusates, or pharmaceutical compositions are prepared by uniformly and intimately combining the polynucleotide with liquid carriers or finely divided solid carriers, or both.

[0258] Liquid dosage forms of the compositions, perfusates, or pharmaceutical compositions described herein include pharmaceutically acceptable emulsions, microemulsions, solutions, and suspensions.In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

[0259] In addition to the polynucleotide or vector, the suspension may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof.

[0260] Pharmaceutical compositions suitable for parenteral administration or administration into perfusates may contain one or more polynucleotides, vectors, or nanoparticles described herein in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted immediately before use into sterile injectable solutions or dispersions, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes that render the composition isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers that can be used in pharmaceutical compositions of the present 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. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0261] These pharmaceutical compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms on the polynucleotides, vectors, or nanoparticles described herein can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like.

[0262] Depot forms can be made by forming microencapsulated matrices of the polynucleotide or vector in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of polynucleotide or vector to polymer and the nature of the particular polymer used, the release rate of the polynucleotide or vector can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Formulations can also be prepared by entrapping the polynucleotide or vector in liposomes or microemulsions that are compatible with organ tissues.

[0263] Regardless of the route of administration selected, the polynucleotides, vectors, or nanoparticles described herein and / or pharmaceutical compositions of the present disclosure can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. The actual dosage level of the polynucleotide or vector in a pharmaceutical composition can be varied to obtain an amount of polynucleotide or vector that is effective to achieve the desired therapeutic response for a particular organ or patient, composition, and mode of administration, without exhibiting unacceptable organ or patient toxicity.

[0264] For example, the dosage level of polynucleotide, vector, or nanoparticle in the perfusion solution will depend on a variety of factors, including the activity of the particular polynucleotide, vector, or nanoparticle used, the type or organ being perfused, the method of perfusion, the time of perfusion, and / or other drugs, compounds, and / or substances used in combination with the particular polynucleotide, vector, or nanoparticle used, as well as the age, sex, weight, condition, general health, and prior medical history of the subject from whom the organ was harvested, and the age, sex, weight, condition, general health, and prior medical history of the subject receiving the organ.

[0265] In some embodiments, the polynucleotides or vectors described herein are administered to cells and / or organs by a variety of methods, including, but not limited to, incorporation into vehicles such as iontophoresis, liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. In some embodiments, microemulsification techniques can be utilized to improve the bioavailability of the polynucleotides or vectors described herein and, for example, to enhance the bioavailability of the polynucleotides or vectors by directing absorption into the lymphatic system of an isolated organ rather than the circulatory system.

[0266] In some embodiments, the formulation contains micelles formed from a polynucleotide or vector described herein and at least one amphiphilic carrier, wherein the micelles have an average diameter of less than about 100 nm. In some embodiments, micelles having an average diameter of less than about 50 nm are used with the polynucleotides or vectors described herein. In some embodiments, micelles having an average diameter of less than about 30 nm, or even less than about 20 nm, are used.

[0267] While any suitable amphiphilic carrier is contemplated, currently preferred carriers are those with Generally Recognized as Safe (GRAS) status. Examples of amphiphilic carriers include saturated and monounsaturated polyethylene glycolated fatty acid glycerides, such as those derived from various fully or partially hydrogenated vegetable oils. Such oils can advantageously consist of tri-, di-, and mono-fatty acid glycerides and di- and mono-polyethylene glycol esters of the corresponding fatty acids; particularly preferred fatty acid compositions include capric acid 4-10%, capric acid 3-9%, lauric acid 40-50%, myristic acid 14-24%, palmitic acid 4-14, and stearic acid 5-15%. Another useful class of amphiphilic carriers includes sorbitan and / or sorbitol partially esterified with saturated or monounsaturated fatty acids (SPAN-series) or their corresponding ethoxylated analogs (TWEEN-series).

[0268] Commercially available amphiphilic carriers may be useful, including the Gelucire series, Labrafil, Labrasol, or Lauroglycol (all manufactured and sold by Gattefosse Corporation, Saint Priest, France), PEG-mono-oleate, PEG-di-oleate, PEG-mono- and di-laurate, lecithin, polysorbate 80, and the like.

[0269] Suitable hydrophilic polymers for use with the polynucleotides or vectors described herein are those that are readily soluble in water, can be covalently attached to vesicle-forming lipids, and are tolerated in vivo without toxic effects (i.e., are biocompatible). Suitable polymers include polyethylene glycol (PEG), polylactic acid (also called polylactide), polyglycolic acid (also called polyglycolide), polylactic acid-polyglycolic acid copolymers, and polyvinyl alcohol. In some embodiments, the polymer has a molecular weight of about 100 or 120 daltons up to about 5,000 or 10,000 daltons, or about 300 daltons to about 5,000 daltons. In some embodiments, the polymer is polyethylene glycol having a molecular weight of about 100 to about 5,000 daltons, or about 300 to about 5,000 daltons. In some embodiments, the polymer is 750 dalton polyethylene glycol (PEG(750)). Polymers may also be defined by the number of monomers therein; in some embodiments, polymers of at least about three monomers are used, for example, PEG polymers consisting of three monomers (approximately 150 daltons).

[0270] Other hydrophilic polymers that may be suitable for use in the present disclosure include polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses, such as hydroxymethylcellulose or hydroxyethylcellulose.

[0271] In some aspects, the compositions of the present disclosure comprise a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic acid esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, cellulose, polypropylene, polyethylene, polystyrene, polymers of lactic and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acid, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

[0272] In some embodiments, the compositions of the present disclosure comprise a cationic polymer. In some embodiments, the cationic polymer comprises a polyethyleneimine (PEI) backbone. In some embodiments, the PEI backbone is linked to a lipid or polyethylene glycol. In some embodiments, the cationic polymer comprises cationic dextran, cationic chitosan, cationic gelatin, cationic cellulose, or cationic cyclodextrin.

[0273] In some embodiments, the compositions of the present disclosure comprise liposomes. Liposomes consist of at least one lipid bilayer membrane surrounding an aqueous interior compartment. Liposomes can be characterized by membrane type and size. Small unilamellar vesicles (SUVs) have a single membrane and typically range from 0.02 to 0.05 μm in diameter, while large unilamellar vesicles (LUVS) are typically larger than 0.05 μm. Large oligolamellar vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 0.1 μm. Liposomes with several non-concentric membranes, i.e., several smaller vesicles contained within a larger vesicle, are called multivesicular vesicles.

[0274] In some embodiments, the lipid bilayer of the liposome contains a lipid derivatized with polyethylene glycol (PEG), such that the PEG chains extend from the inner surface of the lipid bilayer into the interior space enclosed by the liposome and from the exterior of the lipid bilayer into the surrounding environment. Polymer-derivatized lipids, such as PEG lipids, can act to inhibit micelle / membrane fusion. The active agent, e.g., a polynucleotide or vector, contained within the liposomes described herein is in a solubilized form. Aggregates of surfactant and active agent (e.g., emulsions or micelles containing polynucleotides or vectors) can be encapsulated within the interior space of liposomes according to the present disclosure.

[0275] Liposomes according to the present disclosure can be prepared by any of a variety of techniques known in the art. See, for example, U.S. Patent No. 4,235,871, published PCT application WO 96 / 14057; New RRC, Liposomes: A practical approach, IRL Press, Oxford (1990), pages 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 a lipid derivatized with a hydrophilic polymer into a preformed liposome, e.g., by exposing the preformed liposome to micelles composed of lipid-grafted polymers, at a lipid concentration corresponding to the final molar percentage of derivatized lipid desired in the liposome. Liposomes containing hydrophilic polymers can also be formed by homogenization, lipid-field hydration, or extrusion techniques, as are well known in the art.

[0276] In some embodiments, the active agent is first dispersed in lysophosphatidylcholine or other low CMC surfactant (containing polymer-grafted lipid) by sonication. The resulting micellar suspension of the active agent is then used to rehydrate a dried lipid sample containing an appropriate mole percent of polymer-grafted lipid or cholesterol. The lipid and active agent suspension is then formed into liposomes using extrusion techniques well known in the art, and the resulting liposomes are separated from the non-encapsulated solution by standard column separation.

[0277] In some embodiments, liposomes are prepared to have a substantially uniform size within a selected size range. One effective sizing method involves extruding an aqueous suspension of liposomes through a series of polycarbonate membranes with selected uniform pore sizes, the pore sizes of which will approximately correspond to the largest size of liposomes produced by extrusion through the membrane. In some embodiments, reagents such as DharmaFECT® and Lipofectamine® can be used to introduce polynucleotides or vectors into cells.

[0278] The release characteristics of the formulations of the present disclosure depend on the encapsulation material, the concentration of the encapsulated polynucleotide or vector, and the presence of a release modifier. Release can be engineered to be pH-dependent, for example, by using a pH-sensitive coating that releases only at low or higher pH. Coatings can be used to prevent release from occurring until after transplantation of the organ containing the formulation into a subject. For example, multiple coatings or a mixture of coatings can be used to encapsulate the polynucleotides or vectors described herein used in perfusion solutions or compositions, such that the encapsulated polynucleotides or vectors are taken up by cells of the perfused organ, but the polynucleotides or vectors are released only in the presence of low pH, such as occurs with metabolic acidosis when the function of a transplanted kidney declines.

[0279] Release can also be manipulated by including salts or pore-forming agents, which can increase the uptake or release of water from the composition by diffusion from the encapsulation. Excipients that alter the solubility of the composition can also be used to control the release rate. Agents that promote matrix degradation or release from the encapsulation matrix can also be incorporated. The agent can be added to the composition as a separate phase (i.e., as microparticles) or co-dissolved in the polymer phase, depending on the composition. In some embodiments, the amount is 0.1 to 30 percent (w / w polymer). Types of degradation-promoting agents 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; organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine; and surfactants such as Tween® and Pluronic®.

[0280] In some embodiments, the encapsulated particles are coated with a cell adhesion polymer. Examples include chitosan, cellulose, and polymers with free carboxyl groups, such as polyacrylates, among others (as used herein, polyacrylate refers to polymers containing acrylate groups and modified acrylate groups, e.g., cyanoacrylate and methacrylate).

[0281] In some embodiments, the polynucleotide or vector can be formulated to be contained within or adapted to be released by a surgical or medical device or implant. Examples of medical devices include vascular stents that are implanted before organ transplantation, e.g., stents that are implanted in organ arteries or organ ureters to maintain patency. In some embodiments, the implant can be coated or otherwise treated with the polynucleotide or vector. For example, hydrogels or other polymers, e.g., biocompatible and / or biodegradable polymers, can be used to coat the implant with the compositions described herein (e.g., the use of hydrogels or other polymers can make the composition suitable for use in medical devices). Polymers and copolymers for coating medical devices with drugs are well known in the art.

[0282] VI. Organs and Perfusion Systems Provided herein are organs and methods for preparing them for transplantation. In some embodiments, the organ is removed from a donor. In some embodiments, the organ is removed from one subject (donor), subjected to the materials and methods described herein, and transplanted into another subject (recipient). In some embodiments, the organ is removed from one subject (donor), subjected to the materials and methods described herein, and transplanted into the same subject (the donor and recipient are one subject). In some embodiments, the donor is a human subject. In some embodiments, the recipient is a human subject requiring an organ transplant. In some embodiments, the organ is an organoid generated in vitro. In some embodiments, the organoid is rejuvenated using the methods described herein before being transplanted into a recipient. In some embodiments, the organoid comprises a vasculature including blood vessels and lymphatic vessels, and is perfused with the perfusion solution described herein through a tube connected to the blood vessels and / or lymphatic vessels.

[0283] In some embodiments, the organ is subjected to perfusion before, during, and / or after being subjected to the materials and methods described herein. In some embodiments, the organ is subjected to cold perfusion before, during, and / or after being subjected to the materials and methods described herein. In some embodiments, the organ is subjected to normothermic perfusion before, during, and / or after being subjected to the materials and methods described herein. In some embodiments, the organ is subjected to cold perfusion before being subjected to the materials and methods described herein, and normothermic perfusion during and / or after being subjected to the materials and methods. In some embodiments, the organ is subjected to cold perfusion before and during being subjected to the materials and methods described herein, and normothermic perfusion after being subjected to the materials and methods. In some embodiments, the cold perfusion and / or normothermic perfusion are performed using a perfusion system.

[0284] In some embodiments, the perfusion system is a normothermic mechanical perfusion system. In some embodiments, the perfusion system is a hypothermic (cold) mechanical perfusion system.

[0285] In some embodiments, a perfusion system is used in the methods described herein. In some embodiments, the perfusion system is a Hugo Sachs / Harvard Apparatus, a Kidney Assist™ system, an OrganOX system, a Radnoti system, an ARK Kidney system, or an Aferetica PerLife® system.

[0286] In some aspects, the organ is operably connected to the perfusion system such that perfusion fluid moved by the perfusion system enters the organ through the organ's blood vessels. In some aspects, the organ is operably connected to the perfusion system such that perfusion fluid moved by the perfusion system enters the organ through the organ's arteries. In some aspects, the organ is operably connected to the perfusion system such that perfusion fluid moved by the perfusion system enters the organ through the organ's veins.

[0287] In some embodiments, the organ is a kidney and is operably connected to a perfusion system such that perfusion fluid moved by the perfusion system enters the organ through the ureters of the organ.

[0288] In some aspects, the organ is operably connected to a perfusion system via a perfusion fluid reservoir, whereby the organ is contained in the perfusion fluid reservoir and the perfusion system moves the perfusion fluid through blood vessels, ureters, or lymphatic vessels to enter or permeate the organ.

[0289] In some embodiments, the organ is perfused with a perfusion solution comprising at least one of albumin, dextran, and an extracellular electrolyte composition, hi some embodiments, the perfusion solution comprises human serum albumin, dextran, and an extracellular electrolyte composition.

[0290] In some embodiments, the organ is perfused with a perfusion solution comprising a polynucleotide, vector, and / or nanoparticle described herein and at least one inorganic salt, hi some embodiments, the organ is perfused with a perfusion solution or composition comprising a phosphate bicarbonate buffer.

[0291] In some embodiments, the organ is perfused with a perfusion solution comprising at least one of sodium chloride, calcium chloride, potassium chloride, magnesium chloride, sodium bicarbonate, monopotassium phosphate, or dibasic sodium phosphate. In some embodiments, the organ is perfused with a perfusion solution comprising sodium chloride, calcium chloride, potassium chloride, magnesium chloride, sodium bicarbonate, monopotassium phosphate, and dibasic sodium phosphate.

[0292] In some embodiments, the organ is perfused with a perfusion solution comprising at least one vasodilator. In some embodiments, the organ is perfused with a perfusion solution comprising at least one tonicity agent. In some embodiments, the organ is perfused with a perfusion solution comprising at least one of a buffer, an inorganic salt, an amino acid, a nutrient, a cytokine, a growth factor, a hormone, an antioxidant, an anti-inflammatory agent, an immunosuppressant, an anticoagulant, or an antibacterial agent.

[0293] In some embodiments, the organ is perfused with a perfusion solution comprising an oxygenator that is plasma-free, leukocyte-depleted red blood cells, hemoglobin, a synthetic hemoglobin-based oxygen carrier, an acellular oxygen-carrying medium, a polymerized hemoglobin-based oxygen carrier, or a pyridoxylated hemoglobin. In some embodiments, the organ is perfused with a perfusion solution comprising an acellular oxygen-carrying medium selected from the group consisting of Lifor™, Aqix® RS-I, STEEN Solution™, Hemarina®, and a perfluorocarbon.

[0294] In some embodiments, the organ is perfused with a perfusion solution that includes a tonicity agent that is dextrose, glycerin, mannitol, potassium chloride, or sodium chloride.

[0295] In some embodiments, the organ is perfused with a perfusion solution containing a vasodilator that is carbon monoxide, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin receptor blocker, a calcium channel blocker, prostacyclin, hydralazine, minoxidil, or nitroglycerin. In some embodiments, the organ is perfused with a perfusion solution containing a carbon monoxide-releasing molecule (e.g., CORM-3, CORM-A1) (see, e.g., Csongradi et al. Curr Pharm Biotechnol. 13:819-26, 2012). In some embodiments, the organ is perfused with a perfusion solution to which carbon monoxide has been added. In some embodiments, the organ is perfused with a perfusion solution containing about 20 ppm to 250 ppm carbon monoxide. In some embodiments, the perfusion solution does not contain carbon monoxide as a vasodilator.

[0296] In some embodiments, the organ is perfused with a perfusion solution containing a polynucleotide, polypeptide, lipid nanoparticle, polymer nanoparticle, or ligand-conjugated lipid or polymer nanoparticle described herein. In some embodiments, the organ is perfused with a perfusion solution containing a regeneration factor-protein transfer domain fusion protein. In some embodiments, the organ is perfused with a perfusion solution containing a viral vector containing a polynucleotide encoding at least one regeneration factor described herein. In some embodiments, the organ is perfused with a perfusion solution containing an AAV vector, an adenoviral vector, a retroviral vector, or a lentiviral vector. In some embodiments, the organ is perfused with a perfusion solution containing a regeneration factor polypeptide.

[0297] In some embodiments, the organ is a kidney and is perfused with a perfusion solution comprising at least one of human serum albumin, dextran, and an extracellular electrolyte composition. In some embodiments, the kidney is perfused with a perfusion solution comprising at least one of human serum albumin, dextran, and an extracellular electrolyte composition.

[0298] In some embodiments, the organ is a kidney and the perfusion system used to perfuse the kidney is a normothermic perfusion system. In some embodiments, the organ is a kidney and the perfusion system is a Hugo Sachs / Harvard Apparatus, a Kidney Assist™ system, an OrganOX system, a Radnoti system, an ARK Kidney system, or an Aferetica PerLife® system. In some embodiments, the kidney is perfused using a normothermic perfusion system with a perfusion solution comprising a dextran / albumin solution, packed red blood cells, calcium gluconate, heparin, antibiotics, vasodilators, amino acids, glucose, insulin, and lactated Ringer's solution.

[0299] In some embodiments, the organ is a liver and the perfusion system used to perfuse the liver is a normothermic perfusion system. In some embodiments, the organ is a liver and the perfusion system is a Hugo Sachs / Harvard Apparatus, a Kidney Assist™ system, an OrganOX system, a Radnoti system, an ARK Kidney system, or an Aferetica PerLife® system. In some embodiments, the liver is perfused using a normothermic perfusion system with a perfusion solution comprising packed red blood cells, colloidal solution, sodium bicarbonate, calcium gluconate, heparin, antibiotics, vasodilators, amino acids, glucose, and insulin, and an isotonic saline solution containing sodium taurocholate.

[0300] In some embodiments, the organ is a kidney and is perfused with a perfusion solution containing the polynucleotides, polypeptides, vectors, and / or nanoparticles described herein. In some embodiments, the kidney is characterized by at least one of aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, viral-induced hepatitis, alcohol, or fibrosis not associated with any known cause prior to exposure to the perfusion solution described herein. In some embodiments, the aged or damaged kidney is contacted with the perfusion solution for different periods of time and / or with perfusion solutions containing different components, depending on the age and / or type and degree of injury. In some embodiments, the aged or damaged kidney can be exposed to the perfusion solution for days or weeks before transplantation into a recipient. In some embodiments, aged or damaged kidneys can be evaluated by diagnostic tests and / or biopsies before and / or after exposure to the perfusion solution described herein to determine whether signs of aging or damage, including, but not limited to, inflammatory cell infiltration, tubular damage, interstitial edema, or fibrotic changes, are reduced after exposure to the perfusion solution compared to before exposure to the perfusion solution. In some embodiments, aged or damaged kidneys can be exposed to the perfusion solution for one or more additional periods. In some embodiments, the composition of the perfusion solution can be adjusted depending on the aging or damage present in the kidney before perfusion. In some embodiments, the composition of the perfusion solution can be adjusted depending on the damage remaining in the kidney after a first perfusion period to perfuse the kidney for a second or more perfusion periods.

[0301] In some embodiments, the kidney is perfused with a perfusion solution described herein at a temperature of about 2°C to about 38°C, or at about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22°C, or at about 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38°C.

[0302] In some embodiments, the organ is a liver and is perfused with a perfusion solution containing the polynucleotides, polypeptides, vectors, and / or nanoparticles described herein. In some embodiments, the liver is characterized by fibrosis not associated with aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, viral-induced hepatitis, alcohol, or any known cause prior to exposure to the perfusion solution described herein. In some embodiments, the aged or damaged liver is contacted with the perfusion solution for different periods of time and / or with perfusion solutions containing different components, depending on the age and / or type and / or degree of injury. In some embodiments, the aged or damaged liver can be exposed to the perfusion solution for days or weeks before transplantation into a recipient. In some embodiments, the aged or damaged liver can be evaluated by diagnostic tests and / or biopsies before and / or after exposure to the perfusion solution described herein to determine whether signs of aging or damage, including, but not limited to, inflammatory cell infiltration, tubular damage, interstitial edema, or fibrotic changes, are reduced after exposure to the perfusion solution compared to before exposure to the perfusion solution. In some embodiments, the aged or damaged liver can be exposed to the perfusion solution for one or more additional periods. In some embodiments, the composition of the perfusion solution can be adjusted depending on the aging or damage present in the liver before perfusion. In some embodiments, the composition of the perfusion solution can be adjusted depending on the damage remaining in the liver after the first perfusion period to perfuse the liver for a second perfusion period.

[0303] In some embodiments, the liver is perfused with a perfusion solution described herein at a temperature of about 2°C to about 38°C, or at 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 at 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.

[0304] VII. Method Provided are methods of modulating (e.g., inducing) partial cell reprogramming, cell rejuvenation, cell regeneration, tissue repair, tissue regeneration, tissue rejuvenation, partial tissue reprogramming, organ regeneration, organ rejuvenation, partial organ reprogramming, or any combination thereof, the methods comprising contacting a cell, tissue, or organ with a polynucleotide, oligonucleotide, polypeptide, compound, vector, or nanoparticle described herein. In some embodiments, the method comprises contacting the cell, tissue, or organ ex vivo. In some embodiments, the method comprises contacting the cell, tissue, or organ with a polynucleotide, such as an engineered polynucleotide encoding at least one regenerative factor.

[0305] In some embodiments, the method includes organ regeneration. In some embodiments, the method includes organ rejuvenation, comprising contacting an organ ex vivo with a polynucleotide, oligonucleotide, polypeptide, compound, vector, or nanoparticle described herein, wherein the polynucleotide, oligonucleotide, polypeptide, compound, vector, or nanoparticle comprises or encodes at least one regenerative factor, wherein the at least one regenerative factor is expressed or present in the organ, and the organ is rejuvenated. In some embodiments, the method includes organ rejuvenation characterized by at least one of fibrosis not associated with aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, viral-induced hepatitis, alcohol, or any known cause.

[0306] In some embodiments, the method comprises administering to a subject (recipient) in need of an organ transplant comprising a polynucleotide, oligonucleotide, polypeptide, compound, vector, or nanoparticle described herein.

[0307] In some embodiments, the method comprises contacting an organ ex vivo with a polynucleotide comprising a polynucleotide encoding Oct4. In some embodiments, the method comprises contacting an organ ex vivo with a polynucleotide comprising a polynucleotide encoding Sox2. In some embodiments, the method comprises contacting an organ ex vivo with a polynucleotide comprising a polynucleotide encoding Klf4. In some embodiments, the method comprises contacting an organ ex vivo with a polynucleotide comprising a polynucleotide encoding c-Myc.

[0308] In some embodiments, the method comprises contacting the organ with a first polynucleotide encoding Oct4. In some embodiments, the method further comprises contacting the organ with a second polynucleotide encoding Sox2. In some embodiments, the method further comprises contacting the organ with a third polynucleotide encoding Klf4. In some embodiments, the method further comprises contacting the organ with a fourth polynucleotide encoding c-Myc.

[0309] In some embodiments, the method comprises contacting the organ with a polynucleotide encoding Oct4, Sox2, and Klf4. In some embodiments, the polynucleotide further encodes c-Myc. In some embodiments, the method comprises contacting the organ with a polynucleotide comprising a polycistronic cassette comprising polynucleotides encoding Oct4, Sox2, and Klf4. In some embodiments, the method further comprises contacting the organ with a polynucleotide encoding c-Myc.

[0310] In some embodiments, the method includes contacting the 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 embodiments, the method includes contacting the organ with a polynucleotide comprising the first inducible promoter, wherein the first inducible promoter is a tetracycline-inducible promoter and is operably linked to a polynucleotide encoding at least one of Oct4, Sox2, and / or Klf4. In some embodiments, the method further includes contacting the organ with a polynucleotide comprising a second inducible promoter operably linked to c-Myc. In some embodiments, the method further includes contacting the organ with an inducible compound such that the inducible promoter promotes expression of at least one of Oct4, Sox2, Kl4, and / or c-Myc.

[0311] In some embodiments, the method comprises contacting the organ with a polynucleotide comprising a first inducible promoter that is a tetracycline-inducible promoter and operably linked to a polynucleotide encoding at least one of Oct4, Sox2, and / or Klf4. In some embodiments, the method further comprises contacting the organ with a second polynucleotide comprising a second inducible promoter that is a coumermycin-inducible promoter operably linked to c-Myc. In some embodiments, the method comprises contacting the organ with a polynucleotide comprising a first inducible promoter that is a tetracycline-inducible promoter and operably linked to a polynucleotide encoding at least one of Oct4, Sox2, and / or Klf4, the polynucleotide further comprising a second inducible promoter that is a coumermycin-inducible promoter and operably linked to c-Myc.

[0312] In some embodiments, the organ is incubated 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, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 19 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 45 hours, 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, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 22 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, The organ is contacted with a polynucleotide encoding at least one regeneration factor for about 1 minute to about 72 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours, about 120 hours, about 132 hours, about 144 hours, about 156 hours, or about 168 hours, or any period therebetween. In some embodiments, the organ is contacted with the polynucleotide encoding at least one regeneration factor for about 1 minute to about 72 hours, followed by a non-contacting period and another period during which the organ is contacted with the polynucleotide for about 1 minute to about 72 hours. In some embodiments, the organ is repeatedly contacted with the polynucleotide described herein, with periods of non-contacting in between. For example, in some embodiments, an organ is contacted with a polynucleotide encoding at least one regeneration 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 another 12-hour non-contact period, followed by another contact period. In some embodiments, the period of contact with the polynucleotide and the non-contact period are of different lengths. In some embodiments, the periods of contact with the polynucleotide are of different lengths and the non-contact periods are of the same length. In some embodiments, the periods of contact with the polynucleotide are of different lengths and the non-contact periods are of different lengths. In some embodiments, organ function is measured after contact with the polynucleotide, and the non-contact period and subsequent contact periods are based on measurements of organ function.

[0313] In some embodiments, the contacting occurs immediately after the organ is donated, or 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, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, or about 9 weeks after organ donation.

[0314] In some embodiments, the interval between contacting the organ with a polynucleotide comprising at least one regeneration factor operably linked to an inducible promoter and contacting the organ with a compound that activates the inducible promoter is 0 minutes, or from about 10 minutes to about 9 weeks or more, e.g., 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, or about 18 hours. about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or 9 weeks.

[0315] In some embodiments, the organ is contacted intermittently with the compound 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.

[0316] In some embodiments, the organ is irradiated about 2 to 10 times over a period spanning about 1 week to about 6 weeks, about 2 to 10 times over a period spanning about 1 week to about 5 weeks, about 2 to 10 times over a period spanning about 1 week to about 4 weeks, about 2 to 10 times over a period spanning about 1 week to about 3 weeks, about 2 to 10 times over a period spanning about 1 week to about 2 weeks, about 2 to 10 times over a period spanning about 2 weeks to about 6 weeks, about 2 to 10 times over a period spanning about 3 weeks to about 6 weeks, about 2 to 10 times over a period spanning about 4 weeks to about 6 weeks, about 2 to 10 times over a period spanning about 5 weeks to about 6 weeks, or any combination thereof. is intermittently contacted with the compound about twice in a period spanning from about 1 week to about 6 weeks, about three times in a period spanning from about 1 week to about 6 weeks, about four times in a period spanning from about 1 week to about 6 weeks, about five times in a period spanning from about 1 week to about 6 weeks, about six times in a period spanning from about 1 week to about 6 weeks, about seven times in a period spanning from about 1 week to about 6 weeks, about eight times in a period spanning from about 1 week to about 6 weeks, about 9 times in a period spanning from about 1 week to about 6 weeks, about 10 times in a period spanning from about 1 week to about 6 weeks, or about 2, 3, 4, 5, 6, 7, 8, 9, or 10 times in any period spanning from about 1 week to about 6 weeks.

[0317] In some embodiments, the organ is contacted for a period of time with a perfusion fluid that does not contain any polynucleotides, polypeptides, vectors, or nanoparticles, and subsequently contacted with a perfusion fluid that contains a polynucleotide, polypeptide, vector, or nanoparticle described herein.

[0318] In some embodiments, the method further comprises contacting the organ containing the polynucleotide, nanoparticles comprising the polynucleotide, or vector comprising at least one regeneration factor polynucleotide operably linked to an inducible promoter with an inducing compound.

[0319] In some aspects, the step of contacting the organ with a compound described herein that induces an inducible promoter present in the organ comprises administering the compound that induces the inducible promoter to a subject that has received the organ.

[0320] In some aspects, the methods include contacting an organ containing a polynucleotide, a nanoparticle comprising a polynucleotide, or a vector described herein with an inducer compound prior to transplantation of the organ into a recipient. In some aspects, the methods include contacting an organ containing a polynucleotide, a nanoparticle comprising a polynucleotide, or a vector described herein with an inducer compound after transplantation of the organ into a recipient, wherein the compound is administered to the recipient.

[0321] In some embodiments, the method comprises administering the organ to the recipient 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, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 15 hours, 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, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 19 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 45 hours, 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 In some embodiments, the method comprises contacting the organ with the inducer compound for about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours, about 120 hours, about 132 hours, about 144 hours, about 156 hours, or about 168 hours, or any period therebetween. In some embodiments, the method comprises contacting the organ with the inducer compound multiple times over the above periods after transplantation into the recipient.

[0322] In some embodiments, the method comprises contacting the organ for the period of time described above after transplantation into the recipient, followed by a non-contacting period. In some embodiments, the method comprises contacting the organ for the period of time after transplantation into the recipient, followed by taking a biopsy of the organ and measuring organ function, and optionally contacting the organ for another period of time based on the results of the biopsy and organ function measurement. In some embodiments, the organ biopsy and function measurement are repeated periodically, for example, monthly, every two months, three months, four months, five months, or six months, and based on the results of each biopsy and function measurement, the organ is either contacted or not contacted with a polynucleotide described herein for the period of time described herein.

[0323] In some embodiments, the organ is contacted with a compound that activates an inducible promoter for the stated amount of time on one day per week, and the organ is not contacted again with the inducer compound for six days of that week. In some embodiments, the organ is contacted with an inducer compound for the stated amount of time on two days per week, and the organ is not contacted again with the inducer compound for five days of that week. In some embodiments, the organ is contacted with an inducer compound for the stated amount of time on three days per week, and the organ is not contacted again with the inducer compound for four days of that week. In some embodiments, the organ is contacted with an inducer compound for the stated amount of time on four days per week, and the organ is not contacted again with the inducer compound for three days of that week. In some embodiments, the organ is contacted with an inducer compound for the stated amount of time on five days per week, and the organ is not contacted again with the inducer compound for two days of that week. In some embodiments, the organ is contacted with the inducer compound for the stated time period six days a week, and the organ is not contacted again with the inducer compound on one day of the week.

[0324] In some embodiments, the days of the week that the organ is contacted with the inducing compound may or may not be consecutive, for example, the organ may be contacted with the inducing compound for the recited time on days 1, 3, and 5 of the week, but not on days 2, 4, 6, and 7 of the week.

[0325] In some embodiments, the method further comprises contacting the organ with a first inducer compound that activates a first inducible promoter. In some embodiments, the first inducible promoter is a tetracycline-inducible promoter and the first inducer compound is tetracycline. In some embodiments, the method further comprises contacting the organ with a second inducer compound that activates a second inducible promoter. In some embodiments, the second inducible promoter is a coumermycin-inducible promoter and the second inducer compound is coumermycin. In some embodiments, the method further comprises contacting the organ with the first inducer compound for a period of time that is different from the period of contacting the organ with the second inducer compound. For example, in some embodiments, the method comprises contacting the organ with tetracycline for two days per week for about 1-10 weeks, and contacting the organ with coumermycin 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 embodiments, the method comprises contacting the organ with tetracycline for two days per week for 1-10 weeks, and contacting the organ with coumermycin 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 embodiments, the method comprises contacting the organ with tetracycline for two days per week, and contacting the organ with coumermycin one day per week, where the organ is contacted with coumermycin on the day the organ is also contacted with tetracycline. In some embodiments, the method comprises contacting the organ with tetracycline two days per week and with coumermycin one day per week, wherein the organ is contacted with coumermycin on days when the organ is not contacted with tetracycline.

[0326] In some embodiments, the method comprises contacting the organ with tetracycline 4 days per week for about 1-10 weeks and contacting the organ with coumermycin 1-4 days per week for about 1-10 weeks. In some embodiments, the method comprises contacting the organ with tetracycline 4 days per week and contacting the organ with coumermycin 1-4 days per week, where the organ is contacted with coumermycin on days where the organ is also contacted with tetracycline. In some embodiments, the method comprises contacting the organ with tetracycline 4 days per week and contacting the organ with coumermycin 1-3 days per week, where the organ is contacted with coumermycin on days where the organ is not contacted with tetracycline.

[0327] In some embodiments, the organ is contacted with the first inducer compound for a longer period of time than with the second inducer compound at each contacting. In some embodiments, the organ is contacted with the first inducer compound and the second inducer compound for the same period of time. In some embodiments, the organ is contacted with the first inducer compound multiple times and the organ is contacted with the second inducer compound once. In some embodiments, the organ is contacted with the first inducer compound once and the organ is contacted with the second inducer multiple times.

[0328] In some embodiments, the organ is contacted with a polynucleotide encoding at least one of Oct4, Sox2, and / or Klf4 operably linked to a first inducible promoter and a second polynucleotide encoding c-Myc operably linked to a second inducible promoter. In some embodiments, the method includes contacting the organ with a composition comprising a first inducing compound for a first period of time and adding a second inducing compound to the composition for a second period of time. In some embodiments, the first period of time and the second period of time are the same length. In some embodiments, the first inducing compound is added to the composition at the same time as the second inducing compound. In some embodiments, the first inducing compound is added at a different time than the second inducing compound. In some embodiments, the first inducing compound and / or the second inducing compound is added to the composition weekly for, e.g., two, three, or four days per week for two months. In some embodiments, the first inducing compound and / or the second inducing compound are added to the composition every week for, e.g., 2, 3, or 4 days per week for a month. In some embodiments, the first inducing compound and / or the second inducing compound are added to the composition every other week for, e.g., 2, 3, or 4 days per week for a month. In some embodiments, the first inducing compound and / or the second inducing compound are added to the composition every other week for, e.g., 2, 3, or 4 days per week for a month.

[0329] In some embodiments, the methods include administering to a subject who has received an organ comprising a polynucleotide, vector, or nanoparticle described herein a compound that induces an inducible promoter present in the polynucleotide or vector, and further comprising administering to the subject an additional agent, e.g., an immunosuppressant. In some embodiments, immunosuppressants include, but are not limited to, steroids (e.g., prednisone), non-steroids (e.g., sirolimus (Rapamune, Wyeth-Ayerst Canada), tacrolimus (Prograf, Fujisawa Canada), anti-IL2R antibodies such as daclizumab (Zenapax, Roche Canada), 15-deoxyspergualin, cyclosporine, methotrexate, rapamycin, rapamune (sirolimus / rapamycin), FK506, lisofylline (LSF), or mycophenolate mofetil, antithymocyte globulin, belatacept, or everolimus.

[0330] In some embodiments, the methods include administering to a subject who has received an organ comprising a polypeptide described herein an immunosuppressant, e.g., a steroidal (e.g., prednisone), a non-steroidal (e.g., sirolimus (Rapamune, Wyeth-Ayerst Canada), tacrolimus (Prograf, Fujisawa Canada), an anti-IL2R antibody such as daclizumab (Zenapax, Roche Canada), 15-deoxyspergualin, cyclosporine, methotrexate, rapamycin, rapamune (sirolimus / rapamycin), FK506, lisofylline (LSF), or mycophenolate mofetil, antithymocyte globulin, belatacept, or everolimus.

[0331] In some embodiments, the method includes contacting an inducing compound with an organ that has previously been contacted with a polynucleotide encoding at least one regeneration factor operably linked to an inducible promoter.

[0332] In some embodiments, the method comprises administering an inducer compound to a recipient who has received an organ transplant that was previously contacted ex vivo with a polynucleotide encoding at least one regeneration factor operably linked to an inducible promoter. In some embodiments, the method comprises administering an inducer compound to a recipient who has received an organ transplant that includes an exogenous polynucleotide encoding at least one regeneration factor operably linked to an inducible promoter. In some embodiments, the recipient has received an organ transplant that was previously contacted with a polynucleotide encoding at least one of Oct4, Sox2, and / or Klf4 operably linked to a first inducible promoter and a second polynucleotide encoding c-Myc operably linked to a second inducible promoter. In some embodiments, the method comprises administering a first inducer compound to the recipient for a first period of time and administering a second inducer compound to the recipient for a second period of time. In some embodiments, the first period of time and the second period of time are the same length. In some embodiments, the first inducer compound is administered simultaneously with the second inducer compound. In some embodiments, the first inducer compound is administered at a different time than the second inducer compound. In some embodiments, the first inducer compound and / or the second inducer compound are administered, for example, 2, 3, or 4 days per week for 1-10 weeks every 3 months. In some embodiments, the first inducer compound and / or the second inducer compound are administered, for example, 2, 3, or 4 days per week for 1-10 weeks every 4 months. In some embodiments, the first inducer compound and / or the second inducer compound are administered, for example, 2, 3, or 4 days per week for 1-10 weeks every 6 months. In some embodiments, the first inducer compound and / or the second inducer compound are administered, for example, 2, 3, or 4 days per week for 1-10 weeks every 8 months. In some embodiments, the first inducer compound and / or the second inducer compound are administered, for example, 2, 3, or 4 days per week for 1-10 weeks every 10 months.In some embodiments, the first inducer compound and / or the second inducer compound are administered, for example, 2, 3, or 4 days per week for 1-10 weeks every 12 months.

[0333] In some embodiments, for each administration of a first compound and a second compound to a recipient, the first inducer compound is administered for a longer period of time than the second inducer compound. In some embodiments, the first inducer compound and the second inducer compound are administered to the recipient for the same period of time. In some embodiments, the first inducer compound is administered for a shorter period of time than the second inducer compound. In some embodiments, the first inducer compound is administered to the recipient multiple times and the second inducer compound is administered once. In some embodiments, the first inducer compound is administered to the recipient once and the second inducer compound is administered multiple times.

[0334] In some aspects, the method comprises: (i) contacting an organ with a polynucleotide comprising a first inducible promoter operably 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, resulting in expression of at least one of Oct4, Sox2, or Klf4; (iii) contacting the organ with a polynucleotide comprising a second inducible promoter operably linked to a polynucleotide encoding c-Myc; and (iv) contacting the organ with a second inducer that induces activity of the second promoter, resulting in expression of c-Myc. In some aspects, step (ii) of the described method is performed multiple times 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 multiple times and step (iv) is performed only once. In some embodiments, step (ii) is performed 2 to 100 times and step (iv) is performed only 1 to 10 times. In some embodiments, step (iv) is performed once every 2 to 5 times that step (ii) is performed. In some embodiments, step (iv) is performed once every 1 to 5, 6 to 10, 11 to 15, 16 to 20, 21 to 25, 26 to 30, 31 to 35, 36 to 40, 41 to 45, or 46 to 50 times that step (ii) is performed.

[0335] In some embodiments, steps (ii) and (iv) are performed multiple times during a one-month period and not performed during a subsequent three-month period. In some embodiments, steps (ii) and (iv) are performed multiple times during a one-month period and not performed during a subsequent six-month period. In some embodiments, steps (ii) and (iv) are performed multiple times during a one-month period and not performed during a subsequent nine-month period. In some embodiments, steps (ii) and (iv) are performed multiple times during a one-month period and not performed during a subsequent twelve-month period.

[0336] In some embodiments, the method includes (i) contacting an organ with a polynucleotide comprising a first inducible promoter operably 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 operably linked to a polynucleotide encoding c-Myc, (ii) contacting the organ with an inducer compound that induces activity of the first promoter, and (iii) contacting the organ with a second inducer compound that induces activity of the second promoter. In some embodiments, step (ii) of the described method is performed multiple times and step (iii) is performed once. In some embodiments, step (ii) is performed 2 to 100 times and step (iii) is performed 1 to 10 times. In some embodiments, step (iii) is performed once every 2 to 5 times that step (ii) is performed. In some embodiments, step (iv) is performed once for every 1 to 5, 6 to 10, 11 to 15, 16 to 20, 21 to 25, 26 to 30, 31 to 35, 36 to 40, 41 to 45, or 46 to 50 times that step (ii) is performed.

[0337] In some embodiments, steps (ii) and (iii) are performed multiple times during a one-month period and not performed during a subsequent consecutive three-month period. In some embodiments, steps (ii) and (iii) are performed multiple times during a one-month period and not performed during a subsequent consecutive six-month period. In some embodiments, steps (ii) and (iii) are performed multiple times during a one-month period and not performed during a subsequent consecutive nine-month period. In some embodiments, steps (ii) and (iii) are performed multiple times during a one-month period and not performed during a subsequent consecutive twelve-month period.

[0338] In some embodiments, the organ contacted ex vivo with the compositions described herein is an organ characterized by at least one of aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, virally induced hepatitis, alcohol, or fibrosis not associated with any known cause.

[0339] In some embodiments, the organ is an aging organ. In some embodiments, the organ is 70 years old or older (i.e., the organ is removed from a subject who is 70 years old or older). In some embodiments, the organ is over 60 years old. In some embodiments, the organ is 70-79 years old. In some embodiments, the organ is 60-69 years old. In some embodiments, the organ is 50-59 years old. In some embodiments, the organ is 40-49 years old. In some embodiments, the organ is under 60 years old. In some embodiments, the organ is 50 years old or younger. In some embodiments, the organ is 6-50 years old.

[0340] In some embodiments, the organ is a kidney or a liver.

[0341] In some embodiments, the kidney is 70 years or older. In some embodiments, the kidney is over 60 years old, 70-79 years old, 60-69 years old, 50-59 years old, or 40-49 years old. In some embodiments, the kidney is under 60 years old. In some embodiments, the kidney is 50 years old or younger. In some embodiments, the kidney is 6-50 years old.

[0342] In some embodiments, the kidney is from an elderly donor. In some embodiments, the kidney is from a donor aged 70-79 years, 60-69 years, 50-59 years, or 40-49 years. In some embodiments, the kidney is from a donor with an age-related disease or condition. In some embodiments, the age-related disease or condition is a history of hypertension. In some embodiments, the age-related disease or condition is a history of atherosclerosis. In some embodiments, the age-related disease or condition is a history of toxic renal injury. In some embodiments, the age-related disease or condition is a history of renal immune injury. In some embodiments, the age-related disease or condition is a history of renal perfusion injury. In some embodiments, the age-related disease or condition is fibrosis. In some embodiments, the kidney is from a donor with a serum creatinine of 1.5 mg / dL or greater. In some embodiments, the kidney is from a donor who meets any two of the following criteria: a history of high blood pressure, a creatinine of 1.5 mg / dl or higher, or death from a stroke. In some embodiments, the kidney is from a donor under the age of 50. In some embodiments, the kidney is from a donor under the age of 50, where the donor was brain dead (e.g., due to traumatic injury or other causes such as stroke). In some embodiments, the donor is a cardiac-beating donor (donation after cardiac death (DCD)). In some embodiments, the donor is a brain-dead donor (donation after brain death (DBD)). In some embodiments, the kidney is from a DCD donor who died from uncontrolled circulatory death (also known as Maastricht II category, i.e., cardiac arrest due to unsuccessful resuscitation). In some embodiments, the kidney is from a donor who died from controlled circulatory death (also known as Maastricht III category, i.e., cardiac arrest occurring in a controlled manner within a hospital).

[0343] In some embodiments, the kidney is from a living donor. In some embodiments, the kidney is from a deceased donor.

[0344] In some embodiments, the liver is 70 years of age or older. In some embodiments, the liver is over 60 years of age, 70-79 years of age, 60-69 years of age, 50-59 years of age, or 40-49 years of age. In some embodiments, the kidney is from a donor with an age-related disease or condition. In some embodiments, the age-related disease or condition is steatosis. In some embodiments, the age-related disease or condition is liver fibrosis.

[0345] In some embodiments, the liver is characterized by steatosis. In some embodiments, the liver is from a non-heart-beating donor (donation after cardiac death (DCD)). In some embodiments, the liver is from a brain-dead donor (donation after brain death (DBD)). In some embodiments, the liver is a split liver.

[0346] In some embodiments, the liver is from a living donor. In some embodiments, the liver is from a deceased donor.

[0347] Organ function can be characterized before and after transplantation using assays and tests known in the art. In some embodiments, the organ is characterized by at least one of aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, viral-induced hepatitis, alcohol, or fibrosis not associated with any known cause. In some embodiments, the aged or damaged organ is evaluated by diagnostic tests and / or biopsy before and / or after exposure to a perfusion solution described herein to determine whether signs of aging or injury, including, but not limited to, inflammatory cell infiltration, tubular injury, interstitial edema, or fibrotic changes, are reduced after exposure to the perfusion solution compared to before exposure to the perfusion solution. In some embodiments, the aged or damaged organ can be exposed to the perfusion solution for one or more additional periods. In some embodiments, the composition of the perfusion solution can be adjusted depending on the aging or injury present in the organ before perfusion. In some embodiments, the composition of the perfusion solution can be adjusted depending on the damage remaining in the organ after the first perfusion period, in order to perfuse the organ for a second or more perfusion periods.

[0348] In some embodiments, renal function is characterized by testing one or more of the following: BUN, pH, bicarbonate, sodium, potassium, serum creatinine (e.g., eGFR (1 week / 1 month / 3 months post-transplant)), protein to creatinine ratio (urine), serum KIM1, serum Klotho, CXCL10 / 9 (e.g., measured in urine), and / or blood pressure.

[0349] In some embodiments, renal function is characterized by detecting and / or measuring urine production in the recipient.

[0350] In some embodiments, renal histological findings are characterized, for example, by examining one or more of the following: Banff score (Masson's Trichrome), tubular necrosis score, proliferation (e.g., by assaying Ki67, PCNA), apoptosis (e.g., by TUNEL assay), and / or immune infiltration (e.g., by assaying CD68, CD3, and / or CD19).

[0351] In some embodiments, the liver is evaluated for liver injury, inflammation, regeneration and / or tissue repair. In some embodiments, liver function is evaluated / characterized by one or more of AST, ALT, bilirubin, MPO level, and / or MDA level. In some embodiments, liver histological findings are characterized by, for example, examining one or more of the following: Ki67, PCNA, HGF and / or TGF-beta; TUNEL and / or caspase 3 assay. H&E staining can be performed to evaluate liver injury.

[0352] In some embodiments, the liver is characterized by (evaluated for) one or more of the following: steatosis score (e.g., steatosis, lobular inflammation, and hepatocyte ballooning) Grade 0 (healthy, <5%), Grade 1 (mild, 5%-33%), Grade 2 (moderate, 34%-66%), and Grade 3 (severe, >66%), necrosis score, and / or presence of immune infiltrate (e.g., assays of CD68, CD3, and / or CD19).

[0353] In some embodiments, liver function is assessed by assaying alkaline phosphatase (ALP), albumin and total protein, bilirubin, gamma-glutamyltransferase (GGT), L-lactate dehydrogenase (LD), and / or prothrombin time (PT).

[0354] In some aspects, organs (e.g., organ biopsies) are characterized using epigenetic clocks. In further embodiments, the DNA methylation age of organs is determined using the Horvath epigenetic clock. The Horvath epigenetic clock can be used as an age estimation method based on DNA methylation at CpG dinucleotide motifs in DNA.

[0355] In some embodiments, renal function is characterized in the recipient by examining one or more of the following: BUN, serum creatinine, serum pH, bicarbonate, sodium, potassium, or lactate levels. In some embodiments, kidneys contacted with a polynucleotide described herein exhibit improved BUN, serum creatinine, serum pH, bicarbonate, sodium, potassium, and / or lactate levels compared to before contact with the polynucleotide.

[0356] In some embodiments, the transplanted liver is evaluated for liver injury, inflammation, and regeneration / tissue repair. In some embodiments, liver function is characterized in the recipient by examining one or more of the following: widely standardized liver pathology parameters (AST, ALT, and bilirubin), apoptosis parameters (caspase 3), hepatocyte proliferation (Ki67 / PCNA, HGF, and TGF-β), liver inflammation (MPO activity for neutrophil accumulation, MDA levels for oxidative stress), and / or liver histology (H&E).

[0357] In some embodiments, livers contacted with the polynucleotides described herein exhibit reduced levels of AST, ALT, bilirubin, caspase 3, increased hepatocyte proliferation markers Ki67 / PCNA, HGF and TGF-β, reduced MPO activity, reduced MDA levels, and reduced histological signs of liver injury compared to before contact with the polynucleotides.

[0358] In some embodiments, the methods of rejuvenating senescent cells, tissues, or organs described herein restore or improve at least one functionality of the senescent cell, tissue, or organ, e.g., mitochondrial function, proteolytic activity, heterochromatin levels, histone methylation, nuclear lamina polypeptides, cytokine levels.

[0359] In some embodiments, contacting a cell, tissue, or organ with a polynucleotide described herein results in a change in the collective methylation state of a collection of individual methylation sites in the cell, tissue cells, or organ, and this collective methylation state measures chronological age (DNA methylation clock).

[0360] In some embodiments, contacting a cell, tissue, or organ with a polynucleotide described herein results in an increase in one or more nuclear and / or epigenetic markers compared to a reference value. In some embodiments, contacting a cell, tissue, or organ with a polynucleotide described herein results in an increase in HP1 gamma expression. In some embodiments, contacting a cell, tissue, or organ with a polynucleotide described herein results in an increase in H3K9me3. In some embodiments, contacting a cell, tissue, or organ with a polynucleotide described herein results in an increase in the expression of the lamina support protein LAP2 alpha. In some embodiments, contacting a cell, tissue, or organ with a polynucleotide described herein results in an increase in the gene expression of SIRT1 protein.

[0361] In some embodiments, contacting a cell, tissue, or organ with a polynucleotide described herein exhibits improved mitochondrial health and function compared to when the cell, tissue, or organ is not contacted with the polynucleotide described herein. In some embodiments, the improved mitochondrial health and function is measured as a change in mitochondrial membrane potential, reactive oxygen species (ROS), or a combination thereof. For example, mitochondrial membrane potential can be measured using tetramethylrhodamine, methyl ester, perchlorate (Thermo), a dye that is sequestered by mitochondria based on their membrane potential (as described in PCT Publication WO2019 / 178296). Mitochondrial ROS can be measured using MitoSOX, a fluorogenic dye that is oxidized by superoxide within mitochondria, as described in PCT Publication WO2019 / 178296.

[0362] In some embodiments, methods are provided for preparing an organ for transplantation. In some embodiments, the method comprises contacting the organ with a composition comprising a polynucleotide, vector, or nanoparticle described herein prior to transplantation. In some embodiments, the composition is a perfusion solution described herein. In some embodiments, the method comprises contacting the organ with a perfusion solution comprising a polynucleotide, vector, or nanoparticle described herein prior to transplantation, thereby rejuvenating the organ. In some embodiments, the method comprises contacting an organ characterized by at least one of aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, viral-induced hepatitis, alcohol, or fibrosis not associated with any known cause with a perfusion solution comprising a polynucleotide, vector, or nanoparticle described herein prior to transplantation, wherein at least one of aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, viral-induced hepatitis, alcohol, or fibrosis not associated with any known cause in the organ is treated, ameliorated, prevented, and / or reversed. In some aspects, the method comprises contacting an organ with a perfusion solution comprising a polynucleotide, vector, or nanoparticle described herein prior to transplantation, wherein at least one of fibrosis in the organ not associated with aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, viral-induced hepatitis, alcohol, or any known cause is ameliorated, prevented, and / or reversed.

[0363] In some aspects, the method comprises contacting the organ with a polynucleotide, vector, or nanoparticle described herein. In some aspects, the contacting is carried out by immersing the organ in a perfusion solution comprising the polynucleotide, vector, or nanoparticle described herein. In some aspects, the contacting is carried out by perfusing the organ with a perfusion solution comprising the polynucleotide, vector, or nanoparticle described herein. In some aspects, the perfusion is achieved by connecting tubing to the blood vessels of the organ. In some aspects, the perfusion is achieved by connecting tubing to the lymphatic vessels of the organ.

[0364] In some embodiments, a method for preparing organoids for transplantation is provided.In some embodiments, organoids are generated from pluripotent stem cells in vitro.In some embodiments, organoids are contacted with polynucleotides as described herein.In some embodiments, organoids contain vasculature.In some embodiments, contacting is carried out by immersing organoids in a perfusion solution comprising polynucleotides, vectors or nanoparticles as described herein.In some embodiments, contacting is carried out by connecting the blood vessels of organoids to a tube, and perfusing the organoid with a perfusion solution comprising polynucleotides, vectors or nanoparticles as described herein.In some embodiments, perfusion is achieved by connecting a tube to the blood vessels of organoids.In some embodiments, perfusion is achieved by connecting a tube to the lymphatic vessels of organoids.

[0365] In some embodiments, organ or organoid is contacted with transfection agent.In some embodiments, transfection agent comprises lipofectamine, LT-1, dextran, calcium phosphate, polybrene.In some embodiments, organ or organoid is transfected by electroporation, microinjection or their combination.

[0366] In some embodiments, the organ or organoid is contacted with a composition comprising a combination therapy.

[0367] In some embodiments, a composition comprises a polynucleotide, polypeptide, vector, or nanoparticle described herein and an enhancer. In some embodiments, a composition comprises a polynucleotide, polypeptide, vector, or nanoparticle described herein, ... (de)isoquinolin-2-yl)-N-hydroxybutanamide), phenylbutyrate (e.g., sodium phenylbutyrate) or other short-chain fatty acids, scriptaid, suramin sodium, APHA compound 8, apicidin, sodium butyrate, pivaloyloxymethyl butyrate (Pivanex, AN-9), trapoxin B, chlamydocin, depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994 (e.g., N-acetyldinaline) and MS-27-275), MGCD0103, NVPLAQ-824, CBHA (m-carboxycinnaminic acid acid)bishydroxamic acid), JNJ16241199, tubacin, A-161906, proxamide, oxamflatin, 3-Cl-UCHA (e.g., 6-(3-chlorophenylureido)caproic acid hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31, or CHAP 50. In some embodiments, the composition comprises a polynucleotide, polypeptide, vector, or nanoparticle described herein and a dominant-negative (e.g., catalytically inactive) form of an HDAC, an siRNA inhibitor of an HDAC, or an antibody that specifically binds to an HDAC.

[0368] VIII. Kit In some embodiments, kits are provided that include compositions or perfusion solutions described herein that include polynucleotides, vectors, and / or nanoparticles and one or more pharmaceutically acceptable carriers, excipients, and / or vehicles. In some embodiments, kits are provided that include a first composition or perfusion solution that includes a polynucleotide, vector, and / or nanoparticle described herein and one or more pharmaceutically acceptable carriers, excipients, and / or vehicles, and optionally a second composition or perfusion solution that includes a second polynucleotide, vector, and / or nanoparticle described herein and one or more pharmaceutically acceptable carriers, excipients, and / or vehicles. In some embodiments, the kit further includes an inducer compound. In some embodiments, the kit includes a first inducer compound and a second inducer compound.

[0369] In some aspects, the kits comprise the compositions or perfusates described herein packaged in a manner that facilitates their use to practice the methods of the present disclosure.

[0370] In some aspects, the kit further comprises instructions on how to administer the composition or perfusate, and tools for preparing and administering the pharmaceutical composition or perfusate in a perfusion system described herein. [Example]

[0371] Example 1: Materials and Methods animal Syngeneic and allogeneic renal transplantation models were performed. In the syngeneic renal transplantation model, 3-month-old male Lewis (LEW, Janvier Labs, Le Genest-Saint-Isle, France) rats were used as recipients of 3-month-old male LEW grafts (Lewin, E. et al., Scand J Urol vol. 27, 1993). In the allogeneic renal transplantation model, male LEW rats were used as recipients of 9-month-old male Dark agouti (DA, Janvier Labs, Le Genest-Saint-Isle, France) grafts. DA and LEW strains differ partially in the major histocompatibility complex and various non-MHC loci, resulting in weaker histocompatibility combinations and graft rejection. Systolic blood pressure, proteinuria, and diuresis were monitored to assess the progression of hypertensive nephropathy.

[0372] Normothermic kidney perfusion and AAV administration A Hugo Sachs / Harvard Apparatus device was used for normothermic kidney perfusion. Briefly, the system consisted of a circular chamber with a water jacket and a glass lid assembly. For organ perfusion, the kidney was connected via the vena cava and aorta using two cannulas. The system included different support components attached to the cannulas to maintain the organ at the most physiological conditions: a membrane oxygenator, a reservoir, a cannula line holder, perfusate, gas, and water lines, a bubble trap, and a manifold for water, gas, and perfusate control. Before organ connection, the perfusate was prewarmed to 37°C and oxygenated for 30 min. The perfusate consisted of Gey's solution (Sigma) and 1% penicillin / streptomycin (Sigma). After organ connection, pressure-controlled perfusion was performed, gradually increasing the pressure over 5 min to reach a mean pressure of 70 mmHg. Upon reaching the target pressure, AAV was administered into the perfusate, and perfusion continued for 1 h after AAV administration.

[0373] Human Kidney Perfusion Human kidneys, such as those not suitable for transplantation due to ischemic, toxic, perfusion or other injury, or lack of suitability due to age, are perfused ex vivo with the polynucleotides, vectors, and / or nanoparticles described herein using a perfusion system. The perfusion solution for human kidney perfusion contained the following: 215 mL of dextran / albumin solution (Steen Solution, XVIVO Perfusion AB, Goteborg, Sweden); 400 mL of packed red blood cells; 2 mL of 10% calcium gluconate; 1300 U / L heparin; 400 mg cefazolin; verapamil (a vasodilator); amino acids, glucose, and insulin; and Lactated Ringer's solution to compensate for urine loss

[0374] The human kidney is connected to a perfusion system and perfused for at least 1 hour prior to addition of the polynucleotides, vectors, and / or nanoparticles described herein.

[0375] Next, human renal function will be assessed in pre- and post-perfusion renal biopsies by determining the Banff score (Masson's trichrome); tubular necrosis score; measurement of proliferation markers (Ki67, PCNA); apoptosis markers (TUNEL); immune infiltration (CD68, CD3, and / or CD19); and single nucleus RNA sequencing (snRNAseq) and methylome analysis.

[0376] Human liver perfusion Human livers are perfused according to the Organox Metra protocol. The perfusion system is primed with 3 units of packed red blood cells and colloid solution (Gelafundin, B Brown 500 mL).

[0377] Before connecting the liver to blood-based perfusion, do the following: The perfusion solution was supplemented with 750 mg cefuroxime (antibiotic); 10,000 IU heparin (anticoagulant to prevent thrombosis within the circuit); sodium bicarbonate (buffer to adjust the pH of the infusion solution before placing the liver in the device); and 10 ml of 10% calcium gluconate (to compensate for citrate-calcium binding). After the human liver was connected and during liver perfusion, the following components were added to the perfusion solution: insulin (200 IU) (to control the level of perfused glucose); heparin (25,000 IU) (to maintain anticoagulation); 2% sodium taurocholate / isotonic saline solution (to compensate for bile salt losses); Flolan® 0.5 mg prostacyclin (vasodilator to optimize microperfusion); and parenteral nutrition solution containing amino acids and glucose for liver maintenance.

[0378] Once the circuit is primed and the infusate is connected, the perfusate is automatically circulated, oxygenated, and heated to 37°C, and a human liver is connected to the perfusion system.

[0379] The rates of glucose and amino acid infusion (parenteral nutrition) were variable and glucose levels were entered manually every 4 hours.

[0380] Alternatively, human liver perfusion systems can use non-RBC oxygen carriers, including HEMOXYCarrier® Hemopure, Hemarina®, or perfluorocarbons.

[0381] Human livers were monitored by measuring AST / ALT, ALP, albumin, bilirubin, prothrombin time, L-lactate dehydrogenase, bilirubin, and gamma-glutamyltransferase in the perfusate. Liver biopsies taken before and at the end of perfusion can be examined by snRNASeq and methylome analysis. Histological examinations are performed to assess proliferation markers (Ki67, PCNA), apoptosis markers (e.g., TUNEL), steatosis score (steatosis, lobular inflammation, and hepatocyte ballooning) grade 0 (healthy, <5%), grade 1 (mild, 5%-33%), grade 2 (moderate, 34%-66%), and grade 3 (severe, >66%), necrosis score, and immune infiltration (CD68, CD3, and / or CD19).

[0382] Tissues were lysed and homogenized, and total RNA was extracted using the Maxwell® RSC Instrument (Promega) with Maxwell® RSC miRNA from Tissue (Promega) according to the supplier's protocol. RNA purity and concentration were assessed using a NanoDrop™ Eight Spectrophotometer (ThermoFisher). cDNA was synthesized from the RNA template using an Invitrogen cDNA synthesis kit according to 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. The primers used in cDNA analysis are listed in Table 2.

[0383] Real-time qPCR was performed on a 384-well plate in a QuantStudio 7 device (ThermoFisher) using SYBR Green PCR Master Mix reagent (Appliedbiosystems) with primers corresponding to the gene of interest. Samples were run in triplicate in 10 μl reactions, and target gene mRNA expression was normalized to GADPH mRNA and expressed as relative gene expression to the control group.

[0384] Adeno-associated virus (AAV) Adeno-associated viruses (AAV) prepared by Vector Biolabs were used to deliver DNA encoding key reprogramming genes under control of a controllable promoter. This allowed for efficient delivery and regulated expression of the reprogramming gene products. Reprogramming factors (Oct4, Sox2, Klf4, and cMyc) were cloned under the Tet-ON promoter, which is activated by the transactivator rtTA in the presence of doxycycline (AAV TRE3G SK shortWPRE SV40 polyA of SEQ ID NO: 26; AAV TRE3G cMyc shortWPRE SV40 polyA + EF1a TetON3G of SEQ ID NO: 27; AAV-TRE3G-hMyc-EF1a-rTta of SEQ ID NO: 28; and the plasmid AAV-CAG-CBRLuc-GFP of SEQ ID NO: 29). The sequences of the polynucleotides and their components are listed in Table 2.

[0385] To take advantage of its broad cell tropism, we used AAVs with a DJ serotype capsid (Grimm, D. et al. J Virol 82, 5887-5911, 2008). After delivery of the programming factors to the organ via AAV, gene expression was activated by the addition of commercially available doxycycline (Dox). From 2 weeks after transplantation until the end of the study, rats were weighed weekly and placed in metabolic cages for 24-hour urine and tail vein blood collection. Blood urea nitrogen (BUN), urine, and serum creatinine (Crea) were measured.

[0386] Luc fluorescence analysis. Seven days after graft perfusion and kidney transplantation, rats were anesthetized and intraperitoneally injected with 1 ml of luciferin (15 mg / mL). Thirty minutes later, luminescence images were captured using a standard approach with an IVIS Bioluminiscence imaging system.

[0387] statistical analysis Statistical analysis was performed using GraphPad Prism 5 statistical software (GraphPad Software Inc.). To evaluate rat survival (time from kidney transplantation to death), univariate analysis using the log-rank test (Kaplan-Meier curve) was performed. The Mann-Whitney test was used to compare two groups. A value of <0.05 was considered significant. Values ​​are shown as the mean ± standard deviation.

[0388] We chose to analyze chronic kidney lesions in kidney transplants 6 weeks after transplantation using a validated score (Remuzzi et al. J Am Soc Nephrol 10:2591-2598, 1999) (Table Z) used to evaluate human donor kidneys, with the following modifications: This global score assesses glomerulosclerosis, arterial stenosis, tubular necrosis, tubular atrophy, and interstitial fibrosis.

[0389] Modified Remuzzi score analysis In renal transplantation, the modified Remuzzi score (Remuzzi et al. J Am Soc Nephrol 10:2591-2598, 1999) was used to analyze chronic kidney lesions 6 weeks after transplantation from human donors, with the following modifications: This global score assesses glomerular sclerosis, arterial stenosis, tubular necrosis, tubular atrophy, and interstitial fibrosis.

[0390] Remuzzi score (Rs) (Remuzzi et al. (supra)) Glomerular Global Sclerosis (G) is scored as follows: 0: No overall hardening 1: <20% total glomerular sclerosis 2: 20-50% total glomerular sclerosis 3: >50% total glomerular sclerosis Tubular atrophy (T) is scored as follows: 0: None 1: <20% of tubules affected 2: 20-50% of the tubules are affected 3: >50% of tubules affected Interstitial fibrosis (I) is scored as follows: 0: None 1: <20% of renal tissue is replaced by fibrous connective tissue 2: 20-50% of the renal tissue is replaced by fibrous connective tissue 3: >50% of renal tissue replaced by fibrous connective tissue Arterial and arteriolar constriction (A) is scored as follows: 0: None 1: Increased wall thickness (but to a degree less than the diameter of the lumen) 2: Wall thickness equal to or slightly greater than the lumen diameter 3: Wall thickness far exceeds the luminal diameter, with severe luminal narrowing or obstruction

[0391] Modifications of the Remuzzi score did not include glomerulosclerosis factors (all glomeruli were normal in all samples because ischemic injury does not affect the glomerular compartment) and arteriolar / arteriolar stenosis (arteries were not observed in some slides). Additionally, acute tubular necrosis (ATN) was calculated according to four different grades: none (0), mild (1), moderate (2), and severe (4).

[0392] Example 2. AAV targeting to kidney GFP / luc When AAV vectors were injected into clamped rat renal veins, weak luciferin expression was detected in both the injected kidney and the uninjected contralateral kidney 7 days after in situ injection (see Figure 1A), indicating systematic leakage of AAV, which may lead to undesirable effects. The weak signal observed in the kidney was attributed to the low transduction efficiency of the AAV vector in the kidney.

[0393] Next, two periods (45 and 90 min) of normothermic mechanical perfusion (NMP) were tested with the AAV-GFP / Luc vector. The titer of the injected virus was approximately 1 × 10 12 The total distribution volume within the perfusion system was estimated to be approximately 60-70 ml. As described in the Materials and Methods section above, kidneys perfused ex vivo for 45 minutes by normothermic mechanical perfusion (NMP) with GFP / luc AAV were transplanted into rats. Luciferin was detected only in the perfused kidney (see the animal on the left, Figure 1B). In contrast, in rats injected intravenously (i.v.) (via the penile vein), luciferin was localized to the liver and not observed in the kidney (see the rat on the right, Figure 1B). Therefore, ex vivo normothermic mechanical perfusion (NMP) of rat kidneys with GFP / luc AAV demonstrated specific delivery to the perfused kidney and no apparent "leakage" to other organs and tissues.

[0394] After 90 minutes of perfusion (flow rate set at approximately 4 ml / min), the perfused left kidney showed in vivo luciferin expression, but no luciferin expression was observed in the lung or contralateral kidney (i.e., the non-perfused right kidney). Minimal luciferin levels were observed in the liver.

[0395] Example 3. OSKM treatment improves renal function and rat survival in aging and allograft models In the kidney, aging is associated with decreased glomerular filtration rate, increased vascular and interstitial pathology, and increased susceptibility to acute injury (e.g., ischemia-reperfusion injury) (Stenvinkel, P. & Larsson, TEAm. J. Kidney Dis. 62, 339-351, 2013; Anderson, S. & Brenner, BMAm. J. Med. 80, 435-442, 1986; and Baylis, C. & Corman, B., J. Am. Soc. Nephrol. JASN 9, 699-709, 1998). Using a clinically relevant model of kidney allotransplantation from aging donors, we examined the effects of ex vivo AAV-delivered Yamanaka factors (Oct-3 / 4, Sox2, Klf4, and c-Myc [OSKM]) on renal graft function and survival in renal-dependent rats (Figure 2A). Within this model, ischemic injury results from organ donation and normothermic perfusion, along with immune damage due to the different genetic backgrounds of the donor (Dark agouti strain) and recipient (Lewis strain).

[0396] Donor kidneys from aged (9-month-old) Dark agouti rats (DA rats) were transfected with inducible AAV-OSK and AAV c-Myc / rtTA vectors (approximately 1 × 10 12 , or a total titer of 2 × 10 12 The kidneys were perfused with NMP normothermic perfusion for approximately 1 hour using AAV GFP / luc (1 × 10). The anti-rejection drug tacrolimus was then administered for 5 days after kidney transplantation to prevent early graft rejection. One week after transplantation, the contralateral kidney was removed. Starting one week after transplantation, OSKM expression was induced two days a week by rtTA activation with drinking water supplemented with doxycycline (Dox). Vehicle controls were perfused with the same titer of AAV GFP / luc (1 × 10). 12) Dox administration was discontinued 87 days after transplantation in the AAV OSKM group (80 days after Dox treatment).

[0397] Histological analysis demonstrated inflammatory infiltrates, tubular injury, and interstitial edema in kidneys from aged donors transplanted with AAV-GFP-perfused kidneys (see Figure 2B). Previous studies have shown that inflammatory infiltrates are primarily due to alloimmune injury and can also result from ischemic insults. Tubular injury occurs more prominently in ischemic injury but can also result from alloimmune injury. Interstitial edema can be observed in both alloimmune and ischemic injury.

[0398] The survival rate of the OSKM-treated group was significantly higher than that of 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 after transplantation. One rat died one week after discontinuation (this rat was observed to have impaired renal function), while the other three rats survived for an additional three weeks, at which point they were examined for analysis (see Figure 2C). Renal function was maintained in the three surviving rats, as indicated by stable serum creatinine levels for three weeks after dox discontinuation (see Figure 2K). Notably, compared with the AAV-GFP / luc-treated control group, serum creatinine levels in the OSKM-treated group were lower and maintained throughout the entire study period. Rats transplanted with OSKM-perfused kidneys showed significantly increased renal function compared with control groups treated with AAV-GFP / luc, with lower blood urea nitrogen (BUN) levels (i.e., indicating better function) 2 weeks after kidney transplantation (see Figures 2D and 2F), stable BUN levels, pH, bicarbonate, sodium, potassium, and lactate levels 3–12 weeks after transplantation (see Figures 2G, 2H, 2I, 2J, 2L, and 2M), and specifically stable BUN levels in surviving rats 3 weeks after dox withdrawal (see Figure 2L). The maximum BUN level was noted to be 120 mg / dL, while normal BUN levels were noted to be in the range of 15–20 mg / dL. Transplanted kidneys were harvested from surviving OSKM-treated rats 100 days after transplantation (control rats had not survived at this time). No evidence of tumors was found within the kidneys (see Figure 2N). Macroscopically, the kidneys of these OSKM-treated animals were enlarged and their surfaces were irregular. These features are typically observed in allograft models due to alloimmune injury. Overall, these results demonstrated that partial reprogramming with OSKM can improve kidney function after ischemic and immune injury.

[0399] The OSKM-treated kidneys were then sectioned and stained with hematoxylin and eosin (see Figure 7). Immune infiltration, vascular lesions, and tubular damage were observed in the sections, consistent with alloimmune damage (transplant rejection) (see Figure 7). OSKM-treated mice were not administered anti-rejection drugs throughout the majority of the study period. The treated kidneys showed no signs of tumors or malignant lesions (see Figure 7).

[0400] To assess the level of immune infiltration in the kidneys of the AAV-GFP / luc control group, kidneys were harvested from two pre-mortem rats (i.e., mice showing signs of imminent death) and examined in the AAV-GFP / luc group 15 and 16 days after transplantation. Hematoxylin-eosin (H&E)-stained sections showed characteristics of allograft immune rejection with extensive immune infiltration, as expected given that the mice were not treated with anti-rejection drugs (unless indicated early in the protocol) (see Figure 8). These results demonstrated that the level of immune rejection (immune infiltration) was lower than expected in the OSKM-treated mice. OSKM-treated mice also survived longer and demonstrated improved renal function, indicating that OSKM treatment has a beneficial effect on preventing allograft rejection.

[0401] Example 4. OSKM treatment ameliorates ischemic injury in a syngeneic kidney transplant model Ischemia-reperfusion injury (IRI) is the primary cause of acute kidney injury and a constant feature of kidney transplant procedures. IRI is tissue damage that occurs when blood supply to an organ is interrupted and then restored. IRI is typically associated with a strong inflammatory and oxidative stress response to hypoxia and reperfusion (e.g., during kidney transplantation), disrupting organ function. Organ ischemia can cause tissue damage by reducing levels of the energy storage molecule ATP. This change leads to the accumulation of harmful reactive oxygen species, damages mitochondrial organelles, and triggers an inflammatory response when oxygenated blood flow is restored to the transplanted organ. These symptoms are particularly acute in high-risk donor organs, which account for an increasing proportion of current organ transplants.

[0402] Organ retrieval, transportation, and transplantation of kidney grafts cause IRI. Cold ischemia leads to IRI and can occur during organ transplantation when the organ is cooled with cold perfusion fluid after procurement and subsequent transportation. Warm ischemia can also contribute to IRI, and can occur, for example, upon death of a donor at cardiovascular death (DCD), in which case donor hypoxia can persist between the time of DCD and the time of organ procurement.

[0403] To understand whether OSKM can overcome ischemic injury, kidney perfusion with OSKM was performed in a syngeneic transplantation model (LEW to LEW, lacking alloimmune injury) (see Figure 3A). As shown in Figure 3A, kidneys from 3-month-old donors were transfected with inducible AAV-OSK and AAV c-Myc / rtTA (1 x 10 vectors each). 12 , total titer 2 x 10 12 ) and tolerated NMP for approximately 1 hour. One week after transplantation, the contralateral (right) kidney was then removed. Starting one week after transplantation, OSKM expression was induced approximately four days per week by rtTA activation with doxycycline (Dox) added to the drinking water. Control kidneys were then perfused but without AAV administration. Dox administration was discontinued 105 days after transplantation in the AAV OSKM group (56 days after Dox treatment). Organs were harvested 105 days after transplantation for analysis.

[0404] In this ischemic model, organ donation surgery and normothermic perfusion (1 hour) were associated with ischemic injury and ischemia-reperfusion lesions in ex vivo perfused kidneys, despite continuous oxygenation of the perfusate. The perfusate used in this model, Gey's balanced solution, lacks oxygen carriers, which is thought to reduce oxygen delivery to cells. Ten of 11 animals in the control group died on or before day 63. Compared to the OSKM-treated group, three animals died during the first 15 days, but six survived until the end of the study (approximately 125 days). Renal function stabilized in the six surviving rats, as evidenced by stable serum creatinine and blood urea nitrogen (BUN) levels observed until the end of the study.

[0405] Ex vivo delivery of OSKM dramatically improved rat survival in a syngeneic kidney injury model, with survival rates of >60% in OSKM-treated animals 100 days after kidney transplantation (Figure 3B). Importantly, serum creatinine levels were lower in the OSKM-treated group (Figure 3D, Figures 9C-9D, and Figure 10C) compared with the control group (Figure 3C, Figures 9C-9D-D, and Figure 10C). This difference was statistically significant (Figure 3E, Figures 9C-9D, and Figure 10C). BUN levels were also lower in the OSKM-treated group (Figure 3G, Figures 9A-9B, and Figure 10A) compared with the control group (Figure 3F, Figures 9A-9B, and Figure 10B) (a desirable effect). This difference was statistically significant (Figure 3H, Figures 9A-9B, Figure 10A, and Figure 10B). Macroscopic examination of kidneys harvested from the OSKM group revealed that no tumors or teratomas were observed in the OSKM-treated kidneys.

[0406] Overall, the results demonstrated that partial reprogramming with OSKM ameliorated ischemic injury in transplanted kidneys. The results also demonstrated preservation of transplanted kidney function after syngeneic transplantation in an ex vivo ischemic transplantation model.

[0407] Example 5. OSKM Treatment in Rat Liver - Short-Term Safety Study The infection and safety of AAV-OSKM were evaluated in vivo in Wistar rats (220-250 g) with healthy livers by AAV-OSKM vector administration (intravenous administration via the penile vein) for up to 1 week. In the control group, rats received PBS via the penile route, followed by doxycycline in their drinking water (diluted to 1 mg / mL in drinking water) on day 6 after PBS administration. On day 7, rats were sacrificed, and then livers, blood, and kidneys were collected (kidneys served as controls in this model). In the vehicle control group, vehicle (AAV-GFP / Luc, 1 × 10 per rat) was administered. 12) was administered via the penile route, and on day 6 after vehicle administration, doxycycline was administered in drinking water (diluted to 1 mg / mL in drinking water). On day 7, the rats were sacrificed, and the liver, blood, and kidneys (as controls) were collected.

[0408] Within the treatment group, AAV-OSK + AAV-cMyc-rtTA vectors (1 × 10 per rat) 12 Each vector) was administered via the penile route. On day 6 after vector administration, doxycycline was administered in drinking water (diluted to 1 mg / mL in drinking water). On day 7, the rats were sacrificed, and the liver, blood, and kidneys were collected (kidneys served as controls). Blood and tissue samples were then collected for examination. The liver was evaluated for liver injury, inflammation, and regeneration / tissue repair. Standardized liver pathology 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. Histological analysis (H&E) was used to evaluate liver injury (10x). Data are presented as mean ± standard error. Statistical analysis was performed using the Kruskal-Wallis test, followed by a post hoc Dunn's multiple comparison test where appropriate. P < 0.05 was considered significant.

[0409] The results of biochemical parameters related to liver damage and functionality (AST, ALT, bilirubin) demonstrated that AST, ALT, and bilirubin levels were similar in all groups studied (see Figures 5A, 5B, and 5D). A reduction in caspase 3 levels was observed in the AAV-OSKM-treated group compared to the control group (see Figure 5C).

[0410] Parameters of liver inflammation (neutrophil accumulation and oxidative stress) determined by MPO and MDA levels were similar in all groups (see Figures 5E and 5F), indicating the absence of liver inflammation when AAV-OSKM was administered (intravenously). Furthermore, parameters of hepatocyte proliferation / liver tissue repair determined by Ki67, PCNA, and HGF levels were similar in all groups (see Figures 5G, 5H, and 5I), whereas a decrease in TGFβ (a potent pro-proliferative and fibrogenic molecule) was observed in the AAV-OSKM-treated group compared to the control group (see Figure 5J).

[0411] No obvious necrotic foci were observed in any of the groups evaluated (see Figures 5K, 5L, and 5M), which was consistent with the biochemical measurements.

[0412] These results demonstrated that short-term in vivo treatment was safe, as determined by measuring parameters of liver injury and functionality, liver inflammation, and liver repair / regeneration.

[0413] Example 6. OSKM Treatment in Rat Liver - Long-Term Safety Study The safety of AAV-OSKM infection was evaluated in Wistar rats (220-250 g) with healthy livers up to 12 weeks after intravenous administration of AAV-OSKM. In the control group, rats received PBS via the penile (interchangeably referred to as penic) route, and doxycycline (diluted to 1 mg / mL in drinking water) was administered for 24 hours starting on day 6 after PBS administration. Oral doxycycline administration was continued once a week for 12 weeks. Then, after 12 weeks (Dox treatment), rats were sacrificed, and liver, blood, pancreas, kidney, adipose tissue, skin, spleen, heart, lung, intestine, and muscle were collected. In the vehicle control group, vehicle (AAV-GFP / Luc, 1 × 10 per rat) was administered. 12) was administered by the penile route, and starting on day 6 after vehicle administration, doxycycline was administered in drinking water for 24 hours (diluted to 1 mg / mL in drinking water). Oral doxycycline administration was continued once a week for 12 weeks, at which point the rats were sacrificed and the liver, blood, pancreas, kidneys, adipose tissue, skin, spleen, heart, lungs, intestine, and muscle were collected.

[0414] Within the treatment group, AAV-OSK + AAV-cMyc vectors (1 × 10 per rat) 12 The rats were administered 1 mg / mL of each vector via the penile route. Starting on day 6 after vector administration, doxycycline was administered in drinking water for 24 hours (diluted to 1 mg / mL in drinking water). Oral doxycycline administration was continued once a week for 12 weeks. After 12 weeks, the rats were sacrificed, and the liver, blood, pancreas, kidneys, adipose tissue, skin, spleen, heart, lungs, intestines, and muscles were collected.

[0415] Biochemical parameters related to liver damage and functionality (AST, ALT, bilirubin), including AST, ALT, and bilirubin levels, were similar across all groups (see Figures 6A, 6B, and 6D). Caspase 3 levels were also similar (see Figure 6C). Parameters of liver inflammation (neutrophil accumulation measured by MPO and oxidative stress measured by MPO (see Figures 6E and 6F)) and parameters of hepatocyte proliferation / liver tissue repair (Ki67, PCNA, HGF, and TGFβ levels (see Figures 6G, 6H, 6I, and 6J)) did not show differences between groups. Liver histology in control and AAV-OSKM-treated rats did not reveal any liver injury at 12 weeks (see Figures 6K, 6L, and 6M). These data demonstrate that intravenous administration of AAV-OSKM and periodic weekly Dox activation over a 12-week period is safe.

[0416] Example 7. Effect of AAV-delivered pre-injury liver reprogramming in rats in warm hepatic ischemia-reperfusion injury, an acute, clinically relevant model of liver injury. The effects of pre-injury OSKM reprogramming were evaluated in a rat model of partial (70%) hepatic ischemia (also known as warm hepatic ischemia-reperfusion injury), generally described in Peralta et al., J Hepatology 59, 1094-1106 (2013).

[0417] Acute warm hepatic ischemia-reperfusion injury was induced by clamping major blood vessels to the liver for 1 hour and then releasing the clamp for various periods of time. This generated warm hepatic ischemia-reperfusion (I / R) injury, modeling the I / R injury observed during liver resection and liver transplantation. Warm hepatic ischemia-reperfusion injury occurs during surgical liver resection because major blood vessels to the liver are clamped during surgery to avoid blood loss during hepatectomy (i.e., hepatectomy), a surgical procedure to remove part or all of the liver. Acute warm ischemia-reperfusion injury is also associated with the anhepatic period (the time from physical removal of the liver from the recipient to recirculation of the graft) during liver transplantation. While the remaining liver after surgical resection can regenerate to replace the removed portion, warm ischemia-reperfusion adversely affects regeneration of the remaining liver after hepatectomy, which can lead to major postoperative complications. Warm ischemia-reperfusion injury associated with liver transplantation can result in negative postoperative outcomes.

[0418] PBS (control), vehicle control (5 × 10 11 AAV-GFP / Luc+5×10 11 AAV-rtTA and OSKM AAV (AAV-OSK + AAV-cMyc vector (5 × 10 11 AAV-OSK+5×10 11 AAV (cMyc / rtTA) was administered to rats via the penile route, thereby delivering AAV or control to the liver, and animals were treated with doxycycline after various periods (1, 3, or 5 days) to induce expression of OSKM, as described below.

[0419] 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 leading to the left and central liver lobes were occluded for 60 minutes. Reperfusion was initiated by removing the occlusion clamp. After 4 hours of reperfusion, the animals were sacrificed, and organs / tissues were harvested for analysis.

[0420] Liver pathology parameters (AST, ALT, and bilirubin), apoptosis parameters (caspase 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 presented as mean ± standard error. Statistical analysis was performed by the Kruskal-Wallis test, followed by a post-hoc Dunn's multiple comparison test where appropriate. P < 0.05 was considered significant.

[0421] One week, four day, and two day protocols were tested.

[0422] In the 1-week protocol, rats were administered PBS control, vehicle control (AAV-GFP / Luc + AAV-rtTA), or AAV-OSKM (AAV-OSK + AAV cMyc / rtTA). On day 6 after administration, doxycycline was added to the rats' drinking water (1 mg / mL diluted in drinking water). On day 7 after administration, hepatic I / R was performed as described above. After 4 hours of reperfusion, rats were sacrificed and organs / tissues were harvested.

[0423] Intravenous administration of OSKM (AAV-OSK+AAV-cMyc+AAV-rtTa) 1 week before induction of partial warm hepatic ischemia-reperfusion resulted in a statistically significant decrease in the levels of biochemical markers of liver injury and functionality: transaminases, namely, AST and ALT, compared with the results obtained with the in vivo control vehicle-based OSKM hepatic I / R effect. However, no differences in bilirubin levels were observed between the two groups. Although more time may be required to detect changes in BUN measurements after injury, this is because such differences are typically observed with longer reperfusion times. Apoptosis parameters (caspase 3), hepatocyte proliferation (Ki67 / PCNA, HGF), neutrophil accumulation (MPO activity), and oxidative stress (MDA levels) were not significantly different in any of these measurements, suggesting that the observed beneficial effects were not due to changes in oxidative stress, neutrophil accumulation, and / or cell proliferation parameters (see Figures 12A-12H).

[0424] In the 4-day protocol, rats were administered PBS control, vehicle control (AAV-GFP / Luc + AAV-rtTA), or AAV-OSKM (AAV-OSK + AAV cMyc / rtTA) as described above. On day 3 after administration, doxycycline was added to the rats' drinking water (1 mg / mL diluted in drinking water). On day 4 after administration, hepatic I / R was performed as described above. After 4 hours of reperfusion, rats were sacrificed and organs / tissues were harvested. Treatment with AAV-OSK + AAV-cMyc + AAV-rtTA in livers subjected to hepatic I / R induced a decrease in the levels of transaminases (assessed by ALT and AST (see Figures 13A-13B)) and oxidative stress (measured by MDA (see Figure 13E)). No effect was observed on caspase 3, MPO, PCNA, Ki67, HGF, and TGFB levels (see Figures 13C-13D and 13F-13I, respectively).

[0425] For the 2-day protocol, rats were administered PBS control, vehicle control (AAV-GFP / Luc + AAV-rtTA), or AAV-OSKM (AAV-OSK + AAV cMyc / rtTA) as described above. On day 1 after administration, doxycycline was added to the drinking water (1 mg / mL diluted in drinking water). On day 2 after administration, hepatic I / R was performed as described above. After 4 hours of reperfusion, rats were sacrificed and organs / tissues were harvested.

[0426] AST and ALT did not decrease in the OSKM-AAV-treated group compared to controls (PBS a...

Claims

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

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

3. 3. The organ perfusion solution according to claim 1, further comprising at least one tonicity agent.

4. 4. The organ perfusion solution according to claim 1, further comprising at least one of a buffering agent, an inorganic salt, an amino acid, a metabolic substrate, a hormone, an antioxidant, an anti-inflammatory agent, an anticoagulant, or an antibacterial agent.

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

6. 6. The organ perfusion solution according to claim 3, wherein the isotonic agent is selected from the group consisting of dextrose, glycerin, mannitol, potassium chloride, sodium chloride, and combinations thereof.

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

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

9. 9. The organ perfusion solution of claim 8, wherein the oxygenating agent is selected from the group consisting of red blood cells, hemoglobin, pyridoxylated hemoglobin, synthetic hemoglobin-based oxygen carriers, and combinations thereof.

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

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

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

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

14. The organ perfusion solution according to any one of claims 11 to 13, wherein the at least one regeneration factor encoded by the second polynucleotide is selected from the group consisting of an Oct family transcription factor, a Sox family transcription factor, a Klf family transcription factor, and a Myc family transcription factor.

15. The organ perfusion solution according to claim 13 or 14, wherein the Oct family transcription factor is selected from the group consisting of Oct1, Oct3, Oct4, Oct6, and variants thereof.

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

17. The organ perfusion solution according to claim 13 or 14, wherein the Klf family transcription factor is selected from the group consisting of Kfl1, Klf4, Klf5, and variants thereof.

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

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

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

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

23. The organ perfusion solution according to any one of claims 1 to 22, further comprising a regeneration factor-protein transport domain fusion protein.

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

25. The organ perfusion solution according to any one of claims 1 to 24, further comprising an Oct family transcription factor, a Sox family transcription factor, a Klf family transcription factor, and / or a Myc family transcription factor.

26. 26. The organ perfusion solution of claim 25, wherein the Oct family transcription factor is selected from the group consisting of Oct1, Oct3, Oct4, Oct6, and variants thereof.

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

28. The organ perfusion solution according to any one of claims 25 to 27, wherein the Klf family transcription factor is selected from the group consisting of Kfl1, Klf4, Klf5, and variants thereof.

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

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

31. 1. A method for rejuvenating an organ ex vivo, comprising: (a) organ donation; (b) contacting the organ ex vivo with a composition comprising a polynucleotide encoding at least one regeneration factor operably linked to an inducible promoter; (c) adding to the composition a compound that induces the inducible promoter and promotes the expression of the at least one regenerative factor, wherein the addition of the compound results in the expression of the at least one regenerative factor and rejuvenates the organ.

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

33. 33. The method of claim 32, wherein the intermittent administration comprises administering the compound once daily for two consecutive days, followed by five consecutive days without administering the agent.

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

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

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

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

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

39. 39. The method of any one of claims 31 to 38, wherein the organ is provided in a perfusion system selected from the Hugo-Sachs system, the Organ Assist system, the OrganOX system, the Radnoti system, the ARK Kidney system, and the Aferetica PerLife® system.

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

41. 1. A method of transplanting an organ in a subject in need thereof, comprising: (a) organ donation; (b) contacting the organ ex vivo with a composition comprising a polynucleotide encoding at least one regeneration factor operably linked to an inducible promoter; (c) adding to the composition a compound that induces the inducible promoter; (d) transplanting the organ into the subject.

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

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

44. 44. The method of claim 43, wherein the intermittent addition of the compound comprises adding the compound once daily for two consecutive days, followed by five consecutive days without adding the compound.

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

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

47. 1. A method of transplanting an organ in a subject in need thereof, comprising: (a) organ donation; (b) contacting the organ ex vivo with a composition comprising a polynucleotide encoding at least one regeneration factor operably linked to an inducible promoter; (c) transplanting the organ into the subject; and (d) administering to the subject a compound that induces the inducible promoter.

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

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

50. 50. The method of claim 49, wherein the intermittent administration of the compound comprises administering the compound once daily for two consecutive days, followed by five consecutive days without administering the compound.

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

52. 52. The method of claim 51, wherein said intermittent administration of said compound occurs about 2 to 10 times every three months.

53. 53. The method of claim 52, wherein said intermittent administration of said compound occurs about 2 to 10 times every six months.

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

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

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

57. 57. The method of any one of claims 31 to 56, wherein the explanted organ is provided in a perfusion system selected from the group consisting of a Hugo-Sachs system, an Organ Assist system, an OrganOX system, a Radnoti system, an ARK Kidney system, and an Aferetica PerLife® system.

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

59. 59. The method of any one of claims 31 to 58, further comprising taking a biopsy from the organ prior to transplantation.

60. 59. The method of any one of claims 31 to 58, further comprising taking a biopsy of the organ after transplantation.

61. 61. The method of any one of claims 31 to 60, further comprising monitoring the subject transplanted with the organ for organ function.

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

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

64. 1. An ex vivo organ for transplantation into a subject in need thereof, said ex vivo organ comprising (i) a perfusion solution, and (ii) a polynucleotide encoding at least one regenerative factor.

65. 65. The ex vivo organ of claim 64, which is damaged by at least one of aging, ischemic injury, hypertensive injury, toxic injury, perfusion injury, immune injury, physical injury, steatosis, virally induced hepatitis, alcohol, or fibrosis not associated with any known cause.

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

67. 67. The ex vivo organ of any one of claims 64 to 66, wherein the perfusion solution intermittently comprises a compound that induces the inducible promoter.

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